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2017 ACMG GENETICS AND GENOMICS REVIEW COURSE SYLLABUS TAMPA, FLORIDA MAY 4 - 7, 2017 ©American College of Medical Genetics and Genomics 2017 8th Edition All Rights Reserved Printed in the United States of America 2017 ACMG GENETICS AND GENOMICS REVIEW COURSE SYLLABUS CONTENTS Course Information Agenda ................................................................................................................vi Course Faculty ................................................................................................viii Faculty Disclosures and HIPAA Compliance Statement .................................x (Note: Educational Credits are Not Available for the Purchase of the Syllabus Only) Thursday, May 4 ABMGG Review & Exam Preparation Tips ..........................................................................................3 Friday, May 5 Cell Biology/Genomics ........................................................................................17 Genomics - Basics ..............................................................................................51 Clinical Cytogenetics .........................................................................................111 Clinical Molecular Genetics ...............................................................................139 Genetic Transmission........................................................................................177 Newborn Screening ...........................................................................................201 Developmental Genetics ...................................................................................227 Cancer Genetics I .............................................................................................259 Saturday, May 6 Genetic Counseling & Risk Assessment ...........................................................285 Biochemical Genetics I ......................................................................................311 Neurogenetics ...................................................................................................341 Reproductive Genetics I ....................................................................................367 Cancer Genetics II.............................................................................................395 Biochemical Genetics II .....................................................................................421 Systems-Based Disorders I ...............................................................................453 Sunday, May 7 Systems-Based Disorders II ..............................................................................475 Reproductive Genetics II ..................................................................................501 Genomic Medicine.............................................................................................531 Appendix Syndromes Every Geneticist Should Know .......................................................551 Writing Exam Questions ....................................................................................635 Sample Exam Questions & Answers .................................................................651 American College of Medical Genetics and Genomics, 7101 Wisconsin Avenue, Suite 1101, Bethesda, Maryland 20814, www.acmg.net, Telephone: (301) 718-9603 ii 2017 ACMG GENETICS AND GENOMICS REVIEW COURSE COURSE INFORMATION Course Description & Course Format The ACMG Genetics and Genomics Review Course offers a 3 day format that provides an intense learning environment with exam preparation lectures that cover a broad range of genetic and genomic topics presented by recognized experts in the field. Topics include: • Cell Biology/Genomics • Genomics - Basics • Clinical Cytogenetics • Clinical Molecular Genetics • Genetic Transmission • Biochemical Genetics • Developmental Genetics • Cancer Genetics • Genetic Counseling & Risk Assessment • Newborn Screening • Neurogenetics • Reproductive Genetics • Systems-Based Disorders • Genomic Medicine The Course will feature a pre-course sample examination, exam preparation and exam taking tips, interactive examination workshops with in-depth coverage of exam content areas. There will be an interactive breakout sessions with faculty on-site. Course Objectives At the conclusion of this activity, participants should be able to: • Identify common genetic syndromes and discuss their clinical features • Interpret standard molecular data and explain how to communicate results to families • Perform simple quantitative genetic calculations and solve related problems • Describe basic cytogenetics and identify features of common chromosomal disorders • Recognize clinical features of selected metabolic disorders and describe their molecular basis and review how to provide counseling about them • Discuss the extents and limits of prenatal tests and explain how to perform routine prenatal counseling • Explain clinical and molecular aspects of inherited cancer syndromes and know how to provide counseling for common human cancers Course Objectives approved by the NSGC for Genetic Counselors At the conclusion of this activity, participants should be able to: • Identify common genetic syndromes • Interpret standard molecular data • Perform simple quantitative genetic calculations • Describe basic cytogenetics • Recognize clinical features of selected metabolic disorders • Explain how to perform routine prenatal counseling • Explain clinical and molecular aspects of inherited cancer syndromes iii Target Audience This course is designed to assist genetics healthcare professionals who are seeking to update and reinforce their general knowledge of medical genetics. This course is designed to assist genetics healthcare professionals who are seeking to update and reinforce their general knowledge of medical genetics. This course also allows ABMGG individuals to prepare for Board Certification or Recertification. Genetic counselors are also welcome. HIPAA COMPLIANCE The ACMG supports medical information privacy. While the ACMG is not a “covered entity” under HIPAA 1996 and therefore is not required to meet these standards, ACMG wishes to take reasonable steps to ensure that the presentation of individually identifiable health information at ACMG-sponsored events has been properly authorized. All presenters have completed a form indicating whether they intend to present any form of individually identifiable healthcare information. If so, they were asked either to attest that a HIPAA-compliant consent form is on file at their institution, or to send ACMG a copy of the ACMG HIPAA compliance form. This information is on record at the ACMG Administrative Office and will be made available on request. Content Validation ACMG follows the ACCME policy on Content Validation for CME activities, which requires: Content Validation and Fair Balance 1. ACMG follows the ACCME policy on Content Validation for CME activities, which requires: a) All recommendations involving clinical medicine must be based on evidence that is accepted within the profession of medicine as adequate justification for their indications and contraindications in the care of patients. b) All scientific research referred to, reported or used in CME in support or justification of patient care recommendations must conform to the generally accepted standards of experimental design, data collection and analysis. 2. Activities that fall outside the definition of CME/CE; “Educational activities that serve to maintain, develop, or increase the knowledge, skills, and professional performance and relationships that a physician uses to provide services for patients, the public, or the profession” (source: ACCME and AMA) will not be certified for credit. CME activities that promote recommendations, treatment, or manners of practicing medicine or pharmacy that are not within the definition of CME/CE or, are known to have risks or dangers that outweigh the benefits or, are known to be ineffective in the treatment of patients. 3. Presentations and CME/CE activity materials must give a balanced view of therapeutic options; use of generic names will contribute to this impartiality. If the CME/CE educational materials or content includes trade names, where available, trade names from several companies must be used. Off-label Uses of Products When an off-label use of a product, or an investigational use not yet approved for any purpose, is discussed during an educational activity, the accredited sponsor shall require the speaker to disclose that the product is not labeled for the use under discussion, or that the product is still investigational. Discussions of such uses shall focus on those uses that have been subject of objective investigation. iv Disclaimer: This review course is designed as an educational resource for medical geneticists and other health care providers. Its use does not, and should not be considered to ensure a successful outcome on the certification examinations offered by the American Board of Medical Genetics and Genomics or the American Board of Genetic Counseling, or any other examinations. The course should not be considered inclusive of all appropriate information or all available sources of information that may be useful in preparing for the examinations or for any other purpose. The ACMG does not endorse, or recommend the use of this educational program to make patient diagnoses, particular by individuals not trained in medical genetics. Adherence to the information provided in these programs does not necessarily ensure a successful diagnostic outcome. The program should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed at obtaining the same results. In determining the propriety of any specific procedure or test, a healthcare provider should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. v 2017 ACMG GENETICS AND GENOMICS REVIEW COURSE AGENDA Thursday, May 4, 2017 4:00 pm – 7:00 pm Registration 7:00 pm - 7:30 pm Introduction to Course Bruce R. Korf, MD, PhD, FACMG 7:30 pm – 8:30 pm ABMGG Review and Exam Preparation Tips Miriam G. Blitzer, PhD, FACMG Gary S. Gottesman, MD, )$$3, FACMG Friday, May 5, 2017 7:30 am – 8:00 am Continental Breakfast/Registration 8:00 am – 9:00 am Cell Biology/Genomics Christa Lese Martin, PhD, FACMG 9:00 am – 10:00 am Genomics - Basics Madhuri Hegde, PhD, FACMG 10:00 am –10:30 am Break 10:30 am – 11:30 am Clinical Cytogenetics Christa Lese Martin, PhD, FACMG 11:30 am – 12:30 pm Clinical Molecular Genetics Madhuri Hegde, PhD, FACMG 12:30 pm – 2:00 pm Lunch (on your own lunch) 2:00 pm – 3:00 pm Genetic Transmission Bruce R. Korf, MD, PhD, FACMG 3:00 pm – 4:00 pm Newborn Screening John A. Phillips, III, MD, FACMG 4:00 pm – 4:30 pm Break 4:30 pm – 5:30 pm Developmental Genetics Tony Wynshaw-Boris, MD, PhD, FACMG 5:30 pm – 6:30 pm Cancer Genetics I Sharon Plon, MD, PhD, FACMG 6:30 pm – 7:30 pm Dinner (on your own) 7:30 pm – 9:00 pm Advanced Genetic Topics Informal Sessions • Quantitative • Biochemistry • Molecular • Cytogenetics • Prenatal • Genetic Counseling This session is not live streamed or recorded vi Faculty Saturday, May 6, 2017 7:30 am – 8:00 am Continental Breakfast 8:00 am – 9:00 am Genetic Counseling & Risk Assessment Pamela L. Flodman, MSc, MS, LCGC 9:00 am – 10:00 am Biochemical Genetics I Gerard Berry, MD, FACMG 10:00 am – 10:30 am Break 10:30 am – 11:30 am Neurogenetics Bruce R. Korf, MD, PhD, FACMG 11:30 am – 12:30 pm Reproductive Genetics I Louise E. Wilkins-Haug, MD, PhD, FACMG 12:30 pm – 2:00 pm Lunch (on your own lunch) 2:00 pm – 3:00 pm Cancer Genetics II Sharon Plon, MD, PhD, FACMG 3:00 pm – 4:00 pm Biochemical Genetics II Gerard Berry, MD, FACMG 4:00 pm – 5:00 pm Systems-Based Disorders I Bruce R. Korf, MD, PhD, FACMG 05:00 pm – 6:30 pm Dinner (on your own) 6:30 pm – 8:00 pm Exam Workshop 8:00 pm – 9:00 pm Networking Reception Gary S. Gottesman0')$$3 FACMG Sunday, May 7, 2017 7:30 am – 8:00 am Continental Breakfast 8:00 am – 9:00 am Systems-Based Disorders II John A. Phillips, III, MD, FACMG 9:00 am – 10:00 am Reproductive Genetics II Louise E. Wilkins-Haug, MD, PhD, FACMG 10:00 am – 10:30 am AM Break 10:30 am – 11:30 am Genomic Medicine Bruce R. Korf, MD, PhD, FACMG 11:30 am – 12:00 pm Course Wrap Up Bruce R. Korf, MD, PhD, FACMG John A. Phillips, III, MD, FACMG vii 2017 ACMG GENETICS AND GENOMICS REVIEW COURSE COURSE FACULTY COURSE DIRECTORS John A. Phillips, III, MD, FACMG David T. Karzon Professor of Pediatrics Professor of Pathology, Microbiology and Immunology and Professor of Medicine Director, Division of Medical Genetics & Genomic Medicine Vanderbilt University School of Medicine DD-2205 Medical Center North Nashville, TN 37232-2578 Tel: (615) 322-7602 [email protected] Bruce R. Korf, MD, PhD, FACMG Wayne H. and Sara Crews Finley Chair in Medical Genetics Professor and Chair, Department of Genetics Director, Heflin Center for Genomic Sciences University of Alabama at Birmingham 1720 2nd Ave. S., Kaul 230 Birmingham, AL 35294 Tel: (205) 934-9411 [email protected] FACULTY Gerard Berry, MD, FACMG Harvey Levy Chair in Metabolism Director, Metabolism Program Division of Genetics and Genomics Boston Children’s Hospital Professor of Pediatrics, Harvard Medical School, Center for Life Science Building, Suite 14070 3 Blackfan Circle Boston, MA 02115 Tel: (617) 355-4316 [email protected] Madhuri Hegde, PhD, FACMG Associate Professor Emory Genetics Lab Scientific Director Sr. Director, Emory Genetics Lab, Molecular Lab Department of Human Genetics Emory University School of Medicine 2165 North Decatur Road Decatur, GA 30033 Tel: (404) 727-5624 [email protected] Pamela L. Flodman, MSc, MS, LCGC Adjunct Professor, Pediatrics School of Medicine Director, Graduate Program in Genetic Counseling Department of Pediatrics University of California, Irvine 101 The City Drive Mail Code: 4482 Orange, CA 92868 Tel: (714) 456-5789 [email protected] Christa Lese Martin, PhD, FACMG Autism & Developmental Medicine Institute Geisinger Health System 120 Hamm drive, suite 2A, M.C. 60-36 Lewisburg, PA 17837 Tel: (570) 522-9427 [email protected] Sharon E. Plon, MD, PhD, FACMG Professor, Pediatrics/Hematology-Oncology Professor, Molecular and Human Genetics Human Genome Sequencing Center Director, Medical Scientist Training Program Department of Pediatrics Baylor College of Medicine Feigin Center Room 1200.18 1102 Bates Street Houston, TX 77030 Tel: (832) 824-4251 [email protected] Gary S. Gottesman, MD, FAA3, FACMG Medical Geneticist Center for Metabolic Bone Disease Shriners Hospitals for Children - St. Louis 2001 S. Lindbergh Blvd. St. Louis, MO 63131 Tel: (314) 872-8305 [email protected] viii Louise E. Wilkins-Haug, MD, PhD, FACMG Division Director, Maternal Fetal Medicine and Reproductive Genetics Brigham & Women's Hospital Professor, Obstetrics/Gynecology Harvard Medical School 75 Francis Street Boston, MA 02115 Tel: (617) 732-4208 Fax: (617) 264-6310 [email protected] CONTRIBUTOR Miriam G. Blitzer, PhD, FACMG Executive Director American Board of Medical Genetics and Genomics 9650 Rockville Pike Bethesda, MD 20814-3998 Tel: (410) 706-1429 [email protected] Tony Wynshaw-Boris, MD, PhD, FACMG James H. Jewell Professor of Genetics Chair, Department of Genetics and Genome Sciences Case Western Reserve University, School of Medicine University Hospitals Case Medical Center One 10900 Euclid Avenue, BRB731 Cleveland, OH 44106-4955 Tel: (216) 368-0581 [email protected] ix FACULTY DISCLOSURES As a sponsor accredited by the ACCME, the American College of Medical Genetics and Genomics must ensure balance, independence, objectivity and scientific rigor in all its sponsored educational activities. All faculty participating in a CME-certified activity are expected to disclose to the audience any relevant financial interest(s) or other relationship(s) with the manufacturer(s) of any commercial product(s), provider(s) of commercial services or any commercial supporters, including diagnostic laboratories, of the activity discussed in an educational presentation. Relevant financial interest(s) or other relationship(s) can include such things as grants or research support, consultancy, major stock holder, etc. The intent of this disclosure is not to prevent a planner or speaker with a relevant financial or other relationship from course planning or making a presentation, but rather to provide learners with information on which they can make their own judgments. It remains for the audience to determine whether the speaker's interests or relationships may influence the presentation with regard to exposition or conclusion. All conflicts of interests have been reviewed and resolved by the education and CME subcommittee. The following have reported disclosures: Bruce R. Korf, MD, PhD, FACMG Dr. Korf receives grant/research support from Novartis. He is on the Advisory Board for Accolade and Genome Medical. He is on the Board of Directors for the American College of Medical Genetics and Genomics and the Children’s Tumor Foundation. He is an advisor for the Neurofibromatosis Therapeutic Acceleration Project, a Founding Member of Envision Genomics. He receives a salary from the University of Alabama at Birmingham. Madhuri Hegde, PhD, FACMG Dr. Hegde is an advisor for Genzyme (Pompe program), PTC (DMD program), Coriell Cell Repositories, and PerkinElmer Genetics Inc. Christa Lese Martin, PhD, FACMG Dr. Martin is employed by Geisinger Health System and is a consultant the The Jackson Laboratory. John A. Phillips, III, MD, FACMG Dr. Phillips is a Site Investigator for the following: 1) PKU: BMN 015 &165, 2) Achondroplasia: BMN 111-901, 201 & 202 Clinical Trials BioMarin Pharmaceutical Inc. & 3) FAOD: UX007 Ultragenyx Pharmaceuticals, Inc. He is Principal Investigator for TN State Genetics Contract & Member TN Genetics Advisory Committee. A Co-PI: Vanderbilt Undiagnosed Disease Network (UDN) Clinical Center and a Co-I: Dr Blackwell’s PPG “Mechanisms of Familial Pulmonary Fibrosis”. Sharon Plon, MD, PhD, FACMG Dr. Plon is an employee of Baylor College of Medicine (BCM) which derives revenue from genetic testing, including whole exome sequencing. BCM and Miraca Holdings Inc. have agreed on a joint venture with shared ownership and governance of the clinical genetics diagnostic laboratories. Dr. Plon will also discuss off-label use and/or investigational use of drug target tumors. The following faculty members have nothing to disclose: Gerard Berry, MD, FACMG Pamela L. Flodman, MSc, MS, LCGC Gary S. Gottesman, MD, FAAP, FACMG Louise E. Wilkins-Haug, MD, PhD, FACMG Anthony J. Wynshaw-Boris, MD, PhD, FACMG Miriam G. Blitzer, PhD, FACMG Jane Radford, MHA, CHCP (ACMG Staff) x Exam Preparation Tips EXAM SKILLS WORKSHOP Gary S Gottesman, MD, FAAP, FACMG Medical Geneticist Center for Metabolic Bone Disease and Molecular Research Shriners Hospitals for Children – Saint Louis Gary S. Gottesman, MD, FAAP, FACMG Center for Metabolic Bone Disease and Molecular Research Shriners Hospitals for Children – Saint Louis 4400 Clayton Ave. St. Louis, MO 63110 (314) 872-8305 Telephone (314) 872-7844 Fax [email protected] 1 2 Examination Skills Workshop Gary S Gottesman, MD Medical Geneticist Center for Metabolic Bone Disease and Molecular Research Shriners Hospital for Children – Saint Louis Disclosure(s) I have no conflicts of interest to disclosure ABMGG Website • Training & Certification • ITE Practice Exam (External Website) • http://www.starttest.com/ITDVersions/11.1.0.1/ITD Start.aspx?SVC=7478ee5d-5cf8-4042-a4790038337ce40b 3 ABMGG Website Exam Tutorial • Overview of Your Test • Tutorial • Exam Section 1 • Exam Section 2 • Exam Section 3 • Exam Section 4 • Total Session Time 1 Hour 15 Minutes Items 5 5 5 5 20 Exam Tutorial • Navigating Throughout Your Exam • Information Panel • Scrolling the Screen • Zooming • Navigation Panel • Keyboard Accessibility • Button 4 Exam Tutorial • Time Management • Section Time Remaining • Session Time Remaining • Examination Overview • Unauthorized Breaks • Taken during test block • Timer continues to run Exam Tutorial • Answering and Marking Items • Click with your choice of answer with the mouse • You may change your answer at any time • You may opt to mark an item with “Mark” check box Exam Tutorial Help Menu • Highlight (Keywords) • Strike Out (Distractors) • Notes Calculator 5 Exam Tutorial • Overview of the Review Screen • Item Review Screen • Can be accessed at any point • Select Review Button • Appears after the last item in the section • If time remains: • You can return to • Incomplete • Marked items Exam Item Formats • Matching Format • This item format consists of a series of items related to a common logic. • Item set may be used for multiple problems. Exam Tutorial • Multiple Item Format • “The following vignette applies to the next 2 items.” • May also be a single option set that is associated with multiple vignettes: • “The response options for the next 4 items are the same. Select one answer for each item in the set.” • “For each description, select the associated disorder (A-E).” 6 Exam Tutorial • End of Examination Survey • End of Session Notice NBME Resources • www.nbme.org • Register with NBME – Customer Access Portal • Find Lessons • Item Writing Manual - 2016 • Download this manual (>90 pages) • Recommend looking at Appendix B • Sample Lead-Ins • Online Interactive Tutorial • http://www.nbme.org/IWTutorial Template for NBME Exam Questions • Stem: • Typically includes a vignette that provides some clinical relevance to the topic being addressed in the question. The vignettes are supposed to be as succinct as possible. 7 Template for NBME Exam Questions • Stem: • The stem ends with a question composed in the following manner: • “Which of the following . . (identify the characteristic that links all the distractors) . . is the best (or most likely) response . . ?” • What it the chance that the . . . (numerical answer) ? Template for NBME Exam Questions • Option Set: A. Options should be lettered from A to E (must have 5 total) B. Place options in alphabetical order based on first word, second word, etc. or numerical order (increasing or decreasing) C. Distractors should all be about the same length as the correct option D. Avoid: “All of the above,” EXCEPT, NOT, Least, etc. E. Avoid multiple-multiple choice problems Template for NBME Exam Questions • Test takers should be able to read the stem and come up with possible answers and the correct answer without ever referring to the option set. 8 Test Taking • READ the stem • Note key words or key information • Highlight them if the stem is long • SIMPLIFY stem • Translate stem into your own words • ANSWER the question • Answer in your own words before looking at options Test Taking • ELIMINATE answers that are unlikely or include absolutes • Never, always, every, at no time, etc. • Answers that mean basically the same thing • COMPUTATION • Determine formula or principle required • Complete computation before looking at options • If your answer is not in option set check your work Test Taking • REMEMBER • Longest answer is often the correct one • Loaded with qualifying adjectives or phrases • Key words from stem in option may identify it as correct • CHOOSE the best answer • Correct choice often contains relative qualifier • Usually, generally, sometimes, often, etc. 9 Test Taking • GUESSING STRATEGY • If two options are opposites one is likely correct • If you cannot identify answer – guess • There is no penalty for guessing. • Always guess the same answer • Unless you can eliminate it Additional Recommendations • Make sure you get a good night’s sleep prior to the exam. • Consider reading a medical school genetics text from cover to cover for general exam preparation. Additional Recommendations • Study photos and common signs and symptoms of genetic disorders from a dysmorphology text and GGRC 100 • Syndromes section (few photos/x-rays). • Check with others who took the test in the past for any study aides they may have used. 10 Additional Recommendations • Arrive at least 30 minutes prior to your exam • Take some snacks to eat during breaks. Genetics Education Resources • Genetics texts: • Thompson & Thompson Genetics in Medicine by Nussbaum, McInnes and Willard • USMLE-type questions • Medical Genetics by Jorde & Carey • medgen.genetics.utah.edu/index.htm • Human Genetics and Genomics (Human Genetics: A ProblemBased Approach) by Bruce R. Korf, MD, PhD and Mira B Irons (Fourth edition) • http://www.korfgenetics.com/ Genetics Education Resources • Genetics Websites • GeneReviews.org • Genetics Education Center (KUMC) • http://www.kumc.edu/gec/ • University of Michigan Medical School http://www.med.umich.edu/lrc/coursepages/M1/humange netics/genetics/humangeneticsquiz.html • Not in NBME format 11 Acknowledgements Debra L. Schindler, PhD, Office of Curricular Affairs, Saint Louis University SOM References www.abmgg.org www.nbme.org Barbara Swanson, I.S.U. Learning Strategies – http://www2.isu.edu/success/strategies/handouts/docs/test_taking_and_money/Systematic%20Method%20of%20Answering%20Multiple%20Choice%20Questions.pdf http://www.studygs.net/tsttak3.htm 12 Cell Biology/Genomics CELL BIOLOGY GENOMICS Christa Lese Martin, PhD, FACMG Geisinger Health System Autism & Developmental Medicine Institute Director and Senior Investigator Christa Lese Martin, PhD, FACMG Autism & Developmental Medicine Institute Geisinger Health System 120 Hamm Drive, Suite 2A, M.C. 60-36 Lewisburg, PA 17837 (570) 522-9427 Telephone (570) 522 9431 Fax [email protected] 15 16 Cell Biology: Chromosomes in Gametogenesis and Cell Division Christa Lese Martin, PhD, FACMG Director and Professor Autism & Developmental Medicine Institute, Geisinger Health System Disclosure(s) Employed by Geisinger Health System Consultant for The Jackson Laboratory Overview • • • • History of Cytogenetics Chromosome Structure Mitosis and Meiosis Numerical and Structural Chromosome Abnormalities 17 History of Cytogenetics 1923: T.S. Painter established human chromosome number as 48 and identified the Y chromosome (XX/XY mode of sex determination) 1953: T.C. Hsu accidentally discovered hypotonic treatment of cells to spread chromosomes 1956: Only 46 chromosomes in humans by Tjio & Levan 1959: Trisomy 21 causes Down Syndrome, by Jerome Lejeune in Paris. 1st chromosome abnormality, birth of clinical cytogenetics 1969-1970: Banding techniques discovered by Caspersson in Sweden (fluorescent Qbanding first, followed by G-banding and others) 1970-75: Amniocentesis shown to be safe and accurate method for prenatal diagnosis 1976: “High-resolution” methods developed by J. Yunis 4 History of Cytogenetics (cont’d) 1976-1980: Fragile sites on human chromosomes re-discovered, including fragile X 1982-87: CVS introduced and shown to be safe and effective for first trimester prenatal diagnosis 1990: FISH – molecular cytogenetics 1990: Genomic imprinting and UPD understood as important mechanisms of human genetic disease 1992: CGH (Comparative Genomic Hybridization) developed for solid tumor studies 1996: 24 color FISH (including SKY, spectral karyotyping) 2001: Cytogenomic microarrays, including array CGH 2010: Cytogenomic microarrays become the first-tier test replacing G-banded karyotype 5 ADD DATE FOR CNV CALLING FROM WES/WGS DATA Chromosome Structure Challenges of DNA packaging: Each human cell contains ~2 meters of DNA if stretched end-end; yet the nucleus of a human cell is only ~6 μm in diameter DNA packaging into a set of chromosomes: • The complex of DNA + proteins is called “Chromatin” • These proteins include: 1. Proteins involved in DNA packaging 2. Proteins associated with gene expression, DNA replication and DNA repair • Each human cell contains two copies of each chromosome (one maternal and one paternal). These pairs are called “Homologous Chromosomes” 18 General model for the many levels of chromatin packaging Molecular Biology of the Cell, 4th edition. Alberts B, Johnson A, Lewis J, et al. Garland Science; 2002 Chromosome Structure Metacentric Submetacentric Acrocentric telomere satellite short arm - p centromere stalk long arm - q chromatid telomere • Metaphase chromosomes are comprised of two sister chromatids connected at the centromere (primary constriction) • All human chromosomes are bi-armed telomere 3 Chromosome Structure Acrocentrics (13, 14, 15, 21, and 22) are a special class of human chromosomes. They have very small p arm comprised of large, tandem arrays of rDNA genes (stalks; stain positive only by silver staining). “Associate” in interphase to form nucleolus (also called nucleolus organizer regions or NORs). At end of stalks are “satellites” (which are made up of highly repetitive “junk” DNA and no known coding sequences) which stain positive by C-banding. Length/size of stalks and satellites are polymorphic. 19 G-banded Karyotype acrocentric Image from ISCN 2013 Courtesy of N.L. Chia G-banding pattern and DNA content G negative (light bands) G positive (dark bands) Higher GC content Lower AT content Lower GC content Higher AT content Rich in SINE and Alu repeats Rich in LINE repeats Early replicating Late replicating Contain housekeeping genes Genes are tissue specific Rich in transcribed sequences Sparse genes Human genome sequencing project supports these findings G-banded Ideograms Left - Computer generated ideogram Right – ISCN 1995 schematic and measurements 20 Centromeres and Telomeres of Human Chromosomes Centromere Structure Alpha-satellite DNA: í 171 bp tandemly repeated sequence clustered at all human centromeres í 300 kb - 5,000 kb present at each human centromere í Sequence divergence in 171 bp repeat allows chromosome-specific alpha-satellite FISH probes (except 13 and 21, 14 and 22, whose sequence are too similar for unique hybridization) í The inner plate of a kinetochore is “seeded” in the centromere, followed by cooperative assembly of the entire group of special proteins that form the kinetochore Telomere Structure TTAGGG - simple sequence repeat: í 3-20 kb (T2AG3) n í Substrate for telomerase (solves the end replication problem) í Shortens with age í True terminal deletions can generate new telomeres TAR (telomere associated repeats); aka “subtelomeric repeats”: í 100-300 kb most chromosome ends í Sequence homologies to subsets of chromosomes Chromosome specific (unique) DNA: Most begin ~200-300 kb from end of chromosome 21 Telomere Structure – cont’d í Highest gene density in human genome (high GC content; G-negative bands) í Very high genetic recombination in females and males, but especially in males (only chromosome regions male recombination is higher than females) í Critical role in meiosis: synapsis of homologous chromosomes begins at telomeres í Polymorphic: evidence of length and sequence polymorphism for some human telomeres (e.g., 16p has 3 alleles; 2q deletion) Mitosis and Meiosis Mitosis growth and development of somatic cells one cell divides to give rise to two identical “daughter” cells – – Meiosis production of germ cells diploid cells of the germline give rise to haploid gametes (sperm/egg) – – Mammalian Cell Cycle alternation between mitosis and interphase (resting state) Mitosis PM AT0 hours G2 25 4 -6 hrs 2n, 4c 5 G1 Phase S Phase 12 – 24 hrs 7 hrs 2n, 2c 20 2n, 3c S - DNA replication occurs (synthesis of RNA and proteins) G2 - interval between S and mitosis (repair occurs) 10 15 Interphase (DNA decondensed): G1 - interval between mitosis and replication Mitosis and Cytokinesis: cell division processes (nuclear and cytoplasimic division) 22 Mammalian Cell Cycle n = haploid chromosome # Mitosis PM AT0 hours c = DNA content G2 25 4 -6 hrs 2n, 4c S Phase 7 hrs 20 2n, 3c 46, each chromosome has 2 chromatids 5 G1 Phase 46, each chromosome has 1 chromatid 12 – 24 hrs 2n, 2c 10 15 Many sources use “n” incorrectly to refer to DNA content; it is important to understand the difference Mitosis •Prophase Chromosomes condense Mitotic spindle/centrioles begin to form Figure 2-10 from Thompson & Thompson, Genetics in Medicine, edition 7, copyright 2007, Elsevier Mitosis •Prometaphase Nuclear membrane disappears Figure 2-10 from Thompson & Thompson, Genetics in Medicine, edition 7, copyright 2007, Elsevier 23 Mitosis •Metaphase Chromosomes fully condensed Chromosomes line up on the “metaphase plate” Spindle fibers begin to contract Figure 2-10 from Thompson & Thompson, Genetics in Medicine, edition 7, copyright 2007, Elsevier Mitosis •Anaphase Centromeres divide in two Spindle fibers pull chromatids toward opposite sides of the cell (centromere first)) Figure 2-10 from Thompson & Thompson, Genetics in Medicine, edition 7, copyright 2007, Elsevier Mitosis •Telophase Cytokinesis (forms two identical daughter cells) Two nuclear membranes form Spindle fibers disappear Chromosomes decondense and return to interphase Figure 2-10 from Thompson & Thompson, Genetics in Medicine, edition 7, copyright 2007, Elsevier 24 Mitosis Summary 1 1 1 2n2c 1 1 2n4c 1 1 1 2n2c 1 1 1 1 1 1 (only showing chr 1) 1 1 Meiosis Meiosis is a specialized form of cell division that occurs only during gametogenesis • comprised of 1 round of DNA replication, but 2 cell divisions -- M I and M II • divides genetic material in half • shuffles genetic material by recombination Meiosis Meiosis I • reduction division (46ї23, 2n ї 1n, diploid ї haploid) • occurs only in meiosis NO INTERVENING DNA REPLICATION Meiosis II • identical to mitosis in somatic cells, except only 23 chromosomes are present 25 Male Meiosis I MI: 2n,2c Male Meiosis II MII 1n,2c 2n,4c 1n,2c 1n,1c From Vogel and Motulsky, Figure 3.34, 4th edition, Copyright 2010, Springer MI: 2n,2c From Vogel and Motulsky 2n,4c 1n,2c From Vogel and Motulsky, Figure 3.34, 4th edition, Copyright 2010, Springer Prophase I: - leptotene: chromosomes begin to condense - zygotene: homologs pair (telomere); Synaptonemal complexes form - pachytene: crossing over occurs - diplotene: homologs separate; remain attached at chiasma - diakinesis: separation of homologs MII: 1n,2c ĺ 1n,1c. 4 gametes in males. Note recombinant and nonrecombinant chromosomes. MII 1n,2c 1n,1c From Vogel and Motulsky, Figure 3.34, 4th edition, Copyright 2010, Springer 26 1 Recombination Only one sister chromatid involved in each crossover event • Centimorgan used to describe recombination frequency; 1 cM equals 1% chance for recombination; translates to ~1 Mb •Avg. 50 chiasmata in male meiosis; Number correlates with length of chromosome arms • ≥ 1 chiasma/chromosome arm is required for normal segregation Consequences of Meiosis Random segregation during Meiosis I makes the likelihood of any two gametes from an individual having the exact same chromosomes equal to 1 in 223 (1 in 8 million) Shuffling of the DNA through recombination makes the above likelihood even smaller That’s why no two people are exactly the same!! Meiosis I Summary – homologs separate (only showing chr 1 and chr 4) 1 11 1 2n4c 1 11 1n2c 11 4 44 4 1 4 1 4 4 44 44 1n2c 1 4 27 Meiosis II Summary – chromatids separate Essentially same as mitosis except only 23 chromosomes are present 1n2c 1n2c 1 1 4 4 1n1c 1n1c 1 1 1 4 4 4 1 4 Female vs. Male Meiosis Male Commences Puberty Duration of meiosis 60-65 days Gametes/ meiosis 4 spermatids Gamete production 100-200 million per ejaculate Female Early embryonic life 10-50 years Only complete after fertilization 1 ovum and 2 (3) polar bodies 1 ovum per menstrual cycle Begins prenatally Arrested in first meiotic prophase = dictyotene (does not occur in males) Female Meiosis Meiosis I completed at ovulation 1n, 2c 2n, 2c 1n, 1c From Vogel and Motulsky, 3rd edition, Springer 28 Male vs Female Meiosis Image from Pearson Education http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-20/20_09.jpg Errors of Meiosis and Mitosis: Nondisjunction • Failure of homologous chromosomes (MI) or sister chromatids (MII, mitosis) to separate; • Trisomy and monosomy can originate from meiotic or mitotic nondisjunction; • Parental origin of the extra chromosome in trisomy is most often maternal and most often a result of an M I error. Mitotic Nondisjunction – Somatic: Results in Mosaicism 46 46 46 46 46 46 46 NORMAL 47 46 47 45 47 45 45 MOSAICISM (45 cell line often not viable) Don’t confuse mosaicism with chimerism! Most common form: two zygotes fuse to form one embryo 29 Meiotic Nondisjunction 1st meiotic division nondisj. nl nl 2nd meiotic division nl. nl nondisj. Meiotic Nondisjunction (cont’d) fertilization MI nondisj. trisomy and monosomy MII nondisj. Normal, trisomy & monosomy Meiotic Origin of Nondisjunction MI MII Molecular genetic polymorphic markers can be used to determine at which stage of meiosis the error occurred. MI errors will show three different homologs. MII errors will show only two different homologs. 30 Parental Origin of Aneuploidy % mat MI MII % pat 92 75 25 8 3 +21 +18 97 31 69 +16 100 100 0 0 XXX 90 78 22 10 XXY 54 54 74 26 46 X 30 30 MI MII 100 70 XXX cases are the same as autosomal trisomies. XXY: mat = pat nondisjunction. 45,X is mainly paternal nondisjunction. Maternal Age Effect – Trisomy 21 Incidence (per 1,000 births) 25 20 15 10 5 0 20 25 30 35 40 Mother’s Age 45 Meiotic Origin of Nondisjunction Studies have shown that aberrant recombination is also associated with nondisjunction: • Absent (achiasmate) or reduced recombination - reported for nearly all acrocentric trisomies, some non-acrocentrics (chr 18 and sex chromosomes) • Suboptimally located crossovers - exchanges among MII nondisjunction tri21 cases clustered at the pericentromeric region (not tel) – suggesting location may also cause susceptibility to MI or MII non-disjunction (Lamb et al., 1996; Lamb et al., 1997; Brown et al., 2000; Garcia-Cruz et al., 2009) 31 Meiotic Errors Leading to Aneuploidy Most errors occur at Meiosis I (MI) Non-disjunction is associated with abberations in both the frequency and placement of chiasmata: 9 Too few recombinations 9 Too close to the centromere 9 Too distal Premature separation of sister chromatids: another mechanism for aneuploidy in meiosis Hassold and Hunt, Nature Vol2:280-292 (2001) Multiple Mechanisms Contribute to Maternal Age Effect Human maternal age effect involves different “hits” that work together to increase frequency of errors in eggs • Decreased interactions between homologous chromosomes in prophase during recombination (females seem to be more prone) • Long prophase arrest in females • Failed repair checkpoints in oocytes with longer arrest time (males seem to maintain better checkpoints and default to spermatocyte “death”) Nagaoka et al. (2012) Nat Rev Genet Frequency of Chromosome Abnormalities • Spontaneous miscarriage (first trimester) 50.0 % • ~15% of all pregnancies result in miscarriage • Stillbirths and perinatal deaths 5.0 % • Livebirths • Congenital anomaly with ID • Congenital heart disease 0.5 - 1% 23.0 % 13.0 % • Couples with multiple spontaneous abortions (balanced rearrangements) 5.0 % 32 First Trimester Spontaneous Miscarriage ~15% of all first trimester pregnancies end as miscarriage: NORMAL CHROMOSOMES 40 % ABNORMAL CHROMOSOMES Autosomal trisomy - most common tri 16 (~16%); never observed in liveborns Monosomy X (~99% abort) Polyploidy Structural abnormalities 60 % 50 % 25 % 20 % <5 % Numerical Chromosome Abnormalities EUPLOID = exact multiple of haploid set • Haploid = normal number in gametes (n=23 in humans) • Diploid = normal number in zygote and somatic cells (2n=46) • Polyploidy = complete set(s) of extra chromosomes Triploidy = 3n = 69/cell in humans Tetraploidy = 4n = 92/cell in humans ANEUPLOID = loss/gain of single chromosomes • Monosomy = chromosome number = 45 • Trisomy = chromosome number = 47 Triploid Karyotype 69,XXY 1 2 3 6 7 8 13 14 15 19 20 9 21 Tetraploid Karyotype 92,XXYY 4 5 10 11 12 16 17 18 22 X 1 2 3 6 7 8 13 14 15 19 20 9 21 4 5 10 11 12 16 17 18 22 X Y Y No increased risk for recurrence 33 Mechanism of Triploidy Dispermy (66%) - most common cause Meiotic failure (34%) - in spermatogenesis (24%) - in oogenesis (10%) Paternal large placenta small fetus partial hydatidiform mole IUGR oligohydramnios congenital heart defect syndactyly Maternal small placenta large fetus early loss Numerical Chromosome Abnormalities EUPLOID = exact multiple of haploid set • Haploid = normal number in gametes (n=23 in humans) • Diploid = normal number in zygote and somatic cells (2n=46) • Polyploidy = complete set(s) of extra chromosomes Triploidy = 3n = 69/cell in humans Tetraploidy = 4n = 92/cell in humans ANEUPLOID = loss/gain of single chromosomes • Monosomy = chromosome number = 45 • Trisomy = chromosome number = 47 Gene Dosage Effects in Cytogenetic Disorders • Normal chromosome and gene copy number is two (one paternal, one maternal) - but can see Copy Number Variants (CNVs) • Whole chromosome aneuploidy (gain or loss) usually lethal (except sex chromosomes) • Monosomy more severe/lethal than trisomy (no viable autosomal monosomies) 34 Gene Dosage Effects in Cytogenetic Disorders • Monosomy and deletions cause more severe phenotypic consequences than trisomy and duplications - No viable autosomal monosomies (only 45,X) • Larger imbalances (more genes) more severe phenotype than smaller imbalances • Imbalance of G-negative bands (gene-rich) more severe than Gpositive bands (gene-poor) (chr 13, 18, 21 – all gene poor) Chromosome Abnormalities in Newborns Abnormality Birth Frequency Trisomy 21 Trisomy 18 Trisomy 13 47,XXY (Klinefelter synd.) 47,XYY 47,XXX 45,X (Turner synd.) 1 in 800 1 in 6000 1 in 10000 1 in 1000 males 1 in 1000 males 1 in 1000 females 1 in 5000 females Most chromosome abnormalities are prenatal lethals 35 Trisomy 21: 47,XX,+21 1 2 3 6 7 8 13 14 15 19 20 9 21 4 5 10 11 12 16 17 18 22 X Y Common Features - Moderate ID - Hypotonia - Almond-shaped eyes, epicanthal folds - Depressed nasal bridge - Brachycephaly (round head shape w/ flattened occiput) - Excess nuchal skin - Simian crease; clinodactyly - Heart defect - 40% (most A-V canal) ~95% free trisomy; others translocation, mosaicism Image from: ttp://2011gtms8f.wikispaces.com/trisomy+13,18,21+Diego+r Trisomy 18: 47,XY,+18 1 2 3 6 7 8 13 14 15 19 20 9 21 4 5 10 11 12 16 17 18 22 Common Features • Severe ID • IUGR, microcephaly • Weak, feeble activity and cry (hypoplastic muscles) • Clenched hand position w/ 2 & 5 over 3 & 4 • Clubfoot or rocker-bottom feet • Small, low set ears • Omphalocele • Early lethality • most die in first month; 5-10 % survive > 1 yr X Y Images from: http://library.med.utah.edu/WebPath/jpeg3/PERI228.jpg medgen.genetics.utah.edu Trisomy 13: 47,XY,+13 1 2 3 6 7 8 13 14 15 19 20 9 21 4 5 10 11 12 16 17 18 22 X Common Features • Severe ID • IUGR, microcephaly, • Midline anomalies • cleft lip/palate, holoprosencephaly, scalp defect, CHD, omphalocele • Polydactyly, postaxial • Early lethality • most die in first month; 5% survive > 6 months Y Image from: Medscaape.com 36 Structural Chromosome Abnormalities Terminal Deletion Interstitial Deletion Duplication Translocation OR Pericentric Inversion Ring Isochromosome Marker Reciprocal translocations Incidence: 1 in 500 Most common: t(11;22)(q23;q11) Most balanced translocations have normal phenotype, unless bkpt disrupts a gene or deletion occurs at bkpt. If balanced de novo translocation in prenatal setting, ~5% risk of some developmental delay or other abnormality. t(2;8)(q33;q24.1) q24.1 der(8) 2 der(2) 8 der(2) 8 q33 der(8) 2 37 -----------Common------------- Theoretical: 25% 25% Empirical: 85-90% -----Rare----- 25% 25% 10-15% Reciprocal translocations Meiosis: Pairing of translocation products to form quadrivalents. Major segregation products are: alternate (normal and balanced) adjacent 1 (two unbalanced forms). Four products with theoretical risk of 50% unbalanced, but empirical risk is 10-15% abnormal. Adjacent 2 products are more rare, and have two identical centromeres in each gamete (probably occurs less often). They represent much greater imbalance (less viable). Robertsonian Translocation • Incidence: 1 in 1,000 • A translocation between two acrocentric chromosomes (13, 14, 15, 21, 22) resulting in loss of the short arms of both chromosomes, but does not affect the DNA content of the long arms. Usually dicentric. • rob(13;14) most common 21 45,XY,der(14;21)(q10;q10) OR 45,XY,rob(14;21)(q10;q10) 14 21 14 38 Robertsonian Translocation In Meiosis, Robertsonian translocations form a trivalent configuration. Alternate segregation produces roughly equal frequencies of normal and balanced gametes. Adjacent 1 segregation produces four potential gametes, although both monosomies are lethal and trisomy 14 in this example is lethal. Although T21 has a theoretical risk of 33% in this situation, the empiric risk is 10-15% in female carriers and 0-2% in male carriers. Risks for UPD – Robertsonian Translocations Homologous and non-homologous Rob. translocation involving chromosome 14 or 15; Inherited or de novo For prenatally identified non-homologous [e.g. rob(13q14q)], risk for UPD is ~0.6%; for homologous risk is ~66%. Shaffer et al., ACMG Statement for UPD Genet Med. (2001), vol 3(3), pg 206-11 39 Inversions A B C D A C B D A B C D E A D C B E per-i-centric par-ACENTRIC (acentric or dicentric products) Neither stable, so risk for abnormal offspring is low. (duplication and deletion products) Viability depends on size of segments involved. Risk for Abnormal Offspring: Translocations vs. Pericentric Inversion Carriers Translocations: The larger the segments involved in the translocation are, the less likely that an unbalanced offspring will be liveborn. Pericentric Inversions: The larger the inversion, the more likely it is to to produce unbalanced offspring. Marker Chromosomes Called a marker chromosome until the origin is identified, then referred to as a derivative chromosome with the origin identified [e.g., der(3)]. Important to determine whether euchromatic material is present or not to define phenotypic consequences. Most common: chr15, chr22 (Cat Eye syndrome) 40 Acknowledgements David Ledbetter, PhD, Katrin Leuer, PhD, and Fady Mikhail, PhD, for some of the slides/content used in this lecture. Additional Slides for Your Reference Banding techniques *G-banding: Giemsa staining after trypsin treatment. AT rich regions stain darkly (G+); GC rich regions stain lightly (G-). By far most used for clinical and research purposes. Q-banding: Quinacrine fluorescence dye. AT rich regions stain positive (fluoresce brightly); GC rich regions stain negative (fluorescence weakly) R-banding: Reverse banding. Reverse pattern to Q or G; used primarily to highlight telomeric regions 41 Banding techniques C-banding: Constitutive heterochromatin. Heterochromatin stains darkly, euchromatin lightly Ag-NOR Silver nitrate (AgNO3) stains proteins at sites of active rDNA synthesis (the “stalks” on acrocentric chromosomes) Polymorphic regions of the human karyotype: •Acrocentric stalks and satellites. •Pericentromeric regions of 1, 9, and 16 vary tremendously in amount of heterochromatin (Cband positive material) and large variants are referred to as 1qh+, 9qh+, etc. •Long arm of Y chromosome highly polymorphic. Chromosome 14 Band Designation p 1 1 2 1 1 1 2 q 3 2 1 1 3 3 4 2 2 1 3 2 2 3 1 2 1 3 1 .1 .2 .3 2 .1 .2 .3 14q32.3 .1 .2 .3 2 3 3 2 .1 .2 .3 .1 .2 .3 .1 .2 .3 .1 .2 .3 4 1 Band Region 1 2 3 4 14q32 Arm 1 1 .1 .2 .3 .2 .11 .12 .13 .31 .32 .33 14q32.33 Cytogenetic Nomenclature 1) Number of chromosomes present 2) Sex chromosome composition 3) Descriptive characters of abnormalities + add del der dic dup gain of a whole chromosome loss of a whole chromosome additional material of unknown origin deletion derivative chromosome dicentric chromosome duplication 42 Cytogenetic Nomenclature inv iso mat pat mos r t rob inversion isochromosome maternal origin paternal origin mosaic ring translocation Robertsonian translocation Cytogenetic Nomenclature 45,XX,-9 45,X 47,XX,+13 47,XXY 46,XY,del(5)(q13q21) 46,XY,t(2;6)(p23;q32) 47,XY,t(3;7)(p21;p15),+22 Inversions 43 Paracentric Inversions par-ACENTRIC (does NOT include the centromere; two breaks in one arm). Cytogeneticists should be able to draw pairing diagram and derive meiotic products (noncyto should understand difference between repro risks of para vs. peri-. Unbalanced products are acentric or dicentric. Neither is stable, so risk for abnl. liveborn low. Pericentric Inversions nl inv dupA, dupD, delD delA Per-i-centric (i-ncludes the centromere in inversion segment; one break in each arm). Each unbalanced product has a duplication and a deficiency. Viability depends on the size of the segments involved as well as what genes are involved. Generally should be treated similarly to a balanced reciprocal translocation in terms of risk of abnormal offspring. Cytogenetics References: General • Thompson & Thompson Genetics in Medicine Nussbaum, McInnes & Willard Elsevier, 7th ed., 2007; 8th edition out 06/15 Basic and clinical chapters • Chromosome Abnormalities and Genetic Counseling Gardner and Sutherland Oxford University Press, 4th ed., 2011 Detailed mechanisms, segregation, recurrence risks Essential for all clinicians, GCs, lab directors 44 Cytogenetics References: Advanced • Human Genetics: Problems and Approaches Vogel and Motulsky Springer-Verlag, 4th ed., 2010 - Chapter 2 - Human Chromosomes Comprehensive chapter on cytogenetics, including history, methods, *meiosis, abnormalities, and clinical features. • AGT Cytogenetics Laboratory Manual Barch, Knutsen, and Spurbeck Lippincott, 3rd ed., 1997 • Detailed laboratory methods and protocols. 45 46 Genomics - Basic GENOMICS - BASIC Madhuri Hegde, PhD, FACMG Associate Professor Emory Genetics Lab Scientific Director Sr. Director Emory Genetics Lab, Molecular Lab Madhuri Hegde, PhD, FACMG Department of Human Genetics Emory University School of Medicine 2165 North Decatur Road Decatur, Georgia 30033 (404)727-5624 Telephone (404)727-3949Fax [email protected] 49 50 Genomics -Basics Madhuri Hegde Emory University School of Medicine • The following relationship(s) exist related to this presentation: – Category of relationship – Advisor, Employment – Name of commercial entity – Genzyme (Pompe program), PTC (DMD program), Coriell Cell Repositories, PerkinElmer Genetics Inc Learning Objectives • Mutations & their effects: – transitions & PTCs – Recombination & gene rearrangements – Trinucleotide repeats & disease • Methods of detecting genetic variation: – Southern blotting, FISH, CGH, PCR & MPLA – Polymorphisms, Linkage, LOH & MSI – Sequencing (Sanger and Next Generation Sequencing) – Gene discovery (Linkage analysis) 51 Gene Structure & Transcription Promoter Enhancer Start Transcription AUG: Start Translation UAG: Stop Translation 4 Gene Structure: Exons & Introns 5 Gene Families & Pseudogenes Expressed genes: transcribed & functional Pseudogenes (ψ): non functional copies Conventional ψ: not expressed & often occur in gene families Expressed ψ: non functional transcripts 6 52 Human Repetitive DNA Sequences SINEs: short interspersed nuclear elements, ie Alu (~300 bp, 106 copies); ~13% of genome) LINEs: long interspersed nuclear elements, ie LINE 1 (~800 bp, ~5x105 copies); LINES ~20% of genome 7 Effects of Mutations • Loss of function causes reduced activity or product (hypomorphs or null alleles) • Haploinsufficiency when ½ normal levels result in phenotype • Dominant negative in heterozygous state causes loss of normal allele function, usually from multimer formation • Gain of function have increased expression levels or product acquires a new function ACMG Genetics Review Course June 4, 2011 Methods of Detecting Genetic Variation Promoter Enhancer Start Transcription AUG: Start Translation UAG: Stop Translation 9 53 Properties of DNA •Double stranded •Denatures with heat •Complementary basepairing •Primers and enzymes to replicate •Large •Negatively charged DNA Isolation Transfer supernatent Spin to collect WBC pellet RBC lysis WBC lysis RNAaseA Proteinase K Protein DNA precipitation precipitation Procedures vary by sample type. Example – cultured cells Original sample conditions are important! Example – freezing Contents of blood collection tube are important! Example - heparin The Known Versus the Unknown One of a few mutations account for a large proportion of mutations in the population Many private mutations or an unknown spectrum of mutations comprise mutant alleles in the population 54 The Known: Targeted Mutation Testing Benefits: - Low cost - Can be high throughput - Pre-defined possible outcomes (Interpretation) Limitation: - Limited number of mutations detected - Other changes will not be detected - Other changes could interfere with the assay Most Appropriate for: - Carrier screening - Population based mutation screening (NBS) Methods to Detect Pathogenic Variants Detection of specific mutations: Restriction Enzyme digest Allele-specific PCR Allele-specific hybridization Oligo ligation assay Detection of any nucleotide change: Full gene sequencing Mutation scanning - DHPLC - SSCP Detection of large copy number mutations: Southern blotting Quantitative PCR MLPA Array comparative genomic hybridization Restriction Enzyme Digest - Bacterial immune / defense system - Enzymes that recognize and cut specific DNA sequences - Commercially purified for laboratory use - Systematic naming convention Dde I – from Desulfovibrio desulfuricans 5’….CTNAG….3’ 3’….GANTC….5’ HbA 55 Manipulation of Target DNA Dde I 5’….CTNAG….3’ 3’….GANTC….5’ http://www.larasig.com/node/4912 Restriction Digest HbA HbS Not specific! 233 178 55 Amplification-Refractory Mutation System (Allele-specific PCR) 3’ 5’ Extension with polymerase A C G T G T T G G T 3’ G C A C A A C C Normal G G A A T G G T C T T G T A 5’ Test for a C>T mutation at this site 3’ 5’ A C G T G T T G A T 3’ G C A C A A C T Mutant G G A A T G G T C T T G T A 5’ Test for a C>T mutation at this site Basic Principle: The 3’ end of the PCR primer must perfectly match the template for amplification to occur 56 Allele-Specific Oligonucleotide Hybridization (Dot Blot) Basic Principle: Normal and mutant probes that differ by as little as a single basepair mismatch are hybridized to test DNA under stringent conditions. A perfect match is necessary for binding. 3’ 5’ A C G T G T T G Genomic DNA hybridized to a membrane: G C C T T Normal Heterozygote Mutant Normal Probe 3’ 5’ A C G T G T T G A C C T T Mutant Probe Oligo Ligation Assay Probe B Probe A - normal Ligation Probe A - mutant Template DNA Labeled PCR primers specific to Probe A and Probe B are used to amplify ligated probes. Ligation and amplification occur only if the 3’ end of probe exactly matches template DNA. Normal and mutant probes can be differentiated by length. Can be multiplexed (MLPA – Multiplex Ligation-dependent Probe Amplification) Can be quantitative. Methods to Detect Mutations Detection of specific mutations: Restriction Enzyme digest Allele-specific PCR Allele-specific hybridization Oligo ligation assay Detection of any nucleotide change: Full gene sequencing Mutation scanning - DHPLC - SSCP Detection of large copy number mutations: Southern blotting Quantitative PCR MLPA Array comparative genomic hybridation 57 Sanger Sequencing PCR region of interest Separate by capillary electrophoresis Perform two sequencing reactions: one with a forward primer, one with a reverse primer Sanger Sequencing Raw sequence: Sequence assembled, analyzed, and displayed by software: Reference for regions of interest Reference sequence Forward sequence Software comparison of reference and patient sequence Reverse sequence Reference sequence 58 Methods to Detect Mutations Detection of specific mutations: Restriction Enzyme digest Allele-specific PCR Allele-specific hybridization Oligo ligation assay Detection of any nucleotide change: Full gene sequencing Mutation scanning - SSCP - DHPLC Detection of large copy number mutations: Southern blotting Quantitative PCR MLPA Array comparative genomic hybridation Single Stand Confirmation Polymorphism Analysis Denaturation of DNA Heat 59 Heteroduplex Analysis C G Allele 2 T A 1. PCR 2. Melt 3. Reanneal Basic Principle: Heteroduplex (mismatch-containing) DNA fragments will have different melting properties than homoduplex DNA C G T A Homoduplex Homoduplex C ^ A Heteroduplex T ^ G Heteroduplex ^ Allele 1 ^ Person heterozygous for a C/T mutation Denaturing Gradient Gel Electrophoresis and variations DHPLC Melting curve analysis Denaturing High-performance Liquid Chromatography (DHPLC) •Heteroduplex DNA elutes earlier than homoduplex DNA • Several denaturing temperatures per fragment must be tested Fragment detection Melting Curve Analysis 60 Methods to Detect Mutations Detection of specific mutations: Restriction Enzyme digest Allele-specific PCR Allele-specific hybridization Oligo ligation assay Detection of any nucleotide change: Full gene sequencing Mutation scanning - DHPLC - SSPC Detection of large copy number mutations: Southern blotting Quantitative PCR MLPA Array comparative genomic hybridation Gene Deletion / Duplication Testing Southern blotting: Benefit: - Technically simple, can be performed in most laboratories Limitations: - High cost, labor intensive - Low resolution: may miss small deletions or duplications Quantitative PCR: Benefits: - Many small regions (exons) can be tested - Accurate Limitations: - Labor intensive - Significant QA/QC investment MLPA: Benefits: - Many small regions (exons) can be tested - Accurate Limitations: - Labor intensive - Significant QA/QC investment - Single base pair changes can interfere with probe binding Array-based technologies: Benefits: - Highly accurate - High resolution Limitations: - Technically sophisticated - Significant platform investment required Most appropriate for: - Individuals in whom a mutation was not detected by sequencing - Gene in which deletions and duplications are common Southern Blot Analysis EcoRV Uncut P - + Mitochondrial DNA as probe Brief protocol: 1. 2. 3. 4. 5. 6. Digest genomic DNA with a restriction enzyme Run on an agarose gel Denature DNA to create single-stranded DNA Transfer to a nitrocellulose membrane Probe with a labeled gene-specific probe (100’s – 1000’s bp) Visualize labeled blot 61 Quantitative PCR F2 F3 Probe 1: Monitors PCR for the gene tested for deletions Probe 2: Monitors PCR for the gene serving as a control Brief protocol: 1. Amplify target sequences in presence of labeled probes for test and control genes. 2. Fluorescence is monitored during each PCR cycle. 3. Compare amplification curve to standard curve (as above) to determine starting concentration of test and control template. 4. If no deletion, test and control template will have the same starting concentration. If one copy of the test template is deleted, the ration of test to control will be .5. Multiplex Ligation-dependent Probe Amplification (MLPA) Concepts: -Binding of probes to known target - Ligation (joining of perfect match probes) - PCR amplification www.MLPA.com Gene Targeted Array CGH Array CGH is a better way to detect deletions and duplications than older methods. DMD deletion of exons 3 – 9 in a male Each point represents a single probe 62 Detection of Abnormal Repeat Expansions Repeat Expansion Disorders 5’UTR Exon Intron 3’UTR Myotonic Dystrophy Autosomal dominant with anticipation CTG expansion in the 3’UTR of the DMPK gene 1:100,000, but as high as 1:10,000 in some populations (Japan, Iceland) Mild Classic Congenital Cataracts Mild myotonia Weakness Myotonia Cataracts Balding Cardiac arrhythmia Others Infantile hypotonia Respiratory deficits Intellectual disability Classic signs present in adults 63 Myotonic Dystrophy – DMPK Gene Phenotype Normal CTG Repeat Size 5-34 Onset Death None Average Premutation 35-49 None Average Mild 50-150 20-70 60 – Average Classical 100-1500 10-30 48-55 Congenital 1000->2000 Birth-10 Neonatal - 45 **Premutation – an allele that can expand to a full mutation in one generation Myotonic Dystrophy - Anticipation Mild Classic Congenital Cataracts Mild myotonia Weakness Myotonia Cataracts Balding Cardiac arrhythmia Others Infantile hypotonia Respiratory deficits Intellectual disability Classic signs present in adults Myotonic Dystrophy – Anticipation Large expansions occur in female germline. Pedigree – Monckton and Caskey Circulation. 1995; 91: 513-520. 64 Myotonic Dystrophy - Anticipation Large expansions do not occur in male germline. Myotonic Dystrophy – Allele Calculation Myotonic Dystrophy – Normal alleles Capillary electrophoresis 65 Myotonic Dystrophy – Normal alleles Zoom in Myotonic Dystrophy – Allele Calculation Fragment size – non-repeat sequence = Repeat size 3 Myotonic Dystrophy – Normal alleles 147 171 147 – 108 = 39 / 3 = 13 CTG repeats 171 – 108 = 63 / 3 = 21 CTG repeats 66 Myotonic Dystrophy – Abnormal alleles 28,000 0 Zoom in 1,000 0 123 – 108 = 15 / 3 = 5 CTG repeats 378 – 108 = 270 / 3 = 90 CTG repeats Myotonic Dystrophy – Abnormal alleles Phenotype Normal Premutation CTG Repeat Size 5-34 Death None Average None Average Mild 50-150 20-70 60 – Average Classical 100-1500 10-30 48-55 Congenital 1000->2000 Birth-10 Neonatal - 45 BglI | | BamHI ________ Probe A 35-49 Onset 3.4kb | CTG repeat | BamHI BglI | 500bp | poly BglI | 67 BglI | | BamHI ________ Probe A 3.4kb | CTG repeat | BamHI BglI | 500bp | poly BglI | BglI ~ 5,510 bp 5510 – 3400 = 2110 / 3 = ~700 CTG repeats ~3400 bp Myotonic Dystrophy – Abnormal alleles Phenotype Normal CTG Repeat Size 5-34 Premutation 35-49 Onset Death None Average None Average Mild 50-150 20-70 60 – Average Classical 100-1500 10-30 48-55 Congenital 1000->2000 Birth-10 Neonatal - 45 PCR – precise, but limited in ability to amplify large alleles Southern – imprecise, but detects large expansions **Premutation – an allele that can expand to a full mutation in one generation Huntington Disease Autosomal dominant – one of the only true human autosomal dominant conditions CAG expansion in the HTT (also known as IT-15 gene) 1:100,000 in most Western populations, as high as 15:100,000 World’s highest prevalence in Maracaibo region of Venezuela Adult onset (most cases): Progressive motor disability - chorea Cognitive decline Psychiatric disturbances Personality changes Depression Juvenile onset (5-10% of cases): Rigidity Seizures Progressive motor disability - chorea Cognitive decline Psychiatric disturbances 68 Huntington Disease Juvenile HD >60 Well-defined repeat size / phenotype correlation – precision critical in sizing repeat Alleles can expand or contract Large expansions occur through paternal germline (stable paternal transmissions also occur) Huntington Disease Precision in the 35-40 CAG range is important due to repeat size / phenotype correlation ALWAYS run 40 (or similar) CAG control If potential for juvenile onset, Southern may be needed 109 – 46 = 63 / 3 = 21 CAG repeats 166 – 46 = 120 / 3 = 40 CAG repeats Friedreich Ataxia Autosomal recessive GAA expansion in intron 1 of the FXN gene (98%) of alleles and rare mutations 1:25,000 – 1:50,000 in Europe, Middle East, India, North Africa, rare elsewhere Carrier frequency: 1:60 – 1:100 Early onset (10-15 years old) slowly progressive ataxia Dysarthria Muscle weakness Spasticity of lower limbs with loss of reflexes Scoliosis Bladder dysfunction 69 Friedreich Ataxia Phenotype GAA Repeat Size Normal 5-33 Premutation 34-65 pure GAA Borderline 44-66 pure GAA Full penetrance 66-1700 (most 600-1200) (GAGGAA)n and (GAAAGAA)n may be stable May be associated with reduced penetrance Friedreich Ataxia Long range PCR Southern blot How would you add this to a large carrier screening panel? Spinal and Bulbar Muscular Atrophy (Kennedy Disease) X-linked CAG expansion in the AR gene – codes for the androgen receptor 1:150,000 males in Europe and Asia, not yet reported in other populations Most common in Japan Gradually progressive degeneration of lower motor neurons - Proximal muscle weakness - Muscle atrophy - Fasciculations Mild androgen insensitivity in affected males - Gynecomastia - Testicular atrophy - Reduced fertility 70 Spinal and Bulbar Muscular Atrophy Phenotype CAG Repeat Size Normal > 34 Unknown / questionable Reduced penetrance 35 Never reported 36-37 Full penetrance >38 Family history and clinical presentation important Up to at least 55 Fragile X Syndrome Head circumference >50th % Prominent forehead and jaw Large ears (>2 st dev for age) Macroorchidism (> 2 st dev for age) Hyperextensible joints Pectus excavatum Pes planus Mitral valve prolapse Intellectual disability (IQ 20-60) ADD / ADHD Autistic behavior Tactile defensive Poor eye contact Hand-flapping Fragile X Mental Retardation 1 (FMR1) CpG Island 5’ X Transcription X (CGG) n ~ 44 repeats Translation 3’ Common ~44-54 repeats ~55-199 repeats > 200 repeats, methylated Male Intermediate Premutation Full mutation Female 1/25 1/12 1/800 1/250 1/4000 1/4000 Most common form of inherited intellectual disability. S. Sherman 71 Fragile X syndrome and associated disorders Fragile X related tremor/ataxia syndrome (FXTAS) 58,30 75 62,29 90,31 70,30 >200 Ovarian insufficiency 1% POF 15% POF 2 yrs 5 yrs 22 yrs Fragile X syndrome 40 51 46 Age at menopause S. Sherman Fragile X – Allele sizing by PCR CGG repeat size analysis - In-house developed PCR and capillary electrophoresis - Large alleles will be not amplified Commercially available kit for amplification of full mutations Fragile X – Allele sizing by PCR Normal female Normal male 72 Fragile X – Allele sizing by PCR Abnormal male Fragile X – Assessment of methylation by msPCR Neg male Pos male Neg female 271 bp unmethylated allele 223 bp methylated allele Bisulfite treat DNA Amplify with methylated and unlemtylated-specific primers Validated to ~5% methylated – can detect methylation mosaics Fragile X – Southern Blot EcoRI | 2.7kb Probe Patient Male Normal 2.7 Female 2.7 5.2 XhoI* | 2.4kb EcoRI | | CGG repeat Premutation >2.7 2.7 + >2.7 5.2 + > 5.2 Full mutation* >5.2 2.7 + Methylated* >5.2 5.2 + >5.2 * Xho I methylation sensitive If Xho I does not cut EcoRI fragment is ~ 5.2kb plus length of CGG repeats 73 Fragile X – Southern Blot Fragile X – Mosiacism Mosiacism of large expansions not uncommon Mosiacism of premutation and full mutation expansions is consistent with a diagnosis of Fragile X syndrome Gene identification Familial aggregation Twin and adoption studies Is the trait influenced by genetic factors? Linkage analysis Identifying mutations that determine trait Identifying the position of the gene(s) in the genome Precise location Identifies candidate genes in region Specific gene structure, function and effect of mutation 74 Gene Identification for Mendelian Traits • These diseases usually involve a rare, highly penetrant mutation • Environmental component usually minor • They are often phenotypically distinct, which reduces locus heterogeneity Where do we start looking? Linkage Analysis of AD Trait: Microsatellite 1, 2 3, 4 1, 3 1, 4 2, 3 2, 4 1 2 3 4 Determines if DNA variations near gene of interest cosegregate with a phenotype in a family Steps: 1) Get needed samples, 2) Is the study informative? 3) Do you see co segregation? & 4) Infer status (Allele 4 co segregates with the AD trait) 74 Microsatellite Instability (MSI) & Mismatch Repair Failure in HNPCC Blood MSI Normal Tumor 1 Microsatellite Instability Tumor 2 Tumor 3 Change in nucleotide repeats MSI Tumor 4 75 75 Evolution of Sequencing • 1st Gen: 2 reads (Forward and Reverse) Sanger Sequencing Technique: ABI, Beckman • 2nd Gen: Millions of reads Next Generation Sequencers: Roche 454, Junior ABI SOLiD Illumina (HiSeq and MiSeq) • 3rd Gen: Single molecule sequencing, nanopore sequencing Ion Torrent, Pacbio, The MinION Dideoxy DNA Sequencing Homozygous Point Mutation Heterozygous Heterozygous Point Mutation Insertion/Deletion/Fusion 77 Multiple Options for Sequencing NextGen Targeted Panel Exome • Target Enrichment • PCR based • In Solution • Microarray • Target Enrichment • Nimblegen SeqEZ • Illumina Truseq • Agilent SureSelect • Sequencing • Sequencing Genome • Random Shearing of Genome • No target enrichment needed • Sequencing 76 Next Generation Sequencing Process Genomic DNA Fragment Randomly shear DNA + end repair + size select Enrichment Amplification Addition of adaptors Modification of products NextGen Sequencing Raw Data Amplification Enrichment NGS Sequencing Illumina – sequencing by synthesis 77 Interaction between panels and Exome/Genome Informs design of panels • Bioinformatics • Phenotype Driven Analysis Sequencing panels Exome • Functional Analysis Genome • Gene Expression analysis Clinical – 2nd Testing Clinical – 2nd Testing Drop in cost of sequencing Technological advances- sequence once read often Targeted panels/ exome / genome Targeted panels Exome Genome Smaller target region Allows interrogation of entire coding region of the genome(~92% coverage); Exome is 1-2% of the genome Targets the 85% of the genome; 15% are drak regions (repeats, pesudogenes etc) 100X coverage (clinical grade) 100X coverage (clinical grade) 30X (clinical grade) uniform coverage across all targeted regions; 30-200 genes Enhanced coverage only across disease associated region (5300/22,000). All 22,000 genes sequenced but coverage enhanced over disease assoc regions*(see note) Near uniform coverage across entire genome (85% sequencable genome) Difficult for detection of CNVs, Trinucleotide repeats, pseudogenes Difficult for detection of CNVs, indels Trinucleotide repeats, Pseudogenes Cannot detect deep intronic mutations unless specifically targeted in the design Can detect CNVs (both genomic and intragenic), deep intronic mutations, and indels Difficult to detect Trinucleotide repeats, Pseudogenes but new algorithms are ebing developed No incidental findings (Allelic changes) Need to address incidental findings Need to address incidental findings Supplemental Study Slides 78 Components of the Human Genome 85 Transcription: Cis & Trans Acting Factors CAAT box binds NFI & CBF transcription factors & regulates the amount of transcription GC (Sp1) modulates transcription with CAAT TATA box binds TFIID & determines transcription start site (not in all promoters) Initiator (Inr) (PyA+1NT/APyPy) complements TATA to localize transcription start site Inr 86 Promoter Mutation That Affects Transcription (Hemophilia B Leyden) 87 79 Alternative Transcription Start Sites DNA Transcription Start Transcription Start Site 1 Site 2 5㵭 2 B 1 A G 1 A 2 B 3 C 3 C 3㵭 4 D G 3 C 4 D AAAAAAAA 4 D AAAAAAAA Different mRNAs are produced from different transcription start sites 88 Processing & Translation Promoter Enhancer Start Transcription AUG: Start Translation UAG: Stop Translation 89 Splicing Consensus Sequences 90 80 Splicing Mutation Causing Exon Skip 91 Alternative Splicing (AS) Can Give Multiple Transcripts 92 Alternative Splicing of CFTR mRNAs Regulated by IVS8 5T/7T 93 81 㵰Silent Changes㵱 Can Perturb SEs • Splicing enhancers (SEs) can be in exons (ESEs) or introns (ISEs) • SEs bind Serine Rich (SR) proteins & other factors to select splice sites • ESEs can encode AAs(GAAGAA= GluGlu) but also regulate splicing • Synonymous substitutions (silent mutations) in ESEs can encode the same AA but derange splicing 94 ESE Mutation Causes Exon Skip Growth Hormone Deficiency I 1 1 2 ? ? 2 3 4 & 6 5 II A/A A/G A/A 1 3 2 III A/G IV agGAAG A G A/G A/A 1 2 A/G A/G A 95 Translation: Nonsense Mediated Decay (NMD) • NMD degrades mRNAs with premature termination codons (PTCs) • Destroys truncated proteins from PTCs • Efficiency of NMD lessens as PTC moves 5㵭 to 3㵭 • No NMD for PTCs in last 50 bp of next to last, last exon, or single exon genes 96 82 Translation: MicroRNAs (miRNAs) • Short RNAs not mRNAs • Hundreds are known • miRNA (~20 nts long) loads the RNA Induced Silencing Complex (RISC) • RISC represses mRNA translation Translational dsRNA Small RNA duplex miRNA/ siRNA RISC siRNA function mRNA 97 destruction repression Translation: MicroRNAs (miRNAs) MicroRNA Expression in Malignancy • miRNAs are important in regulating gene expression in development • miRNAs are also biomarkers of malignancies 98 Translation: mRNA Editing Apo B gene CAA TAA 5’ 3’ CAA > UAA editing No editing Apo B mRNA CAA UAA UAA Apo B100 protein- Liver UAA Apo B48 protein- Intestine • ApoB100 isn㵭t edited in liver • ApoB48 translated in small intestine from edited ApoB100 mRNA • Editing changes CAA > UAA (Term) & this converts ApoB100 to ApoB48 mRNA without changing the gene 99 83 Silent (Synonymous) Mutations mRNA AUG GAA GCU AGU Protein Met Glu Ala Ser mRNA AUG GAG GCU AGU Protein Met Glu Ala Ser Don’t alter an amino acid ie Glu109Glu Be Careful these may create splice sites, affect ESEs, or alter mRNA secondary structure (Science 314: 1930, 06) 100 Missense (Nonsynonymous) Mutations mRNA AUG GAA GCU AGU Protein Met Glu Ala Ser mRNA AUG GAC GCU AGU Protein Met Asp Ala Ser Changes codon base 1: almost always, 2: always & 3: sometimes causes missense Missense changes amino acid ie Glu10Asp Be Careful the amino acid change may be harmful or neutral 101 Nonsense (Termination) Mutations mRNA AUG GAA GCU AGU Protein Met Glu Ala Ser mRNA AUG TAA GCU AGU Protein Met Stop Ala Ser Changes an amino acid to premature termination codon (PTC) ie Glu132Ter RT -PCR Be careful PTCs can cause 1 2 exon skips, encode truncated 1 WT, 2 Missense & 3 PTC proteins, or trigger NMD 102 84 Frameshift Mutations mRNA AUG GAA GCU AGU Protein Met Glu Ala Ser mRNA AUG Protein Met AGC UAG Ser Stop In/del of anything but (3)N bases alters reading frame & amino acid sequence ie 2 bp Del, 185GA Be Careful frameshift may not produce truncated protein if NMD occurs 103 Splicing Mutations • Can destroy/create splice site, ESE/ISE & cause exon skips, activate cryptic splice sites, change alternative splicing or cause read through • Be careful difficult to predict the effects of mutations on mRNA splicing & translation 104 Hb E Mutation: Splicing & Missense 105 85 Transitions = Pu Purine Pu or Py Py Transversions = Pu Py Transitions Adenine AdenineTransitions Guanine Guanine Transversions Transversions Cytosine Pyrimidine Cytosine ACMG Genetics Review Course June 4, 2011 Uracil Uracil Transitions Thymine Thymine Methylation of CGs Causes C to T & G to A Transitions 107 Repetitive Sequences & Recombination ACMG Genetics Review Course June 4, 2011 86 How do Trinucleotide Repeat Expansions (TNREs) Cause Disease? AUG (CGG)n TAA (GAA)n (CAG)n (CTG)n Coding TNREs (ie HD) can cause polyglutamine runs that affect protein function Non coding TNREs (ie FXS) can affect gene expression by triggering methylation TNREs are associated with anticipation in pedigrees ACMG Genetics Review Course June 4, 2011 Slipped Mispairing Causes TNR Expansions ACMG Genetics Review Course June 4, 2011 Properties of DNA •Double stranded •Denatures with heat •Complementary basepairing •Primers and enzymes to replicate •Large •Negatively charged 87 Ways to Visualize DNA - + Basic concept: separation based on chemical and physical properties - Negative DNA migrates toward positive cathode - Smaller pieces get through gel faster Ways to Visualize DNA – Dyes and Labeling EtBr – ethidium bromide DNA run on an agarose gel containing EtBr shown under and UV lamp *intercalating agent: reversible inclusion of a molecule between two other molecules Ways to Visualize DNA – Dyes and Labeling 23 kb 2 kb Genomic DNA – large pieces of DNA 88 Regions of Interest (Exploiting) Complementary Basepairing 5’ 3’ A C G T G T T G G replication / extension primer / probe / oligonucleotide binding T G C A C A A C C G G A A T G G T C T T G T A 3’ 5’ Enzyme - protein that catalyzes chemical reactions of other substances without itself being destroyed or altered upon completion of the reactions. Enzymes Used in Molecular Biology Enzyme - protein that catalyzes chemical reactions of other substances without itself being destroyed or altered upon completion of the reactions. Polymerase – used to replicate DNA Restriction enzymes – used to cut DNA at specific sequences Ligase – Joins two pieces of DNA 89 Properties of DNA •Double stranded •Denatures with heat •Complementary basepairing •Primers and enzymes to replicate •Large •Negatively charged Ways to Visualize DNA of Interest Amplification – polymerase chain reaction (PCR) PCR video Advantages – amplification of area of interest - design for specificity (18-25 bp) Disadvantages - variability in DNA can interfere with primer binding - assay designs may not work as intended - some regions of the genome are not unique - relatively limited range (4 – 10 kb max) - repetitive regions can interfere with amplification Ways to Visualize DNA of Interest 1500 bp 1000 bp 500 bp Resolution: 10’s to 10,000’s of basepairs (even whole chromosome) Fast, relatively cheap, run and visualize any piece of DNA 90 Ways to Visualize DNA – Dyes and Labeling Incorporation of fluorecently labeled primers 3’ 5’ A C G T G T T G G T 3’ G C A C A A C C G G A A T G G T C 3’ C G T G T T G G T 3’ G C A C A A C C G G A A T G T G G T C T T A 5’ A T C A T T Incorporation of fluorecently labeled dNTPs 5’ C A G T G T A 5’ Ways to Visualize DNA – Dyes and Labeling Capillary electrophoresis Basic concept: separation based on chemical and physical properties - Negative DNA migrates toward positive cathode - Smaller pieces get through polymer faster Resolution: 1 to 1,000’s of basepairs Relatively expensive, run labeled DNA Ways to Visualize DNA – Dyes and Labeling Capillary electrophoresis An array is a systematic arrangement of objects, usually in rows and columns. 91 Ways to Visualize DNA – Dyes and Labeling smaller bigger Targeted Mutation Testing The Known: Mutation Detection Methods Restriction digestion of PCR-amplified DNA Allele-specific amplification Allele-specific hybridization Allele-specific ligation 92 High-throughput screen for a common mutation Factor V Leiden Thrombophilia - Caused by a specific mutation in the F5 clotting factor gene (c.1691G>A (p.R506Q)) - Increased risk of venous thromboembolism and other thrombotic events - Can be considered autosomal dominant or autosomal recessive: Heterozygote: 3-8-fold increased DVT risk Homozygotes: 9-80-fold increased DVT risk - Carrier frequency: 1/12 – 1/33 -Prevalence of homozygotes: 1/5,000 - Lifetime penetrance for heterozygotes: ~10% Melting curve analysis of a PCR product with an “extra” probe over mutation site The Known: Targeted Mutation Testing Benefits: - Low cost - Can be high throughput - Pre-defined possible outcomes Limitation: - Limited number of mutations detected - Other changes will not be detected and could interfere with the assay Most Appropriate for: - Carrier screening - Population based mutation screening (NBS) 93 Cystic Fibrosis Most common genetic disease in the Caucasian population (~1 / 3,300) Autosomal recessive inheritance Dysfunction of ion transport in epithelial cells results in a multisystem disorder www.genetests.org Moskowitz et al., (2008) Genet Med 10: 851-868 The Known: Cystic Fibrosis CFTR gene mutations ACMG Recommendations for CFTR Mutation Selection: A carrier screening mutation panel should include: - mutations present in > 0.1% of CF patient chromosomes - only mutations associated with classic CF ACMG Recommendation: - 25 mutations recommended for carrier screening in 2001, revised to 23 mutation in 2004 based on additional data - Addition of mutations may be appropriate in the future. ACMG Practice Guidelines: Grody et al., 2001 Genet Med 3: 149-153 Watson et al., 2004 Genet Med 6: 387-391 39 Mutation Panel dF508* dI507* 3120+1G>A* G85E* R117H* W1282X* Y122X R334W* V520F R347P* R347H A455E* G542X* S549R S549N A559T G551D* R553X* R560T* Y1092X M1101K R1162X* S1255X N1303K* 394delTT 621+1G>T* 711+1G>T* 1717-1G>A* 1898+1G>A* 1898+5G>T 2184delA* 2307insA 3849+10kbC>T* 1078delT 2183AA>G 2789+5G>A* 3659delC* 3876delA 3905insT * ACMG recommended panel 94 c.443T>C (p.I148T) - Rare variant - Originally identified in individual with CF - Eventually found to be more common in controls than CF patients CFTR Mutation Detection Rate Estimated detection rate Racial/ethnic group ACMG 23 39 mutation panel Ashkenazi Jewish 94% 94% Non-Hispanic Caucasian 88% 90% Hispanic American 72% 74% African American 65% 68% Asian American 49% 49% CFTR mutation detection in different populations Racial/ethnic group Detection rate Ashkenazi Jewish 94% Non-Hispanic Caucasian 90% Hispanic American 74% African American 68% Asian American 49% Percentage of CF patients carrying at least one mutation 99.6% 99.0% 93.2% 89.8% 74.0% 95 Allele Specific Primer Extension Wild type Mutant a A T 3’ 3’ 3’ G C 3’ PCR-amplified target DNA b Two universally-tagged ASPE primers whose 3’ end defines the allele 3’ Tag 2 3’ T c Only correctly hybridized primer will extend and incorporated a biotin-dCTP A T 3’ Tag 1 B A T 3’ Tag 1 3’ C 3’ B G C G C Tag 2 B Allele Detection After Primer Extension Mutant Wildtype Fluorescent signal SA PE B Tag 1 Anti-tag 1 ID read by machine Bead 1 Tag 2 Anti-tag 2 Bead 2 Data from Allele Detection 96 CFTR Assay Positive Results 37 Out of 39 Mutations Observed dF508* dI507* 3120+1G>A* G85E* R117H* W1282X* Y122X R334W* R347P* R347H A455E* V520F G542X* S549R S549N A559T G551D* R553X* R560T* Y1092X M1101K R1162X* S1255X 394delTT 621+1G>T* 711+1G>T* N1303K* 1078delT 1717-1G>A* 1898+1G>A* 1898+5G>T 2183AA>G 2184delA* 2307insA 2789+5G>A* 3659delC* 3849+10kbC>T* 3876delA 3905insT * ACMG recommended panel Unexpected Results: p.R117H / p.R117H by NBS Negative carrier screen - mother’s report only - test method unknown - most likely a carrier panel p.R117H / p.R117H by GA NBS 97 Unexpected Results: p.R117H / p.R117H at Screening Negative carrier screen p.R117H / p.R117H Elevated IRT - Large deletion (not detected by mutation panel or sequencing) - Allele drop out (can occur with any PCR-based method) - Sequence interfering with assay (can occur with any PCR-based method) - Sample mix up - Mom’s screening result incorrect - Information about mom’s screening result incorrect p.R117H / p.R117L Reference: Patient: G A/T CGC – Arg CAC – His CTC – Leu Unexpected Results: R117H / R117H at Screening p.R117H p.R117L p.R117H / p.R117L Elevated IRT Positive sweat test 98 McArdle Disease - A glycogen storage disorder (also known as GSD type V) - Autosomal recessive, caused by mutations in the PYGM gene - Characterized by myopathy, exercise intolerance, rapid fatigue, myalsia , muscle cramps - Myoglobinuria can result in acute renal failure - Mutations common in some populations PYGM Mutation Detection Rate Panel 1 p.R50X Panel 2 p.R85X p.G205S p.F710del European 50% 25% 10% 0% Japanese 0% 0% 0% 65% What percent of affected individuals of European decent would have at least one mutation detected by panel 1? What percent of affected individuals of Japanese decent would have at least one mutation detected by panel 2? PYGM Mutation Detection Rate Panel 1 p.R50X Panel 2 p.R85X p.G205S p.F710del European 50% 25% 10% 0% Japanese 0% 0% 0% 65% What percent of affected individuals of European decent would have at least one mutation detected by panel 1? 97.75% What percent of affected individuals of Japanese decent would have at least one mutation detected by panel 2? 87.75% 99 Populations with Common Mutations Ashkenazi Jewish Sephardic Jewish North America: Old Order Amish Old Order Mennonite Dutch-German Mennonite Bethren in Christ French Canadian First Nations Finland The Netherlands Iceland Any isolated population Common Mutations in the Ashkenzi Jewish Population - 1 in 4 to 1 in 5 individuals carry at least one “common” mutation -Guidelines from the ACMG (Gross et al., 2008 Genet Med 10: 54-56) - 8 disorders - Specific mutations - Many panels offer more diseases and more mutations - Some include Sephardic Jewish mutations as well - Preconception / prenatal carrier screening Common Mutations in the Other Populations Larger carrier screening panels are being designed and offered Not all common mutations are tested for in carrier screens – from Strauss and Puffenberger (2009) about the Clinic for Special Children in Strausburg, PA: Before the Clinic was established, Amish children with nemaline rod myopathy were repeatedly subjected to invasive and costly interventions (e.g., muscle biopsies, nerve conduction testing, electromyography, magnetic resonance imaging, echocardiograms, etc.) that collectively cost the community millions of dollars. A thoughtful physician informed with the right genetic diagnosis can help a family determine appropriate limits of medical care while also protecting the child from futile interventions and the common miseries of hunger, thirst, dyspnea, and pain. 100 Spinal muscular atrophy – technically difficult to screen for - Severe neuromuscular disease caused by degeneration of the anterior motor neurons - Severe progressive hypotonia, muscle weakness - Three types: Type I – severe, lethal in infancy Type II – childhood form Type III – mild - Caused by mutations in the SMN1 gene: 95% deletions, 5% point mutations - Carrier frequency: 1 in 35 Caucasian 1 in 41 Ashkenazi 1 in 53 Asian 1 in 66 African American 1 in 117 Hispanic - Recommendation to offer carrier screening by ACMG, ACOG, and AMP SMN1 – telomeric – active copy SMN2 – centromeric – less active copy SMN1 deletion detection by TaqMan assay Fluorescent tag Quencher Amplify with fluorescent probe for SMN1 and a reference gene ** Add a non-fluorescent, non-hydolysable probe specific to SMN2 - competes for binding Sugarman et al., 2012 Eur J Hum Genet 20: 27-32 101 SMN1 and SMN2 Alleles SMN1 Alleles SMN2 Alleles [1+0] [1+1] [2+0] [2+1] [1+0] [1+1] [1+2] [1+3] [2+0] [2+2] [2+3] Etc. Sugarman et al., 2012 Eur J Hum Genet 20: 27-32 Large scale population-based screening for SMN1 deletions: n= 68,471 Examples of differences by ethnicity: 1-copy 2-copies >3-copies Caucasian ~2.0% ~90.9% ~7.1% African-American ~1.0% ~51.9% ~47.1% 2 copies: [0+2] OR [1+1]? Sugarman et al., 2012 Eur J Hum Genet 20: 27-32 102 How Southern Blots Work Palindrome 157 Southerns: TNR Expansion Size & Methylation • Methylation sensitive enzymes 2 2 • Separates active & inactive FMR1 gene of females • Premutations (1) are not methylated • Methylation of full mutations (2) inactivate the FMR1 gene 1 158 DNA Methylation Silences FMR1 Expression (CGG) GENE RNA n PROTEIN Mild Severe Severe GENE + METHYLATION 159 103 PCR of TNR: Huntington Chorea Allelic Drop out 160 Missed Deletions: Multiplex Ligation dependent Probe Amplification (MLPA) Sequence of cDNA Exon 1 BMPR2 genes Exon 3 MLPA Control Exon 2 Del ACMG Genetics Review Course June 4, 2011 Gene Fusions: Quantitative PCR of BCR/ABL 11 copies of BCR/ABL / 100,000 cells BCR-ABL/BCR= 0.00012 8,421 copies of BCR/ABL / 100,000 cells BCR-ABL/BCR= 0.11275 162 104 Types of Polymorphisms Single nucleotide polymorphisms (SNPs): substitution of a single base Short tandem repeats (STRs): tandem bi, tri or tetra nucleotide repeats such as (TG)n, (CAA)n or (GATA)n (aka microsatellite markers) Variable number of tandem repeats (VNTRs): includes STRs but usually refers to unstable minisatellites (repeats of 9-65 bases) Restriction fragment length polymorphisms (RFLPs): changing a restriction site or an internal repeat or in/del alters fragment length 163 Single Nucleotide Polymorphism (SNP) TGC/TGC CGG/TAC Exon 1 Intron Exon 2 GTC/TGC Intron ACG/TAC Exon 3 SNP: single base substitution (does not include insertions or deletions) Occur ~1300 bp so there are millions Have a population frequency of at least 1% SNPs may or may not affect gene function ACMG Genetics Review Course June 4, 2011 The Unknown: Exome Sequencing Benefits: - Interrogates all* genes in the genome - Uses sophisticated bioinformatics - Highly automated data proccessing Limitations: -Difficult to rule out genes - Amplification or capture required - High cost for infrastructure - Requires sophisticated bioinformatics -Indel detection - Gene discovery in a clinical setting? -Identification of mutation (truncating vs. missense) in new gene of unknown function (e.g. biochemical) - Reagent cost vs. cost of the test - Complicated interpretation and reporting - How will the reports be written? 105 Whole Exome Sequencing (WES) Ng et al Nat Genet 42: 30-36, 2010 Filter NS/SS/I Fam 1 Fam 1+2 Fam 1+2+3 2,362 1,810 1,525 Not dbSNP129 53 25 21 Not HapMap8 46 7 4 Neither 9 1 1 Predict Damaging 1 0 0 DHODH: 10 missense & 1 bp del in 6 Kindreds ACMG Genetics Review Course June 2-5, 2011 㵰DNA is Important!㵱 OT Avery 1944 167 106 Clinical Cytogenetics CLINICAL CYTOGENETICS Christa Lese Martin, PhD, FACMG Geisinger Health System Autism & Developmental Medicine Institute Director and Senior Investigator Christa Lese Martin, PhD, FACMG Autism & Developmental Medicine Institute Geisinger Health System 120 Hamm Drive, Suite 2A, M.C. 60-36 Lewisburg, PA 17837 (570) 522-9427 Telephone (570) 522 9431 Fax [email protected] 109 110 Clinical Cytogenetics Christa Lese Martin, PhD, FACMG Director and Professor Autism & Developmental Medicine Institute, Geisinger Health System Disclosure(s) Employed by Geisinger Health System Consultant for The Jackson Laboratory Overview • • • • Molecular Cytogenetics/genomics Techniques Microdeletions/Microduplications Syndromes Recurrent Genomic Disorders X Chromosome Abnormalities 111 Clinical indications for cytogenetic analysis Intellectual disability Evolution of G-banding to Molecular Cytogenetics G-band designation (subjective) 7q34 (+/- a band) vs. Array mapping (objective) 7q35 – q36.1 Karyotype courtesy of N.L. Chia ISCN2009 G-banding resolution Standard idiograms for 400, 550, and 850 band stages of resolution per haploid genome High-resolution (850-1000 band stage) usually requires cell synchronization methods or the addition of chemical agents to prevent chromosome condensation. Smallest detectable imbalance (deletion, duplication) by Gbanding ~2-3 Mb But, analytical sensitivity at 5 Mb probably only ~70% 112 Copy Number Variation (CNV) • Class of mutation resulting from the loss (deletion) or gain (duplication) of genomic material • > 1 kb in size • Recurrent – common breakpoints mediated by underlying mechanism, such as segmental duplications (e.g., 16p11.2) • Non-recurrent – variable breakpoints throughout the genome CNVs can be observed in normal populations or cause disease Human Disease Normal Individuals • Common cause of normal variation • Identified in ~35% of human genome (Iafrate et al., 2004) • In general: – Smaller in size – Contain fewer genes – Highly variable regions (e.g., pericentromeric DNA, segmental duplications) – Often inherited • One of most common causes of human disease • Diagnostic yield of 10-20% in DD, ID, ASD, birth defects • In general: – Larger in size – Contain more genes – Located in unique regions of the genome – Often de novo Cataloging CNVs in Shared Databases Normal Variation DGV Database of Genomic Variants dbVar Database of Genomic Structural Variation Human Disease ClinVar DECIPHER Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources OMIM Online Mendelian Inheritance in Man 113 Pathogenic vs. Benign Imbalances 1. Evidence from literature/databases - known del/dup or Mendelian disorders OMIM, DECIPHER, ClinGen - known CNV in normal population DGV, dbVar - comparison with patient population data, case reports PubMed, DECIPHER, ClinGen/ClinVar 2. Genomic/Gene Content - correlates with size and location UCSC, Ensembl 3. Inherited or de novo ACMG Guidelines for CNV interpretation • PATHOGENIC - Reported as pathogenic in multiple publications/databases, rarely identified in controls, and/or high genic content • UNCERTAIN - LIKELY PATHOGENIC • UNCERTAIN CLINICAL SIGNIFICANCE • UNCERTAIN - LIKELY BENIGN • BENIGN - Reported in multiple publications/databases as benign or is a known polymorphism Kearney et al. (2011) Genet Med Methods for Copy Number Detection Genome Size and Resolution Human genome Chromosome Metaphase band (500 bands) Prometaphase band (1,000 bands) FISH resolution array/PCR/MLPA Mb 3,000 kb 3,000,000 150 150,000 # Genes ~20,000 1,000 6 6,000 50 3 3,000 25 20-100 <1-100 1 1 114 Copy Number Tests G-banding + + + +/- + - - - Array CGH + - - + - - - +/- SNP Array + +/- - + - + + +/- Exon-level Arrays - - - - - - - + Confirmation Studies Different methods used to confirm copy number array results include: FISH Q-PCR (quantitative-PCR) MLPA (multiplex ligation-dependent probe amplification) Only FISH can determine the mechanism of the imbalance – important for recurrence risk FISH Analysis 1 2 3 6 7 8 13 14 15 19 20 9 21 4 5 10 11 12 16 17 18 22 X Utilizes a DNA probe specific to a targeted chromosomal region (e.g., 22q11.2) Probe binds to complementary DNA in the cell Visualized by fluorescent tag attached to probe Y 115 FISH (Fluorescence In Situ Hybridization) Patient Cells G A T T Patient Cells Hybridize Probe/clone DNA Denature probe DNA ds ssDNA Denature target DNA FISH Probes Whole Chromosome “Painting” Probe Centromere Probe LocusSpecific Probe Metaphase FISH 116 Interphase FISH BENEFITS: Cells do NOT need to be cultured Metaphase cells not necessary LSI 13 LSI 21 CEP 18 CEP X CEP Y FISH Analysis 1 2 3 6 7 8 13 14 15 19 20 9 21 4 5 10 11 12 16 17 18 22 X Resolution: Size of probe (~100 kb); but not equal across entire genome Y Requires at least 500-600 evenly spaced DNA probes to match the power of the karyotype!!! Human Telomere Clone Set cen gene rich 3 -20 kb 100 - 300 kb Unique Telomere Probe Subtel. Repeats (TTAGGG) n 117 Telomere FISH Analysis 1ptel del 1p tel green 1q tel red Most common: 1p del, 9q del, 22q del Knight, Lese et al. (2000) AJHG Unexplained Intellectual Disability ~2.5 - 5% clinically significant 60% of unbalanced translocations were inherited from parent with balanced form of rearrangement Biesecker (Am J Med Genet 2002 107:263-266) Reviewed 14 studies – 1,718 patients with ID, 2-29% yield Conclusion – G-banding alone is insufficient Array-based Copy Number Microarrays Molecular cytogenetic method to detect copy number imbalances (also called copy number variations, or CNVs) 2005 BAC arrays used clinically 2007 - genome-wide interrogation using oligonucleotide and SNP arrays Objective method compared to routine cytogenetic and FISH analyses 118 All microarrays are NOT created equal! Depends on: 1) Purpose of Microarray: Gene expression Sequence changes Copy number imbalances 2) Content of Microarray cDNA Single nucleotides Pieces of DNA (genomic clones, oligonucleotides) Targeted vs Whole Genome Array Focus on specific areas Telomeres Centromeres Microdeletion/duplication regions ~400-2,000 BAC clones or oligo probes Analyze entire genome Tiling path BAC arrays – 32,000 overlapping BACs Oligo arrays – 44,000 to 2M probes SNP arrays – 500K to 2M probes Array-based CGH Patient DNA Genomic Probes Loss: ratio < 0.8 Normal: ratio 0.8 - 1.2 Gain: ratio > 1.2 Control DNA Will not detect balanced chromosome rearrangements or polyploidy (triploidy, tetraploidy) 119 FISH Copy Number Arrays Patient Cells Clone DNA (1 probe) Patient DNA Microarray with Genomic Clones (probes) Control DNA aCGH = hundreds of FISH probes SNPs allow AOH and UPD to be detected Future Directions • Copy Number Variants are now being called from whole exome and whole genome sequencing data • Identification of sequence and structural variants can be achieved in one assay Example Array Results Normal results Chr 4 Trisomy 21 120 Targeted Coverage: PW/AS Region arr 15q11.2q13.1(20,249,886-26,884,937)x1 PWS/AS deletion Targeted Coverage: PW/AS Region arr 15q11.2q13.1(20,249,886-26,884,937)x1 PWS/AS deletion Atypical deletion UBE3A 45 kb loss Previous nl: Methylation UBE3A seq. 22q tel. Understand limitations/purpose of testing Unbalanced translocation between 5p and 17p GAIN 5p LOSS 17p Arrays can identify imbalances, but not determine mechanisms 121 Nomenclature for Reporting arr 16p11.2(29,649,997-30,199,855)x1 array cytogenetic band genomic coordinates x1 = loss (and genome x3 = gain build) Miller et al. (2010) Array Types and Diagnostic Yield Targeted Low Targeted with Backbone Whole Genome High Higher resolution studies increase the yield: Telomere FISH + 2-3% (over G-banding) Targeted Array + 7-11% Genomic Array + 9-20% compared to 2-3% by karyotype! Miller et al. (2010) 122 Array Analysis Now First-Tier Cytogenetic Test Microdeletion/microduplication syndromes Complex phenotypes due to dosage imbalance of multiple, unrelated genes which happen to be contiguous on chromosome. In some cases, clinical syndrome defined before genetic basis known. AKA Contiguous gene syndromes Segmental aneusomy syndromes Genomic Disorders (subset mediated by segmental duplications – seg dup) Mechanisms include deletion, duplication, and UPD = any deviation from normal, biparental inheritance. Microdeletion/duplication Syndromes Wolf-Hirschhorn Cri-du-Chat Williams Langer-Giedion Wilms tumor-aniridia (WAGR) Beckwith-Wiedemann Prader-Willi/Angelman Smith-Magenis Miller-Dieker DiGeorge/VCFS 4p16.3 5p15 7q11.23 8q24 11p13 11p15 15q11-13 17p11.2 17p13.3 22q11.2 123 Microdeletion/duplication Syndromes Mechanism Wolf-Hirschhorn Cri-du-Chat Williams Langer-Giedion Wilms tumor-aniridia (WAGR) Beckwith-Wiedemann Prader-Willi/Angelman Smith-Magenis Miller-Dieker DiGeorge/VCFS 4p16.3 5p15 7q11.23 8q24 11p13 11p15 15q11-13 17p11.2 17p13.3 22q11.2 non-rec non-rec seg dup non-rec non-rec non-rec seg dup seg dup non-rec seg dup Mechanisms of structural rearrangements Recurrent rearrangements (e.g., microdeletions/duplications and translocations) in human are mediated by large blocks of DNA sequence homology (25-400 kb) with very high sequence identity distributed along chromosomes. Mispairing between non-allelic copies produces unequal crossing over and deletion or duplication. = Non-Allelic Homologous Recombination (NAHR) “Segmental duplications” represent ~5% of total genomic sequence in human Segmental duplications (Low Copy Repeats; Direct Repeats) 100-400 kb, >99% sequence identity Unequal Recombination (non-allelic homologous recombination or NAHR) Deletion and Duplication Primary mechanism for common, recurring microdeletion/ microduplication syndromes in humans (Genomic Disorders) 124 Genomic Disorders in Humans Disorder Type Size Williams; autism Location 7q11.2 del/dup 2 Mb Duplicon 100 kb PWS/AS; aut mat 15q11-q13 del/dup 4 Mb 450 kb 24 kb CMT; HNPP 17p12 del/dup 1.5 Mb SMS; PLS 17p11 del/dup 5 Mb 200 kb NF 1 17q11.2 del 1.5 Mb 100 kb DGS/VCF 22q11.2 del/dup 3 Mb 200 kb Male infertility Sotos Yq del 3.5 Mb 200 kb 5q35 del 2.2 Mb 140 kb see review by Mefford and Eichler (2009) Curr Opin Genet Dev del(22)(q11.2) DiGeorge syndrome/VCFS: ~1/4,000 – most common mdel syndrome Thymus hypo/aplasia → cellular immunodeficiency Parathyroid hypo/aplasia → hypocalcemia DD, ID Cardiovascular: Conotruncal heart defects, aortic arch defects Dysmorphic features: Micrognathia, ear anomalies, cleft palate, short palpebral fissures, short upper lip del(22)(q11.2) Image from www.thelancet.com Image from www.pediastaff.com 22q Foundation - www.22q.org 125 16p11.2 Deletions • Most exhibit developmental and/or psychiatric disorders (e.g., autism spectrum disorder, intellectual disability) • Macrocephaly • Obesity • Seizures Zufferey et al. (2012) JMG; Shinawi et al. (2010) JMG; Hanson et al. (2015) Biol Psych 16p11.2 Duplications • Most exhibit developmental and/or psychiatric disorders (e.g., intellectual disability, ADHD) • Microcephaly • Seizures Zufferey et al. (2012) JMG; Shinawi et al. (2010) JMG; Hanson et al. (2015) Biol Psych Miller-Dieker syndrome (MDS) Lissencephaly, type I - complete agyria (absent gyri) or w/ limited pachygyria (broad gyri) severe ID, spasticity, seizures Microcephaly, bitemporal narrowing, vertical furrows on forehead prominent forehead, short nose, upturned nares, protuberant upper lip, thin vermillion border, small jaw Isolated lissencephaly sequence (ILS) - same or milder brain malformation with normal or subtle facial features 126 17p13.3: Normal vs. Lissencephalic Brain Image from Bill Dobyns MDS and ILS genetics del(17)p13.3 including the LIS1 gene (variable or random breakpoints, no “hotspots”) MDS: visible cytogenetic deletion in ~50% deletion by FISH in 100% ILS: normal high-resolution cytogenetics deletion by FISH in 30-40% point mutations found in LIS1 in ~40% non-deletion cases LIS1 gene - b subunit of platelet activating factor acetylhydrolase, brain isoform Ib del(4p) (Wolf-Hirschhorn syndrome) IUGR, microcephaly, hypotonia, severe ID Dysmorphic facial features hypertelorism,prominent glabella, arched eyebrows, nose broad or beaked,CL/P, short upper lip Other scalp defect, hypospadias, heart defect, seizures, preauricular pit Most de novo, 10-15% from balanced carrier parent 127 del(5p) (Cri du Chat syndrome) Cat-like cry in babies (hypoplastic larynx) IUGR, microcephaly, hypotonia, ID Hypertelorism, round face, epicanthal folds, downslanting palpebral fissures, strabismus Heart defect Transverse palmar creases Most de novo, ~15% from balanced carrier parent Smith-Magenis syndrome • First microdeletion syndrome with blocks of duplicated material flanking deletion • Reciprocal duplication product identified • Three blocks of sequence, SMS-REPs, mapped to 17p11-12 • SMS-REPs are >200 kb in length Recurrent Deletions Deleted Region Syndrome/Phenotype 1q21 TAR syndrome 1q21.1 ID/Microcephaly 3q29 3q29 deletion syndrome 5q35 Sotos syndrome 7q11.23 Williams syndrome 8p23.1 8p23.1 deletion syndrome 15q11.2-q13 PW/Angelman (BP1/2-3) 15q13.2-q13.3 ID/Epilepsy (BP4-5) 16p13.11 Autism/ID/Schizophrenia 16p11.2 Autism 17p12 HNPP 17p11.2 Smith-Magenis syndrome 17q12 Renal cysts and Diabetes; Autism/ID/SCZ 17q21.31 17q21 deletion syndrome 22q11.2 22q11.2 Deletion syndrome 128 Recurrent Duplications Duplicated Region Syndrome/Phenotype 1q21 TAR region 1q21.1 ID/Autism 3q29 Variable phenotype 5q35 Short stature, microcephaly 7q11.23 Autism 8p23.1 Variable phenotype 15q11.2-q13 Autism (BP1/2-3) 15q13.2-q13.3 Psychiatric disease (BP4-5) 16p13.11 Variable phenotype 16p11.2 Autism 17p12 CMT1A 17p11.2 Potocki-Lupski syndrome 17q12 Epilepsy 17q21.31 Behavioral problems 22q11.2 Variable phenotype; LD Recurrent CNVs Many show broad phenotypic presentation (some observed in apparently normal individuals) due to incomplete penetrance and variable expressivity Duplications tend to have more variable phenotypes than deletions Many being identified across multiple neurodevelopmental disorders (ID, ASD, Schizophrenia) Significant Phenotypic Variability in CNV disorders (as in all chromosome disorders) Lancet Neurol 2013 129 X chromosome abnormalities Sex chromosome aneuploidy Karyotype Incidence Name 45,X (1/3000) Turner syndrome 47,XXX (1/1000) Trisomy X 47,XXY (1/1000) Klinefelter syndrome 47,XYY (1/1500) 47,XYY syndrome X-Inactivation There are many important genes on the X chromosome… So, how can males, with only one X chromosome, and females, with two X chromosomes, not differ in the products encoded by most of these genes??? Explained by X-inactivation resulting in dosage compensation. 130 The Lyon Hypothesis - 1 In female somatic cells: •One X chromosome is active; •The second is inactive and remains condensed and appears in interphase cells as the Barr body. Barr Body Chromosome Complement 45,X; 46,XY; 47,XYY 46,XX; 47,XXY; 48,XXYY 47,XXX; 48,XXXY; 49,XXXYY 48,XXXX; 49,XXXXY 49,XXXXX Number of Barr Bodies 0 1 2 3 4 # Barr Bodies = n-1, where n=# X chromosomes The Lyon Hypothesis - 2 z X-Inactivation occurs early in embryonic life. ~2 weeks after fertilization, at several hundred cell stage. Note: The inactive X must become re-activated in the female’s germ line so that each egg can receive an active X chromosome. z X-inactivation is random. The inactive X may be either the paternal or the maternal X; with a mix of cells, females are mosaics for the X chromosome. z X-inactivation is clonal. After one X chromosome has become inactivated in a cell, all of that cell’s descendants have the same inactive X. 131 XX XX XX Paternal X Maternal X XX XX XX Random Inactivation XX XX XX X X X X X X Clonality X X X X X X X X X X X X X X X-Inactivation is Incomplete Subject to Inactivation Inactivation Escape Pseudoautosom al Genes Carrel et al. (1999) PNAS 96:1440-1444 Copyright (1999) National Academy of Sciences, U.S.A. X Chromosome Mechanism XIST gene (X-inactivation specific transcript) The XIST gene is located in the X inactivation center of Xq13 and is transcribed only from the inactive X chromosome. XIST mRNA transcripts are only detected in normal females, not normal males. But, the RNA transcript is not translated into a protein, rather it remains in the nucleus and coats the inactive X chromosome, which affects replication (inactive chromosome is late replicating) and condensation. 132 Properties of the Inactive X chromosome • • • • • • transcriptionally inactive late replicating in the cell cycle most genes inactivated undergoes inactivation from a specific initiation region stable and remains inactive inactivation “reset” in oocytes X Chromosome Inactivation Inactive X chromosome forms Barr body, associated with nuclear membrane If one normal X and one abnormal X chromosome (deleted, duplicated), abnormal X will be inactive (by selection in most cases) In balanced X;autosome translocations, the normal X is inactive 5-10% of normal females demonstrate extreme skewing of inactivation pattern – “unfortunate Lyonization” or “skewed X-inactivation” for X-linked disease gene. Pseudoautosomal Regions (PAR) X and Y chromosomes share TWO regions of homology which undergo very high levels of genetic recombination PAR1 is at distal Xp/Yp; obligatory cross-over in 2. 6 Mb region required for proper pairing and segregation of X and Y SRY is just proximal to PAR1 in Y specific region; unequal recombination leads to XX males and XY females PAR2 in distal Xq/Yq 133 Pseudoautosomal Region: •Acts like an autosome; •Exchanges with the PAR on Xp. SRY gene (Sex-determining Region on the Y chromosome) •Expressed during embryonic development; •Encodes product that interacts with other genes to initiate development of undifferentiated embryo into a male. Heterochromatic Region SRY X Y SRY = XX Male = XY Female Acknowledgements David Ledbetter, PhD, and Fady Mikhail, PhD, for some of the slides/content used in this lecture. 134 Clinical Molecular Genetics CLINICAL MOLECULAR GENETICS Madhuri Hegde, PhD, FACMG Associate Professor Emory Genetics Lab Scientific Director Sr. Director Emory Genetics Lab, Molecular Lab Madhuri Hegde, PhD, FACMG Department of Human Genetics Emory University School of Medicine 2165 North Decatur Road Decatur, Georgia 30033 (404)727-5624 Telephone (404)727-3949Fax [email protected] 137 138 Clinical Molecular Genetics Madhuri Hegde Emory University School of Medicine භ The following relationship(s) exist related to this presentation: භ Category of relationship – Advisor, Employment භ Name of commercial entity – Genzyme (Pompe program), PTC (DMD program), Coriell Cell Repositories, PerkinElmer Genetics Inc Learning Objectives භ Describe testing methods for types of mutations, limitations, and how to interpret results for the following types of disorders: භ Mutation Nomenclature භ Sequence interpretation භ Sequencing as a clinical tool භ Detection of different types of mutations භ Test validation භ Future 139 The Human Genome ¾ ~3 billion base pair locations Gaps in sequence still exist Exact position of a specific base changes with builds Current build – Hg19 ¾ Variation Millions of single nucleotide polymorphisms (SNPs) Copy number variants (CNVs) ¾ Nomenclature Standard Historic Standard Nomenclature ¾ Use of standard nomenclature allows for the precise, unambiguous identification of a genomic position ¾ Citing a specific reference sequence is critical for longterm understandability of results ¾ Necessary for targeted testing and proper interpretation of family studies Standard Nomenclature ¾ Recommendations by the Human Genome Variation Society ¾ DNA / RNA / protein identity Genomic denoted as “g.” Coding denoted as “c.” Mitochondrial denoted as “m.” RNA denoted as “r.” Protein denoted as “p.” ¾ Recommendations for Single basepair changes Small deletions, duplications, insertions Large rearrangments Intronic changes Nearly any scenario that has ever been reported http:// www.hgvs.org/mutnomen/ 140 Standard Nomenclature: Recommendations by the Human Genome Variation Society Nomenclature for Variation from Reference Sequence Type of Variation Genomic cDNA Protein Missense change g.5248232T>A c.20A>T p.Glu7Val or p.E7V g.20763582C>A c.139G>T p.Glu47* or p.E47X Deletion g.117199646_ 117199648delCTT c.1521_1523delCTT p.Phe508del or p.F508del Intronic change g.103234177C>T c.1315+1G>A - Nonsense change ¾ Specifying the reference sequence is critical These changes are meaningless without a reference Different references result in different correct ways to describe a variant http:// www.hgvs.org/mutnomen/ Standard Nomenclature The most common sickle cell disease mutation Reference Hemoglobin S Historic HbS dbSNP:rs334 dbSNP:rs77121243 (retired) HBB: Glu6Val NC_000011.9:g.5248232T>A NP_000509.1:p.Glu7Val NM_000518.4:c.20A>T Standard What is a mutation? MedicineNet.com: Mutation: A permanent change, a structural alteration, in the DNA or RNA. mu·ta·tion The FreeDictionary.com: n. 1. The act or process of being altered or changed. 2. An alteration or change, as in nature, form, or quality. 3. Genetics a. A change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the parental type. b. The process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome. c. A mutant. Pathogenic variant is now the preferred term in clinical genetics 141 Mutation Nomenclature භ Database reference: RefSeq (curated) භ Largest transcript; NM_012654.3 භ Note numbering starts at 㵬start site㵭, usually ATG භ Nucleotide substitution භ g (genomic), c (coding), m (mitoDNA), r (RNA) භ g.1162G>A භ c.621+1G>T or IVS4+1G>T (intronic) භ r.957a>u භ Amino acid substitutions: p (protein) 1 or 3 letter code භ p.R117H or p.Arg117His භ Frameshifts: p.Arg83fs or p. Arg83SerfsX15 භ Deletions and insertions භ p.F508del භ c.6232_6236delATAAG භ g.409_410insC භ Report all variants to appropriate database Human Genome Variation Society Human Variome Project www.hgvs.org www.humanvariomeproject.org Gene: Disease Evidence Gene Evidence Disease Variant Function (LoF) Spectrum Effect Penetrance Expressivity Does not change interpretation Does not change disease causality 142 The Growing Complexity…changing the fundamentals Many genes/ one disorder One gene/ many disorders One gene/ one disorder One gene/ AR, AD Somatic (cancer) One gene/ unrelated disorders LGMD and Other Muscular Dystrophies More than 25 LGMD types are linked to specific gene loci Variable expressivity 143 Sequence variant interpretation Pathogenic Likely pathogenic Unknown Likely benign Benign Richards et al., ACMG Recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med 2008: 10(4): 294-300. Maddalena et al., Technical standards and guidelines: molecular genetic testing for ultrarare disorders. Genet Med 2005: 10(8):571-583. Reviewed and Revised 2009. The ACMG Laboratory Practice Committee Working Group, ACMG Recommendations for Standards and Interpretation of Sequence Variants. Genet Med 2000: 2(5): 302-303. Pathogenic Likely pathogenic Unknown Likely benign Benign 1. Variants predicted to result in the loss of protein function (may or may not have been previously reported in patients with disease) a. frameshift (an insertion or deletion that is not a multiple of 3 nucleotides) b. nonsense (introduction of a premature stop codon) c. splice junction (at positions +1,+2, -1 and -2 in an intron) d. change in an initiation codon e. change in the termination codon 2. Variants predicted to result in an amino acid replacement (missense) with one of the following conditions met: a. variant demonstrated to result in reduced protein function (loss of function), or aberrant protein function (gain of function) in an appropriate functional assay b. common disease causing pathogenic variant in a specific population based on evidence in the literature c. variant reported in multiple affected individuals and demonstrated to segregate with disease in multiple families 3. Variants demonstrated to result in aberrant splicing in an appropriate functional assay (eg. intronic or silent) http://genetics.emory.edu/egl/emvclass/EGLClassificationDefinitions.php The Unknown: DMD Full Gene Sequencing - Duchene / Becker muscular dystrophy - X-linked - 79 exons – amplify each exon by PCR and sequence Example: exon 32 144 The Unknown: DMD Full Gene Sequencing c.4405C>T (p.Q1469X) Kabuki Syndrome • Autosomal dominant භ Characteristic facies භ Long palpebral fissures භ Lower lateral eyelid eversion භ Dispersed lateral one-third of eyebrows භ Widely spaced teeth භ Depressed nasal tip භ Malformed / prominent ears භ භ භ භ Skeletal anomalies Dermatolyphic abnormalities Mild to moderate intellectual disability Postnatal growth deficiency භ Caused by pathogenic variants in KMT2D Adam, Hudgins Clin Genet 2004: 67: 209-219 Missense Variants KMT2D: c.15536G>A (p.R5179H) Control Patient Reported in Ng et al., (2010) in two patients, demonstrated de novo Reported in Hannibal et al., (2011) in one additional individual 145 Pathogenic Likely pathogenic Unknown Likely benign Benign 1. Recessive conditions: (all the following conditions must be met) a. diagnosis has been confirmed by biochemical testing or patient phenotype is specific for disease b. variant located on opposite chromosome from a known disease causing pathogenic or likely pathogenic variant c. variant occurs at an evolutionarily conserved nucleotide and/or amino acid d. variant not present in dbSNP, EVS (Exome Variant Server) or other publically available database at a frequency consistent with being a benign variant 2. Dominant conditions: (all of the following conditions must be met) a. variant segregates with phenotype in the family being tested or b. testing parental samples demonstrates that the variant occurred de novo c. variant not present in dbSNP, EVS (Exome Variant Server) or other publically available database at a frequency consistent with being a benign variant Pathogenic Likely pathogenic Unknown Likely benign Benign MSUD is caused by the inability to metabolize branched-chain amino acids - 1 / 185,000 live births - 1 / 175 live births in the Mennonite population - Autosomal recessive - Caused by mutations in one of three genes: BCKDHA, BCKDHB, DBT c. 670G>T (p.E224X) DBT: c. 670G>T (p.E224X) – nonsense mutation c.752T>C (p.V251A) – unknown c.752T>C (p.V251A) DBT: c. 670G>T (p.E224X) – nonsense mutation c.752T>C (p.V251A)– likely pathogenic KMT2D: c.10740G>A (p.Q3580Q) 1 year old Reference Proband Exon 38 Intron 38 146 KMT2D: c.10740G>A (p.Q3580) Reference Reference Father Mother 1 year old Reference Proband Exon 38 Pathogenic Likely pathogenic Intron 38 Unknown Likely benign Benign Likely benign variant (one of the following conditions must be met) 1. Variant found heterozygous (for dominant) or homozygous (for recessive) in multiple unaffected individuals. 2. Variant found in cis with a pathogenic variant in multiple unrelated individuals 3. Variant found in an unaffected family member (for dominant) Pathogenic Likely pathogenic Unknown Likely benign Benign Benign variant (one of the following conditions must be met)* 1. Variant reported in dbSNP, EVS, locus specific databases or EGL database at a population frequency higher than expected given the prevalence of the disease and mode of inheritance. 2. Variant reported in a control population at a frequency inconsistent with being causative of disease 3. Other evidence from published literature that indicates the variant has no effect on function *Benign variants are interpreted as described above and not reported in clinical reports. A list of these variants is available upon request. 147 Benign variation GJB2 ¾ The NHLBI GO Exome Sequencing Project Large scale next-generation sequencing project Focus on heart, lung, and blood disorders European and African American populations ¾ GJB2: c.-34C>T European allele frequency: African allele frequency: 0.08% (A=7 / G=8587) 23.38% (A=1030 / G=3376) ¾ GJB2: c.79G>A (p.V27I) European allele frequency: African allele frequency: 0.21% (A=18 / G=8582) 0.34% (A=15 / G=4391) http://evs.gs.washington.edu/EVS/ Benign variation GJB2: c.79G>A (p.V27I) 1.2 Genotype frequency 1 0.163 0.186 0.8 0.395 0.6 1 0.442 1 0.4 0.2 0.442 0.372 HapMap-HCB (86 alleles) HapMap-JPT (172 alleles) A/A G/A G/G 0 HapMap-CEU (120 alleles) HapMap-YRI (120 alleles) Genotype frequencies of the c.79G>A (p.V27I) variant in HapMap populations. CEU - Utah Residents, YRI - Yoruba in Ibadan, Nigeria, HCB - Han Chinese in Beijing, China, JPT - Japanese in Tokyo, Japan http://www.ncbi.nlm.nih.gov/projects/SNP (rs2274084) http://hapmap.ncbi.nlm.nih.gov/ Pathogenic Likely pathogenic Unknown Likely benign Benign Variant of unknown clinical significance (VOUS) (if unable to classify the nucleotide change in one of the four categories above, it will be classified as a VOUS) a. not reported in HGMD, locus specific databases, published literature, dbSNP or EVS b. reported in dbSNP or EVS, but at an allele frequency insufficient to rule out clinical significance based on mode of inheritance and severity of disorder. c. reported in a single individual with insufficient segregation and/or functional data d. reported in a single individual with inadequate clinical information. Note: In the presence of conflicting data the term of variant of uncertain significance may be used. 148 KMT2D: c.2428_2508del81 (p.810del27) c.2428_2508del81 / + c.2428_2508del81 / + exon 10 c.1539 c.2797 exon 10 c.1539 c.2716 Rare variant vs. incomplete penetrance Variant Proficiency- Programs භ VITAL (Variant Interpretation Testing Across Laboratories) භ CAP NGS program PT; CAP Variant Assessment Programභ භ භ භ CAP-accredited Molecular Pathology laboratories: 821 International Mol Path: 169 CAP-accredited NGS laboratories: 280 International NGS: 51 භ Train the Trainer (R25 grant; Richard Haspel; Beth Deaconess) (Pathologist +Geneticist) 149 1 st Exome (2012)>Reanalysis;2013>2015) Disorders Requiring Sequencing භ Rare and ultra-rare disorders භ Common inherited disorders as 2nd tier strategy if: භ Targeted mutation panel (CFTR) fails to identify mutation in affected individual භ Point mutations are a less frequent type of mutation (DMD) භ Gene has many novel/nonrecurring mutations භ Limitations: will not detect gross alterations භ Scanning used to screen for mutations prior to sequencing භ dHPLC භ HRM analysis Clinical Sequencing One or a few pathogenic variants account for most cases in a population Targeted genotyping assays Many private pathogenic variants or an unknown spectrum of mutations in the population Full gene sequencing 150 The Known Versus the Unknown Genetic Disorder Unknown Gene(s) Known Gene(s) Single Gene One or a few mutations account for most cases in a population (The Known) Single gene Multiple Genes Many private mutations or an unknown spectrum of mutations in the population (The Unknown) Sequencing panels Exome Genome Clinical Information භ 10-day-old male patient භ භ භ භ භ Passed away in 2009 Hypotonia Polyhydramnios due to inability to swallow Reduced fetal movement Diagnosed with congenital muscular dystrophy in 3rd trimester භ type unknown භ Mother is 38 years old භ Now pregnant: 24 weeks භ Reports some bleeding in 1st trimester and feeling a lot of movement භ Normal family history 151 Previous Testing භ 46,XY (G-banded chromosome analysis) භ Normal microarray භ X-linked myotubular myopathy: MTM1 gene sequencing was normal භ Mother was tested for myotonic dystrophy: 5 and 12 CTG repeats භ SMA - normal භ Muscle biopsy – was abnormal but non-diagnostic (increased connective tissue suggestive of a congenital muscular dystrophy) Patient’s Exome Stats භ Total genes covered: 19,160 භ Total exons covered: 212,786 (66,474 HGMD) භ Total low coverage: 6,842 HGMD exons (11%) භ Total variants detected: 29,493 භ Total variants selected for confirmation: 11 භ True positive variants that match phenotype: භ RYR1 gene: c.12612G>A p.W4204X + c.14416A>G p.N4806D Pedigree p.N4806D p.W4204X p p.W4204X p.N4806D 24 weeks 46,XY Normal chromosomes via CVS Normal microarray 152 RYR1 gene භ Chr 19q13.3 භ 106 exons භ Encodes Ryanodine receptor 1 protein භ Form Ca2+ channels in skeletal muscle cells භ Involved in muscle contraction භ Mutations cause different diseases: භ Malignant hyperthermia (AD) භ Myopathies: භ Central Core disease (AD>AR) භ Multiminicore disease (AR<AD) භ Congenital Fiber-type disproportion (AD) Genomic Medicine: Exome, Whole Genome, Next Gen Panels භ භ භ භ භ භ භ භ භ භ භ භ භ භ The Geneticist is the Genomicist The Exome is now The Genome is still a work in progress to clinical Clinically available Standards & Guidelines in process Panels for multigene disorders Technology addresses single nucleotide changes and CNV Informed consent issues Filtering of data Interpretation issues Cloud computing False Positives/False negatives Cost effective? Decision tree for when to test using this method: Strategy Possibilities if a Sequence Variant is Not Detected භ Patient has a mutation in a different gene භ Patient has a mutation type that is not detected by the testing method due to allele drop-out භ Large gene deletions/duplications require a different test technology භ භ භ භ භ MLPA (multiplex ligation probe amplification) Multiplex PCR & dosage RT PCR, quantitative PCR, long-range PCR FISH Array technologies for CNV භ Patient has a mutation in a regulatory region that was not tested 153 Detection of Single base pair changes and small insertions/deletions භ Forward Allele-specific oligonucleotide (ASO) භ Reverse dot blot hybridization (RDB) භ Amplification Refractory Mutation System (ARMS) භ Oligonucleotide Ligation Assay (OLA) භ Fluorescence Resonance Energy Transfer (FRET) භ Liquid Bead Array භ End-point and real-time PCR analysis (TaqMan) භ Melting curve analysis using FRET hybridization probes Duchenne/Becker MD භ X linked recessive: males affected, females carriers භ One third of cases are new mutations භ Two thirds of patients have carrier mothers භ Incidence: 1 in 3000 male births භ Female carriers can rarely be affected due to skewed Xinactivation භ Extremely high new mutation rate ~ 10-4 භ Alu repeats in introns lead to del/dup භ Dystrophin gene located on Xp21.2 භ Large size of gene - 79 exons >2 million bases භ Deletion hot spots භ Genetic Testing: Deletion/duplication analysis & Sequencing DMD/BMD: Deletion/Duplication භ PCR-Multiplex analysis, qPCR, Southern, MLPA, Array භ Assess presence or absence of exons in DMD: භ 65% Deletions DMD; 85% in BMD භ 5-10% Duplications; 6-10% BMD භ Multiplex detects ~98% del/dup; MLPA,array: 100% භ Use Reading frame rule to predict in-frame (milder BMD) vs. out-of-frame (severe DMD) භ “Out of frame” deletion predicts no functional dystrophin protein and severe disease (DMD) භ “In frame” deletion predicts some dystrophin protein may be present, and less severe disease (BMD) භ Deletions cannot be detected by sequence analysis භ Sequencing to detect point mutations භ DMD-25-30% භ BMD-5-10% 154 Multiplex PCR Analysis P1 = del exons 45-48 P2= del exons 48-51 P3= del exon 44 Assay detects both deletions & duplications when done in log phase Thompson & Thompson Genetics in Medicine, 6th Edition, Nussbaum, McInnes, Willard, eds., p228, Copyright Elsevier (2004) cDNA Probes Detect Exons in DMD Gene 1 2 3 4 5 6 7 Exons 47 52 • Scan entire gene for deletions/duplications • Carrier testing/dosage 48/50 1= Wild-type control 2= Del 49-50 3= Del 49-52 4= Del 47-52 5= Del 49-50 49 6= Del 47-50 48 7= Del 47-50 Tom Prior 51 Principle of MLPA antisense primer labelled sense primer ligation point 㵰stuffer㵱 sequence with variable length • denaturation • probe annealing (O/N) • ligation • PCR amplification (universal primers) • separation by capillary electrophoresis Courtesy, G. Pals 155 Principle of MLPA antisense primer labelled sense primer ligation stuffer sequence with variable length • denaturation • probe annealing (O/N) • ligation • PCR amplification (universal primers) • separation by capillary electrophoresis Courtesy G. Pals Principle of MLPA antisense primer labelled sense primer PCR stuffer sequence with variable length • denaturation • probe annealing (O/N) • ligation • PCR amplification (universal primers) • separation by capillary electrophoresis Courtesy, G. Pals MLPA DMD: Female Carrier Test M. Hegde 156 Gene Targeted Microarray භ Gene centric design භ 60K probes tiled on the array භ Average spacing in coding region = 210 bp භ Average spacing in intronic region = 25 bp භ Length of probes ranges from 45-60 bases; isothermal Tm across array (Tm determines length of probe) භ CGH performed using same sex controls භ Array analyzed using manufacturer software භ Data masking feature - ability to extract data for single gene DMD 385k Male Ex79 Ex1 del Ex17 – Ex44 del Ex48 – Ex52 dup Ex2 – Ex4 DMD/BMD Gene Deletion •2 hot spot regions for deletions •Size of deletion does not determine phenotype •Exceptions exist •Reading frame rule correctly predicts >90% case phenotypes Thompson & Thompson Genetics in Medicine, 6th Edition, Nussbaum, McInnes, Willard, eds., p227, Copyright Elsevier (2004) 157 DMD/BMD Reading Frame Rule EXON 1 Nucleotide triplets encoding functional amino acids will be shifted out of reading frame (to create an unstable, non-functional protein) by deletion of exons containing a total number of nucleotides not divisible by 3 TAG CTA GCT EXON 2 AGC TAG CT EXON 3 T GGC EXON 4 CTA GCT NORMAL DMD (out of frame) BMD (in frame) Germline (Gonadal) Mosaicism භ Mother of affected male with known mutation in dystrophin gene may not have mutation in her somatic cells but may carry the mutation in her germ cells භ Germline (or gonadal) mosaic females may have a second affected child භ Risk is estimated at ~15% භ Lab reports should indicate that a mother of an affected male who has a negative test result for the dystrophin mutation present in her son has a risk of having another affected child. Spinal Muscular Atrophy (SMA) telSMN (survival motor neuron) is the primary SMA-determining gene • SMN1 •5q12.2-13.3 •AR • 1:6000 newborns • Homozygous deletion in SMA Types I, II, & III • Exons 7/8 show differences between SMN1 & SMN2 • Exon 7/8 deletion • Assay is PCR & RE digest Courtesy T. Prior 158 SMA telSMN Deletion Analysis Homozygous deletion of exon 7 in telSMN is diagnostic of SMA telSMN cenSMN 1 2 3 4 5 Uncut ___________DraI ____________ Digests of Exon 7 PCR Courtesy T. Prior 200ng DNA Template; 20 Cycles smnT,smnC 0,3 (Affected) Ratio (0, 0.9) 2,2 (NON-Carrier) Ratio (0.68, 0.64) 1,1 (Carrier) Ratio (0.36, 0.33) 1,2 (Carrier) Ratio (0.36, 0.61) Tom Prior Limitations of SMA Carrier Test භ Non Deletion Mutations භ Lack of Phenotypic Prediction භ De-novo Mutations භ 2 copy Cis SMN1 Chromosomes Tom Prior 159 ͳ ʹ ͳ ͳ ʹ ʹ ȋͳǦȌ ȋʹǦȌ Tom Prior Prader Willi/Angelman Syndromes භ Promoter region of SNRPN gene contains CpG islands which are heavily methylated in the maternally-derived allele and unmethylated in the paternally-derived allele භ PW = only methylated (maternal) allele present භ AS = only unmethylated (paternal) allele present භ Test method: Methylation analysis භ Restriction digest with methylation-sensitive RE and Southern using SNRPN probe භ Genomic DNA is treated with sodium bisulfite, converting cytosine to uracil except where cytosine is methylated. භ methylation-specific PCR භ methylation-specific melting analysis භ Detect 80% AS භ Detects 99% PWS PWS/AS: DNA Methylation Analysis • Patient 2 Patient 1 Normal Ctl Deletions 15q12 AS deletion Maternal PWS deletion paternal Southern blot uses methylation-sensitive enzymes & 5㵭 SNRPN probe Other methods of detection Deletion Ctl AS • • • • maternal paternal 160 DNA Methylation Detected by Methylation Specific PCR (MSP-PCR) …GTCMeGATCMeGATCMeGTG… …GTCGATCGATCGTG… Bisulfite treatment converts unmethylated C residues to U. …GTCMeGATCMeGATCMeGTG… ÅG CTAG CTAG CAC …GTUGATUGATUGTG… CTAGCTAGCACG PCR PCR primer PCR primer Product E. Lyon & R. Mao No product Prader-Willi/Angelman Methylation Methylation-Specific Melting Analysis Lower Tm for unmethylated (≈83°C) = Angelman Syndrome U m Higher Tm for methylated (≈87°C) = Prader-Willi Syndrome E. Lyon & R. Mao The Mitochondrial Genome භ Circular; 16,659 bp භ Polymorphic, 0.3% variation between individuals; 7-10x mutation rate of nuclear genome; limited DNA repair භ 2-10 copies/mt; 100’s to 1000’s per cell භ Unique genetic code 161 Genome Collaboration NuDNA MtDNA >300 genes for 37 genes for Resp chain proteins mtDNA Replication Expression Repair Antioxidant defense Fe homeostasis 13 Resp chain proteins 2 rRNAs 22 tRNAs Import machinery Adapted from SIMD NAMA Mitochondrial OXPHOS System * Totals 13 ~ 77 *DiMauro & Schon, NEJM 348: 2656, 2003 Special issues with Mitochondria භ Heteroplasmy is found in mito disorders භ Not all sample types will have the same proportion of the variant භ Blood for mito deletions භ Urine has been found to be suitable for a number of mito point mutations භ Muscle භ Sample type depends on which mito disorder is being tested භ Testing methods භ භ භ භ PCR-based Southern for deletions Array-based Next Gen භ A larger proportion of variant is generally associated with disease භ Unaffected individuals may have low levels of variant භ When a variant is identified, recommend testing mother and siblings 162 Human Disorders Due to Mitochondrial Mutations භ Kearnes Sayre syndrome (KSS) භ Pigmentary retinopathy, chronic progressive external ophthalmoplegia (CPEO) භ Leber hereditary optic neuropathy (LHON) භ Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) භ Myoclonic epilepsy with ragged red fibers (MERRF) භ Deafness භ Neuropathy, ataxia, retinitis pigmentosa (NARP) භ Subacute necrotizing encephalomyelopathy with neurogenic muscle weakness, ataxia, retinitis pigmentosa (Leigh with NARP) E. Lyon & R. Mao Mitochondrial Mutations Associated with Disease HV 1 HV 2 PH1 MELAS 3243A>G PH2 LHON 14484T>C PL LHON 3460G>A Areas deleted in KSS LHON 11778G>A MERRF 8344A>G NARP 8393T>G E. Lyon & R. Mao Detection of KSS Mitochondrial Deletion Mutation by Southern Blot M M + + PvuII U C U C The restriction enzyme, PvuII cuts once in the circular mitochondrial DNA. M = Mutant Heteroplasmy) + = Normal U = Uncut, No PvuII C = Cut with PvuII E. Lyon & R. Mao 16.6 kb (normal) Deletion mutant Autoradiogram 163 Detection of NARP Mitochondrial Point Mutation (ATPase VI 8993 TĺC or G) by PCR-RFLP U = Uncut, no MspI C = Cut, with MspI MspI U C U C U C 551 bp The presence of the mutation creates an MspI restriction enzyme site in the amplicon. 345 bp 206 bp Mutation present E. Lyon & R. Mao Agarose gel Cancer Tests භ Inherited භ Screening tumor භ Screening patient germline භ Screening family භ Algorithm of strategy භ Somatic භ Testing tumor භ patient therapy භ Patient prognosis භ Difference: Complexities of tumor analysis Hereditary Non-Polyposis Colorectal Cancer (HNPCC): Lynch Syndrome භ භ භ භ භ භ භ භ භ Represents ~3% of patients with CRC Can be confused with FAP – rule out first Autosomal dominant inheritance Family history is big clue (Amsterdam/Bethesda) Due to mutations in mismatch repair genes (MMR) භ MLH1, MSH2 Major භ MSH6, PMS2 Minor Tumors display Microsatellite instability (MSI) IHC can identify which MMR protein to test Testing done by sequencing (+/- scanning) and deletion analysis EGAPP recommends screening all newly diagnosed CRC patients 164 Microsatellite Instability 95% of HNPCC tumors have MSI at multiple loci 10%–15% of sporadic tumors have MSI Marker #1 Marker #2 Patient 1 Tumor Patient 1 Normal Patient 2 Tumor Patient 2 Normal Patient 1= MSI-High Patient 2 = MSS -stable S. Thibodeau Immunohistochemistry MMR Proteins M. Troxell BRAF & methylation for predicting sporadic vs inherited CRC භ Serine/threonine kinase භ Part of Ras/Raf/MEK/MAP signal transduction pathway භ Oncogenic mutations in 66% metastatic melanoma, 36% papillary thyroid cancer and 10% colon cancer භ Most BRAF mutations are V600E භ BRAF V600E mutation affects methylation of MLH1 promoter and is nearly 100% somatic භ Absence of MLH1 protein by IHC in the presence of BRAF V600E is most likely somatic, not HNPCC V600E E. Lyon & C. Vaughn 165 FAP-AFAP-MAP – INDICATIONS FOR TESTING Colorectal cancer diagnosed in an individual younger than 40 years of age Colorectal cancer diagnosed in one or more first-degree relatives Colorectal cancer diagnosed in two or more first- or second-degree relatives of any age FAP/AFAP testing: 10-100 adenomatous polyps with dominant inheritance MAP testing: 10-100 adenomatous polyps with family history suggestive of recessive inheritance Test for APC mutations (Point mutation, deletions/duplications) Positive Test for p.Y165C and p.G382D MutYH mutations One heterozygous mutation detected Negative Diagnosis of FAP/AFAP •Test at-risk family members •Offer genetic counseling Patient may have APC mutation not detected by testing method – Consider MAP testing (Important for patients with no/weak FHx* and fewer polyps) Negative MutYH gene sequencing Biallelic mutations detected Diagnosis of MAP •Offer testing for offspring ( obligate carriers) and relatives One heterozygous mutation detected Biallelic mutations detected Negative •Patient is, at minimum, a carrier of MAP •Patient may have MutYH mutation not detected by testing method – Consider FAP testing •Offer screening for relatives and\or genetic counseling Diagnosis of MAP •Offer testing for offspring (obligate carriers) •Offer testing for relatives and genetic counseling • Screen based on FHx* and clinical presentation • Consider FAP testing *FHx: Family History Lynch Syndrome- INDICATIONS FOR TESTING Colorectal cancer diagnosed in all newly diagnosed CRC* Synchronous, or metachronous, colorectal, or other HNPCC-associated tumors Colorectal cancer diagnosed in one or more first-degree relatives with an Lynch-related tumor, with one diagnosed before 50 years of age Colorectal cancer diagnosed in two or more relatives of regardless of age *see EGAPP recommendation3,4 Microsatellite instability and Immunohistochemical analysis Recommend performing both tests when there is a high index of suspicion for LS* MSS or MSI-L No loss of proteins on IHC MSI-H and loss of expression of MLH1 or PMS2 only or both MLH1/ PMS2 based on IHC Probably not Lynch Syndrome Test for MLH1 promoter methylation and BRAF p.V600E mutation MSI-H and loss of expression of MSH2 or MSH6 only or MSH2/MSH6 based on IHC Test for MSH2 gene mutations No Mutation detected Hyper-methylation of MLH1 and BRAF mutation detected CRC not due to MMR defect Hypermethylation of MLH1 and no BRAF mutation detected Hypermethylation absent in normal tissue: CRC not due to MMR defect Normal methlyation of MLH1 and no BRAF mutation detected Hypermethylation present in normal tissue: Inherited epimutation No mutation detected Test for EpCAM Deletion Mutation detected Lynch Syndrome: Test at-risk family members No Mutation detected Test for MSH6 gene mutations Test MLH1 gene mutations No mutation detected Mutation detected Test PMS2 gene mutations Lynch Syndrome with unidentified mutation No mutation detected Mutation detected Lynch Syndrome: Test at-risk family members Lynch Syndrome with unidentified mutation භGene-targeted therapies භBCR/ABL1 : imatinib, dasatinib, nilotinib භPML/RARα : ATRA, arsenic භHER2/neu : trastuzumab භEGFR : erlotinib, getfitinib භKRAS : panitumumab, cetuximab Mutations may predict drug response or absence of response. Barbara Zehnbauer 166 Gene Drug CYP2C9 warfarin TrimGen Corporation eQPCR LC Warfarin Genotyping PGx Diagnostic Test CYP2C9*2, CYP2C9*3 VKORC1 warfarin TrimGen Corporation eQPCR LC Warfarin Genotyping VKORC1:G-1639A CYP2C19 clopidogrel, esomeprazole, omeprazole, phenytoin, others Infiniti CYP450 2C19 CYP2C19*2, CYP2C19*3, CYP2C19*4, CYP2C19*5, CYP2C19*6, CYP2C19*7, CYP2C19*8, CYP2C19*9, CYP2C19*10 CYP2D6 codeine, fluoxetine, metropolol, risperidone, tamoxifen, others Roche AmpliChip Cytochrome P450 Genotyping test and Affymetrix GeneChip Microarray Instrumentation System CYP2D6*1, CYP2D6*2ABD, CYP2D6*3, CYP2D6*4ABDJK, CYP2D6*5, CYP2D6*6ABC, CYP2D6*7, CYP2D6*8, CYP2D6*9, CYP2D6*10AB, CYP2D6*11, CYP2D6*15, CYP2D6*17, CYP2D6*19, CYP2D6*20, CYP2D6*29, CYP2D6*35, CYP2D6*36, CYP2D6*40, CYP2D6*41, CYP2D6*1XN, CYP2D6*2XN, CYP2D6*4XN, CYP2D6*10XN, CYP2D6*17XN, CYP2D6*35XN, CYP2D6*41XN UGT1A1 irinotecan Invader UGT1A1 Molecular Assay Variants Assayed UGT1A1*28 Test Sensitivity & specificity භ Analytic validity: ability to accurately measure a specific analyte, or identify a mutation of interest in the sample type(s) භ Analytic sensitivity: proportion of samples that have a positive test result and that are correctly classified as positive භ Analytic specificity: proportion of samples that have a negative test result and that are correctly classified as negative. භ Clinical validity: ability to accurately identify individuals who have (or will develop) the disorder or phenotype of interest. භ Clinical sensitivity: proportion of individuals who have (or will develop) the phenotype of interest and who have a positive test result. භ Clinical specificity: proportion of all unaffected individuals identified by the proposed test as being negative Predictive Value of Genetic Testing භ The positive and negative predictive values of testing in the target population measure the ability of the test to give accurate clinical information. භ The positive predictive value is the proportion of positive test results that correctly identify an individual who has the phenotype of interest (number of true positives / true positives + false positives). භ The negative predictive value is the proportion of negative tests that correctly identify an individual who does not have the phenotype of interest (number of true negatives / true negatives + false negatives). 167 Clinical Utility භ Addresses risks & benefits of testing: භ Results of pilot trials භ Quality assurance processes that monitor effectiveness of lab testing භ Adverse effects of testing on health or social consequences භ Follow-up treatment/interventions available for patients based on genetic test results භ Ret mutations guide therapy for MEN-2 patients භ EGFR mutations guide drug use for lung cancer භ Financial costs and economic benefits of testing භ ELSI issues ACCE EGAPP Haddow & Palomaki Current State Screening Symptomatic medicine Precision medicine Future State Screening • • NBS 2nd tier (panel) NBS confirmatory (panel) Symptomatic medicine Precision medicine • • • Single gene testing (pharma) Gene Panels (pharma) Exome/ Genome sequencing 168 Pitfalls in Genetic Testing භ භ භ භ භ භ භ භ භ භ භ PCR: PCR Contamination PCR: SNPs in primers Wrong gene….diagnosis incorrect Linkage: Paternity incorrect; recombination error rate Prenatal: Maternal cell contamination R/O is critical! Incomplete testing….test doesn㵭t detect certain types of mutations Patient has had a bone marrow transplant Mutation in patient is not on panel Potential for false negative results VUS – may be uninformative or misclassified Technical problems භ Sample mix-ups at pre- or analytical stage භ Sample mix-up at blood draw site භ SNPs/rare variants at the primer/probe site භ Human Error Helpful Study Guides භ GeneTests Reviews www.genetests.org භ ACMG Standards & Guidelines භ ACMG Practice Guidelines www.acmg.net භ Human Molecular Genetics, Strachan & Read භ Human Genetics & Genomics, Korf භ Genetics in Medicine, Thompson & Thompson භ CAP/ACMG Proficiency Testing Program (targets most common genetic tests) භ ACMG Genetics Review Course භ CLSI Molecular Methods, 2011 edition Supplemental Study Slides 169 භ BCR/ABL1 is molecular signature of Philadelphia chromosome t(9;22) භ Fusion gene of BCR and ABL1 tyrosine kinase භ Diagnostic for CML භ Prognostic for ALL භ Monitor MRD by quantitative PCR භ TKI therapy targeted to fusion gene – imatinib, dasatinib, nilotinib භ Mutations in ABL in kinase domain detected by sequencing may inhibit binding of drug B. Zehnbauer Proposed mode of action of STI571 -Functions through competitive inhibition of the ATP binding site on the BCR-ABL product -This leads to inhibition of tyrosine phosphorylation of proteins S. Olson involved in signal transduction Druker et al., 2001 EGFR (ERBB1, HER1) භProto-oncogene (7p12) භEncodes a receptor tyrosine kinase භOverexpressed in various cancers (lung, head and neck) භApproximately 10% of patients with NSCLC respond to small molecule inhibitors gefitinib or erlotonib Barbara Zehnbauer 170 Mutations Associated with Sensitivity to EGFR Inhibitors EGF binding EGF TM binding Tyrosine kinase Autophosphorylation Exon 1 28 Exon 18 Exon 19 Exon 20 Exon 21 5% 45% <1% 40-45% G719C ΔE746-750 % of all mutations Examples L858R Zehnbauer Mutations Associated with Resistance to EGFR Inhibitors EGF binding EGF TM binding Tyrosine kinase Autophosphorylation Exon 1 28 Exon 18 Exon 19 Exon 20 % of all mutations 1% 5% Examples D761Y T790M Exon 21 Zehnbauer panitumumab cetuximab Zehnbauer EGFR inhibitors will be ineffective when KRAS is mutated and constitutively active. http://www.kras-info.com/slide_set 171 Normal ITD FLT3 Mutation = poor prognosis In AML dHPLC analysis NPM Mutation = good prognosis In AML Insertion of 4bp = frameshift Special issues with Tumor analysis භ FFPE: formalin-fixed paraffin-embedded slides of tumor/normal tissue භ Requires stained slides & microscopic review to identify regions of normal & tumor භ There is a minimal amount of tumor required for analysis භ Tumor heterogeneity can influence result භ Requires method of detection with high sensitivity to detect small amount of variant in tumor sample භ Tumors are very different from germline analysis! Other considerations…. භ Evidence-based tests භ See requirements of GTR භ Test validation භ Review Guidelines භ Pitfalls of DNA tests 172 Genetic Transmission GENETIC TRANSMISSION Bruce R. Korf, MD, PhD, FACMG Wayne H. and Sara Crews Finley Chair in Medical Genetics Professor and Chair, Department of Genetics Director, Heflin Center for Genomic Sciences University of Alabama at Birmingham Bruce R. Korf, MD, PhD, FACMG Department of Genetics University of Alabama at Birmingham 1720 2nd Ave. S., Kaul 230, Birmingham, AL 35294-0024 (205) 934-9411 Telephone (205) 934-9488 Fax [email protected] 175 176 Genetic Transmission Bruce R. Korf, MD, PhD Professor and Chair, Department of Genetics University of Alabama at Birmingham Disclosure(s) Relationship Entity Grant Recipient Novartis Advisory Board Accolade, Genome Medical Board of Directors American College of Medical Genetics and Genomics Children’s Tumor Foundation Advisor Neurofibromatosis Therapeutic Acceleration Project Founding Member Envision Genomics Salary University of Alabama at Birmingham Objectives • Recognize patterns of Mendelian transmission • Describe deviations from classical Mendelian transmission • Perform basic population genetic calculations • Describe models of multifactorial inheritance • Use odds ratios in risk assessment based on GWAS data 177 Autosomal Recessive A a AA A A A a a Aa unaffected Genotype homozygous Phenotype unaffected heterozygous Aa aa a affected homozygous unaffected affected Counseling 2/3 Consanguinity Aa AA AA Aa Aa AA AA Aa Aa AA aa Identical by descent 178 Recessive Mechanisms Gene Loss of function mutations: deletion, frameshift, stop, missense Protein Complete absence of product or significant reduction of function Heterozygote – sufficient activity to avoid phenotype Homozygote – profound loss of activity results in phenotype Autosomal Dominant Aa a aa a a a A A homozygous Phenotype unaffected heterozygous affected Aa aa Aa aa A aa Genotype aa A Aa Aa aa Aa Aa aa homozygous affected Pseudodominance Aa Aa aa aa 179 Penetrance Fraction of individuals who carry a gene who manifest a specified phenotype Age-Dependent Penetrance Expressivity different modes or degrees of expression of trait in population Neurofibromas in NF1 180 Dominant Mechanisms • • • • • • Haploinsufficiency • Dominant negative • Tumor suppressor Deletion Stop Frameshift Missense Structural Loss of Function Gain of Function • Signaling pathway • Missense Mosaicism • Germ line • Somatic X-linkage Male y A Y a unaffected A Y Aa affected y A y Aa a Female A A unaffected A a unaffected a a affected Aa A y AA Aa a A AA AA a No male to male transmission 181 X-linked Dominant Male transmits to all daughters, not to sons X-linked Dominant Male Lethal Males who inherit mutation die in utero Females who inherit mutation are affected X Chromosome Inactivation 182 Genetic Heterogeneity • Locus • Mutations in different genes result in same phenotype • Allelic • Different mutations in same gene Locus Heterogeneity Allelic Heterogeneity gene aa BB AA bb Compound Heterozygote Genetic Heterogeneity Gene A Gene D Phenotype 1 Gene E Gene B Gene C Phenotype 2 Gene F Gene G Sex Limited Expression autosomal dominant transmission expressed only in one sex get male to male transmission requires sex-specific factor for expression male pattern baldness 183 Epistasis gene interaction results in modification of phenotype A O AB Bombay Phenotype A H precursor B 3 Digenic Inheritance Gene Locus 1 Gene Locus 2 Maternal Transmission 184 Mitochondrial DNA • 16.5 kb circular double stranded DNA • Multiple copies per mitochondrion • Heteroplasmy – mixture of mitochondria with different genotypes in same cell • 13 subunits of mitocondrial proteins, tRNAs, rRNAs • Most mitochondrial proteins encoded in nucleus Genomic Imprinting maternal copy expressed paternal copy not expressed Imprinting: differential expression of maternal and paternal copy of a gene Hereditary Paraganglioma 185 Hardy-Weinberg Equilibrium • Large population [A] = p [a] = q p + q =1 • No mutation • No selection [AA] = p2 [Aa] = 2pq [aa] = q2 • Random mating • No migration frequencies remain stable eggs sperm allele frequency A = a = A a p q AA p p2 aA q pq allele frequency Aa pq aa q2 Autosomal Recessive Cystic fibrosis: 1/2,500 = q2 q = 1/50 2pq = 2(1/50)(49/50) ≈ 1/25 2/3 1/25 risk to offspring = (2/3)(1/25)(1/4) = 1/150 186 Selection AA Aa aa Before p 2 2pq q2 After p2 2pq 0 Reduction in reproductive fitness Genetic Lethal p q 0.5 0.5 0.66 0.33 0.75 0.25 generation 1 aa Aa AA gene pool 2 Aa AA aa gene pool 3 AA Aa aa Genetic Lethal – Change in Allele Frequency q 0.6 0.5 0.4 0.3 0.2 0.1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 generation 187 Mutation-Selection Equilibrium a a mutation A A A A A A A A A A A A A A A A A A A A A A A A A a a A A A A A A selection A A A Mutation-Selection Equilibrium q 0.6 0.5 0.4 0.3 selection 0.2 0.1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 generation mutation Mutation-Selection Balance Autosomal Recessive 2 Before p 2pq Fitness 1 1 After 2 p 2pq 2 q 1-s 2 q (1-s) lose 2sq2 alleles each generation at equilibrium, 2μ = 2sq2 q= μ s 188 Mutation-Selection Balance Autosomal Dominant AA Aa aa Before p2 2pq q2 Fitness 0 1-s 1 After 0 2pq(1-s) q2 lose 2ps alleles each generation at equilibrium, 2μ = 2ps p = μ/s Balanced Polymorphism • Maintenance of deleterious allele in heterozygotes • “Heterozygote advantage” • Polymorphism: occurrence of at least two alleles at locus having frequency of at least 1% Malaria AA Globin Disorder aa AA Aa Genetic Drift • Fluctuation in gene frequency due to small size of breeding population • Fixation or extinction of allele possible 189 Founder Effect • High frequency of gene in distinct population • Introduction at time when population is small • Continued relatively high frequency due to population being “closed” Consanguinity Coefficient of Inbreeding: Probability that two alleles in child are identical by descent; symbolized by “F” Coefficient of Inbreeding 190 Coefficient of Inbreeding Coefficient of Inbreeding (1/2)(1/2)(1/2)+(1/2)(1/2)(1/2) = 1/4 Coefficient of Inbreeding 191 Multifactorial Inheritance • Trait clusters in family • Increased concordance in identical twins • Multiple genetic and/or non-genetic factors Familial Clustering λR = ratio of risk in relatives of type R compared with population risk λs = ratio of risk in sibs compared with population risk Cystic fibrosis: Risk in sibs = 0.25; risk in population = 0.0004 λs = 500 Huntington disease Risk in sibs = 0.50; risk in population = 0.0001 λs = 5000 Disorder Population Frequency Recurrence Risk in First Degree Relatives λ Cleft lip 㫧 palate 0.001 0.049 49 Congenital hip dislocation 0.001 0.035 35 Pyloric stenosis 0.002 0.032 16 Type 1 diabetes mellitus 0.002 0.071 35 Twin Studies • Dizygotic – Genetically equivalent to full sibs – Distinct placentas, chorions, amnions, but placentas/chorions may fuse • Monozygotic – Genetically identical – Fetal membranes may be separate or shared % concordance Disorder MZ Sibs Cleft lip 㫧 palate 40 5 Pyloric stenosis 22 4 Clubfoot 32 3 Congenital hip dislocation 33 4 Diabetes mellitus type 1 36 7 192 Heritability genetic environmental variance variance measurement variance VP = VA + VD + VE + VI + CovGE + VM VA = additive genetic variance VD = deviation due to dominance and epistasis VE = environmental variance VI = interaction variance CovGE = covariance of genetics and environment Heritability in narrow sense Heritability in broad sense VA VP h2 = h2 = VG VP Additive Polygenic Model QTL: quantitative trait locus aabb Aabb aaBb Dominant: 2 cm Baseline: 150 cm AaBb aaBB AAbb AaBB AABb AABB Threshold Model Threshold Number of Individuals Trait Liability 193 Sex Differences females males Common Disease-Common Variant Hypothesis Common diseases accounted for by genetic variants found in 1-5% of population Linkage disequilibrium Case-Control Study ACTAGGA Allele 1 ACTCGGA Allele 2 Asthma No Asthma Allele 2 Present 30 10 Allele 2 Not Present 70 90 Hypothesis: Allele 2 is associated with an increased risk of asthma 194 Odds Ratio Asthma No Asthma Allele 2 Present 30 10 Allele 2 Not Present 70 90 Odds Ratio (used on case-control studies): Odds of disease given allele = 30/10 = 3 Odds of disease given not allele 2 = 70/90 = 0.78 Odds ratio = 3/0.78 = 3.85 Odds Ratio and Risk ܴܴ ൌ ܱܴ ͳ െ ܴܿ ൅ ሺܴܿ ȉ ܱܴሻ ܴ݅‫ ݇ݏ‬ൌ ܴܴ ȉ ܴܿ Say the risk of asthma in the population is 0.1 and we have calculated the OR ଷǤ଼଺ to be 3.86. The relative risk would be = 3.00 ଴Ǥଽାሺ଴ǤଵȉଷǤ଼଺ሻ The risk to an allele 2 carrier of getting asthma would then be (0.1)(3.00) = 0.3 GWAS https://www.ebi.ac.uk/gwas/diagram 195 Missing Heritability 100% 90% Non-genetic factors 80% 70% 60% 50% 40% Calculated heritability Missing heritability 30% Possible Explanations • Heritability overestimated • Rare variants account for some heritability • Non-detected variants (e.g., CNVs) 20% Heritability accounted for by GWAS 10% 0% 196 Newborn Screening NEWBORN SCREENING John A. Phillips, III, MD, FACMG David T. Karzon Professor of Pediatrics Professor of Pathology, Microbiology and Immunology and Professor of Medicine Director Division of Medical Genetics and Genomic Medicine Vanderbilt University School of Medicine John A. Phillips, III, MD, FACMG Division of Medical Genetics Vanderbilt University School of Medicine DD-2205 Medical Center North Nashville, TN 37232-2578 (615) 322-7602 Telephone (615) 343-0959 Fax [email protected] 199 200 Newborn Screening John A Phillips III David T Karzon Prof of Pediatrics Vanderbilt University Medical Center 4/21/2017 Site Investigator: 1) PKU: BMN 015 &165, 2) Achondroplasia: BMN 111-901, 201 & 202 Clinical Trials BioMarin Pharmaceutical Inc. & 3) FAOD: UX007 Ultragenyx Pharmaceuticals, Inc. PI: TN State Genetics Contract & Member TN Genetics Advisory Committee Co-PI: Vanderbilt Undiagnosed Disease Network (UDN) Clinical Center Co-I: Dr Blackwell’s PPG “Mechanisms of Familial Pulmonary Fibrosis”. 4/21/2017 Learning Objectives •Understand the approach used in Newborn Screening (NBS) • Understand clinical presentations, Dx & Rx of selected disorders in the NBS Panel • Be able to access & use ACMG ACT sheets & New England Emergency Protocols 4/21/2017 201 NBS: Key Points • Done by state health programs • Emphasizes disorders with effective, necessary & available early Rx • Samples obtained by hospital, but tests & follow up done by state • Methods (ABR, DNA, enzyme, hormone, HPLC, MS/MS, OAE) & disorders may differ between states 4/21/2017 NBS: Criteria • Serious & has reasonable frequency • Clinical Dx is difficult & requires test • UnRx causes irreversible damage • Test is rapid, sensitive & specific • Feasible intervention is available that improves outcome • NBS program is cost effective 4/21/2017 NBS: Approach • Traditional Medicine- based on signs, symptoms, or FH • NBS- done on all to identify those to Rx • Preclinical-period before disease onset 4/21/2017 202 Sensitivity: Fraction of affecteds who screen positive = TP/(TP+FN) Sample screen = TP+TN+FP+FN 4/21/2017 1- Sensitivity = False Neg Rate Specificity: Fraction of unaffected who screen neg = TN/(FP+TN) 4/21/2017 1- Specificity = False Pos Rate Positive Predictive Value (PPV): Fraction with + screen who are affected =TP/(TP+FP) 4/21/2017 203 Management of NBS Results 96% 1% 4/21/2017 3% Unsatisfactory NBS Samples 4/21/2017 Abnormal NBS Results Mildly abnl (PPV < 10%) • Only one analyte ~cut off • Lab notifies PCP/facility to repeat NBS Highly abnl (PPV > 60%) • Single/multiple analytes >>cut off • Lab notifies PCP ASAP & gives Metabolic Specialist contact info • ACMG ACT sheets & Algorithms, GeneReviews & New England Emergency protocols 4/21/2017 204 ACMG NBS Info (www.acmg.net) 4/21/2017 Phenylketonuria (PKU): Dx & Rx • AR def of Phenylalanine Hydroxylase (PAH) • ~1/15,000 • UnRx IQ < 50 • Dx: Phe > 1200 μmol/l (μM) (20 mg%; Tyr & nl blood Biopterin or urine pterins (Genet Med 16: 188, 14) • Rx: Phe diet if >360 μM 4/21/2017 PKU: NBS History Folling 1934 Guthrie Bacterial Inhibition Assay 1961 MS/MS 2017 4/21/2017 205 PKU: NBS by MS/MS • Sample prep & add internal standard (IS) • Ionize & inject into 1st MS/MS mass analyzer (MA) • Select ions & fragment by argon • Separate fragments by 2nd MA & determine signatures 4/21/2017 PKU: MS/MS Phe & Phe/Tyr ratio 4/21/2017 Case 1: What Should You Do? • It is 4:30 PM on a Friday • You (Baby Gale’s PCP) are called by the State NBS lab • Baby Gale’s Phe level was 410 μM (nl <152) & Phe/Tyr was 3.02 (nl < 2.01) at 24 hrs of age (3 days ago) • What should you do? 4/21/2017 206 ACMG NBS Info (www.acmg.net) 4/21/2017 4/21/2017 ACMG Algorithm http://www.acmg.net 4/21/2017 207 Case 1: What should you do? a) Inform family of NBS result b) Evaluate baby & consult metabolic specialist c) Initiate confirmatory/diagnostic tests with metabolic specialist d) Provide family basic info on PKU e) All of above 4/21/2017 PKU (PAH) vs BH4 Defects 98% 2% 4/21/2017 Hyperphenylalaninemia Category Phe Level μM/L Mutations Classic PKU >1200 (20mg%) PAH Mild/Moderate PKU 360-1200 (6-20mg%) PAH Non-PKU HPA 120-360 (2- 6 mg%) PAH Transient HPA Premature, TPN, Liver disease & Mat HPA None BH4 Deficiency ~2% of those 120>1200 (2-20 mg%) PCD, DHPR, GTPCH & PTPS BH4 Responsive PKU ~400-1100 PAH 4/21/2017 208 Treatment of PKU 4/21/2017 PKU: Treatments • Standard: Phe free formula & low protein diet Goals: Phe 120-360 μM (all ages) Ref: Genet Med 16: 188, 14 • Cofactor: BH4 (Sapropterin™) @ 20mgm/kg/day ź Phe >30% in ~50% May be used in pregnancy 4/21/2017 PKU: Treatments • Competition: Large neutral amino acids • Competes with Phe for uptake by membrane transporters & Lysine impairs Phe absorption • May reduce CNS Phe but compliance critical • Not recommended in pregnancy • Enzyme Substitution: Recomb PEGylated Phe Ammonia Lyase • Phase 3 trial testing safety, effect & dose on Phe levels • Injection site reaction & immune reaction problems • 0.1mg/kg lowers Phe by 62% 4/21/2017 209 Maternal PKU • Phe teratogenic causes miscarriage, IUGR, ID, CHD, microcephaly, CL/P & pyloric stenosis • Phe<360μM 3 months before conception 4/21/2017 Tyrosine: Pathway Tyrosinemia Type II Tyrosinemia Type I 4/21/2017 Tyrosinemia Type I (Hepatorenal) • AR defect in FAH • Sx: liver failure/cirrhosis & hepatic cancer (35-40%) by 5yrs; renal Fanconi syndrome, FTT; neurologic crises • Dx: UOA + succinylacetone; PAA & NBS Tyr & Met; 1/5000 French Canadians • Rx: NTBC & Phe/Tyr diet, liver transplant ? 4/21/2017 210 Tyrosinemia Type II (Oculocutaneous) • Clin: ID, corneal dystrophy, hyperkeratosis & erosions of palms/ soles • AR deficiency of Tyr aminotransferase • Dx: PAA Tyr, UOA 4-OH-phenyl lactate & N-acetyltyr • Rx: low Tyr diet 4/21/2017 Classical Homocystinuria • Cystathionine ɴ Synthase Deficiency • AR ~1/150,000 • NBS for Methionine rises slowly; likely true + if Met > 100 μM • Dx:Ÿplasma Methionine & Homocysteine • źCysteine & urine + nitroprusside 4/21/2017 Classical Homocystinuria • Clin: ID, lens dislocateź thrombosis & osteoporosis • Rx: Pyridoxine (B6) & Folate; prn low Met diet, Betaine, Dipyridamole & ASA • ~½ respond B6 (usually Ile278Thr) vs non responders (Ser307Gly) 4/21/2017 211 Branch Chain AA (BCAA) Catabolism 4/21/2017 Maple Syrup Urine Disease (MSUD) • AR def branched chain (BC) ketoacid decarboxylation • Freq ~1/ 250,000 (Mennonites 1/176) • Clin: Urine maple syrup odor; lethargy, irritability, emesis & coma • NBSŸLeucine; Dx:Ÿblood BCAA (Leucine, Isoleucine &Valine) & Alloiso-leucine (diagnostic); urineŸBC ketoacids & ketones • Rx: Thiamine (B1), diet źBCAA 4/21/2017 ACMG NBS Info (www.acmg.net) NBS often + due to mom’s levels 4/21/2017 212 Medium Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency • AR, most common FAOD ~1/12,000 • Fasting > lethargy, hypoketotic hypoglycemia & SIDS (first crisis fatal in 25% before NBS) • NBS: MS/MSŸC6, C8 & C8/ C10 • Dx: Acylcarnitine profile,źfree carnitine, UOA ŸC610 dicarboxylics, >90% of mutations are Lys304Glu (K304E) • Rx: Carnitine & avoid fasting 4/21/2017 MCAD MS/MS Acylcarnitine Profile Normal 100 % 0 100 MCAD Deficiency % 0 225 250 275 300 325 350 375 400 425 450 475 500 m/z 4/21/2017 NBS for Other FAODs • SCAD: hypotonia & metabolic acidosis; NBSŸC4 & UOA have ethylmalonic acid; common mild variants of ? Significance • LCHAD: cardiomyopathy, hypotonia, rhabdomyolysis & moms have HELLP; NBS ŸC14-OH, C16-OH , C18OH & C18:1-OH • VLCAD: cardiomyopathy, hepatomegaly, rhabdomyolysis & SIDS; ŸC14:1 & C14:1/ C12:1 4/21/2017 213 ACMG NBS Info (www.acmg.net) D D NBS often + due to mom’s D 4/21/2017 3Methylcrotonyl CoA Carboxylase Def (3MCC) • Clin: highly variable; some decompensate but great majority are asymptomatic; metabolic acidosis; Ÿ AG & NH3;źglu • AR, heterodimer MCCA*, MCCB* • NBS:ŸC5-OH, mom’s levels can cause false + infant NBS; UOA show 3-methylcrotonylglycine • Rx with diet (low Leu if needed) & add carnitine & Glycine 4/21/2017 Case 2: What Should You Do? • It is 4:30 PM on a Friday • You (baby Adon’s PCP) are called by State Lab • Baby Adon had a C3 acylcarnitine level of 19.92 (nl <6.35) at 25 hrs of age (two days ago) • What should you do? 4/21/2017 214 ACMG ACT Sheet http://www.acmg.net 4/21/2017 New England Emergency Protocols @newenglandconsortium.org 4/21/2017 Case 2: What Should You Do? a) Inform family of NBS result b) Evaluate ASAP & consult metabolic specialist c) Check BS, AG, ketones & NH3 d) If indicated stop protein, hydrate & Rx chemical imbalances per metabolic specialist e) All of above 4/21/2017 215 Propionic Acidemia Dx 4/21/2017 Rx Normal Newborn Screening Sx Dx Rx Preclinical stage ACMG NBS Info (www.acmg.net) 4/21/2017 Hemoglobin Disorders: HPLC Sickle cell FS anemia (HbSS or HbS/ßº Thal) Hb SC disease FSC (HbS/C) Hb S/beta FSA Thalassemia (HbS/ß+ Thal) 4/21/2017 216 Biotinidase Deficiency 4/21/2017 Cystic Fibrosis • AR, 1/3000 Caucasians (80% neg FH) • NBS: two tier test with Immunoreactive Trypsinogen (IRT); IRT>IRT or IRT>DNA • Dx Iontophoresis (sweat test) or DNA • Rx: CF Clinic, pulmonary Rx, pulmozyme, antibiotics; Kalydeco (ivacaftor for G551D); diet, oral enzymes & genetic counseling 4/21/2017 Hearing: Auditory Brainstem Response (ABR)/ Evoked Otoacoustic Emissions (OAE) ~15% prelingual deaf are either GJB2 (Connexin 26) 35delG homozygotes 98% (AR) or GJB2 35delG/GJB6 (Connexin 30) del double heterozygotes (2%) 4/21/2017 217 Classical Galactosemia (GALT) Galactose Galacitol Galactokinase Galactonic ATP ADP Galactose-1-phosphate GALT UDPGlu UDPGal Epimerase Glucose-1-phosphate Phosphoglucomutase 4/21/2017 Glucose-6-phosphate Classical Galactosemia • AR, ~1/62,000, Galactose 1 PO4 Uridyl Transferase (GALT) deficiency • Clin: emesis, diarrhea, jaundice & E Coli sepsis; ź liver & renal function; cataracts only develop later • NBS:ŸGal / Gal1P & Absent GALT = EMERGENCY. You should immediately: 1) Repeat Gal, Gal 1 P & GALT 2) Start Gal free formula 4/21/2017 ACMG ACT Sheet @ www.acmg.net 4/21/2017 218 Congenital Hypothyroidism • ~1/4,000; 85% thyroid develop, 10% synthetic & 4% hypopit, only~15% AR Pendred: deaf as NB; deaf + adolescent euthyroid goiter • Clin: Poor growth, NB have subtle signs but 2/3 ź IQ if not Rxd • NBS:TSH >24 hrs; Dx T4 ,TSH & TRH • Rx: L thyroxine 4/21/2017 Congenital Adrenal Hyperplasia (CAH) ACTH causes adrenal hyperplasia & over-production of 17 OHP, testosterone & estradiol 4/21/2017 Congenital Adrenal Hyperplasia (CAH) • AR, ~1/12,000; > 90% CYP21OH (salt wasting); 3ɴHSD similar; 11ɴOHŸ BP • Clin:źaldosterone & cortisol cause hyponatremic hyperkalemic dehydration & death;Ÿandrogen virilizes females • NBS:Ÿ17OHP (false + 20 toźbirth wt & < 24 hrs) • Rx: Cortisone & mineralocorticoid 4/21/2017 219 ACMG NBS Info (www.acmg.net) 4/21/2017 Severe Combined Immunodeficiency (SCID) • XL, AR & źADA ~1/50-100,000 • Clin: Cellular & humoral due to B & T cell); presents 1-3/12 with FTT, oral/diaper candidiasis, absent tonsils/lymph nodes, recurrent & persistent infections despite Rx •Gen: NBS T cell receptor excision circles (TRECs) •Rx: Antibiotics (Pneumocystis), IVIg, avoid live viral vaccines; HSCT within 3/12, gene therapy? 4/21/2017 Fabry Disease • Clin: XL ~1/3-4k accumulate globotriaoslyceramide (Gb3). Onset 4-8 yrs: angiokeratomas, acroparesthesia, hypohydrosis, corneal/lens opacities & proteinuria. Adults: LVH/ischemia, TIA/stroke, tinnitus, hearing loss & ESRD. 70% Ƃ affected,variable & later. • NBS: ɲ galactosidase A (GLA), if low WBC GLA in ƃ (not reliable in ֯). Dx: low WBC GLA in ֱ do DNA testing (classical vs later onset variants). In ֯ DNA testing. • RX: Test ֱ for proteinuria q y after 2 y with GFR; ֯ less frequently. ERT agalsidase ɲ/ɴ [Replagal/ Fabrazyme] as needed. 4/21/2017 220 Gaucher Disease • Clin: AR ~1/40-60k; Type 1: Non neuronopathic infancy to adult; anemia & љplatelets; HS megaly, bone disease but nl CNS. Type 2: Acute neuropathic (np) Gaucher cells onset < 2y with CNS љ & death by 2-4 y. (crushed tissue paper) in bone Type 3: Chronic np longer survival. marrow (BM). • NBS: glucocerebrosidase (GBA), if low WBC GBA. Dx: low WBC GBA, N370S/N370S; N370S/L444P; N370S/ 84GG ~65% cases. N370S/? np protective vs L444P/L444P not. • RX: ERT: Cerezyme (Imiglucerase)/VPRIV (Velaglucerase ɲ) or Elelyso (Taliglucerase ɲ) for Type 1, +/- Type 3; not Type 2. Monitor ACE, TRAP & Chito. Substrate reduction: Eligustat (? CYP2D6) or Zaveca (Miglustat) Type 1 adults. 4/21/2017 Krabbe Disease • Clin: AR ~1/100k; Infantile onset (IO) has progressive CNS љ/death < 2 y (85-90%). Irritabile, spastic, blind, deaf & delay with startle <6/12 & become decerebrate. Onset > 1 y slower & variable (10-15%). • NBS: galactocerebrosidase (GALC), if low reflex to psychosine (p), if p high, reflex to 30 kb del (seen in ~45%), if not (del/del) sequence. Dx: 0-5% activity in WBC/ fibroblasts + CSF protein (2), brain MRI (2), NCV (2) & BAER (1). If ш 4 refer for HSCT. • Rx: IO & later onset with mild symptoms, may improve & preserve cognition but many show peripheral nervous system deterioration. Supportive care IO in later stages. 4/21/2017 Niemann-Pick Disease • Clin: AR ~1/250k (Ashkenazi 1/40k) Type A: neonatal onset, hypotonia, FTT, CNS deterioration, deaf, blind (cherry red spot), lung infiltrates, HS megaly & fatal by 1.5 - 3 y. Type B: later onset, HS megaly, interstitial lung & nl intelligence. • NBS: sphingomyelinase, if low, sphingomyelin phosphodiesterase 1 (SMPD1) DNA which may indicate Type A or B. Foam “Niemann-Pick foam cells” in BM. • Rx: supportive, liver/HSC transplantation, or ERT may be considered. ERT is highly complicated. 4/21/2017 221 Pompe Disease (PD)(GSD Type II) • Clin: AR ~1/40k; Early onset:2 m hypotonia, weakness, cardiomegaly/cardiomyopathy, FTT & hearing loss. Usually fatal < 12 m. Late-onset: proximal muscle weakness & respiratory failure; cardiac uncommon. Genotype to determine CRIM status (null alleles) but genotype & phenotype may not correlate. • NBS: acid ɲ glucosidase (GAA), if low urine hexose tetrasaccharide (HEX4), CPK, chest X ray, EKG, ECHO, GAA sequence & CRIM status. • RX: ERT ASAP if WBC GAA low & heart involved. Monitor Ab for immune modulation. CRIM- often develop rhGAA IgG Ab with poor outcome without immune modulation. 4/21/2017 Hurler Disease (MPS1H) & Scheie (MPS1S) •Clin: AR~1/107k; Infants: hernias, recurrent URIs; then growth & development slow, progressive HS megaly, course facies, corneal clouding (>1 yr), hydrocephalus, skeletal & cardiac disease. Fatal by 5-10 y. Scheie is milder, onset adolesence or adulthood. •NBS: ɲ L Iduronidase (IDUA), if low glycosaminoglycans (GAGs) to RO Ɏdeficiency & sequence IDUA •RX: ERT Laronidase (Aldurazyme) for non CNS symptoms; HSCT & ERT for severe genotype < 2y age. 4/21/2017 References ACMG NBS ACT sheets & algorithms @ http://www.acmg.net (ACMG Act Sheets App) Genetic Home References @ https://ghr.nlm.nih.gov GeneReviews @ https://ghr.nlm.nih.gov New England Emergency Protocols @ http://newenglandconsortium.org SIMDNAMA course Vademecum Metabolism (EVM) @ vademetab.org 4/21/2017 222 Developmental Genetics DEVELOPMENTAL GENETICS Tony Wynshaw-Boris, MD, PhD, FACMG James H. Jewell Professor of Genetics Department Chair of Genetics and Genome Sciences Case Western Reserve University School of Medicine University Hospitals Case Medical Center Tony Wynshaw-Borris, MD, PhD, FACMG Dept. of Genetics and Genomic Sciences Case Western Reserve University School of Medicine University Hospitals Case Medical Center One 10900 Euclid Avenue, BRB731 Cleveland, Ohio 44106-4955 (216) 368-0581 Telephone (216) 368-3832 Fax [email protected] 225 226 Developmental Genetics Tony Wynshaw-Boris, MD, PhD Chair, Department of Genetics and Genome Sciences Case Western Reserve University School of Medicine University Hospitals Cleveland Medical Center Disclosure(s) None Congenital Anomalies 1-3% of all newborns Leading cause of neonatal morbidity and mortality 20% of infant deaths 10% NICU admissions, 25-35% of deaths Pediatric admissions 25% to 30% have major birth defect 227 Causes of Congenital Anomalies Congenital Anomalies භ Isolated Anomaly Incidence per livebirths Undescended testes Heart defect Club foot Neural tube defects Cleft lip + cleft palate Hypospadias Polydactyly Cleft palate Craniosynostosis Syndactyly 1:30 1:150 1:300 1:500 1:1000 1:1000 1:1500 1:2000 1:2000 1:2000 Deformation Developmental Process is normal Mechanical force alters structure Examples: Oligohydramnios Breech presentation Bicornuate uterus 228 Deformation Clubbed feet • spina bifida Moore. The Developing Human. Saunders, 1994 Disruption Developmental process is normal, but interrupted Examples: Amniotic band sequence Fetal Cocaine exposure Disruption Porencephaly http://www.neuropat.dote.hu/develop.htm#Porencephaly Amniotic Band Wiedemann and Kunze. Clinical Syndromes. Mosby-Wolfe, 1997 229 Dysplasia Abnormal tissue organization, microscopic structure Examples: Skeletal or connective tissue dysplasias Ectodermal dysplasias Dysplasia Ectodermal Dysplasia Buyse. Birth Defects Encyclopedia. Blackwell Science, 1990; Baraitser and Winter. Color Atlas of Congenital Malformation Syndromes, Mosby-Wolfe, 1996; Bergsma. Birth Defects Compendium, Alan R. Liss, 1979. Malformation Morphological defect from an intrinsically abnormal developmental process Examples: holoprosencephaly, congenital heart disease, neural tube defect 230 Malformation Unilateral Cleft Lip and Palate Moore, Persaud, and Shiota. Color Atlas of Clinical Embryology. Saunders, 1994 Syndrome A recognizable pattern of anomalies presumed to be causally related Genetic: chromosomal, single gene Environmental: alcohol, retinoic acid Complex: more than one genetic and/or environmental factor Syndrome: Environmental Causes Fetal Alcohol Growth retardation Microcephaly Mental retardation Short palpebral fissures Short nose Smooth philtrum Thin upper lip Small distal palanges Hypoplastic finger nails Cardiac defects Clarren and Smith. NEJM 298:1063, 1978 231 Normal Development http://embryo.soad.umich.edu/carnStages/carnStages.html Developmental Pathways and Mechanisms Development 232 Germ Cells and Stem Cells Lineages from Stem Cells: Blood Fate, Specification, and Determination 233 Fate, Specification, and Determination Differentiation CS 7, day 15-17 Gastrulation occurs as cells migrate from the epiblast, to form mesoderm. Mesoderm lies between the ectoderm and endoderm as a continuous layer From the primitive node a tube extends under the ectoderm to form the notochord http://embryology.med.unsw.edu.au/ wwwhuman/Stages/Stages.htm http://www.med.unc.edu/embryo_images/ unit-bdyfm/bdyfm_htms/bdyfm003.htm Pattern Formation CS 10, week 4 Ectoderm: Neural folds fuse Mesoderm: continued segmentation of paraxial mesoderm (4 - 12 somite pairs) http://embryology.med.unsw.edu.au/wwwhuman/Stages/Stages.htm 234 Organogenesis CS 13, week 5 Ectoderm: sensory placode, lens placode, otic vesicle, early nasal placode, forebrain Mesoderm: 30 somite pairs, heart prominence Head: 1st, 2nd and 3rd pharyngeal arch, stomodeum Body: heart, liver, umbilicus, upper limb bud, lower limb bulge http://embryology.med.unsw.edu.au/ wwwhuman/Stages/Stages.htm Organogenesis CS 16, week 6 CS 18, week 7 Nasal pits moved ventrally, auricular hillocks, foot plate Finger rays, Ossification commences http://embryology.med.unsw.edu.au/wwwhuman/Stages/Stages.htm Growth CS 20, week 8 CS 23, week 9 Upper limbs longer and bent at elbow Rounded head, body and limb http://embryology.med.unsw.edu.au/wwwhuman/Stages/Stages.htm 235 Establishment of Body Axes A-P: anterior-posterior (cranial-caudal) [Proximal-distal for limbs] D-V: dorsal-ventral (back-front) L-R: left-right axes Patterning program of the embryo is overlaid onto these axes Rotation of the Proximo-Distal (P-D) to Anterior-Posterior (A-P) axis and Mesoderm Induction Nodal cilia rotate in a clockwise fashion to drive leftward fluid flow Nonaka et al. PLoS Biology 3:e268 (2005) 236 Posterior Localization of Nodal Cilia in a Single Node Cell Hashimoto et al. (2010) Nat Cell Biol. 12:170 Proximal Distal Anterior Dorsal (WNT7A) Ventral AER (FGF4 FGF8) Posterior ZPA (SHH) HOX Genes: Transcription Factors for Positional Information 237 Hox Gene Mutation: Syndromes Anterior - Head HOXA1 Athabaskan Brainstem Dysgenesis Bosley-Salih-Alorainy Syndrome (Duane Syndrome, Deafness, Delayed Motor Milestones, Autism) Posterior - Tail HOXA11 Radioulnar Synostosis with Amegakaryocytic Thombocytopenia HOXA13 Hand-Foot-Uterus Syndrome Preaxial Deficiency, Postaxial Polydactyly and Hypospadius Hox Gene Mutation: Syndromes Posterior - Tail HOXD10 Vertical Talus, Congenital (Rocker-Bottom Foot) HOXD13 Synpolydactyly 1 (Syndactyly, Type II) Brachydactyly, Types D and E Cellular and Molecular Mechanisms During Development 238 Transcriptional Regulation: Model for Wnt Pathway and Pitx2 during Development Kioussi et al. Cell (2002) Morphogens: Sonic Hedgehog (SHH) in Neural Tube and Limb Cell Shape and Organization 239 Neurogenesis: Neuroepithelium vs. Radial Glia The cortex forms by radial and nonradial migration 3V cortex hip caudate, putamen HIP LV NCX LGE PCX MGE globus pallidus Neocortical Layering in the Mouse 240 Neuronal Proliferation & Migration Proliferation Microcephaly AR – multiple loci Migration Lissencephaly Miller-Dieker LIS1 X-linked: DCX (doublecortin) X-linked with abnormal genitalia (ARX) Cobblestone dysplasia (Fukuyama MD, Walker-Warburg, muscle-eyebrain) Heterotopia Periventricular nodular (FLN1) Cortical Organization Pachygyria/polymicrogyria Schizencephaly EMX2 schizencephaly Normal Type I Lissencephaly (Severe) Lissencephaly Normal Severe MR Seizures Early Death Incidence: 1/50,000-1/100,000 241 Isolated Lissencephaly Sequence LP87-001 Isolated Lissencephaly Sequence LP86-003 Miller-Dieker Syndrome LP82-002 Heterozygous Deletions of 17p13.3 in ILS and MDS X-linked Lissencephaly and Subcortical Band Heterotopia LP95-136m LP95-136a1 242 Other Lissencephaly Syndromes Lissencephaly with Cerebellar Hypoplasia Reelin mutation VLDL Receptor mutation Lissencephaly with Abnormal Genitalia ARX mutation Lissencephaly Pathways Programmed Cell Death During Development 243 Developmental Pathways and Mechanisms Developmental Pathways Core Pathways Developmental Pathways Cell Cycle, Proliferation, Apoptosis 244 Developmental Pathways/Processes Developmental Pathways- General Ligand Cell Membrane Receptor Signaling Molecules Transcription Factor Nucleus 245 FGF FGFR Ras-MAPK STAT PI3K, PLCγ Transcription Transcription Factors Factors STAT Fibroblast Growth Factor Signaling Pathway Bonaventure and El Ghouzzi. Expert Rev Mol Med 2003:1-17, 2003 FGFR Craniosynostosis Syndromes Autosomal dominant Genetic heterogeneity Crouzon Phenotypic variability Gain of function mutations, missense and in-frame deletions and insertions, splicesite mutations in 85 to 90% Jackson-Weiss Apert Crouzonodermoskeletal Beare-Stevenson SADDAN Jabs. ed. Jameson, Principles of Molecular Medicine, 1998 246 Fibroblast Growth Factor Receptors Expert Reviews in Molecular Medicine © Cambridge University Press Bonaventure and El Ghouzzi. Expert Rev Mol Med 2003:1-17, 2003 SHH SMOH CHOL Sterol delta-7 reductase PTCH GLI GLI Transcription Factors 247 syndactyly polydactyly upturned nose ptosis cryptorchidism CNS hypoplasia holoprosencephaly dysmorphic features (short metacarpals, rib defects, broad face, dental abnormalities) cancer predisposition (rhabdomyosarcoma, medulloblastoma variable midline defects (single maxillary incisor , hypotelorism, holoprosencephaly, cyclopia) postaxial polydactyly syndactyly hypothalamic hamartomas imperforate anus hypertelorism, syndactyly, preaxial polydactyly with broad thumbs, and great toes 248 Bardet-Biedl Syndrome Bardet Biedl Syndrome Beales et al. J Med Genet 36:440, 2007 Ocular rod-cone dystrophy Hearing loss Anosmia High arched palate Situs inversus Congenital heart defects Liver disease Truncal obesity Renal abnormalities Postaxial polydactyly and Brachydactyly Hypogenitalism in males Cognitive deficits Genetic Heterogeneity in BardetBiedl Syndrome 249 BBS Genes Tobin and Beales. Pediatr Nephrol 22(7):926-936, 2007 Joubert Syndrome AR group of inherited conditions Congenital ataxia Hypotonia, Episodic breathing Mental retardation Associated finding in some patients: Retinal dystrophy Nephronophthisis (renal fibrocystic disease) Molar Tooth Sign: Specific malformation of the brainstem, cerebellum and the cerebellar peduncles, 250 Figure 2. Cystoproteins are proteins of genes that are mutated in cystic kidney diseases of humans, mice, or zebrafish Hildebrandt, F. et al. J Am Soc Nephrol 2007;18:1855-1871 Copyright ©2007 American Society of Nephrology 251 Ligand Cell Membrane Receptor Tyrosine Kinase Adapter RAS RAF ERK/MAPK Transcription Factor Nucleus Ras/MAPK Pathway Kate Rauen Genetic Syndromes of the Ras/MAPK Pathway Neurofibromatosis 1 Cap-AV Malformation Gingival Fibromatosis 1 RTK RasGaps active RAS SOS1 GRB2 SHP2 Costello KRAS HRAS SPRED1 CRAF BRAF MEK1 MEK2 ERK1 ERK2 LEOPARD pERK nucleus Noonan Kate Rauen Legius pERK Cardio-facio-cutaneous 252 254 Cancer Genetics I CANCER GENETICS I Sharon E. Plon, MD, PhD, FACMG Professor, Department of Pediatrics/Hematology-Oncology Molecular and Human Genetics Human Genome Sequencing Center Director, Medical Scientist Training Program Baylor College of Medicine Sharon E. Plon, MD, PhD, FACMG Department of Pediatrics Feigin Center Room 1200.18, 1102 Bates Street Houston, TX 77030 (832) 824-4251 Telephone (832) 825-4276 Fax [email protected] 257 258 Cancer Genetic 1 Sharon E. Plon, MD, PhD, FACMG Professor Baylor College of Medicine • I have the following financial relationships to disclose: • I am a employee of Baylor College of Medicine (BCM) which derives revenue from genetic testing, including whole exome sequencing. • BCM and Miraca Holdings Inc. have entered into a joint venture, Baylor Genetics, with shared ownership and governance of the clinical genetics diagnostic laboratories • I am a member of the BMGL Scientific Advisory Board • I will discuss off label use and/or investigational use in my presentation. Outline of topics – Cancer Genetics 1 • Basic principles • Oncogenes • Somatic cell genetics/targeted therapy • Basic principles • Tumor suppressor genes (LOH) • Familial predisposition syndromes • • • • Retinoblastoma Li-Fraumeni syndrome Telomere Disorders miRNA Disorders (miRNA) 259 Key points regarding mutation mechanisms in Oncogenes • The types of mutations differ significantly between oncogenes and tumor suppressor genes (loss of function variants). • Proto-oncogene - formal name for gene prior to mutation/activation in tumor • Mutations in oncogenes activate the gene product (missense) or cause the gene to be misexpressed or overexpressed (translocations, amplification). • Mutations in oncogenes are frequently somatic, e.g., found in the tumor but not in matched normal DNA from the patient with the second allele unaltered. 1. Activation by Single Nucleotide Variation SPECIFIC MISSENSE VARIANTS frequently alter the normal activity of a protein to become transforming. • RAS proto-oncogene proteins have GTPase activity that is normally regulated. • RAS activity becomes constitutively active due to specific missense mutations that block ATPase function. • HRAS codon 12 is one of the most common mutations in all human cancer. Active Ras-GTP G12V Inactive Ras-GDP Common Somatic Ras Missense Mutations Ras Gene Codon Tumor Type H-ras 12 GGC ÆGTC Bladder K-ras 12 GGT Æ GAT 12 GGT Æ GTT 12 GGT Æ GAT 61 CAA Æ CGA Pancreas, Colon, Lung, Uterine N-ras Leukemia, small number of colon cancers It is not entirely clear why there are different Ras mutations in different tumor types. 260 2. Oncogene Amplification • Increases the copy number of oncogene to be activating due to increased expression – can result in hundreds of copies of the oncogene. • Amplification often results from two overlapping mechanisms that are distinguished by use of FISH: • Double minutes (multiple extrachromosomal fragments) • Homogeneously staining regions (HSR) – seen within chromomsomes • MYCN (n-myc) frequently amplified in high risk neuroblastoma. • Her-2/Neu/c-ERBB2 amplification frequent in human breast cancers. • Therapeutic target of Trastuzumab/Herceptin DzE Amplification - Associated w/ Poor Prognosis in Neuroblastoma 100 MYCN Not Amplified (N = 71) EFS (%) 80 60 P < 0.0001 40 MYCN Amplified (N = 31) 20 0 0 1 2 3 4 5 6 Years from Dx 7 8 Courtesy of Garret Brodeur – Children’s Hospital of Philadelphia Translocations/Fusion Proteins • Often results in oncogenes being abnormally expressed with the creation of fusion proteins. • Many translocations are specific to certain tumor types and can be used to confirm diagnosis. • Initially hallmark of hematopoietic malignancies but now clear that translocations are found in many solid tumors. • Transcription factor or kinase are most common downstream partner of the translocation. • Highly variable geography of translocations • Intrachromosomal deletions and inversions can also result in oncogenic fusion proteins. 261 Rearrangement types • Imprecise translocations result in an oncogene being moved to the proximity of a transcriptionally active gene with varying geography. • Overexpression of the “normal” coding region of the oncogene in a cell type normally silenced. • Precise translocations result in the precise joining of two genes to make a novel fusion gene: • The 5' end of the gene controls the expression pattern +/functional domains and 3' end of the gene often controls function. • Intrachromosomal deletions/inversions • Deletions – P2RY8 exon 1 is fused to CRLF2 exon 1 and activates transcription in high risk ALL and DS-ALL. Imprecise Fusions t(8;14) MYCC proto-oncogene at 8q24 IgG locus at 14q32 Chromosome 8 Chromosome 14 IgG – c-Myc t(8;14) Translocation Activation of MYCC oncogene by juxtaposition of MYCC with the Immunoglobulin locus in lymphoid cells in Burkitt㵭s Lymphoma – no fusion protein is made Precise Translocation: Philadelphia Chromosome in CML Images from R. Naeem, TCH 262 Impact of BCR-Abl Inhibitor – Imatinib/Gleevac • Development of a specific inhibitor against the BCR-Abl protein has resulted in improved treatment of Ph+ leukemia (CML and ALL) with improved survival. • Now also used for tumors which have activation of other related kinases, like gastrointestinal stromal tumors (GIST) with activation of c-Kit kinase. Courtesy – Stephen Kornblau (MDACC) D>ϰͲ><Fusion Results from Recurrent Inversion Chr 2p KD EML4 ALK Normal cell KD ALK Tumor cell Somatic inversion event EML4 KD = kinase domain • Somatic inversion event in ~3% of non-small cell lung cancer. • Results in activation of ALK kinase because the EML4 domain aids in heterodimerization. • FDA approved companion diagnostic test to decide upon use of ALK inhibitors – crizotinib - in treatment of lung cancer. Specific Kinase Alterations in Cancer • JAK2 mutations in high-risk childhood ALL and ALL associated with Down syndrome. • ALK missense and amplication (neuroblastoma), inversion (NSCLC) and translocation (lymphoma) - crizotinib. • FLT3 internal tandem duplication in AML. • EGFR missense mutations in lung cancer associated with sensitivity to tyrosine kinase inhibitors (gefitinib was first of class FDA approved in 2003). • Secondary somatic mutations V843I or T790M then associated with resistance to same class of drugs. • BRAF (B-raf) gene V600E in a substantial percentage of malignant melanoma (multiple inhibitors FDA approved) 263 Metabolic Genes Implicated through Whole Exome Sequencing • Whole exome sequencing of high grade gliomas by Hopkins group (Parsons et al, Science, 2009) led to discovery of specific missense mutations in IDH1/2 in astrocytomas and secondary glioblastoma multiforme. • IDH1/2 also frequently mutated in AML and sarcomas • Functional assays reveals that these are gain of function mutations which alter substrates for chromatin remodeling • Multiple other Krebs cycle proteins implicated in cancer, e.g. fumarate hydratase From Wallace, DC Nat Rev Cancer 2012, 10:685 Sampling FDA approved targeted drugs Drug Target Tumors - off label use in italics Imatinib BCR-ABL and C-KIT CML ,GIST, PH+-ALL Trastuzumab ERBB2/HER2/neu Breast cancer, gastric or GEJ junction cancer Crizotinib EML4-ALK ALK missense mutation NSCLC Neuroblastoma and other ALK+ tumors Vermurafinib BRAF V600E Melanoma Erlotinib EGFR missense Lung cancer Vandetanib RET missense Medullary thyroid cancer Everolimus TSC1/TSC mutant Subependymal giant cell astrocytoma Olaparib BRCA1/2 germline mutation Ovarian cancer (PARP inhibitor) Germline status 264 Genetic susceptibility to cancer Multiple Mechanisms of Cancer Susceptibility • Common alleles with modest risk – GWAS • Numerical chromosomal abnormalities: autosomes and sex chromosomes • Microdeletion/rearrangements • Overgrowth syndromes including imprinting disorders and mosaicism for somatic mutations. • Autosomal dominant disorders – LOF mutations in TSG and activating mutations in proto-oncogenes • Autosomal and X-link recessive disorders Multiple Mechanisms/Same Tumor- Wilms tumor Syndrome Location - Gene Molecular Basis Trisomy 18 Trisomy 18 Trisomy WAGR 11p13 - WT1 Microdeletion MYCN-DDX1 2p24.3 Microduplication Dennys-Drash 11p13 - WT1 Point mutation (AD) Perlman syndrome 2q37 -DIS3L2 Autosomal recessive BeckwithWiedemann syndrome 11p15, IGF-2, H19 p57KIP2, LIT1/ KCNQ10T1 Loss of imprinting, deletion, UPD & mutation 265 GWAS experiments – large case:control cohorts genotyped for common variants Note: Many candidate SNPs have not validated in larger studies Taylor et al Trends Mol Med. 2001 Nov;7(11):507-12. Breast cancer GWAS hits demonstrate the typical modest impact on cancer risk Gene/SNP Chromosome Risk allele frequency Relative Risk per allele FGFR2 10q 0.38 1.26 TNRC9 16q 0.25 1.2 5q 0.28 1.13 LSP1 11p 0.30 1.07 ?/rs13281615 MAP3K1 8q 0.40 1.08 ?/rs13387042 2q 0.50 1.2 CASP8 2q 0.86 1.13 From Pharoah, (2008) N Engl J Med 358;26; 2796 Chromosomal Abnormalities and Cancer Risk – Trisomy 21 • Results in ~20 fold increase in leukemia and also shifts the myeloid: lymphoid leukemia ratio to 40:60. • Transient myeloid proliferation (TMP) which may or may not evolve into leukemia is seen in infants. • Somatic GATA1 mutations found in TMP cells • Acute megakaryocytic leukemia is 400 fold RR. • In DS associated ALL see JAK2 missense mutations and CRLF2 activation due to intrachromosomal deletion. • Little increased risk of other cancer types 266 Pathways towards DS Associated Leukemia TMP GATA1 somatic variant AML Often cytogenetically “normal” Patient with Trisomy 21 ALL JAK2 variant CRLF2 activation (intrachromosomal deletion) Sex Chromosome Abnormalities • Girls with mosaic Turner syndrome or gonadal dysgenesis are at increased risk for gonadoblastoma. • Correlates with presence of Y chromosome containing material. • XXY males have an increased risk of breast cancer. • Long-term survivors of trisomy 18 have increased risk of Wilms tumor. Inherited Structural Chromosome Defects • WAGR – Wilms tumor, Aniridia, Genital abnormalities, mental Retardation • Contiguous gene syndrome on 11p13. • WT1 - Wilms Tumor and GU abnormalities • PAX6 - aniridia • Denys-Drash syndrome caused by missense or nonsense mutations in WT1. • Increased risk of end stage renal failure • Very significant risk of WT (~50%) in patients with WAGR and estimated to be 90% in patients with Denys-Drash • Recommend screening children with deletion by abdominal U/S q 3 months until age 8. 267 Other microdeletions associated with cancer risk through deletion of tumor suppressor genes • Retinoblastoma - ~3% constitutional deletions of 13q14 overlying the RB1 gene. • Deletions of NF1 - ~3-5% of patients. • Earlier onset neurofibromas • Increased risk malignancy (PMNST) • More severe developmental phenotype • Syndromic thrombocytopenia with AML: • Associated with deletions of RUNX1 at 21q22 (see photo from Ramawi et al, Blood 2008). Imprinting Disorders and Tumor Risk • Beckwith-Weideman Syndrome –excessive growth, macroglossia, organomegaly, ear creases, hemihyperplasia • Overall Wilms Tumor risk 2-5% and hepatoblastoma risk ~1-2%. • Cancer risk >> BWS including HH or organomegaly - ~30% risk. • Gain of methylation IC1 (28%), loss of methylation IC2 (2.6%), pUPD11 16%, and CDKN1C mutations (6.7% ) – Maas et al, AJMG, 2016 • Recommend screening kidneys by ultrasound and AFP for hepatoblastoma as per WAGR. Mendelian (high risk) Forms of Cancer Susceptibility • Inherited cancers are a minority of the total number of cases but the proportions can range from 1-60%. • For most tumor types, e.g. breast, the inherited fraction fall in the range of 1-10%. • For an increasing number of rare tumors, adrenocortical carcinoma, rhabdoid tumors, retinoblastoma and optic gliomas are recognized to have a very high inherited fraction (30-60%). • Specific histological subtypes can have higher frequency, e.g. pediatric ALL ~1% but hypodiploid ALL is ~50%. • The majority of conditions result from loss of function mutations in tumor suppressor genes 268 Diagnosis with >10% hereditary form Genetic Loci Retinoblastoma RB1 Adrenocortical or choroid plexus carcinoma, hypodiploid ALL, anaplastic rhabdomyosarcoma TP53 Pheochromocytoma/ paraganglioma VHL, NF1, RET, SDHB, SDHD… Retinal or cerebellar hemangioblastoma, Endolymphatic sac tumor (ELST) VHL Optic pathway tumor, malignant peripheral nerve sheath tumor, JMML NF1 Medullary thyroid cancer RET Atypical teratoid/ malignant rhabdoid tumor SMARCB1/A4 Ovarian small cell carcinoma, hypercalcemic type SMARCA4 Acoustic or vestibular schwannomas NF2 Pulmonary pleuroblastoma DICER1 General Features or Autosomal Dominant Hereditary Cancer Disorders • Multiple generations affected with cancer w/highly variable proportion of de novo mutations. • Transmission through mothers and fathers independent of tissue type. • Earlier age of onset of cancer c/w sporadic cases • Increase in multiple and bilateral tumors • Clustering of specific tumor types w/in family • Variable penetrance with unaffected mutation carriers Autosomal Dominant Cancer Disorders Associated with Inherited Oncogene Mutations Gene Disorder RET Multiple endocrine neoplasia type 2 MET Hereditary papillary renal cell cancer HRAS Costello syndrome with skeletal abnormalities, developmental delay, bladder KRAS Cardio-Facio-Cutaneous syndrome – no known cancer phenotype ALK Hereditary neuroblastoma (specific missense alleles) EGFR Familial lung cancer (V843I or T790M)- germline When somatic these variants associated with acquired resistance to tyrosine kinase inhibitor therapy 269 Multiple Endocrine Neoplasia 2 • Type 2A includes- in this order of diagnosis: • • • • Medullary thyroid carcinoma (MTC) Pheochromocytomas Parathyroid disease Missense mutations in cysteine residues found in >90%. • Type 2B presents in infancy with • Ganglioneuromas of the GI tract, lips and skeletal abnormalities. Can be confused with Hirschsprung’s megacolon. • Onset of MTC in early childhood • Two specific tyrosine kinase domain missense mutations: M918T >> A883F • Familial MTC - without other MEN features. Clinical Evaluation for MEN2 • Genetic testing is standard of care in MEN2 families and anyone with MTC especially at young ages. • Testing is sequencing of either the entire cDNA or regions with recurrent missense mutations in RET. • If mutation positive, recommend prophylactic thyroidectomy (Chen et al., Pancreas, 2010): • Level 1 mutations age 5 – 10 outside conserved cysteine residues • Level 2 mutations by age 5 (cysteine alleles in exon 10-11) • Level 3 mutations by age 1 (including MEN2B alleles). • Specific RET alleles also associated with Hirschsprung’s Tumor Suppressor Genes Many dominant disorders result from loss of function (LOF) mutations in tumor suppressor genes. TSG normally function to: • Inhibit proliferation • Down regulate the cell cycle • Repair DNA • DNA damage checkpoints Active Ras-GTP NF1 Inactive Ras-GDP 270 Retinoblastoma • 1 in 20,000 children affected • Unilateral or bilateral tumors develop in early childhood • Occurs in heritable and nonheritable forms • 15% of unilateral and ~100% bilateral are heritable • 3-4% demonstrate somatic mosaicism for RB1 mutation • Identifying at-risk infants substantially reduces morbidity • Developed concept of tumor suppressor gene and loss of heterozygosity. Features of Zϭ gene • RB1 gene was linked to 13q14.2 by obvious cytogenetic deletions in ~5% of children with Rb, developmental delay and anomalies. • Majority of germline and somatic variants are a variety of LOF alleles (nonsense, frameshift, deletions, splicing – 90%). • RB1 gene encodes a cell cycle regulator. • Inhibits the G1 to S phase by recruiting histone deacetylases to promoters to inhibit transcription of genes required for S phase. • RB1 protein itself regulated by phosphorylation. Familial Retinoblastoma B-Rb dx 1 5y B-Rb Dx 6 mo 7y • Truncating RB1 germline variants results in ~90% penetrance for diagnosis of Rb (not necessarily bilateral). • 80% of patients with bilateral RB results from de novo mutations w/o family history of Rb. • Only 20% of patients with bilateral Rb have a positive family history. • Can see families with incomplete penetrance due to missense or splicing alleles. • Germline mosaicism in parents (fathers > mothers) leads to substantial recurrence risk 271 Recurrence Risk for Rb in absence of testing Clinical scenario Offspring of bilateral cases Retinoblastoma Risk 45% Offspring of unilateral cases 7.5% Sibling of bilateral cases (with unaffected parents) Sibling of unilateral cases (with unaffected parents) 5-7% 1% Concept: Somatic versus Germline Variants • Fundamental aspect of cancer genetics is differentiating inherited from somatic variants that occur as tumors develop. • The same gene may undergo mutation in both the sporadic and inherited forms. • For TSGs you typically see a mixture: • One inherited & one somatic (second hit) • Two somatic (one smaller/point mutation and often second larger event). • One somatic or inherited (dominant negative) • No mutation – silencing by methylation instead Two Hit Hypothesis of TSGs Normal genes (prevent cancer) 1st mutation ( if inherited susceptible carrier) GROWTH 2nd mutation or loss (leads to cancer) 272 Two Hit Hypothesis • Inactivation of both copies of a TSG is required to develop cancer. • Both inactivation events can occur somatically during development of childhood (unilateral tumor) OR • Inherit a single mutation in a TSG and then somatically acquires loss/inactivation of the second normal copy of the gene to develop tumor (more likely to be bilateral). • At cellular (somatic) level these genes are recessive because both copies of the gene are lost in tumors. • Autosomal dominant inheritance of susceptibility to cancer. Loss of Heterozygosity Experiment Using Heterozygous Markers Flanking TSG SNPchip data near TSG Nl Tumor Polymorphic marker near TSG Molecular Basis of Second Hit • Genetic events associated with LOH • Loss of whole chromosome • Loss of chromosome and reduplication of chromosome containing mutation • Loss of whole arm or large interstitial deletion • Mitotic recombination between chromosomes • Not associated with LOH • Point mutation or intragenic deletion • Silencing of gene by methylation of promoter 273 Test Results of Two Unilateral Rb patients Sample Allele 1 Allele 2 Tumor 1 Q347X LOH Blood 1 Q347X Normal Patient 1: Hereditary form of RB Tumor 2 Blood 2 Methylation Promoter Normal 567delAG Normal Patient 2: Sporadic Rb due to somatic mutation and promoter methylation Recurrence Risk for Retinoblastoma w/genetic testing – assume Zϭtesting is 95% sensitive Bilateral Proband • If blood testing reveals RB1 mutation • Offspring and siblings tested for mutation identified. • If blood tested with negative result: • Uninformative • Maximize detection of mosaic results Unilateral Proband • If blood sample is positive • Offspring and siblings tested for mutation identified. • If tumor positive and blood negative - confirmed sporadic result: • Offspring <<1% • Siblings – population risk • If blood only is tested with negative result: • Offspring <0.5% • Siblings – <0.1% Second Malignancies after Rb • Long term survivors of bilateral Rb have a very high rate of second malignancies. • Bone and soft tissue sarcomas are the most common second primary cancer in childhood. • Uterine leimyosarcomas and lung cancer seen in adults • Radiation therapy of RB significantly increases the risk of malignancy, particularly for sarcomas. • Cohort study suggest 68% risk of second primary malignancy through ~age 50 with epithelial tumors, e.g. lung cancer common in adult RB1 mutation carriers. 274 dWϱϯ Tumor Suppressor Gene • Guardian of genome, regulates DNA damage responses (checkpoint, repair and apoptosis) • Somatic TP53 mutations in cancers are very frequent in osteosarcoma, breast, colon, pancreatic and other solid tumors. • Majority of mutations are missense mutations that interfere with functional domains. • Also see deletions other truncating alleles • Accompanying LOH in ~50% of tumors • Constitutional mutations associated with Li Fraumeni Cancer Predisposition Syndrome. Li-Fraumeni syndrome (LFS) • First identified in children with sarcomas and family history of early onset breast cancer. • Autosomal dominant inheritance of cancer susceptibility • Small proportion of germline de novo mutations. • Penetrance nearly 85% for lifetime tumor development: women greater and earlier than men • High breast cancer risk w/ 31 average age of diagnosis (NCCN recommends TP53 testing for breast cancer <age 31) • 15% of TP53 carriers develop more than one cancer LFS Pedigree Breast dx 35 Lung dx 45 Sarcoma dx 35 Sarcoma, Dx 5 Adrenocortical Ca, dx 10 Stomach Breast, dx 38 dx 25 ALL, Dx 3 45 275 LFS Clinical Diagnostic Criteria Can Aid in Decision on dWϱϯ testing • LFS Classic definition • Sarcoma <45 yrs PLUS • 1° relative with any cancer <45 yrs PLUS • Another 1°/2° relative with any cancer <45 yrs OR sarcoma at any age. • High suspicion: Chompret Criteria for LFS Diagnosis • Li-Fraumeni-Like Syndrome (LFL) • Tumor within LFS spectrum <46 yrs PLUS 1°/2° relative with LFS-related tumor <56 yrs OR with multiple tumors OR • Multiple tumors, 2 within LFS spectrum and 1st tumor <46 yrs OR • Any adrenal cortical carcinoma or CPT, regardless of FH • Less stringent parameters Tinat et al, JCO, 2009 Molecular Genetics of LFS • 80% of LFS families have mutations detectable by sequencing TP53. • Missense variants are the most common pathogenic variant • 5-10% have deletions of TP53 • TP53 also on most BRCA and other hereditary cancer panels: • NCCN guidelines: TP53 testing for woman w/ BRCA <age 31 • No other LFS gene has clearly been identified except one paper on POT1 missense variants and LFS with angiosarcomas. • CHEK2 is LFS2 in OMIM but data that causes LFS is very slim. • Mainly associated with breast and colon cancer risk. Common Tumor Types in Li-Fraumeni Syndrome • Sarcomas – both soft tissue and osteosarcoma all ages, not Ewing’s sarcoma. • Particularly rhabdomyosarcoma embryonal anaplastic subtype. • Breast cancer – most common malignancy in LFS families overall • Average age of onset ~ 31. • Leukemias and lymphomas – including hypodiploid ALL • Adrenocortical carcinoma – in children, not necessarily adult onset. • 60% of children with ACC have germline TP53 mutation • Specific Brazilian variant, R337H, with ~10% penetrance for ACC • Brain tumors – in both children and adults. • Choroid plexus carcinoma are highly indicative of TP53 mutations. • GI malignancies including colon cancer, colorectal, laryngeal • Others: kidney, testicular, head/neck cancers, neuroblastoma 276 Modifiers of cancer risk in dWϱϯmutation carriers • p53 pathway (Fang et al PLoS One 2010): • TP53 codon (P72R) - (RR/RP vs PP carriers age of cancer diagnosis (21 vs 34.4 years, P = 0.05) • MDM2 - 309T>G (GG/GT vs TT carriers age of cancer diagnosis (18.6 vs 27.6 years, P = 0.009) • Telomere length –short telomeres assoc. w/ greater risk childhood cancer diagnosis. • Increasing copy number variation assoc. w/cancer risk. Surveillance for dWϱϯMutation Carriers – Toronto Protocol • Demonstrated to improve morbidity/mortality • Evaluations q3-4 months: • Physical exam • Imaging: abdominal ultrasound (ACC) • Blood tests: adrenal androgens, AFP, ɴ-hCG (ACC), CBC (leukemia) • Urine test: Urinalysis (ACC) • Annual brain MRI (brain tumor) • Annual whole body MRI (sarcoma other soft tissue tumors) • Adults include breast MRI (beginning age 18-21) and colonoscopy beginning age 25. Recent update of the Toronto Protocol Outcomes Lancet Oncology, 2016 277 Telomere Disorders: Dyserkatosis congenita • Disorder due to dysfunctional telomeres (telopathy) • Classical clinical triad • Nail dystrophy • Oral leukoplakia • Abnormal (reticulate) skin pigmentation • Causes of DC mortality • Mean age of death 3rd decade • Bone marrow failure or immunodeficiency (60-70%) • Pulmonary complications 10-15% • Malignancy 20% (including AML and head and neck cancer in young adults) From Savage and Bertuch, Genetics in Medicine, December 2010. • Diagnosis: • Clinical Triad (minority of patients) • 1 or 2 classical features + hypoplastic bone marrow • Known pathogenic germline variant • Very short telomeres (<1st %ile) in 3 or more lymphocyte subsets using flow cytometry. • Features of telomere length study • Test of fresh blood sample is now clinically available in several centers. • Needs to be compared with aged matched controls. • Flow cytometry allows separation of the different types of blood cells and then telomere length is measured for each cell type in the blood sample. Telomere components From Savage and Bertuch, Genetics in Medicine, December 2010. 278 DC: Genetic heterogeneity (at least 10 genes) Hoyeraal-Hreidarsson syndrome - DC-related disorder with additional features of IUGR, immunodeficiency, and cerebellar hypoplasia. Associated with DKC1, TERT, TINF2, RTEL1, ACD, or PARN DKC1 (Dyskerin) 30% XLR TINF2 (TIN2) 10-15% De novo AD mutations, Revesz syndrome TERC (RNA component) 5-10% AD TERT (TERT) 5% AD, AR, More minor genes <5% each NHP2 (NHP2) AR NOP10 (NOP10) AR TCAB1/WRAP53 AR (compound het missense) CTC1 AR (Coats Plus syndrome & DKC) PARN AR Pleuro Pulmonaryblastoma (PPB) & /Zϭ • Rare developmental tumor disorder associated with lung cysts and PPB • Patients with DICER1 germline mutations also at increased risk for: • • • • • • • Multicystic goiter Cystic nephroma Ovarian Sertoli-Leydig-type tumors Wilms tumor Rhabdomyosarcomas (including ovarian and CNS) Intraocular medulloepithelioma Pituitary blastoma and Pineoblastoma Foulkes et al., Nat Rev Cancer, 2014 Genetics of /Zϭ Syndrome Pre-miRNA • Autosomal dominant inheritance w/incomplete penetrance and variable expressivity. • Caused by inactivating mutations in DICER1 • First evidence of a cancer susceptibility gene which encodes a miRNA processing enzyme that cleaves premiRNA • 2nd hits missense mutations specifically clustered in the RNAse IIIb domain (not LOF mutations). • Young children found to carry DICER1 mutations undergo screening focused on surveillance of the pulmonary cavity • Somatic mutations in other miRNA components (DROSHA) are also seen in Wilms tumor. • DROSPHA works at an earlier step to cleave pri-miRNA to form pre-miRNA Figure from National Institute of General Medical Sciences: https://ghr.nlm.nih.gov/condition/dicer1-syndrome#genes 279 Genetic Counseling & Risk Assessment GENETIC COUNSELING & RISK ASSESSMENT Pamela L. Flodman, MSc, MS, LCGC Adjunct Professor, Pediatrics School of Medicine Director, Graduate Program in Genetic Counseling University of California Medical Center, Irvine Pamela L. Flodman, MSc, MS, LCGC Department of Pediatrics University of California, Irvine 101 The City Drive Mail Code: 4482 Orange, CA 92868 (714) 456-5789 Telephone (714) 456-5330 Fax [email protected] 283 284 Genetic Counseling and Risk Assessment Pamela Flodman, MSc, MS, LCGC Adjunct Professor and Director, Genetic Counseling Program University of California, Irvine Disclosure(s): Nothing to disclose Overview භ Principles of genetic counseling භ Ethical, legal and social issues භ Community services / advocacy භ Risk assessment භ Carrier screening 285 y Genetic counseling is the process of helping people understand and adapt to the medical, psychological and familial implications of genetic contributions to health and to disease. y This process integrates: • Interpretation of family and medical histories in combination with results of genetic testing, to assess the chance of disease occurrence or recurrence. • Education about inheritance, testing, management, prevention, resources and research. • Counseling to promote informed choices and adaptation to the risk or condition. Non-directive: Help individuals to make their own best decisions. Components of a genetic counseling session • Case preparation: records, lit review, etc. • Contracting – – – – – – Establish rapport Identify client concerns Establish an agreed-upon agenda Assess prior knowledge Understand the cultural context Acknowledge and respond to anxiety, anger, emotion • Obtain and evaluate medical & family history – Recognize relevant symptoms and findings – Identify need for specific records and test results Components of a genetic counseling session • Interpret family history; risk assessment – Determine inheritance pattern – Bayesian modification of risk if necessary – Apply appropriate risk assessment models • Explain diagnosis, natural history, inheritance, risk, and reproductive alternatives • Discuss options for genetic screening & testing – Assess prior perceptions – Decide who and how to test – Discuss testing options and possible outcomes, including risks / limitations, and impact on family – Interpret and explain results – Determine need for further testing 286 Components of a genetic counseling session • Psychosocial assessment & support – Support systems; cultural beliefs and values – Coping strategies and defense mechanisms • • • • Denial; suppression; anger Projection; displacement Rationalization; intellectualization Acceptance • Identifying resources – – – – Educational materials, and address social media Advocacy and support groups Public and private agencies Option to participate in research; clinical trials • Follow-up Genetic counseling techniques • Primary empathy – A continuum – Minimal encouragers (including non-verbal) – Reflection of content and feeling • Advanced empathy – Goes beyond what is explicitly stated – Tentative: explore together with client • Anticipatory guidance • Confrontation – Not commonly used, but can be powerful Professional issues භ Transference and counter-transference භ Ethical and legal issues: භ Privacy and confidentiality භ Informed consent භ Access to resources භ Professional Codes of Ethics භ Ethics boards 287 භ Transference: Occurs when a patient redirects or projects feelings that they have for another person into their relationship with their counselor, without being aware they have done so. Examples: භ Anger භ Mistrust භ Extreme dependence භ Counter-transference: Occurs when a counselor redirects feelings for another person into their relationship with their client. භ Important to be aware when this happens භ Can occur in response to transference GINA: Genetic Information Non-discrimination Act of 2008 • Provides a baseline protection to prevent discrimination in health care coverage and employment based on genetic information. • Some states have even more protective laws. • Prohibits health insurers from requesting genetic information or using it for decisions re: coverage, rates, or preexisting conditions. • Prohibits most employers from using genetic information for hiring, firing, promotion, or any decisions regarding employment. • Does not extend to life, disability, or long-term care insurance. • Employment provisions don’t apply if < 15 employees. http://www.genome.gov/Pages/PolicyEthics/GeneticDiscrimination/GINAInfoDoc.pdf Counseling Issues:Pre-symptomatic testing for adult-onset disease Reasons for seeking evaluation at this time Perceived risk Current emotional well-being and mental health history Emotional response to family history of disease Coping strategies with respect to perceived risk Reactions and responses during counseling Support mechanisms What specifically will the counselee do differently depending on the results 288 Guidelines: Genetic testing in minors For detecting conditions in which treatment or preventative measures exist: • Testing at the earliest age where health benefits accrue For determining genetic risk or diagnosis only for reproductive decision-making: • Minor should be the primary decision maker (assess voluntariness) Parents or minor request testing with no immediate benefit to the minor • Advisable to defer testing to adulthood • No ethical justification for testing before age 11 or 12 Testing for the benefit of another family member Must have a clear medical benefit Both parents and minor must consent / assent Reassess Relevant Practice Guidelines National Society of Genetic Counselors Genetic cancer risk assessment and counseling Counseling of consanguineous couples and their children Genetic counseling and testing for FMR1 mutations Communicating a prenatal or postnatal dx of Down syndrome Position statements on topics including non-discrimination, disability, reproductive freedom, … many others American College of Medical Genetics Carrier screening in individuals of Ashkenazi Jewish descent Genetic counseling for advanced paternal age Genetic testing and counseling for Alzheimer disease Carrier screening for Spinal Muscular Atrophy Non-invasive prenatal screening for fetal aneuploidy Recommendations for utilization of array-based technology … many others Tools for genetic risk calculation: භ Mendelian genetics භ Empiric risk figures භ Hardy-Weinberg Equilibrium භ (Linkage analysis) භ Bayesian calculation 289 Sometimes, there is additional information which must be included in the calculation of genetic risk: Further pedigree information E.g., having unaffected sons may reduce but not eliminate the chance that a woman is a carrier of an XLR mutation. Test results E.g., a test result that reduces but does not eliminate the chance that a person is a carrier (such as a screening test result) Age of unaffected at-risk individuals Reduces but does not eliminate chance the person carries a predisposing mutation Æ Bayesian analysis, a statistical technique for incorporating additional information. Example: Conditional probability භ A man knows that he has been adopted from a region in which ¼ of the population is Ashkenazic Jewish (and ¾ is not). භ A particular mutation is carried by 3% of the Ashkenazic population, and by 1% of the non-Ashkenazic population. භ If the man finds out that he is a carrier of this mutation, what is the probability that he is Ashkenazic? Bayesian analysis: Graphic representation Ashkenazic Not Ashkenazic The population is represented by the rectangle. •¼ is Ashkenazic •¾ is not The proportions carrying the mutation are shown by the shaded areas .03 .25 .75 .01 290 Bayesian analysis: Graphic representation Ashkenazic Not Ashkenazic The proportion of carriers is represented by the area of the shaded boxes: (.03 x .25) + (.01 x .75) = 0.015 Probability of being Ashkenazic given that he is a carrier is: (.03 x .25) (.03 x .25) + (.01 x .75) = 1/2 .03 .01 .75 .25 Bayes’ theorem P(A|B) = P(A ∩ B) P(B) (Statistical statement of Bayes’ theorem) Bayes’ theorem (Another way to state Bayes’ theorem) 291 Bayesian calculations: A method of combining probabilities භ Consider 2 or more alternative hypotheses. භ Start with a prior probability for each. භ Bring in relevant evidence to support or oppose each hypothesis: conditional probabilities. භ Combine these to obtain overall posterior probabilities. Prior probability Hypothesis 1 Hypothesis 2 He is Ashkenazic He is not .25 .75 Hypothesis 1 Hypothesis 2 He is Ashkenazic He is not .25 .75 .03 .01 Conditional probability (carrier) Joint probability Posterior probability Prior probability Conditional probability (carrier) Joint probability Posterior probability 292 Hypothesis 1 Hypothesis 2 He is Ashkenazic He is not .25 .75 .03 .01 .25 x .03 = .0075 .75 x .01 = .0075 Hypothesis 1 Hypothesis 2 He is Ashkenazic He is not .25 .75 .03 .01 .25 x .03 = .0075 .75 x .01 = .0075 Prior probability Conditional probability (carrier) Joint probability Σ= .015 Posterior probability Prior probability Conditional probability (carrier) Joint probability Posterior probability .0075 .015 = 1/2 .0075 .015 Σ= .015 = 1/2 Bayesian Calculations: Steps 1. Write down all possible mutually exclusive hypotheses (one to each column). 2. Assign a prior probability to each. 3. Consider the additional information. For each column, determine the likelihood of this observation IF the hypothesis is true (conditional probabilities). 4. Multiply down the columns Æ joint probabilities. 5. Scale the joint probabilities so that they sum to 1. These are the posterior probabilities. 293 Hypothesis 1 Hypothesis 2 Event “A” “Not-A” P(A) P(Not-A) P(B | A) P(B | Not-A) P(B | A) x P(A) P(B | Not-A) x P(Not-A) Prior probability Conditional probability “B” Joint probability Posterior probability P(A | B) = P(Not-A | B) = P(B | A) x P(A) P(B) P(B | Not-A) x P(Not-A) P(B) X-linked recessive: Example 1 II:1 I:1 I:2 II:2 II:3 : Duchenne muscular (DMD) dystrophy Mrs. Y IV:1 III:1 III:2 IV:2 IV:3 Prior probability III:3 III:4 IV:4 What is the probability that Mrs. Y is a carrier? Hypothesis 1 Hypothesis 2 Mrs. Y is a carrier Not a carrier 1/2 1/2 Conditional probability (3 unaff sons) Joint probability Posterior probability 294 Hypothesis 1 Hypothesis 2 Mrs. Y is a carrier Not a carrier 1/2 1/2 (1/2)3 = 1/8 1 Hypothesis 1 Hypothesis 2 Mrs. Y is a carrier Not a carrier 1/2 1/2 (1/2)3 = 1/8 1 Prior probability Conditional probability (3 unaff sons) Joint probability Posterior probability Prior probability Conditional probability (3 unaff sons) Joint probability 1/2 x 1/8 = 1/16 1/2 x 1 = 1/2 = 8/16 Σ= 9/16 Posterior probability Hypothesis 1 Hypothesis 2 Mrs. Y is a carrier Not a carrier 1/2 1/2 (1/2)3 = 1/8 1 Prior probability Conditional probability (3 unaff sons) Joint probability Posterior probability 1/2 x 1/8 = 1/16 1/16 9/16 = 1/9 1/2 x 1 = 1/2 = 8/16 8/16 = 8/9 9/16 Σ= 9/16 295 For the next example: • Sometimes we need to consider more than two alternative possibilities • For example, if there is information to incorporate from multiple generations X-linked recessive: Example 2 : Duchenne muscular (DMD) Mom A Daughter B C dystrophy What is the probability that Mom is a carrier? That Daughter is a carrier? At conception, a 50% chance that Mom inherited the mutation. Her risk is reduced because she has an unaffected son, and grandson. Mom: Daughter: carrier carrier carrier not carrier not carrier not carrier Prior probability Conditional A is unaff. B is unaff. Joint probability Posterior probability 296 Mom: Daughter: carrier carrier carrier not carrier not carrier not carrier ½x½ ½x½ ½x1 carrier carrier carrier not carrier not carrier not carrier Prior probability ½x½ ½x½ ½x1 Conditional A is unaff. B is unaff. ½ ½ ½ 1 1 1 carrier carrier carrier not carrier not carrier not carrier Prior probability ½x½ ½x½ ½x1 Conditional A is unaff. B is unaff. ½ ½ ½ 1 1 Prior probability Conditional A is unaff. B is unaff. Joint probability Posterior probability Mom: Daughter: Joint probability Posterior probability Mom: Daughter: Joint probability 1 (½)4 = (½)3 = ½ 1/16 1/8 = 2/16 = 8/16 Σ = 11/16 Posterior probability 297 Mom: Daughter: carrier carrier carrier not carrier not carrier not carrier Prior probability ½x½ ½x½ ½x1 Conditional A is unaff. B is unaff. ½ ½ ½ 1 1 Joint probability Posterior probability Mom: Daughter: 1 (½)4 = (½)3 = ½ 1/16 1/8 = 2/16 = 8/16 1/16 11/16 = 1/11 2/16 11/16 = 2/11 8/16 11/16 = 8/11 carrier carrier carrier not carrier not carrier not carrier Prior probability ½x½ ½x½ ½x1 Conditional A is unaff. B is unaff. ½ ½ ½ 1 1 Joint probability Posterior probability 1 (½)4 = (½)3 = ½ 1/16 1/8 = 2/16 = 8/16 1/16 11/16 = 1/11 2/16 11/16 = 2/11 8/16 11/16 Σ = 11/16 Σ = 11/16 = 8/11 P(Mom is a carrier) = 1/11 + 2/11 = 3/11 = 27% P (Daughter is a carrier) = 1/11 = 9% For the previous examples: • We knew that an XLR mutation was segregating in the family, because there were multiple affected individuals • Æ We could start with a Mendelian risk for the prior probability • BUT – in a family in which there is an ISOLATED case of an affected male, we also have to take into account the possibility that this occurred as a result of a de novo mutation 298 X-linked recessive: Example 3 Mom Daughter : Duchenne muscular (DMD) dystrophy Son No other family hx of DMD What is the probability that Mom is a carrier? Mom may be a carrier of a DMD mutation which she passed to her son. Alternatively, her son may have a new mutation. Three fundamental probabilities: ʅ = mutation rate per locus per generation (per meiosis) ʅ is very small N ʅ N 2ʅ 4ʅ (New μ from father or mother) Digression: deriving 4ʅ There are three possible ways that a woman in the general population is a carrier of the XLR mutation, if you know nothing about her parents: New mutation on the X from her mother: ʅ New mutation on the X from her father : ʅ Her mother is a carrier and passed the mutation to the woman. Let C be P(woman in general pop’n is a carrier) C = ʅ + ʅ + 1/2 C 1/2 C = 2 ʅ C=4ʅ 299 X-linked recessive: Example 3 Mom Daughter : Duchenne muscular (DMD) dystrophy Son No other family hx of DMD What is the probability that Mom is a carrier? Mom may be a carrier of a DMD mutation which she passed to her son. Alternatively, her son may have a new mutation. Prior probability Mom is a carrier Mom is not a carrier 4ʅ 1 - 4ʅ у 1 Mom is a carrier Mom is not a carrier 4ʅ 1 - 4ʅ у 1 ½ ʅ Conditional probability 1 aff’d son Joint probability Posterior probability Prior probability Conditional probability 1 aff’d son Joint probability Posterior probability 300 Prior probability Conditional probability 1 aff’d son Joint probability Mom is a carrier Mom is not a carrier 4ʅ 1 - 4ʅ у 1 ½ ʅ 2ʅ ʅ Mom is a carrier Mom is not a carrier 4ʅ 1 - 4ʅ у 1 ½ ʅ 2ʅ ʅ Σ = 3ʅ Posterior probability Prior probability Conditional probability 1 aff’d son Joint probability Posterior probability 2ʅ 3ʅ ʅ 3ʅ = 2/3 Σ = 3ʅ = 1/3 AD w/ age-dependent penetrance : Huntington disease (onset in early-mid 40s) No other family hx of DMD 50 y.o. What is the probability that your 50 y.o. patient has inherited the HD mutation? Cumulative incidence: 75% of individuals with an HD mutation have onset by age 50 301 Prior probability Carrier Not a carrier 1/2 1/2 Carrier Not a carrier 1/2 1/2 0.25 1 Carrier Not a carrier 1/2 1/2 0.25 1 .5 x .25 = .125 .5 x 1 = .5 Conditional probability Unaff at age 50 Joint probability Posterior probability Prior probability Conditional probability Unaff at age 50 Joint probability Posterior probability Prior probability Conditional probability Unaff at age 50 Joint probability Posterior probability Σ = .625 302 Prior probability Conditional probability Unaff at age 50 Joint probability Posterior probability Carrier Not a carrier 1/2 1/2 0.25 1 .5 x .25 = .125 .5 x 1 = .5 .125 .625 = 1/5 .5 .625 Σ = .625 = 4/5 Heterozygote carrier screening and testing: • Bayesian analysis is also used to incorporate the results of carrier testing, when the testing is not fully predictive of carrier status • I.e. when sensitivity and/or specificity is < 1 • With a negative test result, a residual risk may remain Carrier testing in a family with an AR disorder Mr. G had a brother with CF; no molecular testing was done for the brother. Mr. G chooses to have carrier testing. Mr. G The mutation panel detects 93% of CF mutations in the Caucasian population (sensitivity), with a specificity of > 99.9%. Two possible results: : cystic fibrosis Ethnicity: Caucasian Mutation detected Mr. G is a carrier No mutation detected Bayesian calculation needed 303 Prior probability Hypothesis 1 Hypothesis 2 Mr. G is a carrier Not a carrier 2/3 1/3 Hypothesis 1 Hypothesis 2 Mr. G is a carrier Not a carrier 2/3 1/3 0.07 0.999 Hypothesis 1 Hypothesis 2 Mr. G is a carrier Not a carrier 2/3 1/3 0.07 0.999 2/3 x 0.07 = 0.047 1/3 x 0.999 = 0.333 Conditional probability (neg carrier test) Joint probability Posterior probability Prior probability Conditional probability (neg carrier test) Joint probability Posterior probability Prior probability Conditional probability (neg carrier test) Joint probability Σ= 0.380 Posterior probability 304 Hypothesis 1 Hypothesis 2 Mr. G is a carrier Not a carrier 2/3 1/3 0.07 0.999 2/3 x 0.08 = 0.047 1/3 x 0.999 = 0.333 Prior probability Conditional probability (neg carrier test) Joint probability Posterior probability 0.047 0.380 = 12% 0.333 0.380 Σ= 0.380 = 88% Strategies for problem solving Determine whether a Bayesian approach is needed “If”, “Given that”, “Among”, “Conditional on” Lay out the alternate hypotheses Identify all of the relevant information Prior probabilities Conditional information Do the math – avoid the common mistakes Check your answer against common sense Practice working problems through, and identify your local resources! 305 Biochemical Genetics I BIOCHEMICAL GENETICS I Gerard Berry, MD, FACMG Harvey Levy Chair in Metabolism Director, Metabolism Program Division of Genetics and Genomics Boston Children’s Hospital Professor of Pediatrics, Harvard Medical School Gerard Berry, MD, FACMG Harvard Medical School Center for Life Science Building, Suite 14070 3 Blackfan Circle Boston, MA 02115 (617) 355-4316 Telephone (617) 730-4874 Fax [email protected] 309 310 Biochemical Genetics I Gerard T. Berry, MD, FACMG Founding Fellow ACMG Division of Genetics and Genomics Boston Children’s Hospital Harvard Medical School Director of Harvard Medical School BG training program ACMG Genetics and Genomics Review Course June 20-23, 2013 ACMG Genetics and Genomics Review Course June 20-23, 2013 Nothing to disclose Inborn Errors: A Few Principles • Rare monogenic disorders typically with extensive allelic heterogeneity • Complex interactions of involved proteins • Integrate into pathways, cycles, organelles • Genetic and environmental modifiers • Example: varying severity with age of onset • Early onset – “classic” severe phenotype • Later onset – milder variants and phenotypic spectrum 311 Some Terms and Abbreviations • Sx – symptoms • PFVLCSz – poor feeding, vomiting, lethargy, coma, seizures • Dx – diagnosis • Ge – genetics (AR=autosomal recessive; AD - autosomal dominant; XL=X-linked) • Rx – treatment • PAA – plasma amino acids; UOA – urine organic acids; ACP – plasma acylcarnitine profile • IDD – intellectual disability Acute Metabolic Disease – The Newborn Crash • Consider in neonate with presumed sepsis or acidosis; older child with acidosis, lethargy • Usually term infant, good Apgars, well interval • Family Hx usually negative – may see consanguinity, XL pedigree, hx sibling deaths • Sx – generally non-specific with poor feeding, irritability, vomiting, seizures, progressing to lethargy, coma, apnea (PFVLCSz) • Occasionally specific sx/clues – urine odors, skin/hair (see Appendices) Differential Diagnosis • Amino acid disorders - MSUD • Urea cycle disorders - all except arginase • Organic acid disorders • Carbohydrate disorders - congenital lactic acidosis, fructose disorders, occ. GSDs • Mitochondrial disorders, FAO disorders • Other - sepsis, adrenal insufficiency, CHD, asphyxia 312 Acute Metabolic Disease • Initial labs - sepsis w/up with CBC, lytes, anion gap (AG), NH3, lactate, glucose, urine ketones; check the NBS results • STAT PAA and UOA; ACP; genetics consult • Acute rx - high IV glucose; eliminate protein, lactose, fructose; hemodialysis for NH3 >400 to 500 or high leucine; ?vitamins, carnitine • Specific therapy tailored to individual disease Neonatal Hemodialysis Urea Cycle Disorders • All AR except OTC which is X-linked; overall incidence ~1/30,000 • Sx – all can present in neonatal period with NH3 except arginase deficiency; milder partial defects exist • AS lyase deficiency often develop hepatomegaly/fibrosis • Arginase deficiency – spastic diplegia; IDD 313 Urea Cycle Orotic acid NH3 + HCO3 + CAP CPS I OTC N-Ac Glu Citrulline Ornithine NAG synthetase Glutamate ASA synthase Arginase Urea Arginine ASA lyase ASA X-linked Pattern of Inheritance Biochemical Diagnosis in Urea Cycle Disorders භ Dx - NH3, acidosis only late in course භ Plasma amino acids – elevated glutamine; citrulline level is key Cit is absent in CPS/OTC in citrullinemia in AL deficiency භ Urine orotic acid - in OTC and low in CPS භ Confirmation by DNA (or enzyme assay) 314 Urea Cycle Disorders භNBS - cit , arg; some states screen for low ( ) cit or gln/cit ratio to detect OTC, CPS, NAGS deficiency භRx - low protein diet; meds remove toxic NH3 via alternate pathways; arg or cit replacement; liver transplant for OTC භIV ammunol (Na benzoate and Na phenylbutyrate) – for acute hyperammonemia භBuphenyl (Na phenylbutyrate) – oral powder භRavicti (glycerol phenylbutyrate) – liquid NEW! Removal of NH3 via Alternate Pathways (phenylbutyrate) (ravicti) NH3 = From Gelehrter & Collins, Principles of Medical Genetics, 1990, Fig. 7-20 OTC Heterozygotes භMay be symptomatic in late infancy or childhood; can present with lethargy out of proportion to degree of illness භDx - NH3 and orotic acid increased when symptomatic; allopurinol or protein challenge; molecular testing best way to diagnose භRisk of post-partum coma in OTC heterozygotes 315 Urea Cycle Disorders භOutcome - virtually all have MR with neonatal onset; outcome depends on duration of hyperammonemia භPrenatal dx – by DNA for all with known mutations; enzyme assay (CVS, amnio) for AS, AL, arginase; OTC & CPS only expressed liver, intestine Mitochondrial Carrier Family Transporters Involved in the Urea Cycle Aspartate / glutamate Transporter “Citrin” SLC25A13 Mitochondrion NH4 + HCO3 TCA cycle OAT Ornithine Transporter ORNT1 CAP Ornithine OTC Ornithine Urea Arginine Citrulline Citrulline Asp ASA Cytoplasm Other Disorders Related to the Urea Cycle – Transporter Disorders භHHH syndrome භSx - episodic NH3 භDx - ornithine, homocitrulline භGe – AR; gene ORNT1; redundant fx of related genes භRx – low protein; + citrulline භType II citrullinemia (citrin def) භSx – neonatal hepatitis, adult-onset citrullinemia; භDx - orn, NH3 භGe – AR; gene SLC25A13 භ Rx – low protein 316 Disorders of Ornithine Metabolism-Gyrate Atrophy භSx - blindness & chorioretinal degeneration භDx – PAA with orn, nl NH3 භGe - AR defect in OAT enzyme භRx - low arg diet Glutamate 1 Δ - P5C Urea cycle OAT Retina Ornithine Proline Disorders of Ornithine Metabolism – Creatine Deficiency Syndromes භSx - MR, seizures, hypotonia, autism භDx - plasma and/or urine guanidinoacetic acid (GAA); MRI with MRS (Cr); molecular භGe - 2 very rare AR (AGAT, GAMT); XL Cr transporter (SLC6A8; ?~1% XL MR) භRx - Cr (AGAT, GAMT); low arg + orn (GAMT); ?none for XL transporter deficiency AGAT = arginine:glycine amidinotransferase deficiency GAMT = guanidinoacetate methyltransferase deficiency CNS Muscle SAM Creatine Creatine transporter GAA Ornithine GAMT AGAT Glycine Arginine GAMT AGAT SLC6A8 Urine GAA High Low Nl Urine Creatine (Male) Low Low High Nl Nl High High Low Nl Urine Creatine/Creatinine Plasma GAA 317 Alkaptonuria භOne of original inborn errors recognized by Garrod භSx - osteoarthritis; ochronosis භAR; deficiency of homogentisic acid oxidase in tyr pathway භDx - urine dark upon standing; homogentisic aciduria භRx - ?high dose ascorbic acid; ?NTBC Homogentisic Acid MAA HGA oxidase Cystinosis භSx (infantile or nephropathic) – onset <1 yr; FTT, renal Fanconi, low PO4 rickets, fair hair and skin, photophobia, corneal erosions, hypothyroidism (~age 10), nl IQ, delayed puberty, diabetes, male infertility භJuvenile form (onset 5 -10 yrs) - Nl hair & skin භAdult onset – Mild eye disease only Cystinosis භAR; defect in egress of cystine (cys-cys) from lysosome; gene (CTNS) - cystine transporter භDx - +/- nitroprusside; plasma cys nl; assay cys content in WBC; molecular භRx - cysteamine; symptomatic for renal disease භLate sx post transplant (non-renal) include myopathy, CV & GI disease, resp failure; need to continue cysteamine rx 318 Nonketotic Hyperglycinemia භSx (classic infantile) – most common form; intractable seizures (may have prenatal onset), hypotonia, severe IDD භRare later onset (milder) variants as well as transient form resolving by 8 wks භGe - AR; defect in glycine cleavage enzyme (4 subunits – P*, T*, H, L ) Nonketotic Hyperglycinemia භDx - gly in CSF; plasma gly usually ; CSF:plasma gly ratio > 0.08 is diagnostic; molecular භRx භNa benzoate (excrete gly as hippurate) භlow protein diet භdextromethorphan (inhibits CNS NMDA receptors) භnone very effective Glutaric Aciduria Type I භSx - macrocephaly; acute encephalopathic crises produce IDD, dystonia; MRI with basal ganglia changes and cortical atrophy භAR; defect in lys metabolism (glutaryl CoA dehydrogenase) භDx - urine organics with elevated glutaric, 3-OH glutaric; on NBS panels ( C5-DC) භRx - low protein diet, carnitine, riboflavin 319 Canavan Disease භGe - AR; increased incidence Ashkenazi Jewish pop (~1/40 carrier frequency); deficiency ASPA භDx - N-acetylaspartic acid (NAA) on UOA, NAA on MRS; 3 mutations account for~99% Jewish alleles (E285A 84%) භRx -Sx - onset 3-5 mo; hypotonia, progressive leukodystrophy, macrocephaly; later optic atrophy, seizures භ None; Phase II gene therapy clinical trial N-acetylaspartate L-aspartate + acetate Aspartoacylase (ASPA) (reaction occurs in CNS only) Mitochondrial Fatty Acid Oxidation භ During fasting FAO provides up to 80% total body energy needs භ Long chain (LC) fats preferred substrate for cardiac & skeletal muscle භ LC free fatty acids (FA; C18, C16) released from TG in adipose tissue භ Peripheral tissues oxidize FA to CO2 and H2O Liver oxidizes FA to ketone bodies for energy for gluconeogenesis and ureagenesis භ Ketone bodies used as fuel in CNS Mitochondrial Fatty Acid Oxidation Medium Chain FA LC Ac-CoA Carnitine Cycle LC Ac-CoA mitochondrion ß-Oxidation Cycle TCA Ac-CoA Electron transfer Ketone synthesis Four components of fatty acid oxidation: Carnitine cycle ß-oxidation cycle Electron transfer Ketone body synthesis 320 Fatty Acid Oxidation Disorders (FAO) භLiver - glu, ketones, LFTs/NH3, coagulopathy, enlarged liver, fasting intolerance භCardiac - cardiomyopathy, arrhythmias භSudden death - liver (hypoglycemia), cardiac භSkeletal disease භ Acute - rhabdomyolysis භChronic – weakness, fatigue, lactic acidosis භ Risk HELLP (hemolytic anemia with elevated LFTs and low platelets) in females with LCHAD fetus භRx - avoid fasting; low fat diet (Lipistart, Monogen); carnitine Mitochondrial FAO: Carnitine Cycle Carnitine Extracellular fluid Plasma Membrane Carnitine Cycle Defects - Clinical CU Liver Heart CU + + CPTI + CT + + CPTII + + Cytosol Skel muscle Acute Chronic Fatty acid Outer Mito Membrane CPT I (+) Carnitine Acylcarnitine Carnitine Acyl-CoA Inner Mito Membrane CT + (+) + CPT II Acyl-CoA Carnitine Acylcarnitine Carnitine Mito matrix ß-Oxidation cycle Mitochondrial FAO: ß-Oxidation Spiral Clinical phenotype ß-Oxidation Spiral Liver Heart Skel muscle Acute Acyl-CoA Ac-CoA 1 4 3 keto Acyl-CoA Enoyl-CoA 2 3 3 OH Acyl-CoA 1. Acyl-CoA dehydrogenase 3. 3-OH Acyl-CoA dehydrogenase Chronic Acyl-CoA dehydrogenases VLCAD + MCAD + + + SCAD + 3-OH Acyl-CoA dehydrogenases LCHAD SCHAD + + + + + 321 Mitochondrial FAO: Ketone Synthesis ß-OxidationSpiral Acetoacetyl-CoA Clinical phenotype Ac-CoA Onset in infancy or childhood Hypoketotic hypoglycemia 1 HMG-CoA 2 Acetoacetate 3 4 βȕ-Hydroxybutyrate Acetoacetyl-CoA 1. 2. 3. 4. HMG CoA synthase HMG CoA lyase SCOT D-3-OH-butyrate dehydrogenase FAO Genetics භAt least 25 enzymes and 22 distinct disorders භAll AR – MCAD most common (~1/10-15,000); others rare භDx – ACP; UOA (short chains); carnitine levels (<10 in Carnitine Uptake Deficiency, CUD) භMolecular භLCHAD common mutation (>80% alleles) භ2 common SCAD polymorphisms 7% population PA and MMA Metabolism Gut bacteria Ile, Val Met, Thr Odd chain FA Propionate Propionyl CoA PCC, Biotin D-Methylmalonyl CoA L-Methylmalonyl CoA Mutase, B12 Succinyl CoA Adapted from SIMD NAMA 322 Branch Chain Amino Acid Catabolism Leucine Isoleucine Valine Keto-isocaproic Keto-methylvaleric Keto-isovaleric MSUD Isovaleryl-CoA IVA Methylcrotonyl-CoA MCC Methylglutaconyl-CoA 3-OH-3methylglutaryl-CoA Acetoacetate Methylbutyryl-CoA Isobutyryl-CoA Tiglyl-CoA 3OH-Isobutyryl-CoA Methyl-3OH-butryl-CoA 3OH-Isobutyric acid 2-Methylacetoacetyl-CoA Methylmalonic semialdehyde Acetyl-CoA Malonyl-CoA Acetyl-CoA Propionyl-CoA PA Methylmalonyl-CoA MMA Succinyl-CoA Methylmalonic Acidemia (MMA) භ Sx - PFVLCSz; later onset with hematologic sx (macrocytic anemia), altered gait & cognition in some Cbl (B12) defects භ Dx - Metabolic acidosis, NH3; PAA: Gly; homocysteine (cbl C,D,F); UOA: MMA, PA metabolites & ketones භ Rx - like PA; OH-B12 for cbl defects Methylmalonyl-CoA mutase Succinyl-CoA AdoCbl L-Methylmalonyl-CoA Cobalamin Metabolism From OMMBID, Ch155fg12 323 Methylmalonic Acidemia Genetics භMethylmalonyl-CoA mutase - adenosylcobalamin (AdoCbl) co-factor භMut0 - (0 enzyme activity); mut- - residual activity භLocus heterogeneity භcbl A – MMA only භcbl B – MMA only භcbl C - combined MMA & homocystinuria, relatively common භcbl D & F – combined MMA and homocystinuria, rare භcblE, G – homocystinuria only PA and MMA: Some Complications භBone marrow suppression during acute crises and as part of chronic disease භSevere feeding difficulties භProgressive renal disease භCardiomyopathy භBasal ganglia infarcts “metabolic strokes” භPancreatitis භEye and vision problems Treatment of MMA and PA භRx - low protein (Propimex); restrict precursors [Val, Met, Ile, Thr, odd chain FA (VOMIT)]; carnitine, biotin, ?metronidazole, carbiglu 324 Isovaleric Acidemia (IVA) භSx- PFVLCSz; odor of sweaty feet භDx- Metabolic acidosis; Anion Gap; NH3; UOA: isovaleric & 3-OH-isovaleric acid; isovaleryl-glycine; ketones භGe- AR deficiency of Isovaleryl CoA dehydrogenase භRx- low protein + formula (Valex), glycine, (carnitine) Metabolic Disorders of Morphogenesis භGroup of inherited disorders in which major malformations occur prior to birth භImply lack of product or presence of increased substrate or metabolite is teratogenic භExamples භCholesterol biosynthetic disorders (SLOS) භGeneralized peroxisomal disorders (Zellweger syndrome) භSevere MADD/ glutaric acidemia Type II (an FAOD) භPyruvate dehydrogenase deficiency භSome mitochondrial disorders Cholesterol Biosynthetic Disorders භ 10 disorders reported භ Mevalonic aciduria, Hyper IgD syndrome භ CHILD syndrome භ X-linked dominant chondrodsyplasia punctata (CDPX2) භ Lathosterolosis භ Smith-Lemli-Opitz syndrome (SLOS) භ Desmosterolosis භ SC4MOL deficiency භ Lanosterol demethylase deficiency භ (Antley-Bixler) – defect in P450 oxidoreductase භ HEM dysplasia Dx: urine organics (mevalonic aciduria, Hyper IgD); plasma sterol profile for others 325 Smith-Lemli-Opitz Syndrome භMost common disorder භSx – MR/autism, microcephaly, hypotonia, FTT, 2-3 toe syndactyly, ptosis, cataracts, hypogenitalism, cleft palate, occ. CDP භDx – low cholesterol; elevated 7-DHC භRx – cholesterol supplementation may improve growth & behavior; no effect on development From Porter & Herman, Jl Lipid Res., 52: 6-34, 2011. CDPX2; Happle Syndrome භ X-linked dominant, us. male lethal භ Sx - epiphyseal calcification (CDP), asymmetric dwarfing; asymmetric cataracts/ microphthalmia; linear hyperkeratotic erythroderma, alopecia භ Distinct phenotype (MR, hypotonia, szs, CNS & other malformations) in >10 males with hypomorphic mutations භ Dx - plasma sterol analysis; molecular defect in Δ8−Δ7 sterol isomerase (EBP) Disorders of Carbohydrate Metabolism භCongenital lactic acidosis භ(Galactosemia – see NBS) භDisorders of fructose metabolism භGlycogen storage disorders 326 Congenital Lactic Acidosis භIncludes some CHO (F-1,6-bisphosphatase def), disorders of pyruvate metabolism (PDH, PC), & some mitochondrial (mt) disorders භSpectrum of clinical phenotypes භSevere neonatal lactic acidosis +/- congenital malformations (agenesis of corpus callosum) භChronic with MR, szs, hypotonia භEpisodic +/- FTT, MR භDx - elev. plasma/CSF lactate, PAA: ala; normal lactate to pyruvate ratio (PDH/PC); hypoglycemia (PC; F-1,6-BP); molecular Congenital Lactic Acidosis භPDH: Most common cause භ5 proteins in complex (E1ɲ,E1ɴ,E2,E3,X, PDP) භMutations in XL E1ɲ most frequent; most de novo; males with hypomorphic muts or lethal; females often with sx භF-1,6-BP භ½ have 1st sx <1 week of age භDo not need to ingest fructose භSx – acidosis, glu, +ketones, liver with dysfx , FTT, hypotonia භRx - poor outcome for neonatal cases; biotin and high CHO for PC; low CHO (ketogenic diet), thiamine for PDH; limit fructose & rx acute attacks for F-1,6-BP Hereditary Fructose Intolerance භSx - Asymptomatic in the absence of dietary fructose; Nausea, vomiting, hypoglycemia & metabolic acidosis following fructose intake (sucrose, fruits, honey); occ chronic sx - FTT භDx - Controlled fructose load; AR defect aldolase B (A149P accounts for ~70% of mutant alleles) භRx - Fructose avoidance; tolerance may improve with age ATP Fructose Aldolase B F1P DHAP + Glyceraldehyde 327 Glycogen Metabolism & Liver GSDs Glycogen 1,4 1,6 Debrancher GSD III Brancher - GSD IV 1,4 Glycogen synthase Phosphorylase GSD VI GSD 0 G1P Glucokinase G6P G6Pase complex Lactate Glucose Brain & other peripheral tissues GSD I Pyr TCA cycle Glycogen Storage Disease (GSD) - Type I භTwo types: Ia – glucose-6-phosphatase deficiency Ib – translocase deficiency භSx Type Ia – most severe of GSDs with glu, hepatomegaly, short stature; irritable infant with chubby cheeks; epistaxis and bleeding භSx Type Ib – same + neutropenia and chronic ER infections, oral/intestinal ulcerations, chronic G6P G6Pase Glucose + Pi inflammatory bowel disease G6P translocase G6P භLate complications - osteopenia cytosol and fxs; gout; pancreatitis; Glycogen renal disease; hepatic adenoma/Ca GSD - Type I භ Dx - hypoglycemia, elev. TG/chol, lactate, uric acid; no response to glucagon; LFTs usually nl; neutropenia with GSD Ib; molecular dx (liver bx often not necessary now) භ Ge - Both Ia and Ib are AR භ Type Ia (G6PC gene) ~80-90% and Type Ib (SLC374A gene) ~10-20% භ Some common mutations with founder effects භ Rx - freq. feeds of complex carbs, cornstarch/ Beneprotein; avoid lactose, fructose, gal; DDAVP for bleeding, G-CSF for Ib neutropenia; allopurinol for uric acid; monitor renal fx; liver US and AFP for adenomas, liver Ca 328 GSD 1A From OMMBID, Ch71Fg8 GSDs - Type II Pompe Disease භSx - hypotonia, cardiomegaly, large tongue, hepatomegaly; juvenile form with progressive muscle weakness භGe - Lysosomal storage disease (α-glucosidase deficiency); AR භDx - elev. CPK, massive QRS on EKG; enzyme assay or molecular for dx භRx - enzyme replacement therapy භLate sx with ERT in infantile Pompe – hearing loss, osteopenia, motor/speech delay, GE reflux/dysphagia 329 GSD Type III Debrancher Deficiency භSx - hepatic sx like Ia but often milder; muscle involved (hypotonia); liver sx often resolve with puberty; adult cardiac/skeletal myopathy භDx - lactate, uric acid nl; elev. CPK and LFTs; molecular or enzyme assay fibroblasts, liver භGE - AR; most pts with defective enzyme in liver & muscle Glycogen (IIIa);10-15% liver only (IIIb) with nl CPK (ass’d with 2 early frameshift, nonsense 1,6 1,4 1,4 mutations and truncated protein) භRx -like Ia, but can have some fructose, galactose Debrancher deficiency Phosphorylase Related Defects (GSD VI, IX) භDefects of phosphorylase itself (Type VI) or components of phosphorylase kinase (Type IX) භSx - hepatomegaly and short stature; mild hypoglycemia; some with more severe sx භMost common form of phosphorylase kinase deficiency (IX) is X-linked (“short males with pot bellies”); rest AR Glycogen 1,4 1,6 1,4 Glycogen synthase Phosphorylase system G1P භPrognosis good for most & many require no rx Glucokinase G6P Lactate G6Pase complex Glucose Pyr Other forms of GSD in Appendix Disorders of Metal Metabolism - Menkes Disease භX-linked; ~1/100,000 භSx - hypotonia; szs; depigmented steely, brittle hair; FTT; typical facies; hypothermia; lax skin; tortuous vessels; osteoporosis; death by age 2 භCation transporting ATPase (ATP7A gene); defective Cu uptake placenta, gut, BBB භDx - low ceruloplasmin and low serum Cu; occ. elevated lactate භRx - symptomatic; early rx with Cu histidinate improves outcome in less severe cases 330 Menkes Disease Pili torti Wilson Disease භAR; ~1- 4/100,000 භSx - hepatic with jaundice, hepatic failure, hemolysis, bone disease or neurologic with dementia, intellectual loss, dysarthria, basal ganglia sx; Kayser-Fleischer rings (eye) භDx - low ceruloplasmin, increased Cu in urine and liver භLiver ATPase; H1069Q accounts for ~40% N. Europe alleles; >100 mutations identified භRx - penicillamine, Zn, liver transplant for hepatic failure Molybdenum Cofactor Deficiency & Sulfite Oxidase Deficiency භMo complexes to 3 enzymes (sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase); sx with cofactor deficiency or isolated sulfite oxidase deficiency (both AR) භSx – severe progressive szs, dislocated lenses, MR භDx – low uric acid (<2; in the combined deficiency), increased sulfite excretion (sulfocysteine) භRx – Trials for Rx with cyclic pyranopterin monophosphate (cPMP) in MoCD Type A 331 Neurodegenerative Disorders of Iron Accumulation • PKAN pantothenate kinase ass’d neurodegen) • Dystonia, retinopathy, acanthocytosis • Depletion of CoA in mitochondria • PLAN (P’lipase A2 ass’d neurodegen) • Weakness to spasticity, muscle wasting, optic atrophy • BPAN • Neurodegenerative, XL dom with mosaic males, can resemble Rett or Angelman • WDR45 ɴ-propellor protein; fairly frequent APPENDICES Holocarboxylase Synthase (HCS) and the Biotin Cycle භHCS catalyzes the covalent addition of biotin to 4 carboxylases භ3-Methylcrotonyl-CoA carboxylase භPropionyl-CoA carboxylase Holocarboxylase Aposynthase carboxylases භAcetyl-CoA carboxylase භPyruvate carboxylase B භBiotinidase catalyzes recovery of proteinbound biotin B Holocarboxylases Diet Biotinidase proteolysis 66 332 Holocarboxylase Synthase (HCS) Deficiency භSx – PFVLCSz, odor of cat’s urine; skin rash and alopecia in later onset cases භDx – metabolic acidosis & ketosis; +anion gap, NH3, UOA: 3-OH-isovalerate, methylcitrate,3methylcrotonylglycine, propionylglycine, 3-OH-propionate භGe – AR; functional deficiency of all 4 biotin-dependent carboxylases භRx – support during acute crises; 10-50 mg/dy biotin is virtually curative Porphyrias භGroup of enzyme disorders responsible for heme synthesis from gly + succinyl CoA භTwo types of major sx – neurovisceral and/or photosensitivity භExamples of heterozygous enzyme deficiency causing disease භExamples of pharmacogenetic disorders where avoidance of certain drugs may prevent sx Porphyria Cutanea Tarda (PCT) භMost common type භSx - adult disorder; lesions on sun-exposed skin; liver disease (cirrhosis 40%); alcohol and estrogen aggravate sx භDx - urine/stool coproporphyrins; some inherited (AD), most acquired, defects in UROD (uroporphyrinogen decarboxylase) භRx - avoid triggers; phlebotomy 333 Acute Intermittent Porphyria (AIP) භIncidence ~1,20,000 භSx - after puberty, abdominal pain and neuropsychiatric; attacks can be precipitated by certain drugs (dilantin, sulfa drugs, etc) භ80-90% of gene carriers asymptomatic භAD deficiency of PBG deaminase භDx - urine porphyrins; enzyme assay/molecular භRx - high CHO diet; IV glucose and hematin for attacks; avoid offending drugs Other Liver GSDs භGSD 0 - Glycogen synthase deficiency භFasting hypoglycemia in infancy; postprandial lactate භFrequent developmental delay භNormal liver size with reduced hepatic glycogen භAR inheritance; GYS2 mutations භGSD IV - Brancher deficiency (Anderson) භChildhood cirrhosis with hepatosplenomegaly භAbnormal glycogen - Amylopectinosis භAR inheritance; GBE mutations GSD- Type V McArdle’s Disease භAR; muscle phosphorylase deficiency භSx - muscle weakness and cramping, usually ass’d with exercise; sx generally start as adult භDx - myoglobinuria (50%); lack of rise in lactate on ischemic exercise test is virtually diagnostic භRx - avoid precipitating factors; glucose with exercise; B6 334 Other Muscle Glycogen Storage Diseases භ GSD VII - AR; Phosphofructokinase deficiency භSimilar to GSD V plus hemolysis භ GSD X - AR; Phosphoglycerate mutase deficiency භExercise intolerance, cramps, myoglobinuria භ GSD XI - AR; Lactate dehydrogenase deficiency භExercise intolerance, cramps, myoglobinuria Defects of Purine and Pyrimidine Metabolism භLesch-Nyhan syndrome (HGPRT def) භMR, choreiform movements, selfmutilation, gouty arthritis; milder variants භXL; Dx - elevated uric acid, enzyme assay/molecular භAPRT def – renal sx, stones භAdenysuccinate lyase def – MR, szs, autistic features භADA and NP deficiency – AR immunodeficiency syndromes භHereditary orotic aciduria - megaloblastic anemia & orotic acid crystals; +/- MR; rare AR Disorders of Bilirubin Metabolism භUnconjugated hyperbilirubinemia භCrigler-Najar I and II – AR; deficiency of bilirubin-UDPglucuronosyl transferase 1; kernicterus; Type I more severe and don’t respond to phenobarbitol භGilbert – mild with no clinical sx; pts with homozygous promoter mutations භConjugated hyperbilirubinemia භDubin-Johnson – AR; defect in liver anion transporter භRotor syndrome – AR; rare, usually benign; recent demonstration defect is biallelic mutations in linked bilirubin transporter genes 335 • Online Metabolic and Molecular Basis of Disease • Blau et al., Physician’s Guide to Laboratory Diagnosis of Metabolic Disease • Nyhan et al., Atlas of Metabolic Disease • NAMA slide sets from SIMD • Lee and Scaglia, eds., Inborn Errors of Metabolism (2014) ACMG Genetics and Genomics Review Course June 20-23, 2013 336 Neurogenetics NEUROGENETICS Bruce R. Korf, MD, PhD, FACMG Wayne H. and Sara Crews Finley Chair in Medical Genetics Professor and Chair, Department of Genetics Director, Heflin Center for Genomic Sciences University of Alabama at Birmingham Bruce R. Korf, MD, PhD, FACMG Department of Genetics University of Alabama at Birmingham Kaul 230, 1530 3rd Avenue South Birmingham, AL 35294-0024 (205) 934-9411 Telephone (205) 934-9488 Fax [email protected] 339 340 Neurogenetics Bruce R. Korf, MD, PhD Professor and Chair, Department of Genetics University of Alabama at Birmingham Disclosure(s) Relationship Entity Grant Recipient Novartis Advisory Board Accolade, Genome Medical Board of Directors American College of Medical Genetics and Genomics Children’s Tumor Foundation Advisor Neurofibromatosis Therapeutic Acceleration Project Founding Member Envision Genomics Salary University of Alabama at Birmingham Objectives • Formulate genetic differential diagnosis for neurological problems • Recognize indications for genetic testing for neurological disorders • Describe natural history and management for selected neurogenetic disorders 341 Approach to Neurogenetic Disorders Anatomical localization Brain Spinal cord Peripheral nerve Muscle Sensory organs Temporal course Congenital or acquired Static or progressive Continuous or paroxysmal Physiology Developmental mechanisms Intercellular signaling/signal transduction Structure/function Control of cell growth Metabolic Phakomatoses • Neurofibromatosis • NF1 • NF2 • Schwannomatosis • Tuberous Sclerosis complex • von Hippel-Lindau syndrome +/+ -/- +/- Tumor suppressor mechanism Neurofibromatoses NF1 Inheritance/ Penetrance Frequency Features Gene/Protein Function NF2 Schwannomatosis AD/Complete AD/Complete 1:3,000 1:25,000 AD/Incomplete 1:40,000 neurofibromas; café-au-lait macules; learning disabilities; skeletal dysplasia; gliomas; malignant peripheral nerve sheath tumors vestibular schwannomas; other schwannomas; meningiomas; ependymomas; cataracts Schwannomas; pain NF1 – chromosome 17 neurofibromin NF2 - chromosome 22 merlin/schwannomin INI1/SMARCB1 or LZTR1 GTPase activating protein cytoskeletal protein chromatin remodeling 342 NF1 • • • • • • Frequency: 1:3,000 Inheritance: AD, complete penetrance Gene: NF1 Diagnosis: clinical criteria, genetic testing Pathophysiology: tumor suppressor, control of RAS signaling Surveillance: blood pressure, growth, puberty, vision, tumors, malignant peripheral nerve sheath tumor • Treatment: surgery, chemotherapy for low grade gliomas, clinical trials (MEK inhibitor) • Counseling: AD, new mutation, mosaicism NF1 Diagnostic Criteria • At least six café-au-lait macules • 5 mm pre-puberty • 15 mm post-puberty • Skin-fold freckles • Two or more neurofibromas/one plexiform neurofibroma • Two or more iris Lisch nodules • Optic Glioma • Characteristic Skeletal Dysplasia • tibia • orbit • Affected first-degree relative Diagnosis requires fulfillment of two criteria 343 Differential Diagnosis of Multiple Café-au-Lait Spots Condition Gene(s) Comments NF1 NF1 Six or more NF2 NF2 Usually fewer than six; bilateral vestibular schwannomas Legius syndrome SPRED1 Café-au-lait spots, skin fold freckles; no tumors Noonan syndrome Ras pathway genes Dark café-au-lait spots; some with lentigines Mismatch Repair Deficiency Syndrome Mismatch repair genes High risk of malignant tumors Fanconi anemia Various DNA repair genes Congenital anomalies, bone marrow failure Ataxia-Telangiectasia ATM Ataxia, telangiectasia McCune-Albright GNAS1 mosaicism Large, irregularly shaped; precocious puberty; fibrous dysplasia Chromosomal mosaicism Various aneuploidies Irregularly shaped along lines of Blashko Bloom syndrome BLM Short stature, risk of cancer Tuberous sclerosis complex TSC1/TSC2 More often hypopigmented macules NF1 Pathogenesis 23a 9br 1 2 3 4a 4b 5 6 7 8 10a 10b 10c 12a 13 16 17 1819a 20 2 1 22 23-2 48a 24 25 26 27a27b 28 OMGP EVI2B 29 30 31 32 33 34 48 49 EVI2A EGF EGF Grb 36 37 38 39 40 41 42 4344 45 46 Sos NF1 +/- Ras neurofibromin NF1-/- Schwann cell Grb Sos Ras NF1-/Other changes MPNST Neurofibroma NF2 • Frequency: 1:25,000 • Inheritance: AD, complete penetrance • Gene: NF2 • Diagnosis: clinical criteria, genetic testing • Pathophysiology: tumor suppressor, cytoskeletal protein • Surveillance: hearing, brain and spinal imaging, eye exam • Treatment: surgery, clinical trials (bevacizumab) • Counseling: AD, new mutation, mosaicism 344 NF2 Diagnostic Criteria Bilateral vestibular schwannomas, or First degree relative with NF2 and any two of: meningioma ependymoma schwannoma juvenile posterior subcapsular cataract/cortical wedge opacity Schwannomatosis • Frequency: ~1:40,000 • Inheritance: AD, incomplete penetrance • Gene: INI1/SMARCB1 or LZTR1 • Diagnosis: clinical criteria, genetic testing, mosaic staining INI1 in schwannoma (for SMARCB1-related schwannomatosis) • Pathophysiology: tumor suppressor, complex findings in tumors; chromatin remodeling protein(s) • Surveillance: tumor growth, pain • Treatment: surgery, pain management • Counseling: AD, incomplete penetrance, new mutation, mosaicism Schwannomatosis Germline SMARCB1 SMARCB1 NF2 NF2 Tumor SMARCB1 Deletion NF2 Similar mechanism with LZTR1 345 Tuberous Sclerosis Complex • Frequency: 1:6,000 • Inheritance: AD, complete penetrance • Gene: TSC1 (9q34 hamartin); TSC2 (16p13 tuberin) • Diagnosis: clinical criteria, genetic testing • Pathophysiology: tumor suppressor – mTOR inhibition • Surveillance: development, seizures, SEGA, renal, pulmonary, eye • Treatment: everolimus for progressive SEGA or AML • Counseling: AD, new mutation, mosaicism TSC Diagnostic Criteria Major • • • • • • • • • • • Angiofibromas (ш3) or fibrous cephalic plaque Ungual fibromas (ш2) Hypomelanotic macules (ш3, ш5mm) Shagreen patch Multiple retinal hamartomas Subependymal giant cell astrocytoma Subependymal nodules (ш2) Cortical dysplasias (ш3) Cardiac rhabdomyoma Renal angiomyolipoma Lymphangioleiomyomatosis Minor Dental enamel pits (>3) Intraoral fibromas (ш2) Non-renal hamartomas Retinal achromic patch Confetti skin lesions Multiple renal cysts Definite TSC Two major One major, two or more minor Possible TSC one major, or two or more minor TSC Skin Lesions hypomelanotic macule Shagreen patch facial angiofibroma periungual fibroma 346 TSC CNS Lesions TSC Normal TSC Other Lesions Cardiac Rhabdomyoma Levine, D., Barnes, P., Korf, B., Edelman, R. Am. J. Roentgenol. 2000;175:10671069 Angiomyolipoma Renal cysts Lymphangioleiomyomatosis TSC Surveillance • Brain • MRI (repeat every 1-3 years) • Eye • EEG • Ophthalmology exam annually • Developmental screening • Kidney • Heart • Echo on children • ECG all ages (every 3-5 years) • MRI abdomen (repeat every 1-3 years) • Skin – exam annually • Blood pressure annually • Teeth – exam every 6 • GFR annually • Lung (female >18) months • PFT, 6 minute walk test • High resolution chest CT (repeat every 5-10 years) 347 TSC Treatment hamartin tuberin Rheb GDP mTOR SEGA Everolimus Renal AML growth & proliferation Von Hippel-Lindau Syndrome • Frequency: 1:36,000 • Inheritance: AD • Gene: VHL • Diagnosis: clinical criteria, genetic testing (genotype-phenotype correlation) • Pathophysiology: tumor suppressor – vascular response to hypoxia • Surveillance: eye, hearing, brain, kidney, pheochromocytoma • Treatment: some clinical trials • Counseling: AD, 20% new mutation, potential risk for those with sporadic hemangioblastoma Von Hippel-Lindau Syndrome • Hemangioblastoma • cerebellum • retina • spinal cord • • • Pheochromocytoma Renal cell carcinoma Endolymphatic sac tumor HIF VHL O2 angiogenesis 348 VHL Surveillance • Birth • Physical exam/hearing screen • Ages 1-4 • Annual ophthalmological exam • Physical exam • Ages 5-15 • • • • Annual PE and eye exam Annual fractionated metanephrines Every 2-3 year audiology MRI if repeated ear infections • Age 16 and beyond • Annual eye, PE, fractionated metanephrines • Annual abdominal u/s with MRI every other year • Every 2 years brain MRI, audiology • During Pregnancy • Test for pheochromocytoma early, mid, and late pregnancy • MRI without contrast at 4 months brain and spine www.vhl.org Screening for Pheochromocytoma • Screening for PPGLs should always include measurements of plasma-free metanephrines (obtained from a blood sample) or urinary fractionated metanephrines (obtained from a urine sample). • For a blood sample, patients are now required to be supine (lying down) for a minimum of 20 (ideally 30) minutes between the time the needle is inserted and the time the blood is drawn. • Blood sample analysis should be done using norms (a reference standard) from supine tests, not seated tests, to minimize the chance of a false negative result (missing a PPGL that is present) www.vhl.org Epilepsy • Affects 1% population • At least 2 unprovoked seizures • Classification • Focal Onset • Aware/Impaired Awareness • Motor/Non-motor onet • Focal/Bilateral Tonic-Clonic • Generalized Onset • Motor (tonic-clonic, other) • Non-Motor (Absence) • Unknown Onset • Motor • Non-Motor • Monogenic mostly ion channelopathies • Multifactorial: epilepsy with complex genetics • CNVs: 15q11.2, 15q13.3, 16p13 • Epilepsy syndromes • Part of some genetic syndromes 349 Channelopathies Na Channel Na+ Generalized epilepsy with febrile seizures Severe myoclonic epilepsy of infancy Idiopathic childhood epilepsy with Generalized t/c seizures Primary erythermalgia Paroxysmal extreme pain disorder Insensitivity to pain Myotonia Periodic paralysis Empirical Recurrence Risk Counseling for Epilepsy Prasad AN, Prasad C. Genetic Aspects of Human Epilepsy. Emery & Rimoin, Principles and Practice of Medical Genetics, 6th Ed. Movement Disorders • Bradykinesia – slow movements • Dystonia – sustained muscle contraction resulting in abnormal posture • Chorea – sudden involuntary movement • Myoclonus – muscle jerking • Tremor – rhythmic oscillatory movement • Tic – sudden stereotyped motor movement or vocalization By Mikael Häggström, based on images by Andrew Gillies/User:Anaru and Patrick J. Lynch [CC-BY-SA-3.0 (www.creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons 350 Parkinson Disease Tremor, rigidity, bradykinesia 1% >60 yo; 4% >80 yo Most cases sporadic (multifactorial) Glucocerebrosidase heterozygosity Multiple risk alleles by GWAS 5-10% monogenic autosomal dominant SCNA, LRRK2, VPS35 autosomal recessive PINK1, DJ-1, Parkin, DNAJC6 Atypical PD ATP13A2, FBX07, PLA2G6, SYNJ1 By Sir_William_Richard_Gowers_Parkinson_Disease_sketch_1886.jpg: derivative work: Malyszkz [Public domain], via Wikimedia Commons Fragile X Tremor Ataxia Syndrome • Ataxia • Intention tremor • Short term memory loss • Dementia • Parkinson symptoms • Premutation • >50 yrs • Penetrance males older than 50 >33%; females 5-10% • Toxic gain of function of FMR1 mRNA Dystonia • DYT1 • Torsin A(9q34) • AD • GAG deletion most common mutation • Focal to generalized dystonia • DYT5 • AD • GTP cyclohyrolase I/tyrosine hydroxylase • DOPA-responsive By James Heilman, MD (Own work) [CC-BY-SA-3.0 (www.creativecommons.org/licenses/by-sa/3.0) or GFDL (www.gnu.org/copyleft/fdl.html)], via Wikimedia Commons 351 Dystonia Condition Genetics Isolated Dystonias Childhood/Adolescent-onset AD: DYT1 (TOR1A), DYT6 (THAP1), DYT13; AR: DYT2,17 Adult-onset DYT7, DYT21, DYT23 (ClZ1), DYT24 (ANO3), DYT25 (GNAL) (all AD) Combined Dystonias Persistent Dystonia with Parkinsonism Dystonia with Myoclonus Dystonia with Chorea Paroxysmal Nonkinesigenic Kinesigenic Exercise-induced AD: DYT5(GCH1, TH, SPR), DYT12 (ATP1A3); AR: DYT5, 16; XR: DYT3 (TAF1) DYT11, DYT15 (SGCE) (AD) DYT4 (TUBB4) (AD) All AD DYT8 (MR-1), DYT20 DYT10 (PRRT2), DYT19 DYT18 (SLC2A, GLUT1) Neurodegeneration with Brain Iron Accumulation • Clinical features • Movement disorder • Dystonia, choreoathetosis, rigidity • Ophthalmologic signs • Retinitis pigmentosum, optic atrophy • Iron accumulation in basal ganglia • Genetics • PKAN – pantothenate kinase deficiency • PLAN – infantile neuraxonal dystrophy or later onset (PLA2G6) • MPAN – mitochondrial membrane protein (C19ORF12) • BPAN – beta-propeller (WDR45) – X-linked • Aceruloplasminemia (CP) • FAHN – fatty acid hydroxylase (FA2H) • Kufor-Rakeb (ATP13A2) • Neuroferritinopathy (AD)(FTL) • Woodhouse-Sakati (DCAF17) • Idiopathic Curr Opin Neurol. 2016 Aug; 29(4): 486–495. doi: 10.1097/WCO.0000000000000352 Monogenic Choreas Disorder Huntington chorea Gene HTT (CAG expansion) Inheritance AD Notes Dementia, psychiatric disturbance Prion PRNP (octapeptide expansion) AD Dementia, psychiatric disturbance HDL2 JPH3 (CAG/CTG expansion) AD Parkinsonism AD Ataxia, dementia, Parkinsonism SCA17 TBP (CAG expansion) Dentatorubral-pallioluysian atrophy ATN1 (CAG expansion) AD FTD/MND C9orf72 (GGGGCC expansion) AD Dementia, psychiatric disturbance Neuroferritinopathy FTL AD Facial dystonia Seizures, myoclonus, dementia Idiopathic basal ganglia calcification SCLC20A2, PDGFB, PDGFRB, XPR1 AD Basal ganglia calcification Choreoacanthocytosis VPS13A AR MacLeod syndrome XK XLR Neuropathy, cardiomyopathy, inc. CK Ataxia-telangiectasia ATM AR Ataxia, telangiectasia, immune deficiency Ataxia with oculomotor apraxia APTX, SETX, PNKP AR Gordon-Holmes syndrome RNF216 AR Hypogonadism, cerebellar atrophy NKX2-1 related chorea NKX2-1 AD/de novo Hypotonia, learning disabiliites, pulmonary and thyroid dysfunction ADCY5-related chorea ADCY\5 AD/de novo PDE10-A-related chorea PDE10-A AR/de novo Delayed milestones and language GPR88-related chorea GPR88 AR Delayed language/learning disabilities Early infantile epileptic encephalopathy 17 GNAO1 de novo Congenital Rett disease FOXG1 de novo Intellectual disability, microcephaly, abnormal MRI Severe motor delay and intellectual disability SYT1 de novo Intellectual disability, motor delay, no seizures Early infantile epileptic encephalopathy type 13 SCN8A AD/de novo Paroxysmal dystonia/chorea Oromandibular dystonia, neuropathy, inc. CK Neuropathy, hypoalbuminemia, hypercholesterolemia, cerebellar atrophy Diurnal variation, dystonia, myoclonus, axial hypotonia Developmental delay, some with seizures 352 Huntington Disease • Clinical features • psychiatric: depression, mood swings • cognitive: dementia • motor: chorea, bradykinesia • Early onset with rigidity inherited from father • Pathology: caudate atrophy • Genetics • 4p16.3; huntingtin • CAG triplet repeat (polyglutamine) • Age-dependent penetrance • Peak age of onset 3rd to 4th decades • repeat expansion By Frank Gaillard (Own workvia Wikimedia • general population: <26 repeats Commons • intermediate alleles: 27-35 • HD: >36 (arise from intermediate, more often in sperm) • Reduced penetrance 36-39 repeats Triplet Repeat Disorders AUG CGG TAA GAA CAG CTG Friedreich ataxia SCA Fragile X syndrome Huntington disease DRP atrophy Spinal & bulbar atrophy Machado-Joseph disease myotonic dystrophy increasing severity from one generation to next Severity and onset correlate with repeat size Larger size = greater instability Anticipation Paroxysmal Dyskinesias • Episodic abnormal movements • Paroxysmal kinesigenic dyskinesia – PRRT2 • Triggered by voluntary movements • Infantile convulsions, choreoathetosis • Treated with carbamazepine • Paroxysmal exercise-induced dyskinesia – SLC2A1 • Exercise-induced dystonia • Migraine, hemiplegia, ataxia, epilepsy • Paroxysmal non-kinesigenic dyskinesia – PNKD • Triggered by alcohol, stress, caffeine 353 Hereditary Ataxias • Spinocerebellar ataxias • • Multiple SCAs – most AD Some triplet repeat disorders • Friedreich ataxia • Clinical • • • • Genetics • • • • • • autosomal recessive GAA repeat normal 7-34 times Premutation 34-65 repeats (borderline clinical 44-66) Pathological 66-1,700 Other point mutations also may occur Pathogenesis • • • ataxia, impaired position and vibration sensation loss of DTR’s; pes cavus; extensor plantars hypertrophic cardiomyopathy; diabetes mellitus Conjunctival telangiectasia Repeat interferes with transcription Impaired mitochondrial Fe metabolism Ataxia telangiectasia • • • • • ataxia telangiectasia immune deficiency abnormalities chromosomes 7 and 14 (Ig chains and T cell receptor) ATM mutations Metabolic Ataxias Disorder Metabolic Abnormality Clinical Features Treatment Bassen–Kornzweig syndrome Abetalipoproteinemia Acanthocytosis, retinitis pigmentosa, fat malabsorption Vitamin E Ataxia with isolated vitamin E Deficiency of alpha-tocopherol transfer protein Progressive ataxic syndrome Vitamin E deficiency (AVED) Hartnup disease Tryptophan malabsorption Pellagra rash, intermittent ataxia Niacin Riboflavin, CoQ10, dichloroacetate Mitochondrial complex defects Complexes I, III, IV Encephalomyelopathy Multiple carboxylase deficiency Biotinidase deficiency Alopecia, recurrent infections, variable organic aciduria Biotin Pyruvate dehydrogenase deficiency Block in energy metabolism Lactic acidosis, ataxia Ketogenic diet, chloroacetate Refsum disease Phytanic acid, alpha hydroxylase Retinitis pigmentosa, cardiomyopathy, hypertrophic neuropathy, ichthyosis Dietary restriction of phytanic acid Urea cycle defects Urea cycle enzymes Hyperammonemia Protein restriction, arginine benzoate, alpha ketoacids Opal, P, Zoghbi, H. The Hereditary Ataxias. Emery & Rimoin, Principles and Practice of Medical Genetics, 6th Ed. Alzheimer Disease • • Progressive dementia Autosomal Dominant Genes • (ख़2% cases) • Early onset AD • PSEN1 (presenilin-1) • PSEN2 (presenilin-2) • Amyloid precursor gene (APP) • Late onset AD • APOE ε4 allele • 15x risk homozygote; 3x risk heterozygote • Effect varies with sex and ethnicity 354 Prion-Associated Dementia • PRNP mutations • May be familial (AD) or infectious • Disease associated with misfolded proteins • Spongiform encephalopathies • Creuzfeld-Jacob disease • Gerstmann-Straussler-Scheinker disease • Fatal familial insomnia By Lopez-Garcia, F., Zahn, R., Riek, R., Wuthrich, K. & RCSB [Public domain], via Wikimedia Commons Cerebrovascular Disorders • Small vessel disease • • • Large vessel disease • • • Marfan syndrome Arrhythmias Ischemic stroke • • • • Fabry disease Homocystinuria Cardioembolic stroke • • • EDS vascular type Pseudoxanthoma elasticum Mixed small and large vessel disease • • • Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL): NOTCH3 CARASIL: HTRA1 Sickle cell disease Moyamoya (NF1) Collagen IV mutations Hemorrhagic stroke • • • • Cerebral cavernous malformations Hereditary hemorrhagic telangiectasia Cerebral Amyloid antipathy Cerebral venous thrombosis (prothrombin) Hereditary Spastic Paraplegia • Clinical • Progressive weakness and spasticity in lower extremities • Includes bladder disturbance, LE sensory changes • May be “complicated” by other features, including seizures, dementia, movement disorder • Genetics • Multiple subclasses, dominant, recessive, and X-linked • AD: most common are SPAST, ATLI, KIF5A, REEP1 • AR: SPG11 (intellectual disability, thin corpus callosum, axonal neuropathy), many others • XLR: L1CAM, PLP1 355 Anterior Horn Cell Type • Weakness, absent reflexes, fasciculation • neuropathic EMG and muscle biopsy • normal level of alertness Locus Siddique, T, et al. Motor Neuron Disease, Emery and Rimoin Principles and Practice of Medical Genetics, 6th edition. Phenotype OMIM 21q22 SOD1 AD-ALS 105400 2q33 ALSIN Juvenile AR-ALS Juvenile PLS Infantile onset spastic paraplegia 205100 606353 607225 Incorrectly assigned to 18q21, a mutation in FUS (ALS6) has been identified in the family that was used for initial mapping 606640 ALS3 18q21 – ALS4 9q34 SETX AD- Juvenile ALS ALS5 15q21 SPATACSIN AR- Juvenile ALS SPG11 602099 ALS6 16p11 FUS AD- ALS AD-ALS-FTD 608030 ALS7 • Distal spinal muscular atrophies chaparonopathies Gene ALS1 ALS2 602433 20p13 – AD-ALS 608031 ALS8 20q13 VAPB AD distal SMA AD typical and atypical ALS 608627 ALS9 14q11 ANG AD- ALS 611895 ALS10 1p36 TDP43 AD-ALS AD ALS-FTD 612069 ALS11 6q21 FIG4 AD-ALS CMT4J 612577 611228 ALS12 10p15 OPTN ALS linked to chromosome 9 9p21 C9ORF72- AD ALS AD ALS-FTD 105550 ALS linked to chromosome 12 12q24 DAO AD-ALS AD-ALS – 613435 ALS-VCP 9p13 VCP AD-ALS IBMPFTD – 167320 ALSX Xp11.23 UBQLN2 X-juvenile ALS X-Adult ALS X-ALS/Dementia Spinal Muscular Atrophy cen tel NAIPD P44C SMN2 SMN1 NAIP5 P44T 5q11.2-q13.3 inverted, duplicated segment SMN: survival motor neuron base differences in exons 7 & 8 interacts with RNA-binding protein increased dosage of SMN2 correlated with milder phenotype 95-98% homozygous for SMN1 deletion or truncation 2-5% compound heterozygotes for deletion or truncation and SMN1 intragenic mutation Nusinersin – FDA approved treatment- antisense oligonucleotide that includes exon 7 in SMN2 SMN Therapy SMN1 SMN1 SMN2 6 7 8 6 C 7 U 8 6 7 8 7 8 Antisense oligonucletide Splicing 6 8 C Functional protein 6 U Splicing mRNA SMN2 Transcription Transcription 6 7 8 U Non-functional protein Functional protein 356 Peripheral Neuropathy • Absent deep tendon reflexes, weakness, muscle atrophy • Inflammatory demyelinating • Hereditary • motor and sensory (Charcot-Marie-Tooth) • sensory (including familial dysautonomia) • Friedreich ataxia Familial Dysautonomia • Clinical Features • • • • • • • Feeding difficulty Episodic vomiting Autonomic neuropathy Insensitivity to pain Absent tearing Absent fungiform papillae Increased sweating • Genetics • IKBKAP gene • Splicing mutation in Ashkenazi Jewish form Metabolic Neuropathy • Diabetes • • • • • • Uremia Porphyria Pernicious anemia Abetalipoproteinemia Refsum disease Tangier disease (alpha lipoprotein) 357 Hereditary Sensory & Motor Neuropathy Normal Duplication - CMT Deletion -HNPP + Clinical distal weakness, pes cavus, absent DTR’s Genetics AD, XLR, AR CMT1: PMP22 gene duplication, point mutations flanked by 24 kb repeat, with unequal crossing over HNPP: PMP22 gene deletion, truncating mutations other genes: P0, EGR2, connexin 32 (XL) Neuromuscular Junction: Myasthenia Gravis • Transient neonatal (mother with MG) • Immunological • Rare genetic forms By Posey & SpillerCumulus at nl.wikipedia [Public domain], from Wikimedia Commons Muscle Biopsy dystrophic metabolic neurogenic mitochondrial myopathic Pictures from Dubowitz, V. Color Atlas of Muscle Disorders in Childhood, Chicago: Yearbook Medical Publishers, 1989 358 Myotonic Dystrophy CTG repeat 5-35 copies DMPK gene 3’ untranslated region >50 copies DMPK gene • Clinical • Myotonia, weakness • Hair loss, diabetes mellitus, cataract, ECG changes • Genetics • CTG repeat expansion • Maternal transmission of severe neonatal syndrome (>2,000 repeats) • DM2: CCTG expansion in zinc finger protein ZNF9 Muscular Dystrophy progressive; high muscle enzymes; loss of muscle cells by biopsy Duchenne/Becker dystrophy Facio-scapulo-humeral dystrophy Congenital muscular dystrophy Duchenne/Becker Dystrophy • Clinical • High CPK • Prominent calves • Steroids • Genomic Treatments (exon skipping, nonsense suppression) • Genetics • XLR • Dystrophin mutations (2/3 deletions) • Duchenne – loss of dystrophin • Becker – abnormal dystrophin 359 Facioscapulohumeral Dystrophy Clinical Weakness of facial and upper shoulder girdle muscles Other features Retinal vasculopathy 40-60% Sensorineural hearing loss 60% Genetics Abnormal DUX4 expression Deletions in D4Z4 3.3 kb repeat 11-100 repeats normal 1-10 repeats abnormal Laminopathies • Lamin A/C – alternative splicing of LMNA gene • Emery-Dreifuss Muscular Dystrophy • scapula-humero-peroneal • Elbow contractures • Cardiac – arrhythmias, cardiomyopathy • Missense mutations, usually AD, but may be AR • Limb-Girdle Muscular Dystrophy 1B • Pelvic-scapular • Contractures • Cardiac – arrhythmias, cardiomyopathy • Frameshift mutations • LMNA-congenital muscular dystrophy • Diffuse or dropped head syndrome • Contractures • Cardiac – arrhythmias, cardiomyopathy • Respiratory failure • Missense mutations Congenital Myopathy Variable progression; enzymes may be normal Characteristic pathology central core nemaline centronuclear congenital fiber type disproportion 360 Metabolic Myopathy • Periodic paralysis (SCN4A) • hypokalemic • hyperkalemic • Thyroid disorders • Steroid • Glycogen storage • Mitochondrial 361 362 Reproductive Genetics I REPRODUCTIVE GENETICS I Louise E. Wilkins-Haug, MD, PhD, FACMG Division Director, Maternal Fetal Medicine and Reproductive Genetics Brigham & Women’s Hospital Professor of Obstetrics, Gynecology and Reproductive Biology Harvard Medical School Louise E. Wilkins-Haug, MD, PhD, FACMG Division of Maternal Fetal Medicine Brigham & Women’s Hospital 75 Francis Street Boston, MA 02115 (617) 732-4208 Telephone (617) 264-6310 Fax [email protected] 365 366 Reproductive Genetics 1 Louise Wilkins-Haug MFM Division Director Brigham and Women’s Hospital Disclosure(s) - None Objectives 1) Examine current guidelines for aneuploidy screening මCompare serum screening to NonInvasive Prenatal Testing (NIPT, cfDNA, ffDNA) මUnderstand “false positive” and “false negative” 2) Assess the role of fetal ultrasound මUse of “soft markers” මAnalysis for structural anomalies 367 Case Mr and Mrs Smith present to the Obstetrician at 8 weeks. This is their first pregnancy, achieved by in vitro fertilization due to “multifactor etiology” infertility ම She is 37 yo, healthy, no surgeries or contributing family history, Northern European ancestry ම He is 38 yo, healthy, no surgeries or contributing family history, Northern European ancestry What is our risk of Down syndrome? Screening for Down Syndrome Maternal Age and A Priori Risk American College of OBGYN Guidance All women - offer screening and diagnostic testing for aneuploidy ideally in the first trimester (2007) Options – serum screen vs cfDNA ම 2012 ACOG/SMFM ම cfDNA - screening for high risk women, singletons for common aneuploidies ම 2015 SMFM update – women’s autonomy respected, if cfDNA requested by low risk women, pretesting counseling needed, routine screening remains the preferred option ම 2016 ACOG/SMFM – cfDNA an aneuploidy screening method without delineation to maternal age (ACOG and SMFM update, PB 163, May 2016) 368 Down Syndrome Screening in the Second or First Trimester Screen positive Sensitivity PPV 5.0 % 81.0 % 1 / 40 (2.5%) Nuchal lucency 4.2% 76.8% 1 / 50 (2.0%) NL + PAPP- A, hCG 5.0% 87.0% 1 / 25 (4.0%) Quad – MSAFP, estriol, hCG + inhibin (Nicolaides, 2004) Down Syndrome Screening – Combined 1st and 2nd Trimester Sequential Screening – 3 types ම “Integrated” - Nondisclosure ම PaPP-A and NL 1st trimester + Quad second trimester ම Results released 2nd trimester ම Stepwise – disclosure ම NL + PaPP-A + hCG ම Disclosure of high risk results 1st trimester ම All remaining return for quad ම Contingent – disclosure ම NL + PaPP-A + hCG ම Disclosure of high risk results 1st trimester ම Only intermediate risk return in second trimester (Cuckle, 2008) Serum Screening for Down Syndrome (5 % screen positive rate) – 1990-2000 100 % trisomy 21 90 NT+serum 80 Quad NT 70 60 50 Triple 40 4 % PPV 30 20 10 2% PPV Maternal Age 2% PPV 0 First trimester Second trimester (Malone, 2005; Cuckle, 2008) 369 Serum Screening for Down Syndrome (5 % screen positive rate) – 2000-2010 120 % Trisomy 21 100 NT+serum CS CS SS IS SS Triple 60 40 20 Quad NT 80 2 % PPV 4 % PPVV 2 % PPV 2 % PPV Maternal Age 0 First trimester Second trimester (Malone, 2005; Cuckle, 2008) Noninvasive Prenatal Genetic Testing 2010 -2017 Cells pass between mother and fetus ම Extracted, quantified and studied Cell free nucleic acids in adult serum since 1947 ම Fragments of DNA / RNA without cell membranes Increased with cell turnover ම Inflammatory diseases (Lupus, glomerular nephritis, pancreatitis) ම Cancer ම Tissue injury (trauma, stroke, myocardial infarct) (Desai and Cregel, 1963) How does this apply to pregnancy? Presence of fetal DNA in maternal plasma and serum • Lo, Y M, Corbetta, N, Chamberlain, P F, Rai, V, Sargent, IL, Redman, C W, Wainscoat, J S1997 Lancet, 350:845-7 Fetus-derived Y sequences: ම80% (24/30) maternal plasma ම70% (21/30) maternal serum ම17% (5/30) fetal cells 370 Characteristics of cffDNA Comes from the placenta cffDNA is 5-10% total cell free DNA in maternal circulation Present at 5-7 weeks, cleared within hours Levels not altered Levels altered Maternal age Gestational age Race BMI Parity Aneuploidy Mode of conception Smoking Placental volume (Bianchi, 2006, Pergament, 2014) Distinguishing Fetal from Maternal Free Nucleic Acids RhRhRh- Rh+ Rh+ Rh+ 21 21 21 21 Rh+ Fetal gene is different from mother’s gene SRY – fetal sex Father’s genes different from mother’s 21 Maternal free nucleic acids 21 21 Fetal free nucleic acids Fetal gene is the same as mother’s gene Aneuploidy Detection of Aneuploidy Next Generation Sequencing (Massively parallel genomic sequencing) o 10s of millions DNA fragments sequenced at same time o First 36 bases are sequenced o “Binned” by chromosome (Chiu , 2008; Palomaki, 2011,) 371 Screening for Down Syndrome in Women > 35 2nd trim quad 1st trim combined Integrated DNA testing DR 80% 90% 95% 99% Screen Positive Rate 5% 15% 2% 0.2% Chance of true positive 2% 2-3% 4% 80-99 % Complexity 1 Blood draw US and 1 blood draw US and 2 blood draws 1 blood draw Failure Rate <<1% <1% <1% 0.3 – 3% Unanswered Questions with NIPT #1 Should it be used in low risk women? o Advanced screen? o Primary screen? #2 What do discordant results mean ? o“False positives” o“False negatives #3 What should be done with a “no call” result? #4 Should it expand beyond the common aneuploidies? As an Advanced Screen In Women < 35 Years Old ? 2,800,000 women (5,000 trisomy 21 fetuses) Combined 1st trimester screening (75% detection) 1250 trisomy 21 (25% missed) 140,000 positive (3,750 trisomy 21) 140,000 CVS /Amniocentesis Loss of 1,050 normal fetuses 3,750 trisomy 21 (75% detection) cfDNA (3675 tri 21, 740 nl 1374 failed) 5,789 Amniocentesis Loss of 10 normal fetuses 3675 trisomy 21 (73.5% detection) 372 As A Primary Screen in All Women? Fetal fractions, sensitivities and specificity are independent of maternal age Positive predictive value dependent on a priori risk මBallpark estimates Indication Positive predictive value > 35 years old < 35 years old US + serum screening 80% 50% 2-4% Positive Predictive Value for Aneuploidy 120% 100% 80% 60% PPV 40% 20% 0% 45 yo 35 yo 20 yo 12 weeks, trisomy 21 45 yo 35 yo 20 yo 12 weeks, trisomy 18 Serum Screen – “Hidden” Value vs Risk Value – abnormal karyotype not detected by cfDNA but found by 1st US / serum screen – 2% o Reflects abnormal placentation, increased nuchal lucency o 1.0% if exclude US anomalies, karyotype changes without abnormal phenotype Risk- invasive studies for all positive serum screen (5-7% of screened population) o Predominantly pregnancies with normal chromosomes (Norton, 2014; Peterson, 2014) 373 # 2 What About the Discordant Results? “False Positives” (0.1%) “cfDNA” positive මIncreased chromosome specific DNA segments but not its origin Possible origins මPlacenta ම“Vanishing “ twin මMaternal Confined Placental Mosaicism 1-2% of all pregnancies at 10–12 wks Normal fetal karyotype with normal and aneuploid cell lines in the placenta Adverse pregnancy outcomes ම IUGR ම Uniparental disomy Multiple case reports, overall prevalence? (Futch, 2012, Hall, 2013, Pan 2013) “Vanishing Twins” 1-2% of singletons originate as twins Discordancy with vanishing twins ම 2 of 3 of discordant trisomy 13 cases occurred in setting of an early twin demise Impact on NIPT results ම 0.43% of cases with two paternal haplotypes ම Second twin cfDNA detected >8 weeks after demise (Futch, 2013; McAdoo, 2014) 374 Maternal Cancer - “Widespread genomic imbalance” NIPT Results 37yo, NIPT + 13 and -18; amniocentesis, neonate, placental biopsies – all normal ම Postpartum pelvic pain - small cell carcinoma of the vagina ම Majority of cancer cells with trisomy 13 Recent report of 12 cases with various malignancies One study – 0.03% of tests > 2 aneuploidies ම affected fetuses - 4, normal fetuses – 14 ම 5/14 (36%) of women with a cancer diagnosis (Osborne, 2013; Bianchi, 2015, Snyder, 2016) Maternal Aneuploidies Sex chromosome aneuploidies : ම44 yo with an IVF conception ම NIPT - abnormal X chromosome ratios, newborn karyotype normal ම Maternal karyotype 45, X[3]/46, XX[27] ම25 yo, normal height, intellect and fertility ම NIPT positive for triple X, amniocentesis normal ම Maternal karyotype 47, XXX 8.6% of NIPT positive for sex aneuploidy have maternal sex chromosome mosaicism (Nicolaides, 2013; Lau, 2013; Wang, 20 Discordant Results - False Negatives o Liveborn trisomy 13 and 18 fetuses have mosaicism (euploid and aneuploidy) in their placentas o Case reports as source of a false negative for trisomy 18 (Kalousek, 1989; Pergament, 2014) 375 #3 What Should be Done with “no call” Results? (2-8% of reports) Low fetal fraction = higher false negatives 1) Early gestational age ම 60% get a result on redraw 2) Higher BMI and lower fetal fraction ම 20% in women > 250 lbs ම 50% in women > 350 lbs 3) Aneuploidy (13,18,21, triploidy) ම Increased aneuploidy rate with low fetal fraction / no results ම As high as 20% ( 1 in 5 are chromosomally abnormal) (Ashoor, 2013; Pergament, 2014, Williams, 20 #4 Expanded Beyond the Common Aneuploidies? Micro deletions and duplications ම 1/100 neonates but widespread across genome ම 5 most common are 1/1000, most are 22q deletion ම Emerging technology, not currently supported by professional societies Whole fetal exome ම Technically possible at 7 Mb level ම Extends analyses to other aneuploidies ම Emerging technology (ACOG and SMFM statement, May 2016) 35 yo Positive NIPT for 21 Prenatal care 1st trimester මChoose NIPT – positive for 21 මPPV – 83% Continuation rate influenced by path to result ම NIPT ම Amniocentesis ම CVS 42% 33% 8% 376 Antepartum Management Down Syndrome Antepartum risks ම IUFD rate (4/70) ම Average gestational age = 37.0 wks ම Growth restriction in 20.0% ම Not associated with anomalies, or maternal age Surveillance ම 35.0% delivered for new onset nonreassuring fetal testing ම Not associated with anomalies, growth restriction or maternal age Newborn Genetic Follow-up ම Karyotype (not microarray) (Guseh, 2017) 20 yo with NIPT Positive for Trisomy 13 o Ultrasounds normal o Declined invasive testing o Had “arranged for palliative care team” o Positive predictive value=16 % o Could her placenta have CPM for trisomy 13? 40 yo, NIPT “negative” for 21,18,13, X,Y Aneuploidy o NIPT at 11 weeks – negative, NL 5.0 mm o US - early onset IUGR, polyhydramnios o Newborn karyotype - 47,XY, +18 o NIPT is screening with false negatives o True false negative or possible CPM ? 377 35 yo, NIPT “negative” for 21,18,13, X,Y Aneuploidy Ultrasound at 20 wks delayed growth, possible VSD, and club foot Chose NIPT for aneuploidies to exclude severe conditions (+13, + 18) මResult = negative Microarray reveals “Cri du Chat” (46,XX,5p- syndrome) What is the Role of cfDNA? 1) Appropriate first line for advanced maternal age for trisomy 21 screening. මSingletons, not twins මNot validated for microdeletions මDoes not replace diagnostic study for ultrasound anomalies (ACOG and SMFM joint statement, May 2016) What is the Role of cfDNA in Women < 35 years Old? Pros ම High detection, very low false-positives ම Single blood test any time past 10 weeks ම Provides a noninvasive risk assessment Cons ම Calculation of patient’s positive predictive value essential ම Traditional serum screen identifies additional karyotype anomalies ම Cost efficacy remains to be established (Norton, 2016) 378 Resources – ACOG / SMFM / NSGC 1) NIPT/Cell Free DNA Screening Performance Calculator (ACOG endorsed) ම www.perinatalquality.org 2) “Free Webinar: Prenatal Cell-Free DNA Screening” ම http://cfweb.acog.org/obpractice/webinars 3) “Prenatal Cell-Free DNA Screening: Q&A for Healthcare Providers” ම http://nsgc.org/page/non-invasive-prenatal-testing-healthcare-providers (ACOG endorsed) 4) “Abnormal Prenatal Cell-Free DNA Screening Results: What Do They Mean?” ම http://nsgc.org/page/abnormal-non-invasive-prenatal -testing-results (ACOG endorsed) 5) Resources for Women ම "Cell-free DNA Prenatal Screening Test“ (http://www.acog.org/Patients/FAQs/Cell-freeDNA-Prenatal-Screening-Test-Infographic) ම “Prenatal Cell-Free DNA Screening” -FAQ for patients (http://nasgc.org) (ACOG endorsed) Clinical Case – Initiates First Trimester Screening She has her 11 week US A nuchal lucency of 4.0 mm is reported Clinical Case She returns with her husband one week later. Should she have a CVS? මWhat does it mean if it is now smaller? මShould she get NIPT? මRisk of other conditions ? Nuchal lucency = 2.0 mm 379 Should You Cancel the CVS? Trisomy 21 with increased nuchal lucency මMajority (5/6) resolved by 2nd trimester ම1st trimester NT of 10, 7, 5, 5, 4, 8 mm මNot associated with cardiac anomalies මNot predictive of spontaneous loss “If an abnormal fetus is likely to miscarry, shouldn’t I wait until amniocentesis?” මTrisomy 21 - About 10% chance (CVS to Amniocentesis) (Comstock, 2006; Pandya, 1999; Sawa, 2006) Outcomes for Aneuploidies at Midtrimester 1) Trisomy 21 මLoss rate from 24 weeks about 20% 2) Trisomy 18 මLoss rate about 80% to term 3) 45, X මLoss rate lower with mosaicism මCardiac anomalies and nonmosaic 45,X have highest chance of in utero loss If She Gets cfDNA ? Nuchal Lucency and cfDNA Microarray anomalies not addressed මCMA abnormalities associated with increased NL Role of other genetic syndromes මNoonan syndrome මCystic hygroma 16% මIncreased NL 2% (Bakker, 2011, Lee, 2009) 380 Clinical Case Couple get cfDNA – negative for major aneuploidies Decide to wait for 2nd trimester ultrasound and fetal DNA studies (amniocentesis) What other concerns exist? What else do they need to be prepared for? ම Structural abnormality ම Undetected birth defect / genetic condition in child Structural Anomalies and Inherited Disease Cardiac anomalies – 15% risk if NL > 5.5 mm Increased venous pressures, mediastinal compression ම congenital diaphragmatic hernia ම narrow thorax skeletal dysphasia Altered extracellular matrix ම collagen disorders (chondrodysplasias) Impaired fetal movement ම fetal akenesia sydromes Noonan, Smith Lemli Opitz Syndromes Long Term Outcomes with NT > 4.0 Increased nuchal lucency 50% abnormal karyotype 50% Normal karyotype 25% abnormal second trimester US Cardiac and other anomalies Fetal demise 75% Normal second trimester US 94% Normal at birth 94% Developmentally normal 12-36 mths 6% abnormal at birth Cardiac (TGV) Multiple malformaitons 6 % developmentally abnormal NTs 6.3; 6.3 and 12 (Senat, 2002) 381 Ultrasound in the Second Trimester Ultrasound for structural anomalies Ultrasound for aneuploidy screening ම“soft markers” Ultrasound in the Second Trimester : Structural Survey and Aneuploidy Risk Multiple anomalies ම18.8 % Isolated anomalies ම9.3 % Issue of an “isolated” ultrasound anomaly – minor features and dysmorphia difficult to appreciate (Staebler, M., 2005) Classic Presentations – Second Trimester Cardiac Other Growth / Amniotic fluid Trisomy 21 AV canal Absent nasal bone, clinodactyly, sandal gap, double bubble, omphalocele Mild third trimester growth restriction, normal amniotic fluid Trisomy 18 Variety Clenched hands, CNS including NTD, CDH, omphalocele Significant growth restriction (2nd trimester), polyhydramnios Trisomy 13 Variety Midline defects - Midline facial clefting, holoprosencephaly /cyclopia, omphalocele, polydactyly Third trimester growth restriction, normal amniotic fluid 382 Classic Presentations – Second Trimester Cardiac Other Growth / Amniotic fluid 45, X HLHS, coarctation Cystic hygroma, hydrops, horseshoe kidneys Normal growth and amniotic fluid Triploidy Variety CNS, omphalocele, hypertelorism, micrognathia 2nd trimester IUGR (skeleton more than head), 2nd trimester placental thickening or calcification TABLE 1 – isolated ultrasound findings and risks of chromosome abnormality (appendix) 2nd Trimester Ultrasound “Soft Markers”2nd (Reddy, 2014) 2nd Trimester Ultrasound “Soft Markers” Echogenic bowel LR 5.5 – 6.7 fold increase; also CMV, cystic fibrosis, IUGR Absent nasal bone, 80 fold increase Nuchal thickening 11 – 18 fold increase 383 Incorporating NIPT and “soft markers” Occur individually in 1-5% of normal fetuses Emphasis is on isolated findings, multiple “soft markers” should be considered differently (SMFM, 2017) Case – Returns for 18 week ultrasound මWhat is it? මCould it have been seen sooner? මRisk of undiagnosed aneuploidy ? මRisk of genetic etiologies? මHow to proceed? Clinical Case – Abdominal Wall Defect Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities 384 Abdominal Wall Defects Omphalocele Gastroschisis Location of umbilical vessels Into peritoneal covering Lateral to mass Membrane covered Yes No Defect Altered timing of normal physiologic herniation / enlarged internal organs Vascular accident Associations Other malformations, genetic syndromes and aneuploidy Young age, smoking, cocaine Concerns? What is it? - Omphalocele Could it have been seen sooner? මPossibly, can be confused though with physiologic herniation of small bowel into umbilical stalk at 8-10 wks Concerns? Risk of undiagnosed aneuploidy ? මRisk of false negative for the major aneuploidies මLow (< 2.0%) Risk of other chromosome abnormalities ? මAbout 10 - 15% (aneuploidy and micro del/dups) Risk of genetic etiologies ? 385 To Be Continued . . . Thank You for Your Attention Table 1 - Aneuploidy Risks among Isolated Major Anomalies High risks Cystic hygroma Hydrops Holoprosencepahly Complete AV Canal Omphalocele Duodenal atresia Bladder outlet obstruction Risk > 50% > 50% 50% 40% 30% 30% 20% Most Common 45,X 13,21,18, 45,X 13, 18, 18p21 13, 18 21 13, 18 Lower risks Hydrocephaly/ Ventriculomegaly 10% Cardiac defects 10% Meningomyeloceles 7% Anencephaly 2% Encephalocele 10% Limb reduction 8% Clubfoot 6% Facial clefts 1% Minimal risk Gastroschesis-provided differentiated from ruptured omphalocele Hydranencepahly Single umbilical artery Above numbers are estimates which are influenced by gestational age at detection. 21, 13, 18, triploidy 21, 18, 13, 22-, 8, 9 18 18 47,XXY,47,XXX,18,21 13, 18, 22q- References American College of Obstetricians and Gynecologists. Screening for fetal aneuploidy. ACOG Practice bulletin no. 163. (2016) Obstet Gynecol, 127:e123-37. American College of Obstetricians and Gynecologists. Cell-free DNA screening for fetal aneuploidy. ACOG Committee opinion no. 640.(2015) Obstet Gynecol,126:e31-7. American College of Obstetricians and Gynecologists. Ultrasound in pregnancy. ACOG Practice bulletin no. 175. (2016) Obstet Gynecol,128:e241-56. Bianchi, D. W., T. Wataganara, et al. (2006). "Fetal nucleic acids in maternal body fluids: an update." Ann N Y Acad Sci 1075: 63-73 Cuckle, H. S., F. D. Malone, et al. (2008). "Contingent screening for Down syndrome--results from the FaSTER trial." Prenat Diagn 28(2): 89-94. Fan, H. C., Y. J. Blumenfeld, et al. (2008). "Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood." Proc Natl Acad Sci U S A 105(42): 16266-71. Gregg A, Van den Veyver I, Gross S. J., et al. (2014)”Noninvasive prenatal screening by nextgeneration sequencing” Annu Rev Genomics Hum Genet 15:327-47. Gregg AR, Skotko BG, Benkendorf JL, et al. (2016) Noninvasive prenatal screening for fetal aneuploidy. 2016 update: a position statement of the American College of Medical Genetic and Genomics. Genet Med,10:1056-65. Lefkowitz RB, Tynan JA, Liu T, et al. (2016) Clinical validation of a noninvasive prenatal test for genomewide detection of fetal copy number variants.AmJ Obstet Gynecol, 215:227.e116. Malone, F. D., J. A. Canick, et al. (2005). "First-trimester or second-trimester screening, or both, for Down's syndrome." N Engl J Med 353(19): 2001-11 Nicolaides, K. H. (2004). "First-trimester screening for Down's syndrome." N Engl J Med 350(6): 619-21 Norton M, Jelliffe-Pawlowski L, Currier R (2014). “Chromosome abnormalities detected by current prenatal screening and noninvasive prenatal testing” Obstet Gynecol 124(5):97986. Norton, ME, Biggio, JR, Kuller, JA, Blackwell SC, (2017)The role of ultrasound in women who undergo cell-free DNA screening Society for Maternal-Fetal Medicine (SMFM) Consult Series #42 Pergament E, Cuckle H, Zimmermann B, Banjevic M, Sigurjonsson S, et al. (2014) “Singlenucleotide polymorphism-based noninvasive prenatal screening in a high-risk and lowrisk cohort” Obstet Gynecol 124 (2):210-8. Reiff ES, Little SE, Dobson L, Wilkins-Haug L, Bromley B. (2016).What is the role of the 11- to 14-week ultrasound in women with negative cell-free DNA screening for aneuploidy? Prenat Diagn, 36:260-5. Society for Maternal-Fetal Medicine (SMFM) Publications Committee. Consult Series 36 (2015): prenatal aneuploidy screening using cell-free DNA. Am J Obstet Gynecol 212:711-6. Swaminathan, R. and A. N. Butt (2006). "Circulating nucleic acids in plasma and serum: recent developments." Ann N Y Acad Sci 1075: 1-9. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. 1997. Presence of fetal DNA in maternal plasma and serum. Lancet. 350(9076):485-7. 386 Reproductive Genetics: Prenatal Diagnosis Table 1 and References Table 1 - Aneuploidy Risks among Isolated Major Anomalies High risks Cystic hygroma Hydrops Holoprosencepahly Complete AV Canal Omphalocele Duodenal atresia Bladder outlet obstruction Risk > 50% > 50% 50% 40% 30% 30% 20% Most Common 45,X 13,21,18, 45,X 13, 18, 18p21 13, 18 21 13, 18 Lower risks Hydrocephaly/ Ventriculomegaly Cardiac defects Meningomyeloceles Anencephaly Encephalocele Limb reduction Clubfoot Facial clefts 10% 10% 7% 2% 10% 8% 6% 1% 21, 13, 18, triploidy 21, 18, 13, 22-, 8, 9 18 18 47,XXY,47,XXX,18,21 13, 18, 22q- Minimal risk Gastroschesis-provided differentiated from ruptured omphalocele Hydranencepahly Single umbilical artery Above numbers are estimates which are influenced by gestational age at detection. Nyberg D, Mahony B, Pretorius D. Diagnostic Ultrasound of Fetal Anomalies: Text and Atlas. Mosby Year Book, 1990 Sanders R, Hogge W, Spevak P, Wulfsberg E. Structural Fetal Abnormalities-the Total Picture. Mosby, 2002. Shipp, T. D, Benacerraf, B. R., The significance of prenatally identified isolated clubfoot: is amniocentesis indicated? Am J Obstet Gynecol 178 : 600-2. 387 References American College of Obstetricians and Gynecologists. Screening for fetal aneuploidy. ACOG Practice bulletin no. 163. (2016) Obstet Gynecol, 127:e123-37. American College of Obstetricians and Gynecologists. Cell-free DNA screening for fetal aneuploidy. ACOG Committee opinion no. 640.(2015) Obstet Gynecol,126:e31-7. American College of Obstetricians and Gynecologists. Ultrasound in pregnancy. ACOG Practice bulletin no. 175. (2016) Obstet Gynecol,128:e241-56. Bianchi, D. W., T. Wataganara, et al. (2006). "Fetal nucleic acids in maternal body fluids: an update." Ann N Y Acad Sci 1075: 63-73 Cuckle, H. S., F. D. Malone, et al. (2008). "Contingent screening for Down syndrome--results from the FaSTER trial." Prenat Diagn 28(2): 89-94. Fan, H. C., Y. J. Blumenfeld, et al. (2008). "Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood." Proc Natl Acad Sci U S A 105(42): 16266-71. Gregg A, Van den Veyver I, Gross S. J., et al. (2014)”Noninvasive prenatal screening by nextgeneration sequencing” Annu Rev Genomics Hum Genet 15:327-47. Gregg AR, Skotko BG, Benkendorf JL, et al. (2016) Noninvasive prenatal screening for fetal aneuploidy. 2016 update: a position statement of the American College of Medical Genetic and Genomics. Genet Med,10:1056-65. Lefkowitz RB, Tynan JA, Liu T, et al. (2016) Clinical validation of a noninvasive prenatal test for genomewide detection of fetal copy number variants.AmJ Obstet Gynecol, 215:227.e116. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. 1997. Presence of fetal DNA in maternal plasma and serum. Lancet. 350(9076):485-7. Malone, F. D., J. A. Canick, et al. (2005). "First-trimester or second-trimester screening, or both, for Down's syndrome." N Engl J Med 353(19): 2001-11 Nicolaides, K. H. (2004). "First-trimester screening for Down's syndrome." N Engl J Med 350(6): 619-21 Norton M, Jelliffe-Pawlowski L, Currier R (2014). “Chromosome abnormalities detected by current prenatal screening and noninvasive prenatal testing” Obstet Gynecol 124(5):97986. Norton, ME, Biggio, JR, Kuller, JA, Blackwell SC, (2017)The role of ultrasound in women who undergo cell-free DNA screening Society for Maternal-Fetal Medicine (SMFM) Consult Series #42 388 Pergament E, Cuckle H, Zimmermann B, Banjevic M, Sigurjonsson S, et al. (2014) “Singlenucleotide polymorphism-based noninvasive prenatal screening in a high-risk and lowrisk cohort” Obstet Gynecol 124 (2):210-8. Reiff ES, Little SE, Dobson L, Wilkins-Haug L, Bromley B. (2016).What is the role of the 11- to 14-week ultrasound in women with negative cell-free DNA screening for aneuploidy? Prenat Diagn, 36:260-5. Society for Maternal-Fetal Medicine (SMFM) Publications Committee. Consult Series 36 (2015): prenatal aneuploidy screening using cell-free DNA. Am J Obstet Gynecol 212:711-6. Swaminathan, R. and A. N. Butt (2006). "Circulating nucleic acids in plasma and serum: recent developments." Ann N Y Acad Sci 1075: 1-9. 389 390 Cancer Genetics II CANCER GENETICS II Sharon E. Plon, MD, PhD, FACMG Professor, Department of Pediatrics/Hematology-Oncology Molecular and Human Genetics Human Genome Sequencing Center Director, Medical Scientist Training Program Baylor College of Medicine Sharon E. Plon, MD, PhD, FACMG Department of Pediatrics Feigin Center Room 1200.18, 1102 Bates Street Houston, TX 77030 (832) 824-4251 Telephone (832) 825-4276 Fax [email protected] 393 394 Cancer Genetics II Sharon E. Plon, MD, PhD Professor Baylor College of Medicine Disclosure(s) • I have the following financial relationships to disclose: • I am a employee of Baylor College of Medicine (BCM) which derives revenue from genetic testing, including whole exome sequencing. • BCM and Miraca Holdings Inc. have entered into a joint venture, Baylor Genetics with shared ownership and governance of the clinical genetics diagnostic laboratories • I am a member of the BMGL Scientific Advisory Board • I will discuss off label use and/or investigational use in my presentation. General features of hereditary cancer genetic testing 395 Examples of hereditary cancer syndromes w/ Management specific care guidelines modalities Hereditary breast/ovarian cancer (BRCA1/2) Lynch syndrome (MSH2, MLH1, MSH6, PMS2) Familial adenomatous polyposis (APC) Von Hippel Lindau syndrome (VHL) Tuberous sclerosis complex (TSC) Multiple endocrine neoplasia 2 (RET) Li Fraumeni syndrome (TP53) Imaging Other diagnostic modalities Prophylactic surgery Targeted medications Genetic testing approaches when specifically performing hereditary cancer evaluation Single Genes Very Specific Phenotype Sanger 2-4 Genes – Sanger/NGS Multiple gene panels – Highly variable in size (7 – 150 genes) Performed by a variety of different NextGen sequencing methods w/ copy number analysis Whole exome or whole genome analysis (maybe part of tumor study) – NextGen methods +/- copy number analysis RB1 or VHL TSC1 & TSC2 or APC & MUTYH Hereditary breast cancer or hereditary colon cancer panel Whole exome analysis Outcomes of hereditary panel testing • Simplifies the genetic testing process and it is more efficient for patients. • Can provide “surprising results” with regard to pathogenic variants despite atypical family history or the “right gene: wrong tumor”: • Panel may include genes for which there is limited information with regard to pathogenicity • ClinGen consortium is now systematically reviewing this evidence in what is referred to as “clinical validity framework” (www.clinicalgenome.org) • Using panels increases the likelihood of variants of uncertain significance • In one study with breast panel (30 genes), per patient, the average number of VUS across all genes was 2.1 (Kurian, JCO, 2014) 396 In addition there are many situations were one can infer germline findings from TUMOR ONLY panel or WES/WGS sequencing • Certain specific founder mutations are almost always found in constitutional DNA: • Ashkenazi founder mutations in BRCA1/BRCA2 • BRCA1 loss of function mutations in breast cancer • EPCAM deletion in colon cancer sample • Specific tumor subtypes with high germline yield: • TP53 mutation in hypodiploid ALL (90% TP53; 48% germline) • Medullary thyroid cancer (RET) • Mutation profile associated with germline findings: • Hypermutated colon cancer (POLE) • Chromothripsis in medulloblastoma (TP53 mutation) Zhang J et al. N Engl J Med 2015. DOI: 10.1056/NEJMoa1508054 Distribution of Germline Mutations in Different Gene Categories and Cancer Subtypes in n=1120 childhood cancer patients • 8.5% had pathogenic variants in dominant genes (entire cohort – not unselected tumors) • 5.6% if exclude hypodiploid ALL and ACT • Only one biallelic recessive diagnosis • 8.5% of single recessive P/LP variants Zhang J et al. N Engl J Med 2015. DOI: 10.1056/NEJMoa1508054 397 Data from the MSKCC (adult) analysis of germline findings in IMPACT panel (341 genes) • 246 of 1566 subjects (15.7% 95% CI 14.0% - 17.6%) with germline findings o 198 of 246 individuals, (80.5%; 95% CI, 75.1%-85.0%) of findings in cancer susceptibility genes • Often found P/LP variants in genes not currently associated with the tumor diagnosis of the patient. Summary of germline findings in unselected cancer patients • Multiple studies find ~10-15% of diverse pediatric and adult cancer populations carry P/LP variants in wide range of dominant cancer susceptibility genes. • Mixture of genes with with and without prior association with the patient’s tumor • Another ~6% carry single recessive alleles (unclear whether this is increased over control populations). • Metastatic Prostate cancer patients – 12% carry P/LP variant in DNA repair genes potentially associated with sensitivity to PARP inhibitors. • Initiating genetic testing in prostate cancer patients • Many studies demonstrate that only about 50% of patients with germine findings would meet clinical criteria for genetic testing (e.g. NCCN, Bethesda). • Most existing criteria are designed to be highly specific but not necessarily sensitive for identifying cancer patients with germline cancer susceptibility mutations. Hereditary Colon Cancer and Polyposis Syndromes 398 Risk factors for Colon Cancer Situation Lifetime Risk of CRC General Population 6% Personal history of CRC Inflammatory Bowel Disease Hereditary Nonpolyposis Colon Cancer (HNPCC) Familial Adenomatous Polyposis 15-20% 15-40% 60-80% >95% Familial Adenomatous Polyposis (FAP) • Autosomal Dominant disorder due to loss of function mutations in APC with very high penetrance for polyps and colorecal cancer • Acts as a classic tumor suppressor gene with two hits • >90% are truncating variants; 5-10% result from deletions • Some genotype:phenotype correlations. • Extracolonic symptoms assoc/w variants in long exon 15 in middle of gene. • Attenuated FAP (fewer polyps, later CRC onset) variants cluster at far 5’ & 3㵭ends. • 15-30% of patients with FAP result from de novo mutations. • Adenomatous colonic polyps begin in childhood to adolescence. • Nearly 100% lifetime colorectal cancer risk with 50% risk by age 33. • Extracolonic features often referred to as Gardner Syndrome. Polyposis Associated with FAP Twin A Twin B Typical Adenomatous Polyp from colon of a teenager with FAP Photographs courtesy of M. Finegold, MD – Texas Children㵭s Hospital Colons of twin teenage boys who presented with history of rectal bleeding and abdominal pain and underwent prophylactic colectomy 399 Extracolonic Manifestations of FAP • Desmoid tumors – often abdominal. • Painful and can be difficult to treat. • More likely to occur at sites of surgical resection • Osteomas of the jaw, skull, or other bones • Epidermoid cysts on face or trunk • Congenital Hypertrophy of the Retinal Pigment Epithelium (CHRPE) • present at birth, asymptomatic but useful clinically. • Pediatric hepatoblastoma (~0.5-1% risk) • Some studies suggest that ~10% of hepatoblastoma patients have FAP • Thyroid cancer (1% risk) • Medulloblastoma (<1% risk) Inverse relationship between somatic & germline mutations • Hepatoblastoma and desmoid tumors are associated with two apparently exclusive events: • Somatic activating mutations (exon 3 mutations) of beta catenin (CTNNB1 gene). OR • Germline inactivating mutations in APC. • Only one is needed to upregulate beta catenin signaling so I use somatic information to decide upon need for germline testing for hepatoblastoma and desmoid tumors. • There are other examples of these types of inverse relationships between activating oncogenes in tumor or germline LOF in tumor suppressor genes. Management of W+ Patients • Colonoscopy surveillance beginning age 10–15; continuing every 1-2 yrs. • Colectomy late teens to early twenties (depending on polyp load or dysplasia). • Restorative proctocolectomy with ileal pouch-anal anastomosis • Total colectomy with ileorectal anastomosis with annual surveillance of rectal stump. • Consider Sulindac or NSAID’s for residual stump to decrease size of polyps. • Recent study exploring erlotinib for decreasing polyp burden • Upper GI follow-up by endoscopy for risk of gastric adenomas and duodenal carcinomas beginning late teens or early twenties. • Annual thyroid exam • Hepatoblastoma screening in children controversial. 400 Dhdz,Associated Polyposis • Autosomal recessive polyposis with >100 polyps. • Two MUTYH pathogenic variants, p.Y165C & p.G382D. • The mean ages of CRC diagnosis: • 58 years (homozygous G382D) • 52 years (compound heterozygous) • 46 years (homozygous Y165C) • Conflicting data over whether there is any increased CRC risk in heterozygous patients (~1% allele frequency). • MUTYH encodes base excision repair protein. • Absence of BER leads to increased mutation rate with APC somatic mutations in the polyps. 70 55 CRC dx 66? 48 52 Siblings with >100 polyps and CRC = MUTYH mutation Juvenile Polyposis Coli • Present with bleeding, rectal prolapse, pain. • Childhood onset of juvenile/ hamartomas colonic polyps (>5 polyps). • Mutations in BMPR1A and SMAD4 found in ~50% of patients. • Severe form with deletion including PTEN and BMPR1A • ~1-2% result from PTEN mutations. • CRC rate as high as 50% with average age diagnosis 43 yrs. • SMAD4 > BMPR1A mutation • Remaining JPC loci still not discovered. Photographs courtesy of M. Finegold, MD – Texas Children’s Hospital JPC Management • Monitor for rectal bleeding, anemia, abdominal pain, constipation, diarrhea or change in stool size, shape, and/or color; • CBC, colonoscopy, and upper endoscopy beginning by 15 years of age. • Goal is removal of polyps and then periodic surveillance. • Prophylactic colectomy not recommended but maybe required if polyp burden is very high w/dysplastic changes or excess bleeding. • Some patients with SMAD4 P/LP variants with hereditary hemorrhagic telangiectasia and JPC clinical phenotypes : • Can see severe HHT complications including chronic epistaxis, pulmonary AVMs. • Screening for HHT complications recommended in all JPC patients with SMAD4 mutations (Zbuk and Eng, Nature Practice, 2007) • One study found 6/16 (38%) SMAD4 mutation carriers had evidence of aortopathy (Heald et al., AJMG, 2015) – may result in updated guidelines. 401 Peutz-Jeghers Syndrome • Pigmented spots on lips, buccal mucosa and GI tract • Can fade w/age • Hamartomas of the small and large bowel. • Intussusception – serious complication which can present at later ages than in general population (need to warn families). • 50% deletions/45% truncating mutations in STK11: • Highlights the need for sequence AND deletion analysis • Lifetime cancer risk of 81% (Zbuk & Eng, Nature Practice Oncol, 2007) Photograph courtesy of S. Plon – Texas Children’s Hospital • GI cancers (66%- small intestine, CRC, esophageal & pancreatic) • Breast cancer risk 32% in women. • Surveillance should include breast MRI • STK11 on most BRCA panel tests although rarely positive (small contributor) • Benign ovarian sex-cord tumors with annular tubules and Sertolicell testicular tumors – indication for STK11 testing. 'ZDϭ Polyposis • Described in 2012 as another form of “mixed” polyposis including both adenomatous, juvenile & hyperplastic polyps. • Major Ashkenazi disease allele associated with disease is 40kb duplication upstream of GREM1. • Other rearrangements/duplications have been reported in a few families. • Maybe missed on panels that don’t include copy number. • Recent review of four extended Ashkenazi families demonstrate (Lieberman et al., Gastroenterology. 2017) • GREM1 mutation carriers can phenotypically resemble either FAP or Lynch syndrome. Lynch Syndrome or Hereditary Non-Polyposis Colon Cancer • Autosomal dominant CRC w/o polyposis associated with endometrial ca, bile duct, ovarian, ureteral and gliomas. • ~70% CRC lifetime risk and 50-70% endometrial Ca in classic Lynch. • Right-sided CRC cancer is more frequent. • Better prognosis of CRC stage for stage. • Common to see individuals with 2 or 3 different primary LS-tumors • Due to high mutation rate these patients respond more effectively to immune checkpoint inhibitors • Definitely increasing the interest in identifying LS patients 402 Family History Criteria for HNPCC • Amsterdam Criteria (CRC based) – first exclude FAP • At least 1 CRC < age 50 • 2 affected generations • 3 affected relatives, 2 are 1o relatives of other one • Revised Bethesda Criteria – based on proband characteristics • CRC <50 yrs • Synchronous, metachronous CRC, or other LS tumors. • CRC with the MSI-H† histology‡ in a patient <60.§ • CRC plus 1st relative with LS tumor (<age 50). • CRC in 2 or more relatives with LS tumors. • These criteria have low sensitivity (but high specificity). • Many groups now argue for screening all colorectal cancer patients for tumor features of LS HNPCC- Results from Heterozygous P/LP variants in one of 4 Mismatch Repair Genes • MSH2 and MLH1 mutations make up 80-90% of Amsterdam criteria families. • Most are typical LOF mutations in MMR gene (truncating and deletions); MSH2 inversion • MSH6 (MIS-low tumors) CRC: 44% for men & 22% for women (Baglietto, JNCI 2010). • PMS2 testing difficult due to multiple pseudogenes w/gene conversion events. • Very poorly covered in WES or tests that don’t have focused approach to PMS2. • Lower CRC penetrance BUT ~8% of CRC diagnoses <30 yrs (McKinsey et al., GIM, 2015). • EPCAM deletion (fusion transcript results in epigenetic silencing of adjacent MSH2 gene) /EPCAM Microsatellite Instability (MIS+) in MMR mutant tumors • NCI definition using five markers – MSI - Boland et al., Cancer Research, 1998 o Stable – 0 o Low – 1 unstable o High - >2 unstable • Previously standard approach to evaluating tumors for evidence of Lynch syndrome. • However, most pathology labs more routinely do immunohistochemistry. • MIS studies do not tell you anything about which gene may be responsible for the MIS phenotype. 403 Work-up of CRC patients/specimens More studies arguing to to evaluate all CRC or at least those that meet age cut-off (<70) see Schneider et al., GIM, 2015 and Leenen et al, Genet Med. 2016 CRC +/- age MMR Protein Expression by immunohistochemistry (IHC) MSI Yes MLH1 absent MSH2, MSH6 or PMS2 absent BRAF p.V600E or MLH1 methylation No Consider mutation testing of blood for MMR mutations Yes Sporadic HNPCC Guidelines for mutation positive individuals – NCCN 2016 • Colon cancer: Colonoscopy beginning at age 20–25 y or 2–5 y prior to the earliest CRC (if <25y age of diagnosis) and repeat every 1–2 y. • Unclear how useful aspirin treatment is in cancer prevention • Endometrial and ovarian cancer: • Prophylactic hysterectomy and bilateral salpingo-oophorectomy (BSO) should be considered by women who have completed childbearing. • Dysfunctional uterine bleeding warrants evaluation. • No clear evidence to support further endometrial cancer screening. However, annual endometrial sampling is an option. • Transvaginal ultrasound for ovarian & endometrial cancer not been shown to be sufficiently sensitive or specific to support a positive recommendation. Similar caveats for serum CA-125. • Other LS cancers - no active surveillance other than clinic suspicion is recommended. • LS surveillance has been shown to increase survival. • However, only 50% of close relatives of Lynch syndrome patients undergo genetic testing – we need to do better! WK>ͬWK>ϭAssociated Hereditary CRC • Specific missense variants in POLE/POLD1 exonuclease domain were first identified as somatic mutations in colorectal cancer samples found to be hypermutated (many more somatic mutations c/w MMR deficient tumors). • Mutations increase the polymerase error-prone repair activity of these proteins (not standard tumor suppressor gene with 2 hits). • In a subset of patients variants are germline with hereditary cancer. • • • • • Attenuated or oligo-adenomatous colorectal polyposis (average 16 adenomas) Risk of CRC (~60%) Gastric and duodenal adenomas (57%) Also maybe more responsive to immune checkpoint inhibitors Recent article Belido et al., Genet Medicine, 2016 Apr;18(4):325-32 404 Specific missense variants in the conserved region of these error prone polymerases results in a hyper-mutated tumor which occurs in germline or as a somatic finding. Germline and somatic polymerase İ and į mutations define a new class of hypermutated colorectal and endometrial cancers. Briggs S, Tomlinson I - J. Pathol. (2013) Colon Cancer Molecular Pathology Summary • 85% of CRC demonstrate mutation and LOH of the APC gene. • Initiating events for polyp formation. • 1% due to germline APC mutation • 15% of CRC – demonstrate microsatellite instability or abnl MMR IHC. • 13% Results from silencing of both copies of the MLH1 promoter by methylation. • These patients may not respond to 5-Fluoro-uracil. • 2% due to germline MMR mutations • Rare individuals with hypermutated tumors and germline findings • There are a variety of NextGen CRC panels including: APC, MUTYH, MSH2, MLH1, MSH6, PMS2, STK11, SMAD4, BMPR1A, POLE, POLD1. Constitutional Mismatch Repair Deficiency Syndrome - cMMRD • Turcot Syndrome (original name) – association of brain tumors and colon polyps/cancer in childhood. • Dominant forms with FAP and medulloblastomas or HNPCC and glioblastomas • Autosomal Recessive form due to biallelic inactivation of MMR genes = MMR deficiency – possibly highest pediatric tumor risk of any syndrome • Atypical café au lait spots and axillary freckling Genes Age of Diagnosis of 1st tumor Leukemia Lymphoma Brain Tumors HNPCCTumors MLH1, MSH2 n=20 pts. 3.5y (0.4 -35) 11 4 6 MSH6, PMS2 n=55 pts. 9y (1-31) 16 32 35 Modified from Wimmer & Etzler (2008) 405 cMMRD Management • Skin findings (NF1 like), tumor types and consanguinity key clues to diagnosis: • IHC should demonstrate absence of MMR protein in both normal and tumor DNA. • Surveillance recommendations (Durno et al., Eur J Can, 2015) includes: • GI tract include Colonoscopy (> age 6) and EGD and video endoscopy (>age 8) annually • Brain MRI q6 months (from birth) • Adults – add ultrasound of uterus and urinary tract annually. • New guidelines considering adding whole body MRI as there are a wide variety of other tumor types seen in these children/young adults. Hereditary Breast and Ovarian Cancer Hereditary Breast cancer syndromes • Breast-Ovarian Families – BRCA1>BRCA2 • Li-Fraumeni –average onset 32 yo in TP53 carriers • Peutz-Jeghers Syndrome (STK11)– 32% by age 60 • Hereditary breast cancer – PALB2 (some risk of pancreatic cancer) • Cowden syndrome – PTEN • Hereditary diffuse gastric cancer & lobular breast cancer – CDH1 mutations • diffuse gastric (80% risk) and lobular BRCA (40-50% in women) and cleft lip/palate • RECQL – discovered 2015 in Polish, French Canadian kindreds • Moderate risk alleles RR~2.0 • CHEK2 (1100delC founder), RAD51C • Ataxia telangiectasia (ATM) heterozygotes. 406 Cowden syndrome – WdE Hamartoma syndrome • Breast Cancer risk (~30% lifetime risk). • Thyroid cancer (~10% lifetime risk). • Adult Lhermitte-Duclos disease (LDD), cerebellar dysplastic gangliocytoma • Mucocutaneous lesions • • • • • Trichilemmomas (facial) Acral keratoses Papillomatous lesions Mucosal lesions Pigmented spots on penis • Macrocephaly • Autism Zϭ;17q21) – Hereditary Breast/Ovarian Cancer • Associated with loss of function mutations found throughout large gene. • • • • Most are truncating: frameshift or nonsense mutations. Rare missense alleles in ring finger and BRCT domain Deletions or rearrangements make up 2-5% of disease alleles. Hundreds of different rare missense variants most of which are benign or VUS. • In hereditary cancers follows the two hit hypothesis with loss or inactivation of second allele in tumors. • However, somatic mutations in sporadic breast cancer are almost never found in breast cancer and very rare in other tumors. • Tumors - basal-like gene expression profile. • Triple negative (ER/PR/HER2) more likely. ZϮ – 13q12 • Similar to BRCA1 with LOF mutations spread throughout the gene. • Rare somatic mutations in sporadic breast cancers and other tumors. • Compared with sporadic breast cancer it is difficult to distinguish the specific gene expression profile in BRCA2 mutant tumors. • Notable for significant predisposition to male breast cancer (6%) and pancreatic cancer (~1%) in men and women. • There is genomic instability – referred to BRCAness or HRD (homologous recombination deficient) associated with BRCA1/2 or related DNA repair genes being mutant. 407 Ashkenazi Founder Mutations • Three founder mutations : • Two specific mutations in BRCA1 (185delAG and 5382insC) and one in BRCA2 (6174delT). • Carrier frequency of 2.4% for all three mutations. • Start testing with these three mutations. • Responsible for ~50% of Jewish BRCA families. • If negative for founder mutations recommend full sequence mutation analysis for higher risk families. • Can calculate BRCAPro score as non-Ashkenazi to measure residual risk of BRCA1/2 mutations. • Population screening of Ashkenazi individuals not yet recommended by US guidelines but underway in Israel Panels and Variants of Uncertain Significance (VUS) • VUS in ~3-5% of BRCA1/2 sequence tests. • Predominantly missense mutations in protein regions without known function. • A variety of approaches including conservation, computational predictions, segregation with cancer and population studies are utilized to try and determine the significance. • Different laboratories may report out same variant as a VUS or likely pathogenic or likely benign based on their laboratory’s criteria. • Data sharing through ClinVar and other databases helps to decrease discordance across laboratories. • ENIGMA consortium is a ClinVar Expert Panel (3*) for evaluation of BRCA1/2 variants. • Breast cancer patients – dozens of panel studies: • BRCA1, BRCA2 are the most often seen genes with P/LP variants • Can be used for PARP inhibitor treatment selection • In BRCA1/2 negative about 11% panel positive. CHEK2, ATM some of the most frequently detected (Maxwell et al, Genet Med. 2015 Aug;17(8):630-8) ClinVar Variant View 408 BRCA1, BRCA2, PALB2, RAD51C Proteins • Response to DNA damage regulates homologous recombination • ATM phosphorylates BRCA1; • BRCA1 interacts with Rad51 • BRCA2 interacts with Rad52 and PALB2 • BRCA1 or BRCA2 mutant cells are synthetic lethal with PARP inhibitors (a second back-up path of DNA repair) • Ovarian tumors which are BRCA1 or BRCA2 mutant have increased sensitivity to PARP inhibitors. • Olaparib – FDA approved with positive Myriad Genetics Laboratory companion diagnostic test. • About 11% of men w/metastatic prostate cancers demonstrate HRD much higher than those with localized prostate cancer Risk Prediction Models • There are well established computer models which we can use to predict • Risk of developing breast cancer for someone of 㵰average㵱 risk – Gail Model. • Not appropriate for very high risk families. • Has to be at least 35 years old • Risk of developing breast cancer based on family history of cancer – Claus Tables. • Likelihood that genetic testing will yield a mutation in BRCA1 or BRCA2: • BRCAPro (US model) • BOADICEA (UK model) Cancer risk for Zϭ͕ZϮ͕W>Ϯpathogenic variant carriers Tumor type BRCA1 BRCA2 PALB2 Female breast cancer 50-85% 50-85% 33-58% Male breast cancer <1% 6% Ovarian cancer 15-40% 15-25% Prostate cancer 8-16% 8-16% Pancreatic cancer 1.5-2% Not increased (make up 1% PRCA families) 409 Guidelines for ZϭͬϮ carriers Screening test Interval of Screening Clinical breast exam q6-12 months from age 25 Mammogram Yearly beginning age 25 Breast MRI Yearly beginning age 25 Transvaginal ultrasound Q6 mo beginning age 30 Blood CA-125 level Q6 mo beginning age 30 Comments May defer to age 30 No data available if defers BSO No data available – if defers BSO Prophylactic Surgery Bilateral mastectomy Discuss option with patient Risk reducing Bilateral salpingo-oophorectomy (RRSO) Recommend once childbearing is complete around age 35-40. OVCA develops about 10yrs later so can consider delaying RRSO if patient had bilateral mastectomy No clear pancreatic cancer or melanoma screening guidelines but may be recommended depending on family history Modified from NCCN 2017 guidelines. Multiple Endocrine Neoplasia 1 • MEN1 classic TSG with LOF and 2nd hits; Tumor spectrum includes: • • • • Parathyroid Pancreatic islet cell tumors Anterior pituitary hyperplasia Zollinger-Ellison Syndrome (much higher mortality in sporadic ZES versus MEN1 associated ZES). • Biochemical investigations (yearly) • Serum concentration of prolactin from 5 yo • Fasting total serum calcium calcium from 8 yo • Fasting serum gastrin from 20 yo • Imaging - every 3-5 yrs • Head MRI from 5 yo • Abdominal CT or MRI from 20 yo Screening guidelines in Thakker et al, J Clin Endocrinol Metab 2012 Pheochromocytomas - Paragangliomas • 40-50% of pheochromocytomas/paragangliomas patients carry P/LP variants. • Succinate dehydrogenase subunits (SDHB, SDHD, SDHC, SDHA, SDHAF2) encode mitochondrial proteins. • SDHD imprinted, affected when inherited from father. • SDHB mutations associated with malignant forms of the tumors. • More likely to have extra-adrenal tumors including the chemoreceptor organs (glomus and carotid body tumors) • MAX, KIF1B, TMEM127, EGLN1 • VHL – Von Hippel Lindau syndrome – type 2 (missense mutations) associated with high risk of adrenal and extra-adrenal pheochromocytomas with elevated metanephrines • RET – MEN2 syndrome • Rarely NF1 • Can also see GIST tumors 410 AIP Related Familial Isolated Pituitary Adenomas • Pituitary adenomas usually expressing GH (somatotropinoma), PrL (prolactinoma), TSH (thyrotropinoma), ACTH (corticotropinoma) or non-functioning. Age of onset 20-24 yrs • AD with reduced penetrance resulting from heterozygous mutations in AIP • Aryl hydrocarbon receptor Interacting Protein • Mutation: ~90% sequence & 10% del/dup • Treat by surgery, medical or radiotherapy Genetics of Renal Cell Cancer • Histology typically helps decide which gene to test for or use NextGen panel. • VHL – almost always clear cell histology. • Conversely 80% of sporadic RCC has somatic VHL mutations. • Balanced translocation carriers involving chromosome 3 • A variety of different translocations result in AD RCC (clear cell) • Papillary renal carcinoma – due to activating mutations in c-MET oncogene • Hereditary leiomyomatosis RCC (aka Reed Syndrome): mutations in fumarate hydratase (FH) • Autosomal dominant • uterine fibroids and cutaneous leiomyomata • Birt-Hogg-Dubé Syndrome – chromophobe/oncocytic Birt-Hogg-Dubé Syndrome • Chromophobe/oncocytic histology to RCC • Benign fibrofolliculomas • Colonic polyps • Medullary thyroid cancer • Spontaneous pneumothorax • BHD tumor suppressor gene fibrofolliculomas 411 Autosomal Recessive Disorders • Much rarer disorders associated with increased cancer risk, often during childhood. • Genes involved in either DNA repair or checkpoint response. • Patients are often sensitive to specific DNA damaging agent requiring modification of treatments (see supplemental table). • Specific syndromes frequently have founder mutations in specific ethnic groups, e.g., Bloom syndrome in the Ashkenazi population. Ataxia Telangiectasia • Ataxia, telangiectasias (conjunctiva), immunodeficiency, and lymphomas and leukemias +/- solid tumors. • Cancer can be the first sign of the disease. • Most patients wheelchair bound by the teen-age years. • Cells are very sensitive to ionizing radiation. • A-T patients with cancer need substantial dose reduction to avoid lifethreatening toxicity. • Diagnosis - elevated AFP or increased radiation sensitivity on clonal assay. • Can see t(7;14) translocations in peripheral blood Heterozygous dD mutation carriers • The ATM gene encodes a large DNA damage checkpoint protein which phosphorylates proteins encoded by cancer susceptibility genes. • Mutations in ATM include truncating alleles and missense changes in functional domains. • Decades worth of research in 2-4xfold cancer risk associated with being heterozygous carrier of ATM pathogenic variants: Breast, colon, pancreatic. • Goldgar et al., Br Cancer Treatment, 2011 estimated breast cancer risks for heterozygous rare missense ATM mutations: • Overall, 30% risk of BRCA by age 60 (large CI) • One specific c.7271 C>T p.V2424G mutation in ATM is associated with a significantly higher risk of breast cancer similar to BRCA2. 412 Fanconi Anemia • Autosomal recessive syndrome with 19 different complementation groups • FA – A, B (X-linked), C, D1 (BRCA2), D2, E, F, G, I, J (BRIP1), K, L, M, N (PALB2), O (RAD51C), P (SLX4), Q (ERCC4), R (RAD51A), T (UBE2T), V (MAD2L2), U (XRCC2), • Associated with varying clinical features: • Congenital anomalies • Bone Marrow Failure resulting in pancytopenia with typical onset between 5-10 years treated by BMT. • Significantly increased risk of malignancy (Rosenberg et al. Blood, 2003). • By age 17, 12% FA pts had died from BMF, leukemia or solid tumor. • By age 24, 10% had AML alone. • Overall, solid tumors much more likely in adulthood (including head and neck cancer and GYN malignancies). FA – Complementation Analysis MMC Sensitive FA-A MMC Sensitive FA-B MMC Sensitive or FA-? Fused Cells are MMC Resistant MMC Sensitive + FA-? = Fused Cells are MMC Sensitive MMC Sensitive + FA-? Retrovirus expressing FANCB FA-? FA-A/FA-? = = FA-B/FA-? Infected FA-? Cells are MMC Resistant = Fanconi Type B FA - Congenital Anomalies • Skeletal: radial ray, hip, vertebral • Skin hypo/hyperpigmentation • Short stature • Microphthalmia • Microcephaly • Renal: unilateral aplasia, hypoplasia, horseshoe • Genital: hypogenitalia 413 FA Breakage Assay • Cells from FA patients (both lymphoblasts and fibroblasts) demonstrate increased sensitivity and chromosome breakage after exposure to crosslinking agents: • Diepoxybutane (DEB) • Mitomycin C (MMC) • Used as the gold standard diagnostic test Quadri-radial formations Tri-radial formations in MMC in DEB treated PB from treated PB from FA patient FA patient • When in doubt, order the test, particularly prior to decisions about bone marrow transplant. Images from M. Folsom, R. Naeem, Cytogenetics Laboratory, TCH Core Fanconi Anemia Complex FA Core Complex (in yellow) is required to Monoubiquitinate FANCD2 and FANCI D2 I B E F A M G L C Weak evidence that heterozygous mutations in core complex genes convey cancer risk I D2 Ub P Ub ATM ATM protein phosphorylates FANCD2 after DNA damage FA Proteins and DNA Damage Response D2 I B E F A M G L C I RAD51C/O BRCA2/D1 BRIP1/J PALB2/N D2 Ub P ATM ATM phosphorylates FANCD2 after DNA damage Ub SLX4/P DNA MAD2L2 XRCC2 Genes that encode proteins that play important roles in processing of DNA damage are implicated in both FA and hereditary breast cancer (in green) 414 Summary: Cancer risk of heterozygous carriers for rare FA subtypes Gene Type BRCA2 FA-D1 High risk breast, ovary, pancreatic… Heterozygous cancer risk PALB2 FA-N Moderately high risk breast (2-4 fold increased), pancreatic cancer (familial pancreatic cancer) RAD51C FA-O High grade serous epithelial ovarian cancer; GWAS hit for testicular germ cell cancer; Breast cancer risk unclear BRIP1 FA-J Twofold increased risk of breast cancer SLX4 FA-P No clear breast cancer risk from several large studies XRCC2 FA-U RAD51 paralog; LOF mutations identified in breast cancer families and one FA-like patient cell line RecQ Helicase Disorders Disease Clinical Features Cancer Predisposition Gene/Chromosome location Hereditary Breast Cancer (AD) Autosomal dominant non-syndromic Breast cancer (2015) RECQL 12p12 Bloom (AR) Small stature, photosensitive rash, immunodeficiency Multiple tumor types including leukemia/lympho ma and solid tumors BLM 15q26.1 Werner (AR) Premature ageing, cataracts, diabetes, atherosclerosis Soft tissue sarcomas and skin cancers WRN 8p11 Rothmund Thomson (AR) Poikiloderma rash, sparse hair, radial ray defects, cataracts Osteosarcoma and skin cancers RECQL4 8q24.3 and unidentified gene(s). Osteosarcoma associated with RECQL4 mutation RAPIDILINO (AR) Baller-Gerold (AR) Poikiloderma in RTS • Children born with normal skin (may have absent hair). • Acute phase starts on the cheeks during infancy and spreads to extremities (spares the thorax and buttocks). • The rash persists as stable poikiloderma. • Seen in both Type 1 and Type 2 RTS. C. • Type 2 has extremely high risk of osteosarcoma although response to treatment similar to tumors in general population. • Specific RECQL4 variants associated with lymphoma 415 Phenotypic tests for hereditary cancer syndromes Syndrome Gene Phenotypic test Dyskeratosis congenita Multiple Telomere length by FISH/flow cytometry Bloom BLM Sister chromatic exchange Fanconi Multiple DEB and MMC breakage study Ataxia Telangiectasis ATM, NBN, (and Nijmegen) LIG4 Sensitivity of lymphoblastoid cell lines to ionizing radiation Mosaic Variegated Aneuploidy BUB1B & others Random trisomies in peripheral blood XRCC2 FA-U RAD51 paralog; LOF mutations identified in breast cancer families and one FA-like patient cell line 416 Biochemical Genetics II BIOCHEMICAL GENETICS II Gerard Berry, MD, FACMG Harvey Levy Chair in Metabolism Director, Metabolism Program Division of Genetics and Genomics Boston Children’s Hospital Professor of Pediatrics, Harvard Medical School Gerard Berry, MD, FACMG Harvard Medical School Center for Life Science Building, Suite 14070 3 Blackfan Circle Boston, MA 02115 (617) 355-4316 Telephone (617) 730-4874 Fax [email protected] 419 420 Biochemical Genetics II Gerard T. Berry, MD, FACMG Founding Fellow ACMG Division of Genetics and Genomics Boston Children’s Hospital Harvard Medical School Director of Harvard Medical School BG training program ACMG Genetics and Genomics Review Course June 20-23, 2013 Nothing to disclose Disorders of Organelles & Large Molecules භ Mitochondria Cilia භ Trafficking defects Ly භ Lysosomes භ Peroxisomes Px Nucleus ER 421 Organelle Targeting Signals භ Mitochondria භ Presequence - N-terminal amphipathic helix භ Nucleus භ Internal basic AA di-peptide භ ER භ N-terminal hydrophobic peptide; binds signal recognition particle (SRP) with co-translational import followed by cleavage භ Lysosome භ Mannose-6-P (M6P) added post-translationally භ Peroxisome භ PTS1 -- C-terminal -SKL භ PTS2 -- near N-terminal -RLX5H/QLභ PTS3 - ? Mitochondrial Disease භ~1/5000-8000 overall incidence භNuclear and mt DNA mutations භ>300 nuclear genes involved mt function භ37 mt genes – some resp chain proteins (for all but Complex II), 2 rRNAs, 22 tRNAs භMaternal, cytoplasmic inheritance (for mt DNA) භHeteroplasmy – mitochondria form a population within cells; threshold effect භPhenotypes may vary with age භAffect tissues with high energy demands Symptoms Suggesting a Mt Disorder භCNS - hypotonia, ataxia, IDD, seizures, migraines, dementia, sensorineural hearing loss භEyes – retinitis pigmentosa (RP), optic atrophy, nystagamus, ophthalmoplegia භMuscle - weakness, exercise intolerance, red ragged fibers භCardiac - hypertrophic cardiomyopathy, arrhythmias, heart block භHematologic - macrocytic anemia, pancytopenia 422 Symptoms Suggesting a Mt Disorder භEndocrine - diabetes mellitus, diabetes insipidus, exocrine pancreatic dysfunction, short stature භGI - dysfx, intestinal pseudo-obstruction භLiver - dysfx, failure භRenal - RTA, Fanconi syndrome භMany pts, particularly infants, do not present with classic phenotypes; consider in differential if pt has 2 or more suggestive findings Genetic Defects in Mt Function භ Defects in nuclear genes භSingle function deficiencies භMt biogenesis, complex assembly, and replication defects භ භ Mt DNA point mutations Mt DNA deletions / insertions Many Mt Disorders Caused by Mutations in the Nuclear Genome භ Defects in OxPhos භ ~ 80-90% of the patients have nuclear encoded defects භAll complexes of the e- transport chain have subunits encoded in nuclear genome භ Typically AR inheritance භ Respiratory chain assembly factors භ ACAD9 (Complex I) SURF1 (Complex IV), SCO1&2 (Cu++ homeostasis, complex assembly) භ MNGIE – AR defect in TYMP gene භ(Mt neurogastrointestinal encephalomyopathy) භ CoQ synthesis defects - 5 genes 423 Nuclear Encoded Mitochondrial Genes Recently Identified by Exome Sequencing from Taylor et al., JAMA, 312: 68-77, 2014 (Fig. 2) Leigh Syndrome භ Sx - onset late infancy with regression; MRI abnl with white matter and basal ganglia changes; +/- increased serum lactate භ Heterogeneous with nuclear & mt mutations භ ~50% SURF1 (involved assembly Cyt Oxidase, Complex IV) භ PDH mutations භ Complex I, II, IV deficiency භ NARP (neuropathy with ataxia & RP) mt point mutations භ mt DNA depletion Mitochondrial Depletion Syndromes භAR - ratio of mt/nuclear DNA භGe - heterogeneous and caused by defects in nuclear genes involved in mt DNA replication (POLG1, TK2, DGUOK, TWINKLE, others) භSx - hepatic failure, glu, CNS see Copeland, Ann. Rev. Med. 59: 131-46, 2008 424 Some Disorders Caused by Mt Point Mutations භ LHON - adult onset optic neuropathy; most homoplasmic missense muts භ NARP - Neuropathy, ataxia & RP; most patients with heteroplasmic missense muts in ATP synthase (Complex V) භ Maternally inherited deafness - point muts in mt rRNA; also associated with susceptibility to aminoglycoside ototoxicity භ Several others Some Disorders Caused by Mt Point Mutations භ Mt DNA tRNA mutations භMERRF - heteroplasmic point muts in mt lys tRNA (~80%) භMELAS - heteroplasmic point muts in tRNAleu (most) භ Mechanism not known; ?Sx related to inability to translate several mt proteins and lack of nl processing of transcripts MERFF = myoclonic epilepsy with red ragged fibers MELAS = myoclonic epilepsy with lactic acidosis and stroke MELAS: Myoclonic Epilepsy, Lactic Acidosis & Stroke භEpisodes of metabolic decompensation ass’d with high stroke risk භAcute Rx – arginine භ3243 A>G tRNALeu (~80%); 3271 T>C tRNALeu (~7%) භHeteroplasmic 425 Some Disorders Caused by Mt DNA Deletions &/or Duplications භDiabetes and deafness භPearson syndrome –anemia 2° marrow failure; lactic acidosis; exocrine pancreatic failure; RTA භCPEO – chronic progressive external ophthalmoplegia භKearns-Sayre – PEO, cardiac conduction block, RP, ataxia, lactic acidosis, ataxia; sporadic Laboratory Diagnosis of Mt Disorders භ Lactate, pyruvate (peripheral, CNS), ratio; alanine භ MRI of brain භ Consider muscle and/or liver bx (most involved tissue) භ OxPhos analysis including enzyme assays භ DNA analysis for specific mtDNA/nuclear mutations; mt DNA panels; exome sequencing 3 Parent Embryos to Treat Mt-Encoded Disease from Science, 343: 827, 2014 426 Congenital Disorders of Glycosylation (CDG) භ Glycosylation pathways very complex භ~2% genes encode proteins involved in glycosylation භ>100 human disorders identified to date; most very rare භ Clinical spectrum very broad: CNS, eye, skeletal, skin, clotting, immune , endocrine, GI, liver, & more භ New disorder nomenclature: gene symbol-CDG භCDG1a becomes PMM2-CDG භCDG1b becomes MPI-CDG Classes of Glycosylation Disorders භN-glycosylation defects (ш28 human disorders) භ Amide linkage to asparagine භ N-glycan assembly ER or cytosol; sugars transferred en bloc from dolichol; processing in ER or Golgi භ~50% of all known proteins have ш1 N-gly site භ O-glycosylation defects (ш34) භLinkage through –OH on serine or threonine; transfer single sugars onto growing glycan backbone භIncludes ABO blood groups, Exostoses I & 2 proteins; proteoglycans (with skeletal & connective tissue sx), some congenital muscular dystrophies (POMT1&2, Fukutin, etc) Classes of Glycosylation Disorders භ Combined N- and O-glycosylation defects (ш7) භ Lipid glycosylation defects (ш3) භ GPI-anchor defects (ш12) භ Trafficking defects of Golgi COG complex proteins භ Defects of dolichol synthesis or recycling (ш5) Farnesyl-PP Dolichol-PP (ш13) N-gly ER (3 steps) Dolichol-P Cholesterol O-gly, others Modified from Wolfe et al., AJMG, 160C: 322-8, 2012 427 Summary of Types of Glycosylation PROTEOGLYCANS GLYCOSPHINGOLIPIDS GLYCOPROTEINS from Freeze et al., Inborn Errors of Metabolism, 2015, Fig. 2.1 Classical PMM2-CDG (Ia) Clinical Features භ AR; phosphomannomutase 2 def (most common CDG, 60-70%) භ Multisystem disorder භhypotonia, IDD, szs, ataxia (cerebellar hypoplasia) භRP, strabismus භliver disease, coagulopathy භFTT, inverted nipples, lipodystrophy භ Some die <1 yo; others survive to adulthood; range of cognitive skills භ Abnl transferrin isoelectric focusing Other CDGs භ MPI-CDG (Ib) - Mannose-6-phosphate isomerase def භHepatic/GI sx with vomiting, GI bleeding, protein-losing enteropathy, liver disease, coagulopathy, hepatic fibrosis භMinimal neurologic involvement භOnly form with rx – oral mannose භ SRD5A3-CDG (Iq) – steroid 5-ɲ-3 reductase def (dolichol synthesis defect) භ1st sx between 6 mo – 12 yrs භSevere IDD, ataxia, cerebellar hypoplasia භProminent eye anomalies – coloboma, optic atrophy, cataracts, glaucoma, micro-ophthalmia භHeart defects, liver dysfx, ichthyosis 428 CDG Diagnosis භ Transferrin (Tf) isoelectric focusing – Dx for Nlinked disorders only භ Various mass spec techniques of purified serum proteins (Tf, apoCIII, others) and urine – N- and Olinked disorders භ False positives – young infants (<30 days), galactosemia, HFI, recent EtOH use, liver disease, hemolytic uremic syndrome, Tf protein polymorphisms (sugars don’t bind) CDG Diagnosis: Transferrin Isoelectric Focusing See Jaeken & Matthijs Ann Rev Gen & Hu Genet, 2001 Lysosomal Storage Diseases භLysosomes - cytoplasmic organelles that contain ~50 acidic degradative enzymes භAlso include membrane proteins භTransporters (cys-cys, sialic acid) භSAP activator proteins භDeficiency results in accumulation of macromolecules usually degraded by that enzyme/protein භStored material may cause enlargement of organs and may be visualized in membrane bound vesicles by EM 429 Lysosomal Storage Diseases (LSDs) භTarget organs affected by each disease are determined by normal sites of degradation of each compound භAll are recessive, most are autosomal භMost patients are normal at birth; as material accumulates there is a plateau and then regression භMost disorders are progressive and often fatal භMany have classic infantile (occ prenatal) as well as later onset, milder forms Classification of LSDs භ Mucopolysaccharidoses (Hurler, Hunter) භ Sphingolipidoses (Tay-Sachs, Gaucher) භ Transport disorders (Cystinosis) භ Mucolipidoses – trafficking (I-Cell) භ Glycoprotein (Mannosidosis) භ Neutral Lipid (Wolman) භ Glycogen Storage (Pompe) LSDs : Some General Phenotypic Features භCoarse facies භOrganomegaly (liver, spleen) භEye abnormalities භCorneal clouding භCherry red spot භOptic atrophy භPigmentary retinopathy භSkeletal abnormalities භNon-immune hydrops Cherry red macula Dysostosis multiplex 430 LSDs: General Diagnostic Approach භ භ භ භ භ භ භ භ භ Serum lysosomal enzymes Blood smear Radiologic exam Ophthalmologic exam - fundoscopic & slit lamp Urine mucopolysaccharides and glycoproteins Consider bone marrow Biochemical studies of fibroblast +/- leukocytes Molecular/gene sequencing Other -- depending on specific disorder Mucopolysaccharidoses භ Usually normal at birth භ Gradual slowing of development, regression භ Coarse facies භ +/- Corneal clouding භ Macrocephaly, IDD (intellectual/developmental disability) භ Skeletal involvement ( ROM, claw hand) භ Dysostosis multiplex on x-ray භ Otitis and hearing (sensorineural & conductive) භ Recurrent herniae, thickened mucous භ Late cardiac involvement Hurler Syndrome (MPS I) භPrototypic MPS disorder භAR; incidence ~1/100,000 භSx - onset 6-12 months; death by 5-10 yrs; milder variant w/o CNS (Scheie IS) භDx - +MPS spot test; enzyme assay; DNA භRx - HSCT transplantation with matched donor slows disease if performed early (including CNS; no effect on skeletal sx, corneal clouding); ERT – improved somatic sx, no effect on CNS; protocols now with ERT before HSCT 431 Hurler Syndrome Dysostosis Multiplex Hunter Syndrome (MPS II) භSx - like Hurler but usually no corneal clouding; prominent deafness භXL; ~1/70-150,000; 20% of pts with gene deleted have more severe ID භDx: +MPS spot; enzyme assay; carrier females best diagnosed by DNA භ Rx: ERT gives improved somatic function in pts pts with mild disease (reduces viscero-megaly & GAG excretion, improves joint mobility, preserves linear growth); no effect on CNS; efficacy of HSCT not proven 432 Sanfilippo Syndrome (MPS III) භ4 distinct loci (all AR); A and B most common භSx - more CNS, less somatic features; onset usually 2-4 yrs; chronic diarrhea, insomnia, szs, aggression prominent භDx – MPS may be + or -; enzyme assay/DNA භRx - none; no benefit from HSCT; some enzyme replacement to CNS and gene therapy clinical trials Other MPS Disorders භMorquio (Type IVA & B) භShort-trunk dwarfism with nl IQ; severe odontoid hypoplasia; ERT (elosulfase alfa) භMaroteaux-Lamy (Type VI) භSomatic sx may be as severe as Hurler; usually nl IQ භDefect in Arylsulfatase B; ERT (Naglazyme) භSly (MPS VII) භSevere infantile form like Hurler; prenatal form with hydrops/fetal ascites භDefect in β-glucuronidase Other Features of MPS Disorders භHydrocephalus භObstructive airway disease; difficulty with intubation; excessive secretions භAtlantoaxial instability; odontoid hypoplasia භCardiac disease - valvular, conduction disturbances, EFE, occ cardiomyopathy භPulmonary and systemic hypertension 433 Gaucher Disease භMost common lysosomal storage disease භ3 types based on clinical symptoms භType I - nonneuronopathic; splenomegaly, pancytopenia, bone pain/lytic bone lesions; 1:400-1:1000 US Ashkenazi Jews භType II - acute neuronopathic - rapidly progressive neurologic disease with hepato-splenomegaly; all ethnic groups භType III - subacute neuronopathic - later onset Gaucher Disease භDx - “foam cells” in bone marrow, smear; enzyme assay; molecular for carrier screening in Jewish population භGene (GBA; glucosidase-ɴ acid) on chr 1; nearby pseudogene; certain alleles appear protective (N370S) against CNS disease; L444P usually Type II or III භRx - symptomatic; splenectomy; ERT for Type I pts (no effect Type II); newer substrate reduction therapy (miglustat; D-glu analog) Tay-Sachs Disease (TSD), GM2 Gangliosidosis භAR; incidence ~1/100,000; ~1/4000 in Ashkenazi Jewish pop; also increased in French Canadians භSx classic infantile - onset 6-12 mos; loss of milestones, hyperacusis, apathy; cherry red spot; later onset of szs, blindness, spasticity; death by age 2-5 භMilder juvenile & adult forms 434 Cherry Red Spot භTay-Sachs Disease භSandhoff Disease භSialidase deficiency භNiemann-Pick Disease Type A භGM1 Gangliosidosis Tay-Sachs Disease භMolecular defect of Hexoseaminidase A; defects in Hex B cause Sandhoff Disease (Sandhoff may have some somatic features + CNS) භDx - enzyme assay; DNA භRx - none භPrevention – heterozygote/carrier screening (recommended enzyme + DNA, even in Ashkenazi Jewish pop) Fabry Disease භX-linked; ~1/40-60,000 males; CNS spared භSx (males) – median age of onset 9 yrs; peripheral neuropathy; acroparesthesias; angiokeratomas; lens/corneal opacities; late renal and cardiovascular disease; chr lung disease with fibrosis භAccounts for ~1% chr renal failure & 5% cryptogenic stroke; incidence cardiac variant ~1/3500 435 Fabry Disease භMost females have sx – median age onset 13 yrs; fatigue, stroke & ~10% develop renal failure; can be detected by slit lamp භDx - enzyme assay males (may miss females); heterogeneous mutations; DNA best for females භRx – dilantin/tegretol for neuropathy; renal transplant; ERT may decrease pain, GI sx, slow renal disease; does not proteinuria භAdult male – start ERT at time of dx භPediatric male and all females – start rx at 1st sign of sx Krabbe Disease, Globoid Cell Leukodystrophy භ Sx infantile - onset <6 mos; hypotonia, irritability; optic atrophy; occ. macrocephaly; elev. CSF protein; leukodystrophy on MRI භ Dx - enzyme assay/molecular (GALC gene); pseudo-deficiency can complicate prenatal dx & requires sulfatide loading assay භ Rx - HSCT has some efficacy in later onset cases or if performed very early in infantile cases GALC = galactosylceramidase Lysosomal Processing Defects භ I-Cell Disease (Mucolipidosis II) භ Multiple sulfatase deficiency (Appendix) 436 I-Cell Disease I-Cell Disease භAR defect in targeting enzymes to lysosome via mannose-6-P (2 step Golgi rxn; defect 1st step); MLIII is allelic, milder variant භSx – like severe Hurler; may see neonatal or prenatal onset භDx - plasma activity multiple lysosomal enzymes with deficient activity in fibroblasts; MPS spot භRx - None Lysosomal Transport Disorders භDefective transport out of lysosomes of products of lysosomal degradation භExamples භCystinosis භSialic acid storage disease (infantile and adult forms) භNiemann-Pick Type C (NPC) Disease 437 Niemann-Pick Disease, Type C භAR; ~1/150,000 භSx – like NP A, B; infantile form with neonatal jaundice; later onset forms with ataxia and progressive dementia, psychosis; vertical ophthalmoplegia භDx - nl sphingomyelinase; abn. lysosomal accumulation of unesterified cholesterol භ2 genes identified, NPC1 (95%), NPC2 (~5%); role in intracellular cholesterol trafficking භRx – supportive; clinical trials with drugs to increase chol removal from cells Enzyme Replacement Therapy (ERT) for LSDs භNeed to reach cells with defect භGaucher – macrophages භFabry – endothelial cells භTay-Sachs - neurons භThreshold for effectiveness may differ with each disorder & site (bone resistant in MPS) භMay be most effective early in course of disease (MPS, neuronal involvement) භActs as foreign protein in CRM- patients භCost and availability ERT for LSDs භ FDA approved භ Cerezyme, Vpriv – Gaucher (I,III) භ Fabrazyme, Replagal – Fabry භ Myozyme - Pompe භ Aldurazyme – MPS I (Hurler) භ Elaprase - MPS II (Hunter) භ Naglazyme – MPSVI (Maroteaux-Lamy) භ Elosulfase alfa MPSIVA (Morquio) භ Synageva – Lysosomal acid lipase def (Wolman) භ Ongoing clinical trials භ Sanfilippo IIIA, MLD භ www.clinicaltrials.gov 438 ERT for Type I Gaucher Disease Other Therapies for LSDs භSubstrate reduction භZavesca (miglustat; Gaucher & others) iminosugar analog of glucose; crosses BBB භNeed residual enzyme or alternate pathway භEnzyme enhancement – chaperone therapy භRequire residual activity, stabilize mutant protein භNot specific to a single enzyme, ?mutation specific භCross BBB භCan be complementary & synergistic to ERT Genetic Counseling for LSDs භDx usually relies on enzyme assay with molecular testing for confirmation, carriers &/or prenatal භReliability of biochemical carrier testing variable (excellent for TSD; poor for many) භPseudodeficiency can complicate prenatal dx (MLD, Krabbe) 439 Features of Peroxisomes (Px) M භUbiquitous (except RBCs) භBound by single membrane භContain no nucleic acid; proteins encoded by nuclear genes, translated on free cytoplasmic ribosomes & imported into peroxisome membrane or matrix භSeveral hundred/cell; ~70 enzymes in matrix භPerform a variety of cellular anabolic and catabolic functions ER Medically Relevant Peroxisomal Functions භDegradation of very long chain fatty acids භEarly steps of plasmalogen biosynthesis භDegradation of phytanic acid භSelected steps in cholesterol biosynthesis භDegradation of pipecolic acid, synthesis of bile acid intermediates, glyoxylate metabolism Peroxisome Biogenesis භDivide by fission to form new organelles භBiogenesis and protein import co-ordinated by 16 PEX proteins භImport of matrix proteins uses at least 3 targeting signals භPTS1 - C-terminal SKL - most common භPTS2 - N-terminal sequence භSome proteins use neither of the known signals භTargeting of peroxisomal membrane proteins not completely understood 440 Classification of Peroxisomal Disorders (PSDs) භPeroxisome biogenesis disorders (PBDs) multiple deficiencies භ Zellweger syndrome spectrum භRhizomelic chondrodysplasia punctata (RCDP) භSingle enzyme defects භ X-linked adrenoleukodystrophy භRefsum disease Zellweger Syndrome Spectrum Zellweger syndrome Neonatal ALD Infantile Refsum Disease භ Combined developmental & metabolic disorders භ Overall ~1/50,000 incidence භ Genetically heterogeneous - 12 complementation grps භ 50% defect in PEX1 gene encoding PTS1 receptor Zellweger Syndrome භPrototypic peroxisomal biogenesis disorder භSx - dysmorphic facies, hypotonia, seizures, IDD, neuronal heterotopias, cataracts &/or glaucoma, renal cysts, 50% with epiphyseal calcifications (CDP) භEarly death, usually by 6-12 months භVariants present a bit later, usually w/o dysmorphisms/malformations; survive longer භDx - all peroxisomal functions abnormal; no peroxisomes or ghost membranes seen by EM; biochemical, then molecular 441 Rhizomelic Chondrodysplasia Punctata භ ~1/100,000 භ Skeletal dysplasia, cataracts, ichthyotic skin rash, IDD; occ CHD, cleft palate භ Mutations in PEX7, encoding the PTS2 receptor is most common cause භ Dx – low plasmalogens; elevated phytanic acid භ Phenotype of single enzyme defects in plasmalogen synthesis result in similar phenotype (no phytanic acid) X-linked Adrenoleukodystrophy භ Progressive X-linked neurodegeneration associated with adrenal involvement භ ~1/20,000 prevalence භ Highly variable clinical phenotype භ Childhood cerebral - childhood onset, rapid progression භ Adrenomyeloneuropathy (AMN) - onset 20’s - 30’s with spastic paraparesis භ Adrenal only භ ~50% het female carriers develop mild neurological sx in adulthood භ 20% gait and spinal cord involvement like AMN X-linked Adrenoleukodystrophy භResponsible gene, ABCD1, encodes an ABC transporter in the peroxisome membrane භNo genotype / phenotype correlation VLCFA භSome males with sx & others asymptomatic in same family Px භDefective metabolism of VLCFA භDx – VLCFA; molecular (esp. carrier females) භRx – HSCT early in course for males as soon as MRI changes noted; corticosteroids for adrenal insufficiency 442 X-ALD: Childhood Cerebral Form Age 2 Age 5 Cerebral X-ALD T1 MRI with gadolinium Inflammatory demyelination with perivascular infiltration Diagnostic Abnormalities in the Px Disorders භ PBDs - Zellweger spectrum භ VLCFA භ RBC plasmalogens භ plasma pipecolic acid භ PBDs - RCDP භ RBC plasmalogens භ VLCFA are normal භ Other භ phytanic acid (requires dietary intake) භ bile acid intermediates භ Dicarboxylic aciduria by urine organic acids භ Molecular screening * Steinberg et al, Mol Genet Metab 83: 252, 2004 443 Refsum Disease භSx - Cerebellar ataxia, polyneuropathy & RP; elevated CSF protein භGe - AR deficiency of phytanoyl-CoA hydroxylase භDx - Phytanic acid; molecular භRx - Phytanic acid-restricted diet Clinical Features Suggesting a PX Disorder භ Failure to thrive, developmental delay භ Hypotonia, cerebral atrophy, decreased myelination, neuronal heterotopias භ Dysmorphia similar to Zellweger භ Cataracts, glaucoma, retinitis pigmentosa භ Chondrodysplasia punctata භ Hepatomegaly, renal cysts Thank you and good luck! 444 • Online Metabolic and Molecular Basis of Disease • Blau et al., Physician’s Guide to Laboratory Diagnosis of Metabolic Disease • Nyhan et al., Atlas of Metabolic Disease • NAMA slide sets from SIMD • Lee and Scaglia, eds., Inborn Errors of Metabolism (2014) ACMG Genetics and Genomics Review Course June 20-23, 2013 APPENDICES Nuclear Membrane: The Laminopathies භ Lamins - multifunctional filamentous proteins of the nuclear lamina, just under the inner nuclear membrane භ Three genes - LMNA, LMNB2, LMNB1 භ 13 known disorders, including 11 discrete phenotypes caused by LMNA mutations including: භ Hutchinson-Gilford progeria භ Emery-Dreifuss muscular dystrophy භ Mandibuloacral dysplasia භ Generalized lipodystrophy භ Restictive dermopathy 445 Niemann-Pick Disease,Types A and B භAR - deficiency of acid sphingomyelinase; increased in Ashkenazi Jews (carrier freq ~1:60) භSx - neurodegenerative with spleen > liver; cherry red spot (~50% type A); pulmonary (Type B) භDx - enzyme assay; molecular; sea blue histiocytes in marrow භRx - none SMPD1 gene = sphingomyelinase phosphodiesterase 1 Protein = ASM, acid sphingomyelinase GM1 Gangliosidosis භSx – somatic + CNS affected; hypotonia, szs, MR; ½ pts have cherry red spot භMilder juvenile and adult forms exist භDx – foamy histiocytes in bone marrow; enzyme assay of ɴ-galactosidase; gene (GLB1= galactosidase, beta 1) භVariants භ Morquio Type B pts with residual GM1 activity, skeletal sx, CNS spared භCombined galactosialidosis with protector protein deficiency Tay-Sachs Disease (TSD) TSD = deficiency of Hex A, a+b dimer Sandhoff = deficiency of Hex B – b+b dimer Hex A is heat labile, Hex B is not. Std serum screening assay measures total Hex A+B, then heat inactivates Hex A (or uses low pH) and measures residual Hex B. Then Hex A = Total – Hex B. Molecular testing used to distinguish rare pseudo-deficiency alleles in healthy persons with low HexA Pregnancy, oral contraceptives and some illnesses can make serum screening test inconclusive. 446 Metachromatic Leukodystrophy භSx late infantile - most pts walk; regression before age 2; white matter changes; elev. CSF protein භMilder variants භAR defect in Arylsulfatse A (gene is ARSA) භDx - enzyme assay; pseudodeficiency භGene cloned and 2 single bp changes associated with pseudodeficiency Glycoprotein Disorders භIncludes mannosidosis, aspartylglycosaminuria (AGU), sialidosis, fucosidosis භSx – like mild/mod MPS; fucosidosis has false + sweat test & angiokeratomas; congenital form of sialidosis with fetal ascites භDx – MPS spot neg; enzyme assay; characteristic urine oligosaccharides භAGU common in Finland – C163S missense ~98% Finnish alleles (founder effect) Multiple Sulfatase Deficiency භSx – like severe MPS+ichthyosis, mild skeletal භAR deficiency of Formylglycine enzyme - catalyzes posttranslational modification of conserved cys in all sulfatases; very rare භFeatures of 8 monogenic disorders භ5 MPS - II, IIIA, IIID, IVA, VI භX-linked ichthyosis (steroid sulfatase) භMetachromatic leukodystrophy (Aryl A) භXL rcessive CDP (ARSE) භDx - +urine MPS, enzyme assay(s); molecular (Gene is SUMF1, sulfatase modifying factor 1) 447 Selected Other LSDs භFarber lipogranulomatosis (ceramidase def) – very rare; painful, deformed joints + subcutaneous nodules; IDD භAcid lipase def (Wolman disease) – GI disorder with hepatosplenomegaly, FTT, adrenal Ca++; milder variant is cholesterol ester storage disease භSchindler disease – progressive IDD, blindness, szs, hypotonia; rare cause of neuraxonal dystrophy due to deficiency of lysosomal N-acetylgalactosaminidase භPycnodysostosis –skeletal dysplasia; defect in lysosomal cathepsin K LSD: Sphingolipid Activator Protein (SAPs) භSmall proteins that interact with some lys hydrolases to stabilize them or stimulate activity භ2 SAP Genes භ5q GM2 activator (Tay-Sachs, Sandhoff); gene is GM2A, GM2 ganglioside activator භChr 10 – One gene encodes SAPs A-D; processed to individual proteins (gene is PSAP, prosaposin) භSx – SAP B resembles juvenile MLD; SAP C juvenile Gaucher; All are AR Other Single Function Px Disorders * භ Total at least 13 disorders භ In addition to XALD & Refsum includes: භ Px β-oxidation disorders - most resemble ZS භ Ether phospholipid synthetic defects resemble RCDP භ Mevalonate kinase - classic & Hyper IgD/ periodic fever භ Catalase deficiency - oral ulcers භ Glyoxylate detoxification - Hyperoxaluria type I * Wanders et al, MMBID edt 8, 2001; Wanders & Waterham BBA 1763: 1707, 2006 448 Systems-Based Disorders I SYSTEMS-BASED DISORDERS I Bruce R. Korf, MD, PhD, FACMG Wayne H. and Sara Crews Finley Chair in Medical Genetics Professor and Chair, Department of Genetics Director, Heflin Center for Genomic Sciences University of Alabama at Birmingham Bruce R. Korf, MD, PhD, FACMG Department of Genetics University of Alabama at Birmingham 1720 2nd Ave. S., Kaul 230, Birmingham, AL 35294-0024 (205) 934-9411 Telephone (205) 934-9488 Fax [email protected] 451 452 Systems Based Disorders I Bruce R. Korf, MD, PhD Professor and Chair, Department of Genetics University of Alabama at Birmingham Disclosure(s) Relationship Entity Grant Recipient Novartis Advisory Board Accolade, Genome Medical Board of Directors American College of Medical Genetics and Genomics Children’s Tumor Foundation Advisor Neurofibromatosis Therapeutic Acceleration Project Founding Member Envision Genomics Salary University of Alabama at Birmingham Outline • Skin • GI • Sensory • Psychiatry • Cardiovascular • Epigenetic 453 Pigmentation Skin Eyes Hair Other Gene(s), (Inheritance) Hearing loss Dystopia canthorum Melanocyte Development Waardenburg Syndrome Congenital leukodermia Heterochromia iridis; choroidal hypopigmentation White forelock Piebaldism Patchy hypopigmentation Heterochromia iridis White forelock KIT, SNAI2 (AD) PAX3 (AD) Melanogenesis OCA1 White Decreased acuity White TYR (AR) OCA2 White Decreased acuity White/Yellow OCA2 – tyrosine transporter (AR) OCA3 Copper-red, freckles Nystagmus, strabismus Copper-red TRYP1 (AR) OCA4 White Decreased acuity White OCA5 White Decreased acuity Yellow ? OA1 Normal Decreased acuity Normal GPR143 (XLR) SLC45A2 (AR) Melanocyte Biogenesis/Transport Hermansky-Pudlak Creamy white Decreased acuity White to brown Bleeding, cardiomyopathy, pulmonary fibrosis, colitis, renal failure, absent dense bodies in platelets HPS1 (AR) prevalent in Puerto Rico; other genes Chediak-Higashi Hypopigmentation Decreased acuity Hypopigmentation Hepatosplenomegaly, neuropathy, Intellectual disability, anemia, thrombocytopenia, infections LYST (AR) Griscelli Hypopigmentation Poor fixation Silver-gray Developmental delay MYO5A, RAB27A, MLPH Ectodermal Dysplasia Hypohidrotic Ectodermal Dysplasia Skin Teeth Hair Nails Other Gene(s) (Inheritance) Dry, lack of sweat pores, hypohidrosis Adontia/hypodontia Fine, brittle, sparse Spooned, brittle Depressed nasal bridge, respiratory problems EDA (XLR) EDAR, EDARADD (AD/AR) Odonto-OnychoDermal Dysplasia Hyperkeratosis, erythema, hyperhidrosis Hypodontia Absent, dry, thin Dystrophic Smooth tongue WNT10A (AR) P63 Related Hyperkeratosis Adontia/Hypodontia Sparse Dystrophic Various syndromes: EEC, Hay-Wells, Rapp-Hodgkin, etc. P63 (AD) Clouston Hyperkeratosis, hyperpigmentati on Normal Fine, brittle Dystrophic Short stature, eye anomalies Witkop Normal Small primary, partial/total absence permanent Normal Thin, friable GJB6 (AD) MSX1 (AD) Incontinentia Pigmenti • Clinical • Streaky hyperpigmention (erythema, vesicular verrucous phases) • Abnormal teeth and hair • Neovascularization of retina • Neurological problems (seizures, developmental delay) • Genetics • XLD male lethal • IKBKG gene exons • 65% deletion exons 4-10 454 Ichthyosis • Ichthyosis vulgaris – most common – semidominant – filaggrin • X-linked – steroid sulfatase • AR congenital ichthyosis – collodion membrane • Severe form – harlequin ichthyosis • Other forms – erythroderma and lamellar ichthyosis • Multiple genes – TGM1 50-60%; 90% lamellar; ABCA12 >93% harlequin Epidermolysis Bullosum • Blistering of skin • EB Simplex – splitting in or above basal layer • Types • • • • Localized Generalized Intermediate Mottled Pigmentation Generalized Severe • Genes: Genes: EXPH5, KRT5, KRT14, TGM5 • Junctional EB – AR – within basement membrane • LAMB3 (70%), COL17A1, LAMC2, LAMA3 • Dystrophic EB - scarring below basement membrane • AD or AR – COL7A1 • Kindler syndrome – multiple cleavage planes • kindlin-1 (FERMT1) Alpha-1-Antitrypsin • Clinical • Pulmonary emphysema (heterozygotes and homozygotes • Hepatic cirrhosis (homozygotes, specific alleles) • Panniculitis • Pi type - electrophoretic mobility • inhibitor of neutrophil elastase • M allele in 95% Caucasians • SERPINA1 alleles with decreased production or function • null mutations only associated with emphysema • Z allele: glu to lys at codon 342 • 10-15% Pi activity • impaired release from hepatocytes (liver toxicity) • Treatment • No smoking • Antioxidants (vitamin E) • Transplantation (lung, liver) • Alpha-1-AT intravenous augmentation By Laura Fregonese, Jan Stolk [CC-BY-2.0 (www.creativecommons.org/licenses/by/2.0)], via Wikimedia Commons 455 Hirschsprung Disease • Congenital intestinal aganglionosis • 80% rectosigmoid • 15-20% to sigmoid (long segment) • 5% entire colon Pratap et al. BMC Pediatrics 2007 7:5 doi:10.1186/1471-2431-7-5[CC-BY-2.0 • 5:1 males to females (http://creativecommons.org/licenses/by/2.0) • Major gene RET (AD loss of function) • Many other genes involved rarely (GDNF, NRTN, EDNRB, EDN3, ECE1, NRG1, SEMA3C, SEMA3D) • 12% occur as component of syndrome (some chromosomal, especially Down syndrome) Alagille Syndrome • Clinical • Deficiency/atresia intrahepatic bile ducts • Cholestasis, neonatal jaundice • Skeletal anomalies, ocular anomalies (posterior embryotoxon) • Characteristic facial appearance • Genetics • AGS1- 94% • JAG1 (Jagged-1 is ligand for Notch receptor) • Microdeletion 20p12 in 7% • AGS2 • NOTCH2 Bile Pigment Metabolism • Loss of bilirubin conjugation – Crigler-Najjar syndrome • UGT1A1 deficiency • CN1 – bili 20-40 mg/dl • CN2 – bili 5-20 mg/dl (partial loss of function mutations) • Gilbert syndrome – reduced expression due to promoter polymorphism mild/intermittent hyperbili • Dubin-Johnson syndrome – benign MRP2 (ABCC2) deficiency • Progressive familial intrahepatic cholestasis (ATP8B1, ABCB11, ABCB4) 456 Hemochromatosis • Excessive Fe absorption • Fe overload in tissues • cirrhosis • diabetes mellitus • heart failure • more severe manifestations in males • Treat with phlebotomy • HFE • Autosomal recessive, 1/400 Caucasians • 85% C282Y • Affected genotypes: C282Y/C282Y, C282Y/H63D • TRF2 • Younger age of onset • Juvenile hemochromatosis: HFE2, HEPC Familial Hypercholesterolemia • Deficiency of LDL receptor – LDLR -60-80% • Other genes: APOB, PCSK9 liver • Collectively 1:200 – 1:250 • Heterozygotes: hypercholesterolemia, atherosclerosis, xanthomas B-100 cholesterol • Homozygotes: xanthomas, premature atherosclerosis LDL cholesterol amino acids • Treatment with diet, statins, other cholesterol lowering drugs, PCSK9 inhibitors Lipoprotein Disorders Disorder Gene(s) Hypercholesterolemia LDLR, APOB, PCSK9, AD/AR LDLRAP1 Inheritance Hypercholesterolemia, xanthomas, atherosclerosis; A/hypobetalipoproteinemia MTP, APOB Diarrhea, vomiting, abdominal distension, deficiency of fat soluble vitamins Chylomicron retention SAR1B Tangier disease ABCA1 AR LCAT Deficiency LCAT AR Chylomicronemia LPL, APOC2 Hypertriglyceridemia APOA5, LMF1, GP1HBP1 AR Elevated triglycerides Dysbetalipoproteinemia APOE2 AR Elevated cholesterol and triglycerides; vascular disease Cholesterol ester storage disease (Wolman) LIPA AR Hypercholesterolemia, hepatomegaly – Wolman disease is severe childhood form with hepatosplenomegaly, steatorrhea, anemia – lysosomal acid lipase deficiency AR Phenotype Unable to synthesize intestinal apoB-48; like hypobetalipoproteinemia Orange tonsils, hepatosplenomegaly, corneal opacities, coronary artery disease, neuropathy; lack of HDL Anemia, renal failure, corneal opacities, low HDL Abdominal pain, nausea, vomiting, pancreatitis, hepatosplenomegaly; elevated TG 457 Retinitis Pigmentosum • Clinical • Degeneration of photoreceptors or retinal pigment epithelium • Night blindness, progressive visual loss • Abnormal ERG and visual fields • Posterior subcapsular cataracts • Genetics • Multiple genes/modes of inheritance • RHO 20-30% AD • RPGR 80% X linked • Digenic inheritance – PRPH2, ROM1 Other Retinal Disorders • Usher Syndrome • RP plus congenital sensorineural hearing impairment, vestibular dysfunction • Type 1 – profound congenital deafness, later onset RP (MYO7A, USH1C, CDH23, PCDH15, USH1G, C1B2) • Type 2 – mild – moderate congenital deafness, adolescent/adult RP (ADGRV1, WHRN, USH2A) • Type 3 – slowly progressivevariable sensorineural hearing loss, RP (CKRB1, HARS) • Leber Congenital Amaurosis • Onset first year • AR – multiple genes • Poor vision, RP, eye rubbing • Gyrate Atrophy • Patches of choroidal, retinal atrophy • Increased ornithine, deficient ornithine-ketoacid aminotransferase • Choroidermeia • X-linked • Stippling, atrophy of fundus • Cone-Rod Dystrophy • Loss of cone function, reduced rod function • Alstrom, Bardet-Biedl, neuronal ceroid lipofuscinoses, Joubert syndrome Red-Green Color Blindness • X-linked recessive • 8% European males, 3-4% African ancestry males • Severe • Protanopia – no red cones • Deuteranopia – no green cones • Milder • Protanomaly – anamolous green cones • Deuteranomaly – anomalous red cones 458 Deafness • Sensorineural vs. conductive • 50% prelingual deafness genetic, 30% of which nonsyndromic • Syndromic • AD: Waardenburg, Branchio-oto-renal, Stickler, NF2 • AR: Usher, Pendred, Jervell & Lange-Nielsen, Biotinidase def, Refsum • XL: Alport, Mohr-Tranedjaerg (deafness, dystonia, optic atrophy) • Mitochondrial • Nonsyndromic • DFNA: AD • DFN3A: GJB2 • DFNA3B: GJB6 • DFNA11: MYO7A (also Usher syndrome and DFNB2 AR deafness) • DFNB: AR • DFNB1: GJB2 (may also be compound het for GJB2 mutation & GJB6 del) • DNFB2: MYO7A Autism Spectrum Disorders: DSM 5 • Autism spectrum disorder – no subgroups • Symptom domains • Social communication impairment • Restricted interests/repetitive behaviors • Symptoms can be present or reported in past history • Describe in terms of genetic cause, level of language and intellectual disability, presence of other medical problems • Social Communication Disorder – no repetitive behaviors Autism-Spectrum Disorders • Associated Features • Regressive onset in 30% • Seizures 25% • Dysmorphology 15-20% • Microcephaly 5-15% • Macrocephaly 30% • Genetics • Genetic cause found in 20-25% • If cause unknown, sibling risk 5-10% for ASD, 10-15% for milder abnormalities 459 ASD: Chromosomal Abnormalities • Cytogenetically visible change 5% • 15q11-q13 duplication maternally derived: 1-3% (supernumerary isodicentric 15q) • Trisomy 21 (7%) • 45,X Turner syndrome • CNVs • 2q37 • 7q11.23 (Williams syndrome) • 8q • 15q11 (Prader-Willi syndrome) • 16p11.2 • 17p11.2 deletion (Smith-Magenis) • 17p11.2 duplication (Potocki-Lupski) • 22q13.3 (Ohelan-McDermid – SHANK3) • Xp22.3 ASD: Single Gene Disorders • Syndromes • Fragile X • PTEN macrocephaly syndrome • Sotos syndrome • Rett syndrome • Tuberous sclerosis complex • Metabolic disorders (mitochondrial, PKU, adenylosuccinate lyase, creatine deficiency disorders, Smith-Lemli-Optiz • Isolated single gene mutations Psychiatric Disorders • Schizophrenia • Delusions, hallucinations, disorganized speech and behavior, flattened affect, poverty of speech, avolition, failure to maintain daily functions of life • Affective disorders • Major depression (MDD): dysphoria, changes in sleep, appetite, loss of sense of selfworth, guilt, suicidal ideation, no manic episodes • Bipolar disease (BPD): periods of mania and depression • Genetics • Multifactorial • Some candidate genes may overlap between disorders • 22q11.2 deletion and schizophrenia • CNVs in families with psychiatric disorders • Genetic counseling based on analysis of patient and family history • Pharmacogenetic considerations for treatment 460 Other Neuropsychiatric Conditions • Specific reading disability • unexplained difficulty in learning to read/write • Familial clustering – multiple candidate loci • Attention deficit/hyperactivity • Excessive inattention, hyperactivity/impulsivity • High heritability (0.76) • Candidate loci in catecholamine and serotonin systems • Addictive disorders • Heritability 40-70% • Multiple candidate loci (MAOA and COMT) Cardiomyopathy • Clinical • Decrease in cardiac output • Heart failure and arrhythmia • Dilated vs. hypertrophic • Genetics • Mutations in contractile apparatus proteins • Genetic and non-genetic etiologies • All modes of inheritance • Infectious • Storage disorders (hemochromatosis) • Treatment • ACE inhibitors • Beta blockers Hypertrophic Cardiomyopathy • Hypertrophy of myocardium • Enlarged myocytes, disarray, fibrosis • Signs/Symptoms – age of onset varies • Congestive heart failure • Chest pain • Arrhythmia • Sudden death • LV hypertrophy by echo/ECG • AD inheritance • Contractile apparatus – MYH7, TNNT2/3, MPBPC3, TPM1, ACTC1, MYL3, MYL2, others 461 Dilated Cardiomyopathy • Congestive heart failure • Arrhythmias/abnormal conduction • Thromboembolic events (left ventricular mural thrombus) • Various modes of inheritance • Surveillance every 2-5 years for first degree relatives (exam, ECG, echocardiogram) Syndromic vs. Non-syndromic • Syndromic • Hemochromatosis • Muscular dystrophies • Carvajal syndrome (palmoplantar keratoderma & wooly hair) – AR • Barth syndrome (neutropenia, lactic acidosis, growth retardation, 3-methylglutaconic acid – Xlinked • Mitochondrial Genetics of DHM • More than 30 genes account for 40-50% cases • TTN mutations in 20% cases • Copy number changes in some genes • AD: TTN, LMNA, MYH7, MYH6, SCN5A, MYBPC3, TNNT2, BAG3, ANKRD1, RBM20, TMPO, LDB3, TCAP, VCL, TPM1, TNNI3, TNNC1, ACTC1, ACTN2, CSRP3, DES, NEXN, PSEN1, PSEN2, SGCD, EYA4, PLN, DSG2 • XL: DMD, TAZ 462 Long QT Clinical Syncope, cardiac arrest Associated with increase in sympathetic activity Treated with β-blockers, pacemakers, sympathetic ganglionectomy Genetics Romano-Ward: KVLQT1 Homozygous mutations cause Jervell & Lange-Nielsen syndrome (hearing loss) Other genes: HERG (K channel) SCN5A (Na channel) minK (K channel) MiRP1 (K channel) Hereditary Hemorrhagic Telangiectasia • Clinical • Cutaneous and visceral AV malformations • Epistaxis • GI bleeding • Pulmonary AVM – stroke and brain abscess • Genetics • AD • ENG, ACVRL1, SMAD4, GDF2 Proteus syndrome • Progressive segmental overgrowth • Skeleton • Skin • Adipose tissue • CNS • AKT1 mosaic mutations in 90% • C.49G>A (p.Glu17Lys) • sporadic By Darryl Leja, NHGRI [Public domain], via Wikimedia Commons 463 Epigenetics Normal development One male, one female pronucleus Failure to develop Two male pronuceli Two female pronuceli DNA Methylation Gene Ac vator Promoter methyltransferase cytosine Me CG Me Me CG CG Promoter 5-methylcytosine Gene 464 Methylation Marks Erased in Germ Cells Rett Syndrome • Clinical • Developmental regression • Seizures • Loss of motor coordination • Stereotypies • Atypical Rett syndrome – variable severity • Genetics • XLD • MeCP2 loss of function mutations Willard and Hendrich, Nature Genetics 23, 127 - 128 (1999) Prader-Willi and Angelman Syndromes 465 PWS/AS Mutations Beckwith-Wiedemann Syndrome • Clinical • Macrosomia • Macroglossia • Omphalocele • Hemihyperplasia • Dysmorphism • Risk of tumors • Hepatoblastoma • Wilms’ tumor • Genetics • 85% sporadic • Some AD • 1:13,000 births BWS Region Chromosome 11 466 IC1 Mutations in BWS IC2 Mutations in BWS UPD in BWS 467 Paternal 11p Duplication Russell-Silver Syndrome • Clinical • Low birth weight • Relative macrocephaly • FTT in infancy • Delayed growth & bone age • Skeletal asymmetry • Urogenital anomalies • Some with developmental delay • Genetics • Usually sporadic • <5% Maternal UPD7 • 11p imprinting disorder 11p Duplication in RSS 468 Hypomethylation in RSS GNAS ֱ ֯ NESP55 NESP55 XL Sɲ XL Sɲ Exon1A Exon1A Gs ɲ Exons 2-13 Gs ɲ Exons 2-13 tissue-specific expression Albright hereditary osteodystrophy (intellectual disability and subcutaneous calcification): paternal transmission of Gsɲ mutation Pseudohypoparathyroidism-Ia (AHO and resistance to multiple hormones): maternal transmission of Gsɲ mutation Pseudohypoparathyroidism-Ib (renal parathyroid resistance): loss of maternal methylation at exon 1A Transient Neonatal Diabetes • Clinical • IUGR • Severe neonatal diabetes that regresses around 12 weeks • May relapse at times of stress • Genetics • TNDM1 locus on 6q24: PLAGL1 and HYMA1 • Paternally expressed, maternally methyated • UPD(6)pat – 40% • Dup(6q24)pat – 32% • Maternal hypomethylation TNDM1 – 28% 469 470 Systems-Based Disorders II SYSTEMS-BASED DISORDERS II John A. Phillips, III, MD, FACMG David T. Karzon Professor of Pediatrics Professor of Pathology, Microbiology and Immunology and Professor of Medicine Director Division of Medical Genetics and Genomic Medicine Vanderbilt University School of Medicine John A. Phillips, III, MD, FACMG Division of Medical Genetics Vanderbilt University School of Medicine DD-2205 Medical Center North Nashville, TN 37232-2578 (615) 322-7602 Telephone (615) 343-0959 Fax [email protected] 473 474 Systems Based Disorders II John A Phillips III David T Karzon Prof of Pediatrics Vanderbilt University Medical Center Site Investigator: 1) PKU: BMN 015 &165, 2) Achondroplasia: BMN 111-901, 201 & 202 Clinical Trials BioMarin Pharmaceutical Inc. & 3) FAOD: UX007 Ultragenyx Pharmaceuticals, Inc. PI: TN State Genetics Contract & Member TN Genetics Advisory Committee Co-PI: Vanderbilt Undiagnosed Disease Network (UDN) Clinical Center Co-I: Dr Blackwell’s PPG “Mechanisms of Familial Pulmonary Fibrosis”. 4/21/2017 Learning Objectives • Pulmonary: CF, HPAH, IPF & Primary Ciliary Dyskinesia • Immunogenetics: ADA, CVID, Hyper IgE/M & SCID • Hematology: Hemophilia A/B, SS, ɲThalassemia/XL ID & ɴ Thalassemia • Endo: Androgen Insensitivity Syndrome, XL Adrenal Hypoplasia, Antley Bixler Syndrome, CAH, Familial Hyperinsulinism, Hypophosphatasia, GnRH def & PROP1 Deficiency • Connective: Achondroplasia, Hypo/Pseudo-achondroplasia; MED, COL (OI; Achondrogenesis, Kniest, SED & Stickler); CHH, Diastrophic Dysplasia, EDS, Cutis Laxa, ɎX Elasticum & Marfan/LDS • Renal: BOR, Lowe Syndrome, Polycystic Kidney Disease & AD Tubulointestinal Diseases 475 Pul: Cystic Fibrosis (CFTR) • Clin: Respiratory, exocrine pancreas, intestine, vas deferens, hepatobiliary & sweat glands • 15-20% neonates meconium ileus • >95% CF males infertile • CAVD in men w/o pulm or GI problems • DX: 2 sweat Cl > 60 mEq/L OR 2 CFTR disease causing muts (AR); ȴF508 ~0.7 of ~1500 CF alleles • Rx: antibiotics, bronchodilators, mucolytic (pulmonzyme, mucomyst), ? Kalydeco/ivacaftor G551D & chest PT; avoid smoking, resp viruses & dehydration. Pul: Cystic Fibrosis (CFTR) Pul: Cystic Fibrosis- Mutation Specific Rx 476 Pul: Heritable Pulmonary Arterial Hypertension (HPAH) • Clin: dyspnea (60%), fatigue (19%), chest pain (7%), palpitation (5%) or edema (3%); PA pressure>25 mmHg (rest)/ >30 (exercise) & other causes of PAH excluded; • Increased PA pressure causes right heart failure & death within 3 yrs of Dx Pul:Heritable Pulmonary Arterial Hypertension (HPAH) • Gen: ~6% of PAH cases familial, AD with ~10% penetrance, variable age onset & ? anticipation, 2.4 females/male; ~75% BMPR2 & ACVRL1, BMPR1B, CAV1, ENG & SMAD9 all rare • Rx: sc/iv Treprostinil; po Bosentan, Sildenafil; neb Iloprost & iv epoprostenol; avoid hypoxia, amphetamines & estrogens Pul: Idiopathic Pulmonary Fibrosis (IPF) • Clin: Bibasilar reticular anomalies/nodules on high res CT, abnl lung func (VC), Dx usually 50-70 yrs; +/- lung Ca; 30-50% 5 yr survival • Gen: TERT, TERC & RTEL1 (short telomeres) or SFTPC in 8-15% multiplex & 3% simplex; all AD reduced penetrance; PF also occurs in Hermansky Pudlak (AR) & Dyskeratosis Congenita (AD, AR, XL) • Rx: Supp O2, lung transplant & no smoking 477 Pul: Primary Ciliary Dyskinesia (PCD) • Clin: Abnl situs, sperm motility & cilia structure/function; chronic otosinopulmonary disease, > 75% have ‘neonatal respiratory distress’ & adults have bronchiectasis. Males usually infertile.Situs inversus totalis 40%-50% & heterotaxy ~12%. • Gen: AR, Dx: clinical, ciliary structure or DNA (~2/3 have 2 variants in 1/32 PCD causing genes. • Rx: Immunizations (influenza/pneumococcal vaccines); hand washing, avoid sick contacts, clean respiratory devices; antibiotics for respiratory. Monitor lung function, sputum cultures & hearing. Avoid: cough suppressants, smoking & air pollutants. Learning Objectives • Pulmonary: CF, HPAH, IPF & Primary Ciliary Dyskinesia • Immunogenetics: ADA, CVID, Hyper IgE/M & SCID • Hematology: Hemophilia A/B, SS, ɲThalassemia/XL ID & ɴ Thalassemia • Endo: Androgen Insensitivity Syndrome, XL Adrenal Hypoplasia, Antley Bixler Syndrome, CAH, Familial Hyperinsulinism, Hypophosphatasia, GnRH def & PROP1 Deficiency • Connective: Achondroplasia, Hypo/Pseudo-achondroplasia; MED, COL (OI; Achondrogenesis, Kniest, SED & Stickler); CHH, Diastrophic Dysplasia, EDS, Cutis Laxa, ɎX Elasticum & Marfan/LDS • Renal: BOR, Lowe Syndrome, Polycystic Kidney Disease & AD Tubulointestinal Diseases Immun: Adenosine Deaminase Def • Clin: SCID with FTT, opportunistic infections, marked lymphocytopenia; absent humoral & cellular; usually Dx< 6/12 • Gen: <1% ADA activity (purine metabolism) or 2 known ADA causing muts (AR) • Rx: antibiotic, antifungal, IV immunoglobulin (IVIg), Pneumocystis prophylaxis; bone marrow/stem cell transplant; PEG ADA ERT 478 Immun: Common Variable Immune Def (CVID) • Clin: Humoral immun def after 2yrs (often young adults), sinopulmonary (Strep, H flu, Kleb pn), meningitis after bacterial infections, chronic diarrhea, malabsorption,+/- lymphoid hyperplasia, autoimmune, lymphomas • Gen: IgG<100 mg/dL to low, poor response Pneumovax; loss TAC1, CD19, BAFFER protein; TNFRSF13B(TAC1) (10-15%), ICOS (<1%) muts (AD, AR) • Rx: Immune globulin (IVIg), antibiotics, monitor lymphoma, thyroid function Immun: AD Hyper IgE Syndrome • Clin: Boils, cyst forming pneumonia & very high IgE; characteristic face, Chiari malform,+/- eczema, candiasis, osteopenia, fractures, scoliosis, arterial tortuosity & aneurysms • Gen: IgE >2000 IU/mL (~15x); STAT3 (AD) • Rx: antibiotics to prevent Staph absecess/pn Immun: X Linked Hyper IgM Syndrome • Clin: IgG & A low; IgM due to abnl B & T cell function; 50% onset by 1yr, >90% by 4yr, recurrent respiratory bacterial, recurrent diarrhea with FTT; neutro & thrombopenia, anemia; 10-15% CNS infections; liver, GI, pancreatic tumors; lymphoma (Hodgkins) & EBV • Gen: CD40LG (aka TNFSF5/CD154) muts in 95% affected males (XL) • Rx: Allogenic hematopoietic cell transplantation (HCT), recombinant granulocyte stim factor (G-CSF) for neutropenia, antibiotics & Pneumocystis prophylaxis 479 Immun: XL SCID • Clin: Severe combined cellular & humoral immuno-deficiency due to absent B & T cells, present 1-3/12 with FTT, oral/diaper candidiasis, absent tonsils & lymph nodes, recurrent & persistent infections despite Rx • Gen: NBS (TRECs) in 34 states, IL2RG muts in >99% of affected males • Rx: Antibiotics (Pneumocystis), IVIg, avoid live viral vaccines; bone marrow transplantation ASAP, gene therapy? Learning Objectives • Pulmonary: CF, HPAH, IPF & Primary Ciliary Dyskinesia • Immunogenetics: ADA, CVID, Hyper IgE/M & SCID • Hematology: Hemophilia A/B, SS, ɲThalassemia/XL ID & ɴ Thalassemia • Endo: Androgen Insensitivity Syndrome, XL Adrenal Hypoplasia, Antley Bixler Syndrome, CAH, Familial Hyperinsulinism, Hypophosphatasia, GnRH def & PROP1 Deficiency • Connective: Achondroplasia, Hypo/Pseudo-achondroplasia; MED, COL (OI; Achondrogenesis, Kniest, SED & Stickler); CHH, Diastrophic Dysplasia, EDS, Cutis Laxa, ɎX Elasticum & Marfan/LDS • Renal: BOR, Lowe Syndrome, Polycystic Kidney Disease & AD Tubulointestinal Diseases Hem: Hemophilia A • Clin: Prolonged oozing after trauma, tooth extractions or surgery, age at DX relates to F8 activity, severe joint /deep muscle bleeds <2yrs; 10% carriers bleed • Gen: Low F8 clotting activity with nl von Willebrand factor level; F8 muts in 98% of affected males (XL), IVS 22 inversion in 48% of severe cases & dels/dups in 6% • Rx: Hemophilia center, IV F8, DDAVP; avoid ASA, IM injections, impact sports & activities & always Rx before circumcision 480 Hem: Hemophilia B • Clin: Prolonged oozing after trauma, tooth extractions or surgery, age at DX relates to F9 activity, severe joint /deep muscle bleeds <2yrs, 10% carriers bleed • Gen: Low F9 clotting activity; F9 muts in ~100% of affected males (XL), dels/dups 3% • Rx: Hemophilia center, IV F9; avoid ASA, IM injections, impact sports & activities; always Rx before circumcision Ryan White Hem: Sickle Cell Disease • Clin: Intermittent vaso-oclusive events & hemolytic anemia; dactylitis, splenic infarction/asplenia, cholelithiasis, PAH & leg ulcers • Gen: HBB muts ɴS (Glu6Val), ɴC,ɴPunjab, ɴOArab (AR); SS 60-70%; SS & SC <3.6% & Sɴthal >3.6% Hb A2 • Rx: Hydration, transfusion, penicillin, hydroxyurea; Rx PAH phosphodiesterase inhibs/nitric oxide; monitor CBC, retics, Fe; liver & renal function 481 Hem: Alpha Thalassemia • Clin: Significant Hb Bart hydrops fetalis (Hb Bart Syn) & HbH disease with 90 vs 5% Hb Bart • Gen: Hb Bart syn, HbH, ɲ thal trait, Silent carrier & nl have deletions of 4, 3, 2, 1 & 0 ɲ globin genes, respectively; dels 90% & 10% point muts; ɲ thal moderates SS • Rx: Hb Bart syn fatal, HbH transfuse prn, avoid excess Fe Rx & sulphonamides Hem: Alpha Thalassemia XL Intellectual Disability Syndrome (ATRX) • Clin: Microcephaly, telecanthus, coarse facies, genital anomalies, hypotonia, ID & ɲ thal. Global delays in infancy & may never walk or develop speech. • Gen: PE, ID, hypotonia, HbH, FHx suggesting XL with 95% sequence & 5% del/dup ATRX variants. • Rx: High caloric formula; anticholinergics, botulinum toxin injection of salivary glands &/redirection of submandibular ducts for excessive drooling; antibiotic prophylaxis & vaccinations (pneumococcal/meningococcal) in those with asplenia. Anemia rarely requires treatment. Hem: Beta Thalassemia • Clin: Reduced ɴ globin causes microcytic hypochromic anemia & HbA; ɴ thal major > severe anemia & hepatosplenomegaly <2 yrs ; marrow expansion • Gen: RBC indices, Hb A & months; nucleated RBCs Hb F >12 • Rx: Regular transfusion, Fe chelation, bone marrow transplant, folic acid & splenectomy? ;monitor endocrine function; avoid ETOH & iron meds 482 Learning Objectives • Pulmonary: CF, HPAH, IPF & Primary Ciliary Dyskinesia • Immunogenetics: ADA, CVID, Hyper IgE/M & SCID • Hematology: Hemophilia A/B, SS, ɲThalassemia/XL ID & ɴ Thalassemia • Endo: Androgen Insensitivity Syndrome, XL Adrenal Hypoplasia, Antley Bixler Syndrome, CAH, Familial Hyperinsulinism, Hypophosphatasia, GnRH def & PROP1 Deficiency • Connective: Achondroplasia, Hypo/Pseudo-achondroplasia; MED, COL (OI; Achondrogenesis, Kniest, SED & Stickler); CHH, Diastrophic Dysplasia, EDS, Cutis Laxa, ɎX Elasticum & Marfan/LDS • Renal: BOR, Lowe Syndrome, Polycystic Kidney Disease & AD Tubulointestinal Diseases Endo: Androgen Insensitivity Syndrome • Clin: undermasculinization external genitalia at birth, abnl secondary sexual devel in puberty & infertility with 46,XY karyotype. AIS spectrum: Complete (CAIS), with typical female external genitalia, Partial (PAIS) with predominantly female, male, or ambiguous genitalia & Mild (MAIS) with typical male external genitalia • Gen: 46,XY; XL due to AR variants. Dx: external genitalia, spermatogenesis, absent/rudimentary Müllerian structures, normal or testosterone (T), dihydroT & luteinizing hormone (LH). • Rx: CAIS removal of testes?, vaginal dilation; assignment of sex of rearing. Endo: XL Adrenal Hypoplasia Congenita (AHC) • Clin: Acute adrenal insufficiency by 3 wks in ~60%; vomiting, hypoglycemia & salt wasting with hyperkalemia & ACTH; +/- crytorchidism; • Gen: NROB1(DAX1) dels in 100% with glycerol kinase def +/- DMD; but point mutations in nearly all isolated AHC • Rx: IV glucose & NaCl; gluco & mineralocorticoids; oral NaCl 483 Endo: Cytochrome P450 Oxidooreductase Def (Antley Bixler) • Clin: Steroidogenic defect ranging from cortisol deficiency to Antley Bixler syndrome (ABS) with ambiguous genitalia, craniosynostosis, choanal atresia, radio humeral synostosis & cortisol • Gen: sterol/steroid abnormalities, POR muts (AR) • Rx: Cortisol, tracheostomy, surgery for craniosynostosis & hypospadias Endo: Congenital Adrenal Hyperplasia • Clin: > 90% CYP21OHD, impaired cortisol synthesis by adrenal cortex, simple virilizing (25%) & salt wasting (low cortisol AND inadequate aldosterone) (75%), NBS of neonates lowers risk for initial fatal salt wasting crisis • ACTH causes adrenal hyperplasia & over-production of 17 OHP & sex hormones • Gen: CYP21A2 sequencing panel or del/dups detects 80-98% (AR) • Rx: glucocorticoid (increase with stress), salt wasting add mineralocorticoid & NaCl Endo: Familial Hyperinsulinism • Clin: Hypoglycemia (ranges from severe neonatal to mild childhood onset) • Gen: ~45% ABCC8 (97% in Ashkenazi Jews) & 5% KCNJ11 (AR), ~5% GLUD1 & 5% HNF4A (AD with anticipation) • Rx: IV glucose, diazoxide, diet , pancreatic resection & avoid fasting 484 Endo: Hypophosphatasia • Clin: Prenatal: hypomineralization bone +/or teeth & limb deformaties. Range: stillbirth to lower extremity fractures in adults. Six forms: 1) Perinatal (severe) respiratory insufficiency & hypercalcemia, 2) Perinatal (benign) prenatal skeletal changes slowly improve, 3) Infantile onset birth-6/12 of FTT, rickets & Sx, 4) Childhood (juvenile) low bone density, fractures & premature loss of teeth, 5) Adult stress fractures of lower extremities in middle age & 6) Odontohypophospha-tasia premature loss of primary teeth +/or severe caries without skeletal problems. • Gen: AR serum alkaline phosphatase (ALP) & 1-2 pathogenic ALPL variants &ј phosphoethanolamine. • Rx: Infantile/childhood types: ERT (asfotase alfa), respiratory care, Rx hypercalcemia, sz with B6 & craniosynostosis. Other types: dental care by 1 yr; NSAIDs for osteoarthritis, bone pain & osteomalacia. Avoid: Bisphosphonates & excess vit D. Endo: Isolated Gonadotropin Releasing Hormone (GnRH) Def • Clin: Low testosterone in males & estradiol females; LH & FSH with hypogonadism, +/- micropenis/ cryptorchidism, small testes, absent puberty, 60% anosmia (aka Kallmann or KS), bimanual synkinesis • Gen: CHD7, FGF8, FGFR1, KAL1, ROK2, PROKR2 muts ~25% (AD), KAL1 ~10% KS (XL); > 15 genes cause ~1/2 of Normosomic isolated GnRH Deficiency • Rx: Testosterone, hCG in males & estrogen, progestins in females to induce pubertal changes Endo: WZKWϭ Related Combined Pituitary Hormone Def • Clin: Combined Pituitary Hormone Deficiency (CPHD) with GH, TSH, LH, FSH & PrL +/- ACTH deficiencies; short stature, FTT in childhood • Gen: PROP1 muts > 98% (AR) Nature Gen 18: 147-149, 98 • Rx: GH until 17 yrs, L thyroxine, +/testosterone or estrogens, +/hydrocortisone 485 Learning Objectives • Pulmonary: CF, HPAH, IPF & Primary Ciliary Dyskinesia • Immunogenetics: ADA, CVID, Hyper IgE/M & SCID • Hematology: Hemophilia A/B, SS, ɲThalassemia/XL ID & ɴ Thalassemia • Endo: Androgen Insensitivity Syndrome, XL Adrenal Hypoplasia, Antley Bixler Syndrome, CAH, Familial Hyperinsulinism, Hypophosphatasia, GnRH def & PROP1 Deficiency • Connective: Achondroplasia, Hypo/Pseudo-achondroplasia; MED, COL (OI; Achondrogenesis, Kniest, SED & Stickler); CHH, Diastrophic Dysplasia, EDS, Cutis Laxa, ɎX Elasticum & Marfan/LDS • Renal: BOR, Lowe Syndrome, Polycystic Kidney Disease & AD Tubulointestinal Diseases Conn Tissue: Achondroplasia & Hypo-chondroplasia (&'&Zϯ) • Clin: Rhizomelic short stature, macrocephaly/ ICP, obstructive apnea; kyphosis, lordosis; narrowing interpedicular distance; trident hand, genu varum; cranio-cervical compression (CC) & spinal stenosis • Dx: Signs & X rays; FGFR3: Achondroplasia 98% G>A transition due to CpG results in Gly380Arg vs Hypochondroplasia 49% Asn540Lys (C>A) & 21% C>G • Rx: CNS shunt for ICP; sleep & CC apnea; otitis; orthopedics for gibbus, genu varum & spinal stenosis; avoid trampoline & gymnastics Conn Tissue: Achondroplasia & Hypo-chondroplasia (&'&Zϯ) 486 Conn Tissue: Pseudoachondroplasia (KDW) • Clin: Nl length at birth, nl facies, decline growth ~ 2 yrs, brachydactyly; loose hands, knees ankles but restricted elbows & hips; joint pain & ~1/2 lumbar lordosis • Dx: Signs & X rays show delayed epiphyses ossification & anterior beaking of vertebrae; COMP point muts • Rx: Surgery for lower limb malalignment, scoliosis & C1-2 fixation; watch for odontoid hypoplasia; avoid trampoline & gymnastics Conn Tissue: COL1A1/2 Related Osteogenesis Imperfecta (OI types 1-4) • Clin: Fx after min trauma, +/- dentinogenesis imperfecta (gray/brown) & hearing loss (adults) • OI type 1: Non-deforming with blue sclerae • OI type 2: Perinatal lethal • OI type 3: Progressively deforming • OI type 4: Variable OI with nl sclerae • Dx: Family Hx (fx/signs), X rays (fractures, wormian bones, codfish vertebrae & osteopenia) & COL1A1/2 molecular testing (90% sensitivity) & / or biochemical analysis of type 1 collagen (98% sensitivity) in OI types 1-4 Conn Tissue: COL1A1/2 Related Osteogenesis Imperfecta 487 Conn Tissue: COL1A1/2 Related Osteogenesis Imperfecta • Gen: Molecular testing COL1A/2 detects > 90% of OI types 1- 4 (AD, > 95% seq changes & 2% del/dup); biochemical detects 90, 98, 84 & 84% of OI types 1- 4 & 60, 100, ~100% of types 1-3 de novo, respectively • Rx: Orthopedic & Otolaryngology management, periodic dental & hearing eval; bisphosphonates, oral alendronate or risedronate & GH may reduce fractures, increase bone density & improve growth Conn Tissue: Osteogenesis Imperfecta Types 5-10 • OI Types 5-7: Fx; no dentinogenesis (D) or hearing loss (HL); abnl vertebrae & hyperplastic callous; OI Type 5: IFITM5 (AD); OI Type 6: SERPINF1 (AR); OI Type 7: rhizomelic shortening of all limbs; CRTAP (AR) • OI Type 8: Fx, no D or HL; short limb dwarfism; gracile long bones; LEPRE1 (AR) • OI Type 9: Fx, white/gray sclerae, short limb dwarfism, bowed limbs; PPIB (AR) • OI Type 10: similar to OI Type 1; caused by SERPINH1 (AR) Conn Tissue: Multiple Epiphyseal Dysplasia (AD) • Clin: Joint pain (hips & knees) in early childhood, decreased ROM, early arthritis & adult Ht lower nl or mild short • Dx: Clin & X ray (small, irregular epiphyses but nl spine except for Schmorl nodes); • Gen: COMP, COL9A1-3, MATN3 but 10-20% neg • Rx: Orthopedic, avoid sports involving joint overload & caution with NSAIDs 488 Conn Tissue: Multiple Epiphyseal Dysplasia (AR) • Clin: Joint pain (hips & knees), malformations (hands, feet & knees) & scoliosis; 50% had clubfoot, clinodactyly or CP at birth; adult Ht 150-180 cm • Dx: Clin & X ray; Gen: SLC26A2 seq variants in ~100% (AR) • Rx: Orthopedic, avoid sports involving joint overload & caution with NSAIDs Conn Tissue: Type II Collagenopathies (Achondrogenesis 2, Kniest, SED & Stickler) • Achondrogeneis Type 2 (Langer Saldino): micromelic dwarfism, CP, short ribs & abnl vert; stillborn/neonatal death; COL2A1(AD) de novo • Kniest: Short stature & trunk (platyspondyly); hearing loss (HL); myopia & retinal detach (MRD) & cataract; COL2A1 (AD) • Spondyloepiphyseal Dysplasia (SEDC): flat face/CP, MRD, abnl vert, cervical myelopathy; COL2A1 (AD) • Stickler: Flat face, MRD & cataract; HL, CP +/- Robin; COL2A1, COL11A1-2 (AD); COL9A1-3 (AR); 80-90% COL2A1, 10-20% COL11A1, others rare Conn Tissue: Type 2 Collagen Disorders Achondrogenesis Kniest / SED Stickler 489 Conn Tissue: Cartilage Hair Hypoplasia (Anauxetic Dysplasia Spectrum) • Clin: Severe short limbs, joint hypermobility, fine silky hair, immunodeficiency, anemia, GI dysfunction & risk for malignancy • Gen: metaphyseal dysplasia, +/- epiphyseal & vertebral dysplasia; RMRP (AR) • Rx: Transfusions, surgery cervical vert & kyphosis, antibiotics (neutropenia), immunoglobulin if IgG low, BMT (SCID); avoid live vaccines; varicella can be lethal; 11% malignancies Conn Tissue: Diastrophic Dysplasia • Clin: short limbs, nl skull, hitchhiker thumbs, spine (scoliosis, lordosis, kyphosis), joint contractures & osteoarthritis; CP 1/3, cystic ears 2/3, clubfeet • Gen: Clinical & radiologic confirmed by SLC26A2 >90% have sequence variants (AR) • Rx: PT, casting, ortho surgery with caution as deformities tend to recur, watch C spine for cord compression Conn Tissue: Ehlers Danlos Syndrome • Classic (I & II): skin hyperext, abl wound healing & joint hypermobility • Hypermobility (III): soft skin, dislocations, pain +/- aortic dilation • Vascular (IV): thin, translucent skin; bruising, vascular rupture (12% death secondary to arterial/Uterine rupture in pregnancy); GI perforation • Kyphoscoliotic (VI): friable, hyper extensible skin; scars, bruising, hypotonia, progressive scoliosis & fragile sclerae 490 Conn Tissue: Ehlers Danlos Syndrome Ehlers Danlos Syndrome Gen & Rx • I & II ~50% have COL5A1/2 seq, del/dup (AD) • III TNXB haploinsufficiency very rare (AD) • IV COL3A1 seq >95%, del/dup 2% (AD) • VI deoxypyridinoline/pyridinoline ratio in urine (HPLC) due to deficient lysyl hydroxylase 1 (PLOD1) activity (fibroblasts); PLOD1 seq?, del/dups ~18% (AR) • Rx: I/II care with sutures, ascorbic acid? avoid ASA & contact sports (CS); III watch aorta & avoid CS/joint hyperextension; VI ortho, opthal, pregnancy risks & avoid CS; IV avoid CS & arteriography Conn Tissue: Cutis Laxa • Clin: Skin is “doughy”, furrowed especially neck, axillae & groin, droopy on face & extends without hyper elasticity; myopia & hernias • Gen: ATP6VOA2 (AR) thick cortex & cerebellar anomalies; EFEMP2 aka FBLN4 (AR) arterial tortuosity & aneurysms; FBLN5 (AR, AD) pulmonary emphysema & peripheral PS • Rx: avoid smoking 491 Conn Tissue: Pseudoxanthoma Elasticum • Clin: Affects elastic tissue of skin, eye, CV & GI systems; skin (papules); retina (streaks or hemorrhage); GI bleeds, angina/ claudication • Gen: Skin & eye findings & skin biopsy; ABCC6 seq ~90% both alleles with dels in 5-30% (AR) • Rx: Intraocular injections for macular degeneration; avoid contact sports, ASA, NSAIDs; retinal exams in pregnancy Conn Tissue: Marfan Syndrome • Clin: Ocular (myopia; ectopia in 60%); skeletal (dolichostenomelia, pectus, scoliosis); aortic dilation/ tear/ rupture/ MV prolapse; Systemic Score (thumb, wrist, pectus, pneumothorax, etc) • Gen: Fam Hx, exam (especially ectopia lentis & aneurysm) FBN1 seq 70-93%, dels/dups? (AD) 25% de novo (mosaicism?) • Rx: Lens surg, ɴ blockers/losartan for aortic dilation, surgery 5 cm or if 1 cm/yr; yearly opthal & ECHO; avoid contact sports, isometric; CV stimulants & LASIK; CV risk with pregnancy Conn Tissue: Loeys Dietz Syndrome • Clin: Vascular (CNS, thoracic & abdominal arterial aneurysms/dissections) & skeletal (pectus excav/ carin; scoliosis, lax joints, arachnodactyly & clubfeet. • Continuum; 75% LDS type I have (hypertelorism, bifid uvula/CP & craniosynostosis) & 25% LDS Type II lack craniofacial findings • Gen: TGFBR1/2 in 95% (AD), ¾ de novo • Rx: Aortic dissection at smaller diameters than Marfan, ɴ blockers, C spine instability; avoid contact sports & CV stimulants 492 Learning Objectives • Pulmonary: CF, HPAH, IPF & Primary Ciliary Dyskinesia • Immunogenetics: ADA, CVID, Hyper IgE/M & SCID • Hematology: Hemophilia A/B, SS, ɲThalassemia/XL ID & ɴ Thalassemia • Endo: Androgen Insensitivity Syndrome, XL Adrenal Hypoplasia, Antley Bixler Syndrome, CAH, Familial Hyperinsulinism, Hypophosphatasia, GnRH def & PROP1 Deficiency • Connective: Achondroplasia, Hypo/Pseudo-achondroplasia; MED, COL (OI; Achondrogenesis, Kniest, SED & Stickler); CHH, Diastrophic Dysplasia, EDS, Cutis Laxa, ɎX Elasticum & Marfan/LDS • Renal: BOR, Lowe Syndrome, Polycystic Kidney Disease & AD Tubulointestinal Diseases Renal: Branchiootorenal Spectrum Disorders • Branchiootorenal (BOR) ear pits, tags, anomalies causing deafness > 90%; branchial fistulae/cysts & renal hypoplasia, dysplasia or agenesis • Branchiootic Syn (BOS) is BOR without renal anomalies • Gen: Clin criteria EYA1 40% (AD); SIX5 & SIX1 ~5% (AD) • Rx: Excision fistulae/cysts, ear surgery, aids, cochlear implants Renal: Lowe Syndrome • Clin: Eyes: cataracts, glaucoma & poor vision; CNS: hypotonia, absent DTRs & 75-90% mild to severe ID & Renal: Fanconi, RTA, renal rickets, phosphaturia, aminoaciduria & renal failure by 10-20 yrs • Gen: OCRL seq variants in 95% affected males & carriers (XL); enzyme in fibroblasts • Rx: Remove cataracts, GERD; oral Na/KHCO3, PO4 & calcitriol; avoid contact lens 493 Renal: Polycystic Kidney Disease, AD • Clin: Late onset, bilateral renal cysts; liver & pancreatic cysts; CNS/aortic aneurysms & MVP; renal pain, hypertension, renal failure by 60 yrs • Dx: Renal US ш 3 cyts PPV = 100%; Gen: 85% PKD1 & 15% PKD2 most seq but few del/dups (AD); contiguous PKD1 & TSC2 del > PKD in utero & Tuberous Sclerosis; rare early onset PKD with neg FH due to hypo morph PKD1 in trans • Rx: Hypertension, pain, cyst decompression, nephrolithiasis, clip small & aortic replacement for large aneurysms; avoid nephrotoxic, caffeine & smoking Renal: Polycystic Kidney Disease, AD Renal: Polycystic Kidney Disease, AR • Clin: Neonates with enlarged echogenic kidneys: ~50% hepatomegaly, dilated bile ducts & echogenicity; pulmonary hypoplasia with 30% dying by 1 yr of resp insuff & >50% have renal failure in first decade • Dx: Clin findings without renal cysts in parents; ~80% PKHD1 seq variants, dups/dels seen • Rx: Resp failure, hypertension; avoid NSAIDS, aminoglycosides & caffeine 494 Renal: AD Tubulointestinal Kidney Disease • Clin: Hyperuricemia & gout from renal excretion uric acid, hyperuricemia & gout as early as teens; creatinine 5-40 yrs & renal failure >40 yrs; isosthenuria may exacerbate bouts of dehydration • Gen: UMOD, REN or MUC1 (AD) • Rx: Allopurinol/probenecid for gout; nephrology, peritoneal dialysis, renal transplant; avoid nephotoxic meds, dehydration & meat References Genetic Home References @ https://ghr.nlm.nih.gov GeneReviews @ https://ghr.nlm.nih.gov OMIM @ https://www.omim.org 495 496 Reproductive Genetics II REPRODUCTIVE GENETICS II Louise E. Wilkins-Haug, MD, PhD, FACMG Division Director, Maternal Fetal Medicine and Reproductive Genetics Brigham & Women’s Hospital Professor of Obstetrics, Gynecology and Reproductive Biology Harvard Medical School Louise E. Wilkins-Haug, MD, PhD, FACMG Division of Maternal Fetal Medicine Brigham & Women’s Hospital 75 Francis Street Boston, MA 02115 (617) 732-4208 Telephone (617) 264-6310 Fax [email protected] 499 500 Reproductive Genetics 2 Louise Wilkins-Haug MFM Division Director Brigham and Women’s Hospital Disclosure(s) - None Case – Returns for 18 week ultrasound මWhat is it? Omphalocele මCould it have been seen sooner? Possibly මRisk of undiagnosed aneuploidy? About 10% මRisk of genetic etiologies? මHow to proceed? 501 Beckwith Wiedemann Syndrome LGA, macroglossia, omphalocele, hypertrophy, increased risk of cancer මIsolated omphalocele on fetal US ම20% have BWS (Wilkins-Haug 2009) Beckwith Wiedemann Syndrome Several causes – 50% due to imprinting error on chromosome 11 ම Will require specialized testing Association with Assisted Reproductive Technologies (ART) ම 6 fold increase in ART (5% vs 1%) ම Among ART pregnancies, 1/4000 risk of BWS ම Majority have an imprint abnormality ම Loss of methylation on maternal 11p.15 (DeBaun, 2003;Maher, 2003; Gicquel, 2003; Halliday, 2004) Female germ cell development Preimplantation Ovarian stimulation Oocyte freezing IVF Imprints completed at MII Imprints completed by birth Male germ cell development Preimplantation (Denomme M, 2012) 502 Role of ART – Animal Studies Animal studies suggest a contribution of the ART ම Large offspring syndrome ම Ovulation induction, oocyte manipulation, culture media ම LOS associated with birth defects Extremes of imprint deregulation in cloned animals Imprinting assures male/female contribution / biologic diversity Alternative Hypothesis – Imprinting Associated with Subfertility Parental Questionnaire - children with Angleman, Beckwith Weidemann and Prader Willi Syndrome and controls Children ART Subfertile AS, BWS, PWS Control children 6.4% 2.1% 6.8% 3.5% Same 3 fold higher rate for subfertility without ART among children with imprinting disorders (Doornbos, M, 2007) Epimutations, Imprinted Genes and “Adult Onset” Disease Developmental Origins of Health and Disease (DOHaD) ම“Fetal programming of adult onset disease” මNutritional state and exposures during pregnancy increase adult onset disease – metabolic syndrome, diabetes, cardiovascular disease මDue to placental and fetal genetic reprogramming ම“Barker hypothesis” (Batcheller A, 2011) 503 Epimutations, Imprinted Genes and “Adult Onset” Disease ART children/teens are at higher risk of cardiometabolic disorders, specifically ම Elevated systolic and diastolic blood pressure ම Higher fasting glucose, triglycerides ම Increased body fat composition Persists when ම Sibling groups compared ම Corrected for parental variables (age, BMI, medical conditions) ම Corrected for newborn birth weight (Batcheller A, 2011; Liu, 2015; Pontisilli, 2015; Varoom, 2016 ) Epimutations, Imprinted Genes and “Adult Onset” Disease Mouse models මWider spread epigenomic changes produced by media changes මAssociated with superovulation in some but not all studies මART in mouse models produces specific epimutations leading to endothelial damage (Ramierz-Perez, 2014, Varoomam, 2016) Epimutations, Imprinted Genes and “Adult Onset” Disease Placenta මFour of the 6 imprinted genes (CEBPA, MEST, NNAT and SERPINF1) with aberrant hypomethylation following ART මlinked to adipocyte development, insulin signaling and/or obesity Cord blood – epigenome alterations Blastocysts ම> 50% had epimutations of imprinted genes (Katari S, 2009; Sakain, 2015. Estill, 2016, White 2015) 504 Case – How to get to an answer ? Diagnostic Modalities Chorionic villus sampling Amniocentesis Percutaneous umbilical blood sampling Risks of Invasive Fetal Diagnostic Procedures I - MISCARRIAGE III - TECHNICAL II - FETAL MORBIDITY Rupture of membranes Malformation Infectious Isoimmunization No sample obtained Misdiagnosis Risk of Miscarriage – CVS and Amniocentesis Compared Cochrane review of risks මTransabdominal CVS = 2nd trimester amniocentesis මTranscervical CVS = slightly higher risk of miscarriage Recent systematic review - miscarriage මLoss rates transabdominal CVS compared to amniocentesis Loss within 2 wks Loss up to 4 wks Loss duration over pregnancy CVS - TA 0.7% 1.3% 2.0% Amniocentesis 0.6% 0.9% 1.9% Publications Committee of the Society for Maternal Fetal Medicine, 2012 505 Risk of Miscarriage – CVS/ Amniocentesis vs No Procedure o Nonrandomized observational studies o TA CVS or amniocentesis loss rates similar to no invasive procedures o Amniocentesis loss rates appear no higher than 1/3001/500 or even 1/1000 o May be even lower in experienced centers Publications Committee of the Society for Maternal Fetal Medicine, 2012 Risk of Miscarriage Why was CVS cited with higher loss rates ? ම CVS less commonly performed, numerous centers to get sample size (> 30) ම Each with small numbers ම Learning curve - high rate of “difficult” procedures, > 1 insertions ම Comparison to amniocentesis ම No correction for earlier gestational age at CVS ම Adjusting for confounding variables, and data > 1998, CVS loss rate not significantly increased above the background Publications Committee of the Society for Maternal Fetal Medicine, 2012 Patient Specifics and Amniocentesis Loss Retrospective cohorts, <34 yo, loss < 28 wks Loss < 28 wks Prior loss (> 3) Prior vaginal bleeding No predisposing factors Cases (n=3,910) 2.1 % Controls (n=5,324) 1.5 % P 6.9% 3.5% <0.04 5.9% 3.8% <0.04 0.96% 0.93% NS <0.01 (Antsaklis, 2000) 506 Invasive procedures - Morbidity Limb-reduction defects after CVS මCVS between 10 and 13 weeks does not increase the risk of limb reduction defects Hemangioma මMay be increased following TC - CVS Invasive procedures - Morbidity Leakage of amniotic fluid(AF) after amniocentesis 1.7% leak AF (0.4% controls) Most reseal in 1 week oAF volume normal in 3 weeks oDelivery < 37 weeks 50%; IUGR 30% oPerinatal survival in > 90% of cases Spontaneous 2nd trimester premature rupture of membranes oHigh fetal loss rate (80%), low chance of survival (< 10%) oNo clear benefit to amniotic “plugging” and fluid replacement (Borgida,2000) Invasive procedures - Morbidity Fetomaternal hemorrhage: Placental disruption from the invasive testing මSmall relative to the total fetoplacental blood volume මUnsensitized Rh negative - receive anti-D immunoglobulin මAmniocentesis a better alternative with isoimmunization මRisk of worsening with CVS 507 Invasive procedures - Morbidity Infectious risks oHBV transmission – low unless active disease oHCV is unknown oHIV transmission lowest with antiretroviral therapy (HAART) oAmniocentesis only on HAART, preferably when the viral load is undetectable oCVS – theoretically avoid Technical Risks - “Misdiagnosis" on CVS Evaluation only by direct analysis මSynchiotrophoblast and mesenchymal core cells may differ Maternal cell contamination Confined placental mosaicism ම1-2% of CVS samples ම1/3 are fetal mosaicism, 2/3 are confined to placenta මRisks of uniparental disomy, fetal growth restriciton Twins Misdiagnosis on Amniocentesis Slow growing cultures Maternal Cell Contamination Sampling technique Maternal DNA polymorphisms FISH studies Direct DNA analyses 1) Discard 1.0 ml - MCCԜ>Ԝ5% in 26% vs 2% in first 1 ml discarded 2) Avoid placenta Weida J, 2016 508 Analysis of Fetal DNA Whole chromosome aneuploidy detection Microarray del/dup analysis Disease specific genetic panels, specialized studies Whole exome sequencing Microarray Utility in Obstetrics -Ultrasound Abnormalities (Hillman, 2013) CMA and specific anomalies Anomaly % with CMA anomaly Isolated - CNS (holoprosencephaly, cerebellar hypoplasia), 15 – 17 % - Skeletal (club foot, hand , other) 13 – 14 % Multiple - Cardiac (HLHS, Tetrology) 20 – 27 % - CNS (posterior fossa) 23 % - Cystic hygroma 17% Shaffer , 2012 509 Microarray Utility – Obstetrics Nondividing Cells SABs මadditional 9.8% with clinical relevance Stillbirths ම13% abnormal with prior normal or unobtainable karyotypes Preimplantation Genetic Diagnosis (Raca, 2009) Microarray Utility – Invasive testing Greater disease detection ම Structural/growth abnormality – 6.0% ම AMA (amniocentesis) ම0.5% known pathogenic changes ම1.2% potential for clinical significance Now supported as first line analysis for invasive prenatal diagnostic testing (ACOG) (Wapner, 2012) Single Gene Disorders – Targeted Panels vs Whole Exome Sequencing Targeted gene panels ම Chondrodysplasia, CNS, arthrogryposis ම Advantage of targeted, more complete analysis of suspected regions ම Disadvantage – requires US findings of syndrome be recognized Whole exome sequencing ම Wider range of detection but in less robust detail ම Advantages in detection of conditions with atypical, unknown prenatal presentations ම Disadvantage – higher rate of variants of unknown significance 510 WES and Prenatal Diagnosis Small series ම 30 fetuses/neonates with congenital abnormalities ම 10% genetic diagnosis in 3; potentially significant sequence variants in 5 more ම 24 fetuses with US anomalies ම 25% total detection rate, definitive diagnosis in five and plausible diagnosis in one Similar to pediatric literature ම 25% of patients with a suspected genetic disorder and prior negative genetic testing (Carrs, 2014; Drury 2014) Case Continued Couple declined invasive testing, infant delivers at 39 weeks and has a normal microarray and negative BWS testing. Undergoes successful repair. 2 years later - questions about another pregnancy What else places them at risk for birth defects? Susan is 39 years old, John is 40 ම Healthy, no prior surgeries, no current medications ම Non-smokers, deny recreational drug use and consume alcohol with meals ම Family history is non contributory for congenital anomalies, notable for diabetes / cardiovascular disease on both sides; both are of Northern European ancestry Exam notable for BMI = 40, otherwise benign Human Teratogen Characteristics (J Wilson, 1959) “Teratology” (David Smith, 1960s) 1) Susceptibility depends on genotype and adverse environmental factors 2) Susceptibility varies with the developmental stage of exposure 3) Teratogenic agents act on developing cells/tissues to initiate sequences of abnormal developmental events 4) The access of adverse influences to developing tissues depends on the teratogenic agent 5) There are four manifestations - Death, Malformation, Growth Retardation and Functional Defect 6) Dose response relationship 511 Human Teratogen Characteristics (T Shepard, 1994) Essential (1,2,3) or (1,3,4) 1) Exposure at critical time in pregnancy ම Coumadin and first trimester exposure 2) Two or more epidemiologic studies of high quality ම Valproic acid and neural tube defect 3) Clinical delineation of specific defects 4) Rare exposure associated with rare outcome ම Cataracts in children exposed to rubella Helpful 5) Teratogenicity in animal studies 6) Makes biologic sense 7) Proof in an experimental system Teratogen References 90% of medications have inadequate data for pregnancy Journals Books –Briggs, Shepard Web-based ම REPROTOX (https://reprotox.org) ම TERIS (Teratogen Information System – online Shepard’s) ම http://depts.washington.edu/terisweb/teris/ ම OTIS (Organization of Teratology Information Specialists) ම http://www.teratology.org/OTIS_fact_Sheets.asp ම www.mothertobaby.org/ Maternal Conditions of Concern for Teratogenicity Obesity Diabetes මType 1 මType 2 Alcohol Use 30% of women entering pregnancy 11 % of women 7.6% pregnant women, 1.4% binge (Reproductive age women - 51%, 15% binge) 512 Obesity and Congenital Anomalies Overweight BMI 25 -30 Obese BMI > 30 NTD 1.2 (1.04 – 1.38) 1.87 (1.62-2.15) Cardiovascular 1.17 (1.03-1.34) 1.3 (1.12 – 1.51) Cleft lip +/- palate 1.0 (0.87-1.15) 1.20 (1.03- 1.40) Risk begins with overweight (Stothard K, 2009,) Risk by Category of Obesity NTD Cardiac defects Orofacial clefts (Class I – III) BMI 3034.9 BMI 35 – 39.9 BMI > 40 1.80 (1.29-2.54) 1.11 (1.03-1.19) 2.07 (1.13-3.48) 1.26 (1.12-1.42) 4.08 (1.87-7.75) 1.49 (1.24-1.80) 1.09 (0.91-1.30) 1.62 (1.26-2.09) 1.90 (1.27-2.86) (Blomberg M, 2010) Altered Folate with Increased BMI Known link of congenital anomalies and folate (NTD, CHD) Increased BMI associated with decreased folate levels ම Altered absorption / metabolism ම Altered dietary intake (High fat diets low in folate) Supplementation studies ම Primary occurrence – obese women, NTD risk remains despite folic acid supplements (400 mcg/d) ම Pre and post food supply fortification with folate - highest BMI quartile had least NTD reduction Recurrence - folate protection only among women < 70 kg (Shaw G,1996, Werler M,1996) 513 Diabetes and Birth Defects Diabetes associated with birth defects මType 1 මInsulin deficient, often childhood onset මType 2 මInsulin resistant Process of hyperglycemia, altered lipid metabolism, oxidative stress and cell death Major Malformations and 1st Trimester HbA1C Joslin Clinic 1984-1992 45 40 HbA1C 35 < 6.1 6.1-9.0 9.1-12.0 12.1-15.0 > 15.0 30 % 25 20 15 10 5 0 RR (CI) 1.0 1.4 2.2 8.6 11.1 (0.8-3.2) (0.9-5.3) (4.2-17.3) (4.8-25.4) (Greene, 1994) Diabetic Embryopathy aOR 95% CI 130 34 -100 Renal Agenesis 12 3-46 Hydrocephaly/ MCA 12 3.7-40 Cardiac / MCA 11 6.2-19 All cardiac 4.6 2.9-7.5 Anencephaly 3.4 1.1-10 Sacral agenesis (Correa, 2008) 514 Diabetes Type 2 and Obesity Type 2 diabetes ම“adult onset diabetes” ම90% of diabetes diagnoses මAssociated with obesity, decreased exercise, increased abdominal girth මEstimated 20% of affected individuals undiagnosed (Langer O, 2000) Diabetes Type 2, Obesity and Birth Defects Significant risk for birth defects present only among obese GDM women Estimated 10-15% of GDM are Type 2 with abnormal fasting ම Associated with obesity (Martinez-Frias M,2005) Diabetes Cohort analyses of congenital anomalies to all diabetic pregnancies (Sheffield, J, 2002,) 515 Alcohol Exposure - FAS, FADS (FAE), ARND, and ARBD Fetal Alcohol Syndrome ම 6 – 9 / 1000 ම facial dysmorphia, delayed growth, central nervous system (CNS) impact Fetal Alcohol Disease Spectrum (Fetal Alcohol Effect) ම 2-5% of school age children ම Intellectual disabilities, behavior and learning challenges and congenital malformations. ම Alcohol – Related Neurodevelopment Disabilities ම Alcohol-related Birth Defects (ARBD) (May, P 2014; Cannon, 2016; CDC 2016) Alcohol screening, prevention ම ACOG - screen first prenatal visit and preconception ම Increase overall awareness ම 3 in 4 women “who want to get pregnant” consume alcohol (Behavioral Risk Factor Surveillance System (BRFSS), 2013) Alcohol as a Teratogen Absolute Alcohol per Day in Ounces 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 re ad in g pt s) (5 -7 ම Binge drinking (> 4 drinks in 2 hours or 1 day) ම Early, first trimester exposure IQ w ei gh t bi rth FA S ounces of absolute alcohol Drivers of alcohol induced effects on offspring 516 Alcohol as a Teratogen : Genetic Susceptibility Presentation varies by exposure levels but also between women with same exposure levels Dizygotic twins similarly exposed with discordant phenotypes In mice, strain specific phenotypic effects can be replicated Viral Infection as a Teratogen Common features include CNS involvement, placental infection and subtle longer term concerns in live-borns Toxoplasmosis – uncommon in US Rubella – uncommon post vaccination era CMV - omnipresent Zika - epidemics of 2015 – 2016 Shepard's Criteria and Zika Essential 1) Exposure at critical time in pregnancy - met ම 1st trimester microcephaly ම 3rd trimester exposure – IUGR, IUFD, CNS complications evolving in neonate 2) > 2 epidemiologic studies of high quality - emerging ම Ultrasound anomalies ම Zika + ම Zika - 12/42 (29%) 0/16 ම 1% microcephaly risk in 2013-14 French Polynesian Zika outbreak (50 fold increase) (Rasmussen, 2016) 517 Shepard's Criteria and Zika 3) Specific defects (vs range of congenital anomalies) - met ම Severe microcephaly, CNS calcifications, ocular findings, redundant scalp skin, arthrogryposis ම Fetal brain disruption syndrome with unique scalp redundancy (not usually seen with microcephaly) 4) Rare exposure with rare outcome - met ම Microcephaly a rare event ම Zika not rare in Brazil; disease in women with limited travel to the region is rare (Epes, 2017; Rasmussen, 2016, Moore, 2017) Shepard's Criteria and Zika Helpful 5) Teratogenicity in animal studies – not met මBut shown to be neurotropic 6) Makes biologic sense - met මSimilar to other fetal viral infections මRecovered from fetal CNS tissue, destructive / cell death as a mechanism for microcephaly. (Rasmussen, 2016) Zika and Teratogenicity 1) Delineate full spectrum ම Similar to Rubella and CMV - cataracts, hearing loss & developmental disability 2) Modifiers of infection ම Coinfection with another virus ම Preexisting immune response to another flavivirus ම Genetic background of the mother or fetus ම Severity of infection 3) Prevention ම Avoidance ම Vaccine 518 Return to Case Their biggest question though is since they are doing IVF, should they also have preimplantation genetic testing? a c b d Preimplantation Genetic Diagnosis (PGD) Translocations ම Array CGH replacing FISH Single Gene disorders (over 300 to date) ම Autosomal recessive, dominant, x-linked ම Sex determination for X-linked disorders Genetic disorders of adult onset ම Predisposition to cancer risk (BRCA1/2) HLA compatibility ම Siblings with ALL, AML or Diamond Blackfan anemia requiring bone marrow transplants (Munne, 2012, Curr Genet 13: 463-7) PGS – Preimplantation Genetic Screening With increasing maternal age, increasing aneuploidy ම Day 5-6 (blastocyst) ම 35-37 yo 44.2% aneuploidy ම 41-42 yo 76.3% aneuploidy 3 color FISH First approach – PGS-#1 ම Increasing probes increases “false positive” ම 2007-2010 RCTs – Day 3 (FISH) ම No benefit ම Harm with decreased implantation 5 color FISH 519 Preimplantation Genetic Screening v2 New molecular approaches for comprehensive chromosome screening (CCS) ම aCGH, SNP array, qPCR, next generation sequencing ම Karyomapping ම Simultaneous detection of single gene disorder and molecular aneuploidy in one assay (SNP analysis) Further analysis for mosaicism ම High levels at day 3, a portion correct by day 5 (Dahdouh, 2015, Wells, Fert Sterl, 2014) PGS#2 and Outcomes Meta-analysis 4 RCT and 7 cohort studies ම RCTs - about 250 cycles each arm ම Cohorts – 2,338 women, 729 CCS, remainder controls RCT studies (RR) 95% CI Cohort studies (RR) 95% CI Implantation 1.32 1.18 - 1.47 1.74 1.35 - 2.24 Clinical Pregnancy rate 1.26 0.83 - 1.93 1.48 1.20 - 1.83 Ongoing pregnancy rate 1.31 0.64 - 2.66 1.61 1.30 - 2.00 Livebirth 1.26 1.05 - 1.50 1.35 0.85 – 2.13 (Dahdouh, 2015, Chen, 2015 Wells, 2014) PGS #2 Confounders Data from RCT and cohorts ම Combines indications (AMA, reproductive pregnancy loss) ම Cleavage stage and blastocyst biopsies ම Various molecular techniques not equal in efficacy ම Biased toward women with “good prognosis” ම PGS on day 5 blastocysts favors those who make it to day #5 Analysis based on cycles started ම PGS significantly underperformed compared to non-PGS when starting point was cycle initiated (Gleicher,2014; Kushnir, 2016) 520 Current State of PGS ESHRE Study into The Evaluation of oocyte Euploidy by Microarray analysis (ESTEEM) ම multicentre, randomized double-blind controlled trial with an intention-to-treat analysis including women with advanced maternal age Policy Statements for PGS - ineffective in improving clinical pregnancy rates and decreasing miscarriage (Based on PGS #1) ම American Society of Reproductive Medicine ම European Society for Human Reproduction and Embryology ම British Fertility society Case They call your office 1.5 years later Maternal weight decreased through lifestyle management programs, ceased alcohol consumption Spontaneously conceived and just delivered a healthy girl Thank You For Your Attention [email protected] 521 REFERENCES Alfirevic Z, Gosden CM, Neilson JP. 2000. Chorion villus sampling versus amniocentesis for prenatal diagnosis. Cochrane Database Syst Rev. 2):CD000055. DeBaun MR, Niemitz ELFeinberg AP. (2003). Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet. 72(1):156-60. American College of Obstetricians and Gynecologists. Microarrays and next-generation sequencing technology: the use of advanced genetic diagnostic tools in obstetrics and gynecology. ACOG Committee opinion no. 682. (2016) Obstet Gynecol,128:e262-8 Denomme, M. M. and M. R. Mann (2012) Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies. Reproduction 144(4): 393-409. Antsaklis A, Papantoniou N, Xygakis A, Mesogitis S, Tzortzis EMichalas S. 2000. Genetic amniocentesis in women 20-34 years old: associated risks. Prenat Diagn. 20(3):247-50. Batcheller, A., E. Cardozo, et al. (2011) Are there subtle genome-wide epigenetic alterations in normal offspring conceived by assisted reproductive technologies? Fertil Steril 96(6): 130611. Borgida AF, Mills AA, Feldman DM, Rodis JFEgan JF. 2000. Outcome of pregnancies complicated by ruptured membranes after genetic amniocentesis. Am J Obstet Gynecol. 183(4):937-9. Carss KJ, Hillman SC, Parthiban V, et al. (2014) Exome sequencing improves genetic diagnosis of structural fetal abnormalities revealed by ultrasound. Human molecular genetics.23(12):3269-3277. Doherty AS, Mann MR, Tremblay KD, Bartolomei MSSchultz RM. (2000) Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod. 62(6):1526-35. Doornbos, M. E., S. M. Maas, et al. (2007). Infertility, assisted reproduction technologies and imprinting disturbances: a Dutch study. Hum Reprod 22(9): 2476-80. Dugoff L, Norton ME, Kuller JA. For Society for Maternal-Fetal Medicine (SMFM) (2016)The use of chromosomal microarray for prenatal diagnosis. Consult Series 41. Am J Obstet Gynecol, 215:B2-9. Drury S, Williams H, Trump N, et al. (2015). Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities. Prenatal diagnosis. 35(10):1010-1017. Carmichael, S. L., S. A. Rasmussen, et al. (2010). Prepregnancy obesity: a complex risk factor for selected birth defects. Birth Defects Res A Clin Mol Teratol 88(10): 804-10. Eppes C, Rac M, Dunn J, Versalovic J, Murray K, Suter M, Sanz Cortes M, et al (2017). Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017. Testing for Zika virus infection in pregnancy: key concepts to deal with an emerging epidemic. AJOG March 2017 epub Chang AS, Moley KH, Wangler M, Feinberg AP, Debaun MR. (2005). Association between Beckwith-Wiedemann syndrome and assisted reproduction technology: a case series of 19 patients. Fertil Steril 83(2):349-54 Gicquel, C., V. Gaston, et al. (2003). In vitro fertilization may increase the risk of BeckwithWiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet 72(5): 1338-41. Dahdouh E, Balayla J, Garcia-Velasco J. (2015). Impact of blastocyst biopsy and comprehensive chromosome screening technology on preimplantation genetic screening: a systematic review of randomized controlled trials. Reprod Biomed Online 30(3) 281-9. Gilboa, S. M., A. Correa, et al. (2010). Association between prepregnancy body mass index and congenital heart defects. Am J Obstet Gynecol 202(1): 51 e1-51 e10. Davies M., V. M. Moore, et al. (2012). Reproductive technologies and the risk of birth defects. N Engl J Med 366(19): 1803-13. Hansen M, Kurinczuk JJ, Bower CWebb S. (2002). The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med. 346(10):725-30. Hansen, M., C. Bower, et al. (2005). Assisted reproductive technologies and the risk of birth defects--a systematic review. Hum Reprod 20(2): 328-38. Hansen, M., J. J. Kurinczuk, et al. (2013). Assisted reproductive technology and birth defects: a systematic review and meta-analysis. Hum Reprod Update. Hillman, S. C., S. Pretlove, et al. (2011). "Additional information from array comparative genomic hybridization technology over conventional karyotyping in prenatal diagnosis: a systematic review and meta-analysis." Ultrasound Obstet Gynecol 37(1): 6-14. Hillman SC, Willams D, Carss KJ, McMullan DJ, Hurles ME, Kilby MD. (2015). Prenatal exome sequencing for fetuses with structural abnormalities: the next step. Ultrasound Obstet Gynecol.45(1):4-9. Katari, S., N. Turan, et al. (2009). DNA methylation and gene expression differences in children conceived in vitro or in vivo. Hum Mol Genet 18(20): 3769-78. Langer, O. (2008). Type 2 diabetes in pregnancy: exposing deceptive appearances. J Matern Fetal Neonatal Med 21(3): 181-9. Ludwig, M., A. Katalinic, et al. (2005). Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet 42(4): 289-91. Halliday, J., K. Oke, et al. (2004). Beckwith-Wiedemann syndrome and IVF: a case-control study. Am J Hum Genet 75(3): 526-8. Papantoniou NE, Daskalakis GJ, Tziotis JG, Kitmirides SJ, Mesogitis SA, Antsaklis AJ. 2001. Risk factors predisposing to fetal loss following a second trimester amniocentesis. Bjog. 108(10):1053-6. Reefhuis, J., M. A. Honein, et al. (2009). Assisted reproductive technology and major structural birth defects in the United States. Hum Reprod 24(2): 360-6. Shaw, G. M., E. M. Velie, et al. (1996). Risk of neural tube defect-affected pregnancies among obese women." JAMA 275(14): 1093-6. Shaffer L, Rosenfeld JA, Dabell MP, et al. (2012) Detection rates of clinically significant genomic alterations by microarray analysis for specific anomalies detected by ultrasound. Prenatal Diagnosis.32(10):986-995. Shepard T. (1994) “Proof of human teratogenicity. Teratology 50:97-98. Sheffield, J. S., E. L. Butler-Koster, et al. (2002). Maternal diabetes mellitus and infant malformations. Obstet Gynecol 100(5 Pt 1): 925-30. Stothard, K. J., P. W. Tennant, et al. (2009). Maternal overweight and obesity and the risk of congenital anomalies: a systematic review and meta-analysis.JAMA 301(6): 636-50. Towers CV, Asrat TRumney P. (2001). The presence of hepatitis B surface antigen and deoxyribonucleic acid in amniotic fluid and cord blood. Am J Obstet Gynecol. 184(7):1514-8; discussion 1518-20. Maher E R. (2005). Imprinting and assisted reproductive technology. Hum Mol Genet 14:133-8. van den Veyver IB, Eng CM. (2015).Genome-Wide Sequencing for Prenatal Detection of Fetal Single-Gene Disorders. Cold Spring Harbor perspectives in medicine. 5(10) Martinez-Frias, M. L., J. P. Frias, et al. (2005). Pre-gestational maternal body mass index predicts an increased risk of congenital malformations in infants of mothers with gestational diabetes. Diabet Med 22(6): 775-81. Waller DK, Mills JL, Simpson JL, S et al. (1994). Are obese women at higher risk for producing malformed offspring? Am J OB Gyn: 170:541-8. Wapner, R. J., Martin C.L., et al. (2012). "Chromosomal microarray versus karyotyping for prenatal diagnosis." N Engl J Med 367(23): 2175-84. Wells D. (2014). Next-generation sequencing: the dawn of a new era for preimplantation genetic diagnostics. Fertil Steril 101 (5) 1250-1. Werler, M. M., C. Louik, et al. (1996). "Prepregnant weight in relation to risk of neural tube defects." JAMA 275(14): 1089-92. Wilkins-Haug L., A. Porter, et al. (2009). Isolated fetal omphalocele, Beckwith-Wiedemann syndrome, and assisted reproductive technologies. Birth Defects Res A Clin Mol Teratol 85(1): 58-62. 522 Reproductive Genetics – Preconception References Alfirevic Z, Gosden CM, Neilson JP. 2000. Chorion villus sampling versus amniocentesis for prenatal diagnosis. Cochrane Database Syst Rev. 2):CD000055. American College of Obstetricians and Gynecologists. Microarrays and next-generation sequencing technology: the use of advanced genetic diagnostic tools in obstetrics and gynecology. ACOG Committee opinion no. 682. (2016) Obstet Gynecol,128:e262-8 Anthony S, Buitendijk SE, Dorrepaal CA, Lindner K, Braat DDden Ouden AL. (2002) Congenital malformations in 4224 children conceived after IVF. Hum Reprod. 17(8):208995. Antsaklis A, Papantoniou N, Xygakis A, Mesogitis S, Tzortzis EMichalas S. 2000. Genetic amniocentesis in women 20-34 years old: associated risks. Prenat Diagn. 20(3):247-50. Batcheller, A., E. Cardozo, et al. (2011) Are there subtle genome-wide epigenetic alterations in normal offspring conceived by assisted reproductive technologies? Fertil Steril 96(6): 130611. Blomberg, M. I. and B. Kallen (2010) Maternal obesity and morbid obesity: the risk for birth defects in the offspring.Birth Defects Res A Clin Mol Teratol 88(1): 35-40. Borgida AF, Mills AA, Feldman DM, Rodis JFEgan JF. 2000. Outcome of pregnancies complicated by ruptured membranes after genetic amniocentesis. Am J Obstet Gynecol. 183(4):937-9. Carss KJ, Hillman SC, Parthiban V, et al. (2014) Exome sequencing improves genetic diagnosis of structural fetal abnormalities revealed by ultrasound. Human molecular genetics.23(12):3269-3277. Carmichael, S. L., S. A. Rasmussen, et al. (2010). Prepregnancy obesity: a complex risk factor for selected birth defects. Birth Defects Res A Clin Mol Teratol 88(10): 804-10. Chang AS, Moley KH, Wangler M, Feinberg AP, Debaun MR. (2005). Association between Beckwith-Wiedemann syndrome and assisted reproduction technology: a case series of 19 patients. Fertil Steril 83(2):349-54 Dahdouh E, Balayla J, Garcia-Velasco J. (2015). Impact of blastocyst biopsy and comprehensive chromosome screening technology on preimplantation genetic screening: a systematic review of randomized controlled trials. Reprod Biomed Online 30(3) 281-9. Davies M., V. M. Moore, et al. (2012). Reproductive technologies and the risk of birth defects. N Engl J Med 366(19): 1803-13. DeBaun MR, Niemitz ELFeinberg AP. (2003). Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet. 72(1):156-60. 523 Denomme, M. M. and M. R. Mann (2012) Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies. Reproduction 144(4): 393-409. Doherty AS, Mann MR, Tremblay KD, Bartolomei MSSchultz RM. (2000) Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod. 62(6):1526-35. Doornbos, M. E., S. M. Maas, et al. (2007). Infertility, assisted reproduction technologies and imprinting disturbances: a Dutch study. Hum Reprod 22(9): 2476-80. Dugoff L, Norton ME, Kuller JA. For Society for Maternal-Fetal Medicine (SMFM) (2016)The use of chromosomal microarray for prenatal diagnosis. Consult Series 41. Am J Obstet Gynecol, 215:B2-9. Drury S, Williams H, Trump N, et al. (2015). Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities. Prenatal diagnosis. 35(10):1010-1017. Eppes C, Rac M, Dunn J, Versalovic J, Murray K, Suter M, Sanz Cortes M, et al (2017). Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017. Testing for Zika virus infection in pregnancy: key concepts to deal with an emerging epidemic. AJOG March 2017 epub Gicquel, C., V. Gaston, et al. (2003). In vitro fertilization may increase the risk of BeckwithWiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet 72(5): 1338-41. Gilboa, S. M., A. Correa, et al. (2010). Association between prepregnancy body mass index and congenital heart defects. Am J Obstet Gynecol 202(1): 51 e1-51 e10. Halliday, J., K. Oke, et al. (2004). Beckwith-Wiedemann syndrome and IVF: a case-control study. Am J Hum Genet 75(3): 526-8. Hansen M, Kurinczuk JJ, Bower CWebb S. (2002). The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med. 346(10):725-30. Hansen, M., C. Bower, et al. (2005). Assisted reproductive technologies and the risk of birth defects--a systematic review. Hum Reprod 20(2): 328-38. Hansen, M., J. J. Kurinczuk, et al. (2013). Assisted reproductive technology and birth defects: a systematic review and meta-analysis. Hum Reprod Update. Hillman, S. C., S. Pretlove, et al. (2011). "Additional information from array comparative genomic hybridization technology over conventional karyotyping in prenatal diagnosis: a systematic review and meta-analysis." Ultrasound Obstet Gynecol 37(1): 6-14. Hillman SC, Willams D, Carss KJ, McMullan DJ, Hurles ME, Kilby MD. (2015). Prenatal exome sequencing for fetuses with structural abnormalities: the next step. Ultrasound Obstet Gynecol.45(1):4-9. Katari, S., N. Turan, et al. (2009). DNA methylation and gene expression differences in children conceived in vitro or in vivo. Hum Mol Genet 18(20): 3769-78. 524 Langer, O. (2008). Type 2 diabetes in pregnancy: exposing deceptive appearances. J Matern Fetal Neonatal Med 21(3): 181-9. Ludwig, M., A. Katalinic, et al. (2005). Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples. J Med Genet 42(4): 289-91. Maher E R. (2005). Imprinting and assisted reproductive technology. Hum Mol Genet 14:133-8. Martinez-Frias, M. L., J. P. 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Next-generation sequencing: the dawn of a new era for preimplantation genetic diagnostics. Fertil Steril 101 (5) 1250-1. 525 Werler, M. M., C. Louik, et al. (1996). "Prepregnant weight in relation to risk of neural tube defects." JAMA 275(14): 1089-92. Wilkins-Haug L., A. Porter, et al. (2009). Isolated fetal omphalocele, Beckwith-Wiedemann syndrome, and assisted reproductive technologies. Birth Defects Res A Clin Mol Teratol 85(1): 58-62. 526 Genomic Medicine GENOMIC MEDICINE Bruce R. Korf, MD, PhD, FACMG Wayne H. and Sara Crews Finley Chair in Medical Genetics Professor and Chair, Department of Genetics Director, Heflin Center for Genomic Sciences University of Alabama at Birmingham Bruce R. Korf, MD, PhD, FACMG Department of Genetics University of Alabama at Birmingham Kaul 230, 1530 3rd Avenue South Birmingham, AL 35294-0024 (205) 934-9411 Telephone (205) 934-9488 Fax [email protected] 529 530 Genomic Medicine Bruce R. Korf, MD, PhD Professor and Chair, Department of Genetics University of Alabama at Birmingham Disclosure(s) Relationship Entity Grant Recipient Novartis Advisory Board Accolade, Genome Medical Board of Directors American College of Medical Genetics and Genomics Children’s Tumor Foundation Advisor Neurofibromatosis Therapeutic Acceleration Project Founding Member Envision Genomics Salary University of Alabama at Birmingham Outline • Pharmacogenetics • Genetic Risk Assessment • Genome Sequencing 531 Individuality Experience Culture Environment Ancestry • Risk of disease • Response to treatment • Side effects Pharmacogenetics and Personalized Medicine Administer the right drug at the right dose to the right person at the right time. Drug Metabolism • Absorption • GI • Tissue spaces Phase I • Metabolism • Activation • Inactivation • Excretion • Kidney • Liver Phase II 532 Pharmacogenetic Nomenclature: Diplotypes A1 A2 B1 B2 Haplotypes: A1-B1 or A2-B2 Diplotype: A1-B1/A2-B2 Star System – most common diplotype is *1; other diplotypes *2, *3, etc. Cytochrome Oxidases • CYP2D6 (debrisoquine hydroxylase) – poor, intermediate, extensive, ultrarapid metabolizers • CYP2C9 – Warfarin metabolism • CYP2C19 – clopidigrel prodrug • CYP3A4 • CYP3A5 Class Examples Analgesics Codeine, hydrocodone, tramadol Antiarrhythmics Encainide, mexiletine, propafenone Antidepressants Amitriptyline, desipramine, fluoxetine, fluvoxamine, imipramine, nortriptyline, paroxetine Antihistamines Chlorpheniramine, diphenhydramine, promethazine Antipsychotics Haloperidol, thioridazine Beta Blockers Carvedilol, metoprolol, propranolol, timolol Cough suppressants Codeine, dextromethorphan CYP2D6 excretion Warfarin 7-hydroxywarfarin CYP2C9 polymorphisms S-Warfarin NADH NAD+ VKORC1 Vitamin K oxidized Vitamin K reduced activated clotting factors 533 Clopidigrel Clopidigrel prodrug Cyp2C19 Clopidigrel Active form Thiopurine S-methyltransferase • S-methylation of heterocyclic sulfhydryls • 6-mercaptopurine, 6-thioguanine • Leukemia, autoimmune diseases • TPMT involved in inactivation of drugs • 10% subjects have intermediate activity, 0.3% low activity • Low or intermediate activity associated with increased toxicity and requires lowering of dose N-acetyltransferase 2 • Transfer of acetyl groups to amine and hydrazine substrates p-Aminosalicylic acid • Slow acetylators • ~50% population • Homozygous for allelic variants • Increased toxicity from isoniazid (tuberculosis therapy), hydralazine (hypertension therapy), and other drugs (chemotherapeutics, clonazepam, nitrazepam, procainamide, sulfasalazine) Acetyl CoA p-Aminoacetylsalicylic acid N-acetyltransferase CoA 534 Drug Transporters • Efflux of drugs and toxins from cells • Role in chemotherapeutic drug transport • ABCB1, ABCC1, ABCC2, ABCG2 • OATPs – membrane-bound influx transporters • Antibiotics, cardiac glycosides, chemotherapeutic drugs • OATP1 – SLCO1B1*5 – statin induced myopathy • OCTs • SLC22A gene family • Renal proximal tubule • Metformin, procainamide HLA Variants • HLAB*1502 – Stevens-Johnson syndrome (carbamazepine) • HLAB*5701 – Hypersensitivity to abacavir Malignant Hyperthermia • Sustained muscle contraction, hyperthermia • Triggered by halogenated anesthetics with succinylcholine • Autosomal dominant inheritance • Genes • RYR1 (ryanodine receptor) -70% • Also myopathies • CACNA1S (calcium channel) – 1% also hypokalemic periodic paralysis • others 535 Pharmocogenetics Knowledgebase Clinical Pharmacogenetics Implementation Consortium (CPIC) – PharmGKB and Pharmacogenetics Research Network – peer reviewed guidelines Choice of Therapy • Matching the treatment to pathophysiology of disease • Genetic variants that predict response • Patterns of gene expression that reveal disease subtypes • Examples: • Herceptin and EGFR amplification • Erlotinib or gefinitib and EGFR mutation • Vemurafinib and V600E in melanoma Cancer Genomes Normal Tumor Sequence Difference = cancer-speciÀc genetic changes 536 Treatment of Genetic Disease ivacaftor Potentiator Corrector Everolimus Treatment in TSC hamartin tuberin Rheb GDP mTOR SEGA Everolimus Renal AML growth & proliferation Molecular Therapy Normal Stop Mutation splice switching oligomers Aminoglycoside Readthrough exon skip 537 Genome Editing Non-homologous end joining Mutation at site of repair Homology-directed repair Repair with Donor DNA Clinical Trials Drug Discovery 538 The Diagnostic Odyssey Clinical Problem History & Physical Interpretation Genetic Testing Differential Diagnosis Exome –Capture Technology (2007) Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Fragment and anneal to oligo probes Elute HTS Analyze 27 539 Analysis of Genomic Data Evidence Levels • Pathogenic • • • • PVS1 – very strong PS1-4 – strong PM1-6 - moderate PP1-5 – supporting • Benign • BA1 – stand alone • BS1-4 – strong • BP1-6 – supporting 540 Diversity of category of germline results that can be found WES clinical reports Gene Mutation Example Related to Patient’s Phenotype Other Medically Actionable Pathogenic Pathogenic Pathogenic ARID1A VUS Rare MECP2 missense SCN5A mut mtDNA MELAS mut PCG Genes Recessive Carrier Genes FDA Indication Pathogenic CYP2A mut CFTR ΔF508 Modified from Scollon et al., Genome Medicine, 2014 541 Human Phenotype Ontology Yang et al., :D͕2014 – Description of 2000 proband only WES clinical cases • 1780 (89%) are pediatric patients (<18 yrs) • 1440 (72%) have intellectual disability, seizure disorder or autism • Diagnostic rate ~25% for patients referred for diagnostic WES 542 Molecular Diagnosis Rate 25%: Varies for Different Phenotypic Groups Overall (n=2000) Non-neurologic (n=244) Neurologic plus (n=1147) Neurologic only (n=526) Specific Neurologic (n=83) 0% 10% 20% 30% 40% 50% Diagnostic rate (+/- 95% CI) Yang, et al., JAMA, 2014 Mutations in Positive WES Cases 1.1% 0.8% 0.7% 0.1% 0.1% 0.1% missense frameshift 8.1% nonsense splice 18.9% 48.9% in-frame large deletions start codon stoploss 21.0% promoter region mitochondrial 708 Mutant Alleles in the 504 Positives, 409 (58%) novel at time of reporting Most AD Mutant Alleles Arose de Novo (AD: 74%; XL: 62%) X-LINKED, 13% Mito, 0.2% de novo, 74% AD, 53% AR, 36% unknown, 14% inherited, 11% 543 Genomic Newborn Screening Genomic Prenatal Diagnosis Genomic Carrier Screening 544 Incidental Findings • Constitutional mutations on minimal list should be reported regardless of age of patient • Laboratories should seek and report specific types of mutations on list • Ordering clinician responsible for pre- and post-test counseling • Patients may opt out of learning about incidental findings ACMG Secondary Findings Type Genes Tumor Predisposition Breast/ovarian, Li-Fraumeni, Peutz-Jeghers, Lynch, Polyposis, Von HippelLindau, MEN1/2, Medullary thyroid cancer, PTEN hamartoma syndrome, Retinoblastoma, Paraganglioma/pheochromocytoma, Tuberous sclerosis complex, WT1-related Wilms’ tumor, NF2 BRCA1/2, TP53, STK11, MLH1, MSH2, MSH6, PMS2, APC, MUTYH, BMPR1A, SMAD4, VHL, MEN1 RET, PTEN, RB1, SDHD, SDHAF2, SDHC, SDHB, TSC1, TSC2, WT1, NF2 Connective Tissue Dysplasia Ehlers-Danlos vascular type, Marfan, Loeys-Dietz, Familial aortic aneurysms and dissections COL3A1, FBN1, TGFBR1, TGFBR2, SMAD3, ACTA2, MYH11 Cardiac Hypertrophic cardiomyopathy, dilated cardiomyopathy, Arrhythmia MYBPC3, MYH7, TNNT2, TNNI3, TPM1, MYL3, ACTC1, PRKAG2, GLA, MYL2, LMNA, RYR2, PKP2, DSP, DSC2, TMEM43, DSG2, KCNQ1, KCNH2, SCN5A Metabolic Hypercholesterolemia, Wilson disease, Ornithine transcarbamylase deficiency LDLR, APOB, PCSK9, ATP7B, OTC Malignant Hyperthermia RYR1, CACNA1S Personal Genomes Carrier Status Mendelian Disorder Pharmacogenetics Risk Assessment 545 WGS Workflow 546 Syndromes Every Geneticist Shoud Know SYNDROMES EVERY GENETICIST SHOULD KNOW Originally prepared by Amy Roberts, MD, FACMG Boston Children’s Hospital Updated by Bruce R. Korf, MD, PhD, FACMG University of Alabama at Birmingham 549 550 Syndromes Every Geneticist Should Know Originally prepared by Amy Roberts, MD, Boston Children’s Hospital; Updated by Bruce R. Korf, MD, PhD, University of Alabama at Birmingham 22q11 DELETION SYNDROME CARDIOVASCULAR SYSTEM (DiGeorge, Velocardiofacial syndrome, Shprintzen syndrome) Responsible genes: TBX1 Proteins: T box 1 transcription factor C Cytogenetic locus: 22q11.2 Inheritance: AD; 93% de novo Clinical Features and Diagnostic Criteria: congenital heart disease (74%) (TOF, IAA B, conotruncal defects), immune dysfunction, palate abnormalities (69%), feeding problems, developmental delay, learning problems (70-90%), hypocalcemia (50%), renal anomalies (37%), psychiatric disorders, medial deviation of the internal carotids Clinical Tests: serum Ca, PTH, T/B Cell subsets, Ig’s, post vaccine Ab’s, renal US, video laryngoscopy Molecular Tests: FISH, microarray, or MLPA for DGCR deletion (95%). 3-Mb deletion most common; no clear genotype-phenotype relationship to del size. (A small % with S/Sx 22q11 del without a DGCR deletion have 10p13-p14 deletion) Disease Mechanism: Abnormal development of the pharyngeal arches related to TBX1 dosage Treatment/Prognosis: Standard Tx for CHD, palate repair, pharyngeal flap, Ca replacement, no live vaccines if immunodeficient 22q11 DELETION SYNDROME CARDIOVASCULAR SYSTEM (DiGeorge, Velocardiofacial syndrome, Shprintzen syndrome) (Lin et al, Genet in Med, 2009) 3 551 CARDIOVASCULAR SYSTEM ALAGILLE SYNDROME Responsible genes: JAG1, NOTCH2 Proteins: Jagged 1, Neurogenic locus notch homolog protein 2 Cytogenetic locus (loci): 20p12, 1p13-p11 Inheritance: AD, 50-70% de novo Clinical Features and Diagnostic Criteria: Dx: Bile duct paucity on liver bx + any three of: cardiac defects (most often PA disease, TOF), cholestasis, skeletal abnormalities (butterfly vertebrae), eye (posterior embryotoxin), or characteristic facial features. Also developmental delay, growth failure Clinical Tests: Bile duct paucity on liver bx, Molecular Tests: seq JAG1 (>89%), JAG1 20p12 del FISH (~7%), NOTCH2 seq (1-2%) Disease Mechanism: JAG1:Truncated protein product rendering it unable to bind to the cell membrane resulting in functional haploinsufficiency Treatment/Prognosis: Liver transplant, cardiac and renal anomalies treated in standard manner, evaluate head injuries and CNS symptoms for vascular accidents, fat soluble vitamins, monitor growth and development, 4 ALAGILLE SYNDROME CARDIOVASCULAR SYSTEM HE staining of liver specimen showing paucity of the interlobular ducts (www.cmj.org/Periodical/PaperList.asp?id=LW200) Facial Features: Prominent forehead Deep-set eyes with moderate hypertelorism Pointed chin Saddle or straight nose with a bulbous tip (http://www.icampus.ucl.ac.be/PEDIHEPA/) 5 BRUGADA SYNDROME Responsible gene: SCN5A CARDIOVASCULAR SYSTEM (Pathogenic variants in 22 other genes : ABCC9, CACNA1C, CACNA2D1, CACNB2, FGF12, GPD1L, HCN4, KCND2, KCND3, KCNE5, KCNE3, KCNH2, KCNJ8, PKP2, RANGRF, SCN1B, SCN2B, SCN3B, SCN10A, SEMA3A, SLMAP, and TRPM4, each <1%) Protein: Sodium channel protein type 5 subunit alpha Cytogenetic locus: 3p21 (SCN5A) Inheritance: AD (except KCNE5 XLR) Clinical Features and Diagnostic Criteria: Syncope or nocturnal agonal respiration. STsegment abnormalities in leads V1-V3 on the ECG and a high risk of ventricular arrhythmias and sudden death. Manifests primarily during adulthood (range 2 days to 85 yrs). Mean age of sudden death: 40 yrs. May present as SIDS or the sudden unexpected nocturnal death syndrome (a typical presentation in individuals from Southeast Asia). May have FH sudden cardiac death. Clinical Tests: ECG Molecular Tests: SCN5A scanning/seq (15-30% of cases) Disease Mechanism: Gene mutations cause lack of expression of or acceleration in the inactivation of cardiac sodium channels. Treatment/Prognosis: Implantable defibrillators, isoproterenol, avoid inducing medication (vagotonic agents, alpha adrenergic antagonists, beta adrenergic antagonists, TCA, first generation antihistamines, cocaine, class 1C antiarrhythmic drugs, class 1A agents (procainamide, disopyramide) 6 552 CARDIOVASCULAR SYSTEM BRUGADA SYNDROME 7 CARDIOVASCULAR SYSTEM CARDIO-FACIO-CUTANEOUS SYNDROME Responsible genes: BRAF, MAP2K1, MAP2K2, KRAS Proteins: B-Raf proto-oncogene serine/threonine-protein-kinase, Dual specificity mitogenactivated protein kinase 1 and 2, GTPase KRas Cytogenetic loci: 7q34, 15q22.31,19p13.3, 12p12.1 Inheritance: AD (majority de novo) Clinical Features and Diagnostic Criteria: Cardiac abnormalities (pulmonic stenosis, septal defects, hypertrophic cardiomyopathy, arrhythmia), distinctive facial features, and cutaneous abnormalities (xerosis, hyperkeratosis, ichthyosis, eczema, ulerythema ophyrogenes), mild-moderate intellectual disability, neoplasia in some, most often ALL Clinical Tests: echocardiogram, renal ultrasound, cognitive testing Molecular Tests: gene sequencing Disease Mechanism: sustained activation of the Ras MAPK pathway downstream effectors: MEK and/or ERK Treatment/Prognosis: Standard cardiac care, dermatology consultation, early intervention and individualized education plans 8 CARDIO-FACIO-CUTANEOUS SYNDROME •High forehead with bitemporal constriction CARDIOVASCULAR SYSTEM •Posteriorly rotated ears with think helices •Hypertelorism with down slanting palpebral fissures •Epicanthal folds and ptosis •Depressed nasal bridge with anteverted nares •Highly arched palate •Cupids Bow Lips •More coarse features and more dolichocephaly than Noonan syndrome www.cfcsyndrome.org 9 553 CARDIOVASCULAR SYSTEM COSTELLO SYNDROME Responsible genes: HRAS Proteins: GTPase HRas Cytogenetic loci: 11p15.5 Inheritance: AD (majority de novo) Clinical Features and Diagnostic Criteria: feeding issues, developmental delay, intellectual disability, coarse facial features, loose, soft skin, hypertrophic cardiomyopathy, pulmonary stenosis, arrhythmia, high rate of cancer (bladder cancer, rhabdomyosarcoma and neuroblastoma) Clinical Tests: echocardiogram, neurocognitive testing Molecular Tests: gene sequencing Disease Mechanism: Missense mutations lead to constitutive activation of the abnormal protein product resulting in increased signaling through the Ras MAP Kinase pathway Treatment/Prognosis: Standard cardiac care, dermatology consultation, early intervention and individualized education plans, may require assisted feeding (nasogastric or gastric tube), cancer screeing 10 COSTELLO SYNDROME CARDIOVASCULAR SYSTEM Coarse facial features (full lips, large mouth, full nasal tip) Curly or sparse, fine hair Loose, soft skin with deep palmar and plantar creases Papillomata of the face and perianal region Diffuse hypotonia and joint laxity with ulnar deviation of the wrists and fingers costellosyndromeusa.org Cancer risk 11 CARDIOVASCULAR SYSTEM HEREDITARY HEMORRHAGIC TELANGIECTASIA Responsible genes: ACVRL1, ENG, GDF2, SMAD4 Proteins: Serine/threonine-protein kinase receptor R3, Endoglin, Growth/differentiation factor 2, Mothers against decapentaplegic homolog 4 Cytogenetic loci: 12q11-q14, 9q34.1, 10q11.22,18q21.2 Inheritance: AD Clinical Features and Diagnostic Criteria: nosebleeds, mucocutaneous telangiectases (lips, oral cavity, fingers, and nose), visceral AV malformation (pulmonary, cerebral, hepatic, spinal, gastrointestinal). Hemorrhage is often the presenting symptom of cerebral AVM. Most visceral AVM’s present as a result of blood shunting through the abnormal vessel and bypassing the capillary beds. Clinical Tests: Stool for occult blood, CBC (anemia or polycythemia), contrast echo to find pulmonary AVM, Head MRI for cerebral AVM, US for hepatic AVM, Molecular Tests: Sequence analysis ACVRL1, ENG (60-80%), duplication/deletion analysis (10%); SMAD4 in patients with juvenile polyposis Disease Mechanism: HHT is assumed to be the result of haploinsifficiency Treatment/Prognosis: Transcatherter embolization of pulmonary AVM >3.0mm. OCP can decrease/eliminate bleeding. Liver transplant if hepatic AVM is causing heart failure. 12 554 HEREDITARY HEMORRHAGIC TELANGIECTASIA CARDIOVASCULAR SYSTEM Mucocutaneous telangiectases 13 CARDIOVASCULAR SYSTEM HOLT-ORAM SYNDROME Responsible gene: TBX5, SALL4 (related disorder) Protein: T-box transcription factor TBX5, Sal-like protein 4 Cytogenetic loci: 12q24.1, 20q13.2 Inheritance: AD (85% de novo) Clinical Features and Diagnostic Criteria: Malformation of the carpal bone(s) and, variably, the radial and/or thenar bones (left often more severe than right). 100% have carpal bone abnormality. 75% have CHD, most often multiple ASD or VSD, arrhythmia (even if no CHD) Clinical Tests: hand xray Molecular Tests: TBX5 sequencing (>70%), Del/Dupl analysis (<1%). Rarely due to SALL4 mutations resulting in similar syndrome Disease Mechanism: The TBX5 protein product is a transcription factor with an important role in both cardiogenesis and limb development. TBX5 mutations lead to mutant TBX5 mRNAs that are rapidly degraded or to transcripts with diminished DNA binding- both of which result in decreased gene dosage. Treatment/Prognosis: Pacemaker if severe heart block, standard cardiac surgery, pollicization may be indicated if thumb aplasia/hypoplasia. Annual ECG, annual Holter if h/o abnormal ECG 14 CARDIOVASCULAR SYSTEM HOLT-ORAM SYNDROME Thumb anomaly http://www.emedicine.com/ped/images/1038ASDbj.jpg 15 555 CARDIOVASCULAR SYSTEM Noonan syndrome with Multiple Lentigines (NSML) formerly known as LEOPARD Syndrome Responsible gene: PTPN11, RAF1, BRAF, MAP2K1 Protein: SHP2 , RAF proto-oncogene serine/threonine-protein kinase, B-Raf, mitogen-activated protein kinase 1 Cytogenetic locus: 12q24, 3p25 Inheritance: AD Clinical Features and Diagnostic Criteria: Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of the genitalia, Retardation of growth, sensorineural Deafness. Hypertrophic cardiomyopathy in majority Clinical Tests: Audiogram, ECG, echocardiogram Molecular Tests: PTPN11 sequencing (90%), RAF1 (<5%), others rare Disease Mechanism: Loss of function PTPN11 mutations (versus Noonan syndrome PTPN11 mutations which are gain of function) Treatment/Prognosis: Treat cardiac defects, deafness 16 CARDIOVASCULAR SYSTEM Noonan syndrome with Multiple Lentigines (NS-ML) formerly known as LEOPARD Syndrome Facial Features: -Hypertelorism -Down slanting palpebral fissures -Low set ears -Multiple lentigines (Sarkozy EJHG 2004) NOONAN SYNDROME CARDIOVASCULAR SYSTEM Responsible genes: PTPN11, SOS1, KRAS, RAF1, NRAS, CBL, SHOC2, BRAF, RIT1, SOS2, MAP2K1 Proteins: SHP2, Son of sevenless homolog 1, GTPase KRAS, RAF proto-oncogene serine/threonine-protein kinase, NRAS, CBL, SHOC2, B-raf proto-oncogene serine/threonineprotein kinase, Ric-like protein w/o CAAX Motif 1, Son of sevenless homolog 2 Cytogenetic loci: 12q24.1, 2p22-p21, 12p12.1, 3p25, 1p13.2, 11q23.3, 10q25, 7q35, 1q22, 14q21.3 Inheritance: AD Clinical Features and Diagnostic Criteria: Characteristic facial features, short stature, feeding problems, pulmonary valve stenosis, HCM (RAF1,RIT1 most enriched), cryptorchidism, renal malformation, lymphedema, bleeding disorders, myeloproliferative disorder, incl. risk of leukemia and learning disabilities Clinical Tests: Echocardiogram, renal ultrasound, bleeding studies Molecular Tests: PTPN11 sequencing (50%), SOS1 sequencing (10%), RAF1, RIT1 (10% each), SHOC2 (2%), KRAS, SOS2 (1% each), NRAS/CBL/BRAF/MAP2K1 (<1%) Disease Mechanism: Gain of function mutations that lead to constitutive activation of the Ras MAP Kinase pathway Treatment/Prognosis: Standard cardiac care, orchiopexy, early intervention, GH replacement 18 556 NOONAN SYNDROME GENE PTPN11 CARDIAC >PS CARDIOVASCULAR SYSTEM <HCM SOS1/SOS2 GROWTH >Short Stature >Lower IGF-1 levels DEVELOPMENT SKIN/HAIR OTHER N308D/S mild or no dev. delay Verbal/NV IQ<SOS1 <Short Stature Verbal/NV IQ> PTPN11 <ID CFC-like RAF1 RIT1 >HCM >HCM <Short Stature SHOC2 MVP >Short Stature Inc. pigment Hyperactivity ASD/VSD >GH Deficiency Ichthyosis Hypernasal voice Eczema KRAS More severe delays? NRAS CBL Enlarged LA Ventricular dysrhythmia Mitral Insufficiency Delayed Brain Myelination Sparse, fragile, thin hair CFC-like More severe medical problems? Pre-disposition to JMML Cerebellar Vermis Hypoplasia 19 CARDIOVASCULAR SYSTEM WILLIAMS SYNDROME Responsible gene: Contiguous gene deletion syndrome, ELN in the critical region Protein: Elastin Cytogenetic locus: 7q11.23 Inheritance: AD, majority of cases de novo Clinical Features and Diagnostic Criteria: CV any artery may be narrowed, supravalvar aortic stenosis (SVAS) most common (75%). Distinctive facial features. CT: hoarse voice, hernia, rectal prolapse, joint limitation or laxity. ID. Overfriendly, anxiety d/o, ADD. Endo: hypercalcemia, hypercalciuria, hypothyroidism, FTT infancy Clinical Tests: Serum and urine calcium and creatinine, TFTs, hearing and vision evaluation, renal US, echocardiogram Molecular Tests: FISH, microarray, or MLPA for 7q11.23 critical region (~99%). Point mutations in ELN cause AD isolated SVAS Disease Mechanism: Elastin deletion causes the CV and CT problems, LIMK1 has been linked to the visuospatial construction cognitive deficit Treatment/Prognosis: PT, OT, ST. Monitor adults who are at risk for MVP, AI, arterial stenosis, SNHL, hypothyroidism, DM. Monitor for hypercalciuria. Aggressive management of constipation 20 CARDIOVASCULAR SYSTEM WILLIAMS SYNDROME (www.thefencingpost.com/mary/images/intheairtiny.JPG) (www.wehi.edu.au/media/images/williamskid.jpg) Facial features: Broad brow, bitemporal narrowness, periorbital fullness, a stellate/lacy iris pattern, strabismus, short nose, full nasal tip, malar hypoplasia, long philtrum, full lips, wide mouth, malocclusion, small jaw, and prominent earlobes 21 557 CHROMOSOME BREAKAGE DISORDERS ATAXIA-TELANGIECTASIA Responsible gene: ATM Protein: Serine-protein kinase ATM Cytogenetic locus: 11q22.3 Inheritance: AR (carriers have increased risk of breast, colon and pancreatic) Clinical Features and Diagnostic Criteria: Progressive cerebellar ataxia (onset age 1-4y), oculomotor apraxia, conjunctival telangiectasia, immunodef, choreoathetosis, ionizing radiation sensitivity, risk cancer (lymphoma and leukemia) Clinical Tests: AFP, decreased ATM kinase activity, 7;14 translocation (5-15% of lymphocytes after PHA stimulation) Molecular Tests: ATM sequencing (>95%). Amish founder mutation Disease Mechanism: Most mutations are null leading to no protein product. The normal protein finds double strand dsDNA breaks and coordinates cell cycle checkpoints prior to repair Treatment/Prognosis: IVIG if immunodeficient, PT to reduce contractures, wheelchair usually by age 10, supportive therapy for drooling, choreoathetosis, and ataxia. Avoid ionizing radiation. Regular medical visits to monitor for S/Sx of malignancy 22 CHROMOSOME BREAKAGE DISORDERS ATAXIA-TELANGIECTASIA 23 (http://www.nature.com/embor/journal/v5/n8/images/7400210-f1.jpg) CHROMOSOME BREAKAGE DISORDERS BLOOM SYNDROME Responsible gene: BLM Protein: Bloom syndrome protein Cytogenetic locus: 15q26.1 Inheritance: AR (1/100 carrier freq in Ashkenazi Jewish) Clinical Features and Diagnostic Criteria: IUGR, hyper and hypopigmentation, butterfly distribution sun sensitive telangiectasia, microcephaly, high pitched voice, normal intelligence, immunodeficiency, azoospermia, POF, increased risk of cancer (wide distribution of type and site (colon most common), often multiple primary tumors). Clinical Tests: Chromatid/chromosome breaks; triradial and quadriradial figures Molecular Tests: BLM 2881 del6ins7 (97% mutant allele in AJ) Disease Mechanism: Abnormal DNA replication and repair leading to genomic instability. Treatment/Prognosis: Increased cancer surveillance, decrease exposure to UV light and x-ray, BMT, colon cancer surveillance 24 558 CHROMOSOME BREAKAGE DISORDERS BLOOM SYNDROME Butterfly distribution sun sensitive telangiectasia (www.medicalrealm.net) 25 CHROMOSOME BREAKAGE DISORDERS FANCONI ANEMIA Responsible genes (Protein and Cytogenetic locus): FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG (Fanconi anemia group A, B, C, D2, E, F, and G protein; 16q24.3, Xp22.3, 9q22.3, 3p25.3, 6p22-21, 11p15, and 9p13); FA-D1 - BRCA2 (Breast cancer type 2 susceptibility protein, 13q12.3); BRIP1 (Fanconi anemia group J protein, 17q22); FANCL (E3 ubiquitin-protein ligase FANCL). Inheritance: AR, AD (RAD51), XLR – FA-B Clinical Features and Diagnostic Criteria: Short stature; abnl pigmentation; radial, GU, ear, heart, GI, or CNS malformation; hearing loss, hypogonadism, developmental delay. Progressive bone marrow failure, aplastic anemia, myelodysplastic syndrome, AML, solid tumor of head, neck, esophagus, cervix, vulva, or liver at unusually young age. Clinical Tests: DEB or MMC induced chromosome breakage, macrocytic rbcs, immunoblot assay of FANCD2 monoubiquitination, increased % of cells in G2 arrest by cell sorting. Molecular Tests: Seq and Del/Dup analysis FANCA (66%), Seq analysis FANCB (0.8%), FANCC (9.6%), FANCD1, FANCD2, FANCE, FANCF (~3% each), FANCG (8.8%), FANCL (0.4%) and BRCA2. Numerous additional subtypes with only a few patients described. Disease Mechanism: >5 of the FA proteins are assembled in a nuclear complex. In response to DNA damage, this complex activates monoubiquitination of FANCD2 and FANCI protein with subsequent FA proteins involved in direct DNA repair. Treatment/Prognosis: Androgens, blood transfusions, growth hormone, BMT, cancer prevention (avoid toxic agents and sun exposure), cancer surveillance 26 CHROMOSOME BREAKAGE DISORDERS FANCONI ANEMIA Preaxial polydactyly (radswiki.net) 27 559 CONNECTIVE TISSUE DISORDERS CONGENITAL CONTRACTURAL ARACHNODACTYLY (Beals Syndrome) Responsible gene: FBN2 Protein: Fibrillin-2 Cytogenetic locus: 5q23-q31 Inheritance: AD Clinical Features and Diagnostic Criteria: Marfanoid appearance, long slender fingers and toes, crumpled ears, major joint contracture, muscle hypoplasia, kyphosis/scoliosis, Severe/lethal: aortic dilation, ASD, VSD, IAA, duodenal or esophageal atresia, malrotation Clinical Tests: x-ray, echocardiogram, UGI with SBFT Molecular Tests: FBN2 sequencing (75%) Disease Mechanism: Fibrillin 2 is a glycoprotein of the extracellular matrix microfibrils, it is co-distributed with fibrillin 1 in many tissues. The precise function is not known. Treatment/Prognosis: PT for joint contracture, contracture surgical release, bracing and/or surgical correction of kyphoscoliosis. Echo every 2 years until it is clear the aorta is not involved. Annual exam for scoliosis/kyphosis. 28 CONNECTIVE TISSUE DISORDERS CONGENITAL CONTRACTURAL ARACHNODACTYLY (Beals Syndrome) Crumpled prominent ears Major joint contracture (www.scielo.br) (www.indianpediatrics.net) 29 CONNECTIVE TISSUE DISORDERS EHLERS-DANLOS SYNDROME CLASSIC TYPE (Type I and Type II) Responsible genes: COL5A1 and COL5A2 Proteins: Collagen alpha-1 and alpha-2 (V) chain Cytogenetic loci: 9334.2-q34.3 and 2q31 Inheritance: AD Clinical Features and Diagnostic Criteria: skin hyperextensibility, widened atrophic scars, joint hypermobility, smooth velvety skin, molluscoid pseudotumors (heaped up scar-like lesions over pressure points), subcutaneous spheroids (cyst-like lesions, feel like grains of rice, over bony prominences of legs and arms, they are fibrosed and calcified fat globules), joint sprains/dislocations/subluxations, hypotonia, easy bruising, hernia, chronic pain, aortic root dilation Clinical Tests: Ultrastructural studies by EM suggest disturbed collagen fibrillogenesis (“cauliflower” deformity is characteristic). Molecular Tests: COL5A1 “null” allele testing on cultured fibroblasts (30%), COL5A1 and COL5A2 sequencing (50%) Disease Mechanism: Dominant negative activity of abnormal Collagen alpha-1 or alpha-2 (V) chains (interfere with the utilization of normal protein from the normal allele) Treatment/Prognosis: PT, non-weight-bearing muscular exercise, dermal wounds repaired with two layer closure without tension, if possible avoid joint surgery , baseline echo for aortic root assessment 30 560 EHLERS-DANLOS SYNDROME CLASSIC TYPE (Type I and Type II) CONNECTIVE TISSUE DISORDERS Skin hyperextensibility Joint hypermobility (www.meddean.luc.edu ) (www.nlm.nih.gov) 31 CONNECTIVE TISSUE DISORDERS EHLERS-DANLOS SYNDROME, HYPERMOBILITY TYPE (Type III) Responsible gene: unknown Protein: Unknown Cytogenetic locus: 6p21.3 Inheritance: AD Clinical Features and Diagnostic Criteria: Joint hypermobility, soft or velvety skin with normal or slightly increased elasticity, absence of skin or soft tissue fragility, recurrent joint dislocation/subluxation, chronic joint or limb pain, easy bruising, high narrow palate, dental crowding, and low bone density. Kids less than age 5 are often very flexible and therefore are hard to assess. Reported instances of aortic root dilation and MVP. Clinical Tests: The biochemical etiology is unknown in most cases. Molecular Tests: Not done (rare cases of Tenascin-X deficiency) Disease Mechanism: Abnormal dermal elastic fibers Treatment/Prognosis: Improve joint stability with low-resistance exercise to inc muscle tone, avoid joint hyperextension, avoid high impact sports, wide writing utensils to avoid strain on finger and hand joints, joint bracing, pain management specialist, delay joint surgery in favor of PT and bracing. Baseline echocardiogram 32 CONNECTIVE TISSUE DISORDERS EHLERS-DANLOS SYNDROME, HYPERMOBILITY TYPE (Type III) Beighton Hypermobility Score (www.physiopro.co.za/wp-content/uploads/2012/09/beightonscale.png) 33 561 CONNECTIVE TISSUE DISORDERS EHLERS-DANLOS SYNDROME, VASCULAR TYPE (Type IV) Responsible gene: COL3A1 Protein: Collagen alpha-1 (III) chain Cytogenetic locus: 2q31 Inheritance: AD Clinical Features and Diagnostic Criteria: Major criteria: arterial rupture, intestinal rupture, uterine rupture during pregnancy, FH of Vascular EDS. Minor criteria: thin, translucent skin, easy bruising, thin lips and philtrum, small chin, thin nose, large eyes, aged appearance of hands, small joint hypermobility, tendon/muscle rupture, varicose veins, AV carotid-cavernous sinus fistula, pneumothorax, CHD, clubfoot, gum recession Clinical Tests: Cultured dermal fibroblasts: amount of type III procollagen synthesized, the quantity secreted into the medium, and the electrophoretic mobility are assessed (95% of cases of vascular EDS) Molecular Tests: cDNA or genomic DNA COL3A1 sequence analysis (98-99%) Disease Mechanism: Abnormalities of type III procollagen production, intracellular retention, reduced secretion, and/or altered mobility Treatment/Prognosis: Minimization of surgical exploration and intervention, prompt surgery for bowel rupture, distal colectomy if recurrent bowel rupture, high risk obstetrical care. Minimize lifting and weight training, no contact sports, no arteriograms; investigational treatment celiprolol 34 EHLERS-DANLOS SYNDROME, VASCULAR TYPE (Type IV) CONNECTIVE TISSUE DISORDERS Facial Features: Thin lips Thin philtrum Small chin Thin nose Large eyes Thin skin with visible vessels www.ehlersdanlosnetwork.org 35 CONNECTIVE TISSUE DISORDERS EHLERS-DANLOS SYNDROME, KYPHOSCOLIOTIC TYPE (Type VI) Responsible gene: PLOD1 Protein: Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 Cytogenetic locus: 1p36.3-p36.2 Inheritance: AR Clinical Features and Diagnostic Criteria: Major features: friable, hyperextensible skin, thin scars, easy bruising, generalized joint laxity, severe muscle hypotonia, progressive scoliosis, scleral fragility and rupture of the globe. Minor features: widened atrophic scars, marfanoid habitus, rupture of medium sized arteries, mild to moderate delay of attainment of gross motor milestones Clinical Tests: Crosslinked telopeptides are excreted in urine as a byproduct of increased collagen turnover. Inc ratio of deoxypyridinoline to pyridinoline by urine HPLC is highly sensitive and specific. Enzyme activity in cultured fibroblasts (<25% activity is abnormal) Molecular Tests: PLOD1 seq research only, unknown frequency Disease Mechanism: Enzyme deficiency leads to deficiency in hydroxylysine-based pyridinoline crosslinks in types I and III collagen. Treatment/Prognosis: Surgical correction of scoliosis is not contraindicated), PT, echocardiogram and standard treatment for MVP or aortic root dilation, aggressive control of BP, routine eye exams 36 562 CONNECTIVE TISSUE DISORDERS EHLERS-DANLOS SYNDROME, KYPHOSCOLIOTIC TYPE (Type VI) Kyphoscoliosis www.thefetus.net 37 CONNECTIVE TISSUE DISORDERS LOEYS-DIETZ SYNDROME Responsible gene: TGFBR1, TGFBR2, SMAD3, TGFB2 Protein:TGF-beta recepor type-1 and type-2, Mothers against decapentaplegic homolog 3, Transforming growth factor beta-2 Cytogenetic locus: 9q22.33, 3p24.1, 15q22.33, 1q41 Inheritance: AD Clinical Features and Diagnostic Criteria: vascular findings (cerebral, thoracic, and abdominal arterial aneurysms and/or dissections) and skeletal manifestations (pectus excavatum or pectus carinatum, scoliosis, joint laxity, arachnodactyly, talipes equinovarus). 75% have LDS type I with craniofacial manifestations (ocular hypertelorism, bifid uvula/cleft palate, craniosynostosis); 25% have LDS type II with cutaneous manifestations (velvety and translucent skin; easy bruising; widened, atrophic scars). Clinical Tests: Echocardiogram, MRA or CT scan for arterial aneurysm/tortuosity, spinal xrays Molecular Tests:gene sequencing and del/dup testing Disease Mechanism: data demonstrate increased TGFɴ signaling in the vasculature of persons with LDS Treatment/Prognosis: Regular surveillance imaging of the vasculature, Beta blockers/Losartan for aortic root dilation, bracing/surgery for scoliosis 38 CONNECTIVE TISSUE DISORDERS LOEYS-DIETZ SYNDROME Marked arterial tortuosity (Morris SA and Lacro RV Circ 2011) 39 563 CONNECTIVE TISSUE DISORDERS MARFAN SYNDROME Responsible gene: FBN1 Protein: Fibrillin 1 Cytogenetic locus: 15q21.1 Inheritance: AD Clinical Features and Diagnostic Criteria: Major involvement of 2 body systems and minor involvement of a 3rd. Major Criteria CV: Dilation or dissection of the ascending aorta Skeletal: pectus carinatum or excavatum, reduced upper:lower segment or arm span:ht, scoliosis, pes planus, high palate, reduced elbow extension, protrusio acetabulae, Eye: ectopia lentis, Dura: lumbosacral dural ectasia, FH: pathogenic FBN1 mutation, 1st degree relative with Marfan syndrome. Minor Criteria CV: MV, MR, dilation of main PA, mitral annulus calcification, dilation or dissection of the descending thoracic or abdominal aorta ate age <50yrs, Skeletal: moderate pectus excavatum, joint hypermobility, high palate with crowding of teeth, typical facial features Eye: flat cornea, increased length of globe, decreased pupillary miosis, Lung: pneumothorax, apical lung blebs, Skin: skin striae, hernia Clinical Tests: CXR: apical blebs, Echocardiogram: MVP, aortic measurements, CT or MRI: dural ectasia Molecular Tests: FBN1 seqencing (70-90%) Disease Mechanism: Dominant negative effect of mutant forms of fibrillin Treatment/Prognosis: Beta blockers/Losartan for aortic root dilation, bracing/surgery for scoliosis, annual dilated eye exam, glasses for myopia 40 CONNECTIVE TISSUE DISORDERS MARFAN SYNDROME Positive thumb sign Ectopia Lentis Pectus excavatum www.medstudents.com.br www.chicagohs.org www.gfmer.ch 41 DERMATOLOGIC DISORDERS HIDROTIC ECTODERMAL DYSPLASIA 2 Responsible gene: GJB6 Protein: Gap junction beta-6 protein (Connexin 30) Cytogenetic locus: 13q12 Inheritance: AD Clinical Features and Diagnostic Criteria: malformed, thickened, small nails; hypotrichosis (partial or total alopecia), palmoplantar hyperkeratosis, normal sweating and teeth. Clinical Tests: None Molecular Tests: Four GJB6 mutations (p.Gly11Arg, p.Ala88Val, p.Val37Glu, p.Asp50Asn) account for 100% of identified mutant alleles Disease Mechanism: Helps form a gap junction channel which mediates ion diffusion. Mutations are thought to affect trafficking of the protein and thus the formation of the gap junction Treatment/Prognosis: No specific treatment. Skin emollients for hyperkeratosis 42 564 DERMATOLOGIC DISORDERS HIDROTIC ECTODERMAL DYSPLASIA Plantar Hyperkeratosis vgrd.blogspot.com/2012/09/hidrotic-ectodermal-dysplasia.html 43 DERMATOLOGIC DISORDERS HYPOHIDROTIC ECTODERMAL DYSPLASIA Responsible genes: EDA, EDAR, EDARADD Proteins: Ectodysplasin-A, Tumor necrosis factor receptor superfamily member EDAR, ectodysplasin A receptor-associated adapter protein Cytogenetic loci: Xq12-q13.1, 2q11-q13, 1q42.2-q43 Inheritance: XL (EDA:95%), AD or AR (5%) Clinical Features and Diagnostic Criteria: At birth: peeling skin and perioral hyperpigmentation. Hypotrichosis (sparse scalp and body hair), hypohidrosis (inability to sweat in response to heat leads to hyperthermia), hypodontia (usually only5-7 teeth develop, teeth are smaller with conical crowns. Carriers of XL HED show mosaic pattern of sweat pore function and some degree of hypodontia. Clinical Tests: dental xray. Molecular Tests: EDA sequencing (~95% XL HED), EDAR and EDARADD sequencing Disease Mechanism: Defective ectodysplasin A cannot be activated to mediate the cell-tocell signaling that regulates morphogenesis of ectodermal appendages. Defective EDAR cannot bind ectodysplasin. The protein encoded by EDARADD is co-expressed with EDAR. Treatment/Prognosis: During hot weather maintain hydration and keep down body temp with A/C, “cooling vests”, and/or spray bottle of water. Tooth restoration, orthodontics, and/or dentures may be necessary 44 DERMATOLOGIC DISORDERS HYPOHIDROTIC ECTODERMAL DYSPLASIA Hypodontia and abnormally shaped teeth www.wsahs.nsw.gov.au 45 565 DERMATOLOGIC DISORDERS INCONTINENTIA PIGMENTI Responsible gene: IKBKG (aka NEMO) Protein: NF-kappaB essential modulator Cytogenetic locus: Xq28 Inheritance: XLD (most male fetuses miscarry) Clinical Features and Diagnostic Criteria: Major: Four stages of skin changes: erythema->blister->hyperpigmented streaks->atrophic skin patches. Minor: hypo/andontia, small or malformed teeth, alopecia, woolly hair, nail ridging or pitting, retinal neovascularization causing retinal detachment. ID is rare. Clinical Tests: Free melanin granules if hyperpigmented streak biopsied. Molecular Tests: Southern blot: Exon 4-10 deletion (80%). Skewed X inactivation in females (not diagnostic). Disease Mechanism: Lack of NF-kappa beta activation leads to cells that are sensitive to proapoptotic signals and apopose easily. Treatment/Prognosis: Regular retinal exams in first 1-2 yrs. Cosmetic dentistry. Normal life expectancy. 46 DERMATOLOGIC DISORDERS INCONTINENTIA PIGMENTI IP Stage 2: blistering www.fi.cnr.it IP Stage 4: atrophic patches www.utmedicalcenter.org 47 DERMATOLOGIC DISORDERS OCULOCUTANEOUS ALBINISM Responsible genes: TYR (OCA1), OCA2, TRYP1, SLC45A2, GPR143 (Ocular) Proteins: Tyrosinase protein, P protein, Membrane-assoc. transporter protein, Gprotein coupled receptor 143 Cytogenetic loci: 11q14-q21 (OCA1), 15q11.2-q12 (OCA2) (in PWS/Angelman region), 5p13.2, Xp22.2 (Ocular) Inheritance: AR, XLR (GPR143) Clinical Features and Diagnostic Criteria: OCA1A (no melanin synthesis) nystagmus, dec iris pigment, foveal hypoplasia, dec visual acuity, strabismus, white hair and skin, translucent iris. OCA1B (some melanin synthesis) milder eye and skin manifestation than OCA1. OCA2 ocular problems same as OCA1 but better vision, range of skin and eye pigmentation from minimal to near normal Clinical Tests: Skin and eye exam, VEP Molecular Tests: TYR sequencing (OCA1A: 2 mutations 83%; OCA1B: 2 mutations 37%). 2kb OCA2 deletion testing (most of Sub-Saharin African heritage), SLC45A2 and GPR143 seq Disease Mechanism: Lack of melanin production Treatment/Prognosis: Yearly eye exam, sun screen and monitoring for skin cancer. 48 566 DERMATOLOGIC DISORDERS OCULOCUTANEOUS ALBINISM Hair and skin hypopigmentation http://www.positiveexposure.org/home.html 49 ENDOCRINE SYSTEM X-LINKED ADRENAL HYPOPLASIA CONGENITA Responsible gene: NROB1 Protein: Nuclear receptor 0B1 Cytogenetic locus: Xp21.3-p21.2 Inheritance: X-LR Clinical Features and Diagnostic Criteria: acute onset adrenal insufficiency (hyperkalemia, acidosis, hypoglycemia, shock), cryptorchidism, delayed puberty. Carrier females: may have adrenal insufficiency or hypogonadotropic hypogonadism 1/3 contiguous gene deletion with glycerol kinase, DMD del 2/3 isolated CAH (half are de novo) Clinical Tests: Dec Na+, Inc K+, acidosis, inc ACTH with low cortisol, dec 17 hydroxyprogesterone. If GKD: serum triglyceride, urine glycerol. If DMD: elevated CK Molecular Tests: NROB1 FISH deletion (100%) Disease Mechanism: OB1 is a negative regulator of nuclear receptor pathways Treatment/Prognosis: Treat adrenal crisis, replacement steroids and stress dosing, HRT for hypogonadism, 50 ENDOCRINE SYSTEM 21-HYDROXYLASE-DEFICIENT CAH Responsible gene: CYP21A2 Protein: Cytochrome P450 XXI Cytogenetic locus: 6p21.3 Inheritance: AR Clinical Features and Diagnostic Criteria: virilized female, precocious puberty or adrenarche, childhood virilization in males, infant with Na+ losing crisis at birth. Nonclassic form: moderate enzyme deficiency with variable postnatal virilization, no salt wasting, but rare cortisol def. Clinical Tests: Elevated serum 17-OHD at baseline or after ACTH stim, elevated testosterone and adrenal androgen precursors in females and prepubertal males. Part of NBS (17-OHD level) Molecular Tests: CYP21A2 common mutation and deletion panel detects 80-98% Disease Mechanism: cortisol production pathway is blocked-> accumulation of 17-OHP>shunted into the intact androgen pathway->17,20-lyase enzyme converts the 17-OHP to – androstenedione->converted into androgens. The mineralocorticoid pathway requires minimal 21-hydroxylase activity->salt wasting Treatment/Prognosis: Hydrocortisone (monitor closely: too little will have excess androgen, too much causes Cushing;s, skeletal maturation), stress dose steroids 51 567 ENDOCRINE SYSTEM 21-HYDROXYLASE-DEFICIENT CAH 52 ENDOCRINE SYSTEM ANDROGEN INSENSITIVITY SYNDROME (Testicular Feminization) Responsible gene: AR Protein: Androgen receptor Cytogenetic locus: Xq11-q12 Inheritance: XLR Clinical Features and Diagnostic Criteria: Evidence of feminization (i.e., undermasculinization) of the ext. genitalia, abnl secondary sexual development, and infertility in those with a 46,XY karyotype. Spectrum: complete androgen insens. syndrome (CAIS), with typical female genitalia; partial androgen insens. syndrome (PAIS) with predominantly female, predominantly male, or ambiguous genitalia; and mild androgen insens. syndrome (MAIS) with nl male genitalia. Clinical Tests: impaired spermatogenesis, absent or rudimentary müllerian structures, evidence of normal or increased synthesis of testosterone and its normal conversion to dihydrotestosterone, normal or increased LH, and deficient or defective androgen-binding activity of genital skin fibroblasts Molecular Tests: AR sequence analysis (>95% CAIS, <50% PAIS, unknown % MAIS) Disease Mechanism: Impaired androgen binding Treatment/Prognosis: To prevent testicular malignancy, treatment of CAIS includes either removal of the testes after puberty when feminization is complete or prepubertal gonadectomy accompanied by estrogen replacement therapy. Systematic disclosure of the diagnosis of AIS in an empathic environment 53 ANDROGEN INSENSITIVITY SYNDROME (Testicular Feminization) ENDOCRINE SYSTEM Disorders of Sexual Development (DSD) (Kim and Kim, Korean J Urol, 2012) 54 568 ENDOCRINE SYSTEM KALLMAN SYNDROME TYPE 1 and 2 Responsible genes: KAL, FGFR1 Proteins: Anosmin 1, fibroblast growth factor receptor 1 Cytogenetic loci: Xp22.3, 8p11.1-11.2 Inheritance: XLR, AD Clinical Features and Diagnostic Criteria: Type 1and 2: hypogonadotropic hypogonadism and anosmia. Usually present with delayed pubertal development. Type 1 can also include mirror hand movements, ataxia, GU anomaly, high palate, pes cavus. Type 2 ID, CL/P, cryptorchidism, choanal atresia, CHD, SNHL. Clinical Tests: Low FSH and LH; low testosterone in males; low estradiol in females. MRI: hypo/aplasia olfactory bulbs and tracts. Molecular Tests: Sequencing KAL (5-10%), FGFR1 (8-16%) Disease Mechanism: Lack of anosmin stops olfactory axons from interecting with their target. It is thought that FGFR1 may play a role in olfactory bulb formation and possibly interacts with anosmin Treatment/Prognosis: Normalize gonadal steroid levels. 55 ENDOCRINE SYSTEM KALLMAN SYNDROME TYPE 1 and 2 (humupd.oxfordjournals.org/content/14/4/293/F2.expansion.html) 56 ENDOCRINE SYSTEM KLINEFELTER SYNDROME Clinical Features and Diagnostic Criteria: Tall stature, slightly delayed motor and language skills, inc learning probs, testosterone plateaus age 14, small fibrosed testes, azoospermia and infertility, gynecomastia, inc cholesterol, slightly inc risk of autoimmune disorders and mediastinal germ cell tumors (1% risk). Increased risk of male breast cancer. Clinical Tests: Molecular Tests: karyotype, at least one extra chromosome to a 46,XY Karyotype Disease Mechanism: 1st or 2nd meiotic division nondisjunction of either parent. Maternal>paternal origin. +AMA effect Treatment/Prognosis: Testosterone in mid-late adolescence for bone density, secondary sex characteristic development, muscle mass, cholesterol, increase libido, improved energy. Can do testicular biopsy and use any retrieved sperm for ICSI (inc risk sex chrom abnormality so follow with PGD 57 569 ENDOCRINE SYSTEM KLINEFELTER SYNDROME health.yahoo.com/media/healthwise/nr551770 58 ENDOCRINE SYSTEM MCCUNE-ALBRIGHT SYNDROME Responsible gene: GNAS Protein: Guanine nucleotide-binding protein G(s), alpha subunit Cytogenetic locus: 20q13.2 Inheritance: sporadic Clinical Features and Diagnostic Criteria: polyostotic fibrous dysplasia, pathologic fracture, cranial foramina thickening->deafness and blindness, large irregular café au lait (“coast of Maine”), precocious puberty, hyperthyoidism, inc GH, PRL, or PTH, ovarian cysts Clinical Tests: x-ray, pelvic US, vision and hearing testing, pituitary hormone analysis Molecular Tests: Targeted mutation analysis Disease Mechanism: Activating mutations (a stimulatory G-protein) leads to persistently high cAMP (de-activating mutations cause Albright Heredity Osteodystrophy) Treatment/Prognosis: Aromatase inhibitor to block testosterone, bisphosphonate for fibrous dysplasia, anti-thyroid meds, octreotide (somatostatin analog) and bromocriptine (dopamine receptor agonist) 59 MCCUNE-ALBRIGHT SYNDROME Polyostotic fibrous dysplasia ENDOCRINE SYSTEM Irregular “coast of Maine” café au lait history.nih.gov (brighamrad.harvard.edu) 60 570 ENDOCRINE SYSTEM TRANSIENT NEONATAL DIABETES MELLITUS Responsible genes: HYMAI, PLAGL1 Proteins: unknown (HYMAI), zinc finger protein PLAG1 Cytogenetic loci: 6q24 (HYMAi and PLAG1) Inheritance: UPD isodisomy chromosome 6, paternal 6q24 duplication, or 6q24 methylation defect Clinical Features and Diagnostic Criteria: DM in the first six weeks of life, resolves by 18 months, severe IUGR, dehydration, hyperglycemia. Occassional macroglossia and umbilical hernia. Clinical Tests: High serum glucose and low plasma insulin, no islet cell antibodies, no ketoacidosis. 2% have a visible 6q24 duplication Molecular Tests: UPD6 (35%), 6q24 duplication (35%), imprinting mutation (20%) Disease Mechanism: PLAGL1 and HYMAI are normally only expressed on the paternal allele, unclear why overexpression causes DM. HYMAI may regulate PLAGL1 expression Treatment/Prognosis: Rehydration, IV insulin and then subcutaneous insulin within two weeks, close blood glucose monitoring. Inc risk to later develop type II DM during illness, puberty or during pregnancy 61 ENDOCRINE SYSTEM TURNER SYNDROME Responsible genes: X genes that escape inactivation, SHOX Proteins: SHOX: Short stature homeobox protein Cytogenetic locus: SHOX: Xpter-p22.32 Inheritance: sporadic Clinical Features and Diagnostic Criteria: congenital lymphedema, growth failure, normal intelligence (10% sig delays), coarctation of the aorta, bicuspid aortic valve, HLHS, hyperlipidemia, gonadal dysgenesis (10% 45,X go into puberty), hypothyroidism, diabetes, strabismus, recurrent OM, SNHL, Crohns, renal malformation, osteoporosis. Clinical Tests: echo, renal US, TFTs, GH testing, FISH SRY Molecular Tests: Karyotype Disease Mechanism: SHOX: thought to act as a transcription regulator with many downstream targets that modify growth and stature. SHOX protein has been id’ed in the growth plate from 12 weeks GA to late childhood. Treatment/Prognosis: GH, HRT, gonadectomy if Y chromosome mosaicism (risk for gonadoblastoma). Need lifelong cardiac follow-up, at risk for aortic dilation and dissection with bicuspid aortic valve. 62 TURNER SYNDROME ENDOCRINE SYSTEM Low posterior hairline neck webbing and Hypertelorism and ears low set www.healthofchildren.com www.tsregistry.org/images 63 571 DISORDERS OF HEARING and/or VISION BLEPHAROPHIMOSIS, PTOSIS, and EPICANTHUS INVERSUS Responsible gene: FOXL2 Protein: Forkhead Box Protein L2 Cytogenetic locus: 3q23 Inheritance: AD (50% de novo) Clinical Features and Diagnostic Criteria: blepharophimosis, ptosis, epicanthus inversus, and telecanthus. BPES type I includes the four major features and premature ovarian failure (POF); BPES type II includes only the four major features. Can also see: lacrimal duct anomalies, amblyopia, strabismus, and refractive errors. Minor features: broad nasal bridge, low-set ears, and a short philtrum. Clinical Tests: FOXL2 sequencing, deletion/duplication analysis Molecular Tests: Combination of seq analysis and deletion testing Disease Mechanism: FOXL2 is a transcriptional repressor of granulosa cell differentiation; mutations cause accelerated differentiation of granulosa cells and secondary depletion of the primordial follicle pool Treatment/Prognosis: Surgical correction of eye anomalies, ovum donation if POF 64 DISORDERS OF HEARING and/or VISION BLEPHAROPHIMOSIS, PTOSIS, and EPICANTHUS INVERSUS Facial Features: Blepharophimosis Ptosis Epicanthus inversus Telecanthus http://bpes.blogg.se 65 DISORDERS OF HEARING and/or VISION CONGENITAL HEARING LOSS Connexin 26 and 30 Responsible genes: GJB2 (Cx26), GJB6 (Cx30) Proteins: Gap junction proteins 2 and 6 Cytogenetic loci: 13q11-12 Inheritance: AR Clinical Features and Diagnostic Criteria: Congenital mild-profound SNHL. Rare patients can have AD Cx26 HL which can include skin findings: palmar-planter keratoderma, KID syndrome (keratitis-ichthyosis-deafness) Clinical Tests: Newborn hearing screen, ABR diagnostic, monitor with standard audiometry. Molecular Tests: GJB2: sequencing of exon 2 and exon 1 for splice site mutation (4th most common mutation). 35delG common in Caucasians, 235delC in Asians, 167delT, del35Gand Cx30 gene deletion in Ashkenazi Jewish. GJB6-D13S1830 deletion: deletion that includes Cx30, causes HL if homozygous or combined with single Cn26 mutation. Disease Mechanism: Loss of gap junction prevents recycling of toxic ions and metabolites away from hair cells leading to their death Treatment/Prognosis: No treatment. Some have progressive HL. Habilitation with hearing aids or cochlear implants. 66 572 DISORDERS OF HEARING and/or VISION CONGENITAL HEARING LOSS Connexin 26 and 30 Cochlear Implant http://www.yrsddcd.org.uk/images/cochlea1.jpg &imgrefurl 67 DISORDERS OF HEARING and/or VISION HERMANSY-PUDLAK SYNDROME Responsible gene (protein, cytogenetic locus): HPS1 (10q23.1, HPS 1 protein), AP3B1 (5q14.1, AP-3 complex subunit beta), HPS3,4,5,6,7and 8 (3q24, 22q11.2-q12.2, 11p15-p13, 10q24.3, 6p22.3, 19q13, HPS 3,4,5,and 6 proteins, dysbindin, and biogenesis of lysosomerelated organelles complex -1sununit2), HPS9 (BLOC1S6)(15q21.1) Inheritance: AR Clinical Features and Diagnostic Criteria: Findings of oculocutaneous albanism and a bleeding diathesis: hypopigmentation of the skin and the hair, nystagmus, reduced iris pigment, reduced retinal pigment, foveal hypoplasia, increased crossing of optic nerve fibers. Can develop skin cancer, pulmonary fibrosis, colitis Clinical Tests: Absent platelet dense bodies (sine qua non) on platelet EM. Prolonged bleeding time. Molecular Tests: Del/Dup analysis HPS1 (~75% Puerto Rican HPS), HPS3 (~25% Puerto Rican HPS). Targeted mutation analysis HPS3 (~5% non Puerto Rican HPS) Disease Mechanism: The HPS genes protein products have unknown fcn Treatment/Prognosis: DDAVP prior to dental work, thrombin-soaked gel foam for minor cuts, skin protection, annual eye exam, skin exam, and in adulthood PFT’s. 68 DISORDERS OF HEARING and/or VISION HERMANSY-PUDLAK SYNDROME Hypopigmentation of the skin, hair, and iris http://www.positiveexposure.org/hps/title.jpg 69 573 DISORDERS OF HEARING and/or VISION JERVELL and LANGE-NIELSEN SYNDROME Responsible gene: KCNQ1 and KCNE1 Protein: Voltage-gated K+ channel protein KvLQT1; K+ voltage-gated channel subfamily E member 1 Cytogenetic loci: 11p15.5, 21q22.1-q22.2 Inheritance: AR (Heterozygotes at risk for AD long QT a.k.a. Romano Ward syndrome) Clinical Features and Diagnostic Criteria: Congenital severe-profound bilateral SNHL and prolonged QT interval. At risk for arrhythmia, syncope, and sudden death Clinical Tests: Hearing tests (ABR, audiogram) Molecular Tests: KCNQ1 sequencing (90%), KCNE1 (10%) Disease Mechanism: In cardiac cells: abnormal repolarization of the ventricular action potential. In cochlear cells: abnormal depolarization of the auditory nerve Treatment/Prognosis: Cochlear implants for HL, beta blockers, cardiac pacemakers, and/or implantable defibrillators. Avoid QT prolonging drugs (http://www.arizonacert.org/). If left untreated, over ½ of children with JLNS die prior to age 15 yrs 70 DISORDERS OF HEARING and/or VISION JERVELL and LANGE-NIELSEN SYNDROME http://genedx.com/site/system/files/LQT.jpg 71 DISORDERS OF HEARING and/or VISION LEBER HEREDITARY OPTIC NEUROPATHY Responsible genes: MTND1, MTND4, MTND6 Proteins: Complex I subunits of the mitochondrial respiratory chain Cytogenetic loci: Mitochondrial Inheritance: Mitochondrial Clinical Features and Diagnostic Criteria: Blurred or clouded vision progressing to degeneration of the retinal nerve and then optic atrophy. Fundus: vascular tortuosity of central retinal vessels, circumpapillary telangiectatic macroangiopathy, and swelling of the retinal nerve fibers Clinical Tests: Visual field assessments, ERG, VEP Molecular Tests: Targeted mutation analysis: G11778A (70% cases), G3460A, T14484C (15%) Disease Mechanism: Focal degeneration of the retinal ganglion cell layer and optic nerve Treatment/Prognosis: No treatment available, worsened by smoking or EtOH 72 574 DISORDERS OF HEARING and/or VISION LEBER HEREDITARY OPTIC NEUROPATHY Acute fundal appearance in Leber hereditary optic neuropathy showing disc hyperaemia, swelling of the parapapillary retinal nerve fiber layer and retinal vascular tortuosity. (Yu-Wai-Man P et al. J Med Genet 2009;46:145-158) 73 DISORDERS OF HEARING and/or VISION PENDRED SYNDROME Responsible gene: SLC26A4 (PDS) most common, FOX11, KCNJ10 in rare cases Protein: solute carrier 26A4 Cytogenetic locus: 7q31 Inheritance: AR Clinical Features and Diagnostic Criteria: bilateral severe SNHL, temporal bone abnormalities, vestibular abnormalities, goiter in 75% though only 10% have abnormal thyroid function. Clinical Tests: Hearing test. CT/MRI: dilation of the vestibular aqueduct with or without cochlear hypoplasia (Mondini malformation) Molecular Tests: l236P, T416P, H723R, IVS8+G>A represent 50% of all mutations. SLC26A4 sequencing available. Disease Mechanism: SLC26A4 is a chloride/iodide exchanger in the inner ear and thyroid, mutation leads to inner ear malformation and abnormal iodide processing in the thyroid Treatment/Prognosis: Hearing aids, cochlear implant, monitor thyroid function 74 DISORDERS OF HEARING and/or VISION PENDRED SYNDROME Enlarged vestibular aqueduct in Pendred syndrome (http://www.nidcd.nih.gov/health/hearing/pages/vestAque.aspx#diagram) 75 575 DISORDERS OF HEARING and/or VISION USHER SYNDROME Responsible genes: multiple genes, majority of cases due to MYO7A, USH2A Proteins: Myosin-VIIa, Usherin Cytogenetic loci: 11q13.5, 1q41 Inheritance: AR Clinical Features and Diagnostic Criteria: Type I congenital profound HL, congenital balance problems, retinitis pigmentosa (RP) onset pre-puberty. Type II congenital mild-severe HL, normal balance, RP onset in teens-20’s, Type III progressive later onset HL, progressive balance problems, variable onset RP. Clinical Tests: hearing tests, ERG, eye exam for pigment changes Molecular Tests: Type I MYO7A sequence analysis (40-50%) Type II USH2A sequencing (65%) Disease Mechanism: RP is caused by degeneration of rod and cone functions of the retina. For at least some gene, inner hair cell function and structure are affected in the ear. Treatment/Prognosis: RP is progressive, bilateral, and symmetric resulting in progressively constricted visual fields though not complete blindness. Vitamin A may slow progression. HL is complete in Usher Type I and progressive in types II and III. Cochlear implants and hearing aids for HL 76 DISORDERS OF HEARING and/or VISION USHER SYNDROME webvision.med.utah.edu 77 DISORDERS OF HEARING and/or VISION WAARDENBURG SYNDROME Responsible gene: PAX3 Protein: Paired box protein Pax-3 Cytogenetic locus: 2q35 Inheritance: AD Clinical Features and Diagnostic Criteria: WS1: SNHL, heterochromic irides, white forelock, early graying, leukoderma, dystrophia canthorum, neural tube defect. WS2: WS1 without dystrophia canthorum WS3: WS1 features and limb hypoplasia or contracture, carpal bone fusion, or syndactyly WS4: WS1 with Hirschprung disease Clinical Tests: ABR, audiogram, calculation of W-index to identify dystopia canthorum Molecular Tests: PAX3 gene sequencing (90% WS1) Disease Mechanism: Haploinsufficiency. PAX3 is a homeobox transcription factor involved in melanocyte development. Treatment/Prognosis: Hearing aids or cochlear implants. Folic acid supplementation of pregnancies at risk for WS1 related neural tube defect 78 576 DISORDERS OF HEARING and/or VISION WAARDENBURG SYNDROME White forelock and dystrophia canthorum W Index X = (2a - 0.2119c - 3.909)/c Y = (2a - 0.2479b – 3.909)/b W = X + Y + a/b. WS type I if all affected family members ≥ 1.95. www.emedicine.com 79 HEMATOLOGIC DISORDERS ACUTE INTERMITTENT PORPHYRIA Responsible gene: HMBS Protein: Porphobilinogen deaminase Cytogenetic locus: 11q23.3 Inheritance: AD Clinical Features and Diagnostic Criteria: Onset after puberty, acute attacks, abdominal pain, muscle weakness, neuropathy, hysteria, anxiety, hepatocellular carcinoma, NO CUTANEOUS FINDINGS Clinical Tests: increased urine delta-amonolevulinic acid (ALA) and porphobilinogen (PBG) during acute attack Molecular Tests: HMBS gene sequencing (>98%) Disease Mechanism: toxicity of ALA Treatment: Stop or treat precipitant (medication, infection, EtOH, dehydration, smoking, poor caloric intake); intubate if bulbar paralysis; IV dextrose; IV hemin (repress ALAS-N enzyme activity); pain control; liver transplantation 80 ACUTE INTERMITTENT PORPHYRIA HEMATOLOGIC DISORDERS Photograph of urine from a normal subject (left) and a subject with acute intermittent porphyria (middle). The colors are compared with a dilute aqueous solution of red wine (right). Provided by Shigeru Sassa, MD, PhD http://terrycomeau.com/Porphyria/Urine_in_AIP.jpg 81 577 HEMATOLOGIC DISORDERS ALPHA THALASSEMIA Responsible genes: HBA1, HBA2 Protein names: Hemoglobin subunit alpha 1 and 2 Cytogenetic locus (loci): 16pter-p13.3 Inheritance: AR; if parents Alpha Thal trait, risk for HbH disease if one parent’s mutations are in cis, at risk for HB Bart if both parents in cis Clinical Features and Diagnostic Criteria: HB Bart: loss or dysfunction of all 4 alpha thal alleles, hydrops fetalis, severe hypochromic anemia, death in neonatal period; HbH: loss or dysfunction of 3 of 4 alpha thal alleles, microcytic hypochromic hemolytic anemia, HSM, jaundice Alpha Trait:loss or dysfunction of 2 alpha thal alleles, low MCV, low MCH, nl levels Hgb A2 and F; Alpha “silent” carrier: loss or dysfunction of one alpha thal allele, none or mild thalassemia-like effect Clinical Tests: MCV, MCH, peripheral smear, reticulocyte count, hemoglobin electrophoresis. Prenatal screen at risk populations! Molecular Tests: Targeted mutation analysis for common deletions (90%); gene sequencing (10%) Disease Mechanism: Inability to form normal Hb A (normally composed of two alpha and two beta chains) Treatment/Prognosis: No tx for HB Bart, rec termination due to maternal complications with hydrops; intrauterine blood transfusions, hematopoietic stem cell transplant emerging. Hb H: prbc transfusions during hemolytic crisis, anemia causing cardiac sx, or severe bony changes; splenectomy with abx prophylaxis (if <5y) for splenomegaly 82 HEMATOLOGIC DISORDERS ALPHA THALASSEMIA http://sickle.bwh.harvard.edu/alpha_two.gif 83 BETA-THALASSEMIA HEMATOLOGIC DISORDERS Responsible gene: HBB Protein: Hemoglobin subunit beta Cytogenetic locus: 11p15.5 Inheritance: AR Clinical Features and Diagnostic Criteria: severe anemia and HSM. Without Tx: severe FTT and shortened life expectancy. Thal. intermedia: present later, milder anemia, only rarely requires transfusion; at risk for iron overload due to inc intestinal absorption of iron. The clinical severity of the beta-thal syndromes depends on the extent of globin alpha chain/non-globin alpha chain imbalance. At risk pop’s: Mediterranean, middle eastern, Indian, Thai, Chinese, African, African American. Clinical Tests: microcytic hypochromic anemia, an abnl peripheral blood smear with nucleated RBCs, and reduced amounts of hemoglobin A (HbA) on hemoglobin analysis. Carriers: reduced MCV, MCH, and RBC morphologic changes that are less severe than in affected individuals. Molecular Tests: Mutation scanning/sequencing. In each at-risk population, 4-10 mutations account for the large majority of HBB disease. Compound heterozygosity for a mild/silent mutation and a severe mutation produces a variable phenotype, ranging from thalassemia intermedia to thalassemia major. Disease Mechanism: Absence of globin beta chains. The non-assembled globin alpha chains that result from unbalanced globin alpha chain/non-globin alpha chain synthesis precipitate in the form of inclusions which damage the erythroid precursors in the bone marrow and spleen, causing ineffective erythropoiesis. Treatment/Prognosis: Treat with a regular transfusion program and chelation therapy (to reduce transfusion iron overload), allows for normal growth and development and extends life expectancy into the third to fifth decade; bone marrow transplantation is curative 84 578 HEMATOLOGIC DISORDERS BETA-THALASSEMIA http://web2.iadfw.net/uthman/hemoglobinopathy/thal_pathogenesis.gif 85 HEMATOLOGIC DISORDERS FACTOR V LEIDEN THROMBOPHILIA Responsible gene: F5 Protein: Coagulation factor V Cytogenetic locus: 1q23 Inheritance: AD (moderately inc. risk VTE), AR (significantly inc .risk VTE) Clinical Features and Diagnostic Criteria: inc. risk venous thromboembolism (VTE), most commonly deep venous thrombosis (DVT). Heterozygous: at most modest inc. in VTE recurrence risk, 2-3x inc RR pregnancy loss. Homozygous: Inc. chance VTE recurrence. Arterial thrombosis, MI, and stroke not associated with factor V Leiden. Clinical Tests: APC resistance assay, sensitivity and specificity for factor V Leiden approaches 100% Molecular Tests: F5 G to A substitution at nt 1691 (100%) Disease Mechanism: The G>A substitution affects an APC cleavage site and the mutant factor V Leiden is inactivated 10x more slowly and persists longer in circulation-> inc. thrombin generation Treatment/Prognosis: Risk of VTE compounded by coexisting thromboembolic d/o, malignancy, travel, central venous catheters, pregnancy, OCP, HRT, advancing age, surgery, organ transplant. Heterozygotes with first VTE with no id’ed risk factor or a persistent risk factor require longer course of anticoagulation than those with a transient risk factor (eg surgery). Long term anticoagulation with LMW Heparin or Warfarin if recurrent VTE, multiple thrombophilic d/o, coexistent circumstantial risk factors, and factor V Leiden homozygotes 86 HEMATOLOGIC DISORDERS HEMOPHILIA A Responsible gene: F8 Protein: Coagulation Factor VIII Cytogenetic locus: Xq28 Inheritance: XLR Clinical Features and Diagnostic Criteria: hemarthrosis or intracranial bleed with mild or no trauma; deep muscle hematomas; prolonged or renewed bleeding after trauma, surgery, tooth extraction, nose bleeds, mouth injury, or circumcision, excessive bruising. Clinical Tests: Prolonged PTT, severe hemophilia: <1%, moderate: 1-5%, and mild hemophilia 6-35% Factor VIII activity. 10% of carrier females have Factor VIII activity <35%. Molecular Tests: Severe: F8 intron 22-A gene inversion (45%), F8 intron 1 gene inversion (3%), F8 gene del or rearrangement, frameshift, splice junction, or nonsense mutations (40%), missense mutation (10%). Mild-moderate: missense mutation (97%) Disease Mechanism: Normal Factor VIII circulates as an inactivated clotting cofactor activated by thrombin. Severe mutations lead to absent protein, mild-mod mutations to abnormal protein. Treatment/Prognosis: IV Factor VIII prophylactically 3x/wk in severe cases and after trauma, avoid IM injection. Consider HIV, Hep A, B, and C testing if history of receiving blood products; DDAVP in mild cases 87 579 HEMATOLOGIC DISORDERS HEMOPHILIA A http://scielo.isciii.es/img/revistas/medicorpa/v12n5/10.htm29.gif 88 HEMATOLOGIC DISORDERS HEMOPHILIA B Responsible gene: F9 Protein: Coagulation factor IX Cytogenetic locus: Xq27.1-q27.2 Inheritance: XLR Clinical Features and Diagnostic Criteria: hemarthrosis or intracranial bleed with mild or no trauma; deep muscle hematomas; prolonged or renewed bleeding after trauma, surgery, tooth extraction, nose bleeds, mouth injury, or circumcision, excessive bruising. Clinical Tests: Prolonged PTT, severe hemophilia: <1%, moderate: 1-5%, and mild hemophilia 6-30% Factor IX activity. 10% of carrier females have Factor VIII activity <30%. Molecular Tests: F9 sequence analysis (99%). Large gene deletions, nonsense mutations, and most frameshift mutations cause severe disease. Disease Mechanism: Factor IX activates Factor X which is a critical early step that can regulate the overall rate of thrombin generation in coagulation. Treatment/Prognosis: Recombinant factor IX concentrate 2-3x/wk for severe deficiency and within one hour of trauma. Avoid IM injection. Consider HIV, Hep A, B, and C testing if history of receiving blood products. 89 HEMATOLOGIC DISORDERS HFE-ASSOCIATED HEREDITARY HEMOCHROMATOSIS Responsible gene: HFE Protein: Hereditary hemochromatosis protein Cytogenetic locus: 6p21.3 Inheritance: AR (penetrance is low, a large fraction of homozygotes never develop symptoms. Clinical Features and Diagnostic Criteria: Inappropriately high iron absorption by the GI mucosa leads to excessive iron storage in the liver, skin, pancreas, heart, joints, and testes. Early Sx: abdominal pain, weakness, lethargy, and weight loss. Clinical Tests: Inc. fasting transferrin-iron saturation (men >60%, women >50%; some use >45% as cutoff for both men and women) on at least 2 occasions, inc. serum ferritin concentration (nonspecific for HHC), quantitative phlebotomy to determine iron quantity., liver biopsy, hepatic MRI Molecular Tests: Targeted mutation testing (60-90% C282Y/C282Y; 3-8% C282Y/H63D. Disease Mechanism: HFE protein binds transferrin receptor 1 and is thought to reduce cellular iron uptake- mutation leads to inc. iron uptake Treatment/Prognosis: If untreated: hepatic fibrosis or cirrhosis, increased skin pigmentation, DM, CHF and/or arrhythmias, cardiomyopathy, arthritis, or hypogonadism. Treat with phlebotomy if symptomatic, aim for ferritin <50, transferrin-iron saturation <50% 90 580 DISORDERS OF THE IMMUNE SYSTEM X-LINKED AGAMMAGLOBULINEMIA (Bruton’s Agammaglobulinemia) Responsible gene: BTK Protein: BTK Cytogenetic locus: Xq21.3-q22 Inheritance: X-LR Clinical Features and Diagnostic Criteria: recurrent OM, pneumonia, sinusitis <5yrs; sepsis, meningitis, or cellulitis, paucity of lymphoid tissue Clinical Tests: Low but measureable IgG, <1% B Cells (CD19) Molecular Tests: 90% BTK sequence variant, 10% del/dupl/inv Disease Mechanism: Immune deficiency; BTK protein expressed in myeloid cells, platelets, B lineage cells Treatment/Prognosis: Monthly IV or weekly SC gammaglobulin 91 DISORDERS OF THE IMMUNE SYSTEM X-LINKED AGAMMAGLOBULINEMIA (Bruton’s Agammaglobulinemia) (www.tmd.ac.jp/english/press-release/20120227/) 92 DISORDERS OF THE IMMUNE SYSTEM FAMILIAL MEDITERRANEAN FEVER Responsible gene: MEFV Protein: Pyrin Cytogenetic locus: 16p13 Inheritance: AR Clinical Features and Diagnostic Criteria: Type 1 recurrent febrile episodes with peritonitis, synovitis, or pleuritis, recurrent erysipelas-like erythema, AA type amyloidosis, favorable response to continuous colchicine treatment, at risk ethnic group (Armenian, Turkish, Arab, North African Jewish, Iraqi Jewish, Ashkenazi Jewish). Type 2 amyloidosis as first clinical presentation Clinical Tests: Inc ESR, leukocytosis, inc serum fibrinogen, proteinuria Molecular Tests: MEFV targeted mutation analysis (70-90% depending on panel and ethnicity), MEFV sequencing (90% all ethnic groups) Disease Mechanism: Mutations result in less IL-1beta activation and as a result inc IL-1 responsiveness-> inc inflammatory attacks Treatment/Prognosis: M694V homozygotes or compound heterozygotes with another FMF allele treated with daily colchicine for life. Colchicine decreases inflammatory attacks and deposition of amyloid. 93 581 DISORDERS OF THE IMMUNE SYSTEM FAMILIAL MEDITERRANEAN FEVER (healthlineinfo.com/familial-mediterranean-fever.html) 94 MULTIPLE CONGENITAL ANOMALIES AARSKOG SYNDROME Responsible gene: FGD1 Protein: Rho/Rac guanine nucleotide exchange factor Cytogenetic locus: Xp11.22 Inheritance: XLR (some AR, AD cases reported) Clinical Features and Diagnostic Criteria: hypertelorism, shawl scrotum, brachydactyly, short stature, cryptorchidism, cervical vertebral abnormalities, ID (30%), milder manifestations in females Clinical Tests: xray Molecular Tests: FGD1 sequencing (7-20%) Disease Mechanism: unclear, FGD1/Rho GTPase Cdc42 implicated in cytoskeletal organization, and potentially in skeletal formation and morphogenesis Treatment/Prognosis: orchiopexy, growth hormone trials have not been successful 95 MULTIPLE CONGENITAL ANOMALIES AARSKOG SYNDROME Int. braz j urol. vol.32 no.4, 2006 96 582 MULTIPLE CONGENITAL ANOMALIES ANTLEY-BIXLER SYNDROME Responsible gene: POR Protein: NADPH-cytochrome P450 reductase Cytogenetic locus: 7q11.2 Inheritance: AR Clinical Features and Diagnostic Criteria: Ambiguous genitalia, enlarged cystic ovaries, poor masculinization in males, maternal virilization during pregnancy with an affected fetus. Craniosynostosis, choanal stenosis or atresia, stenotic external auditory canals, hydrocephalus. Neonatal fractures, bowing of the long bones, joint contracture, renal malformations Clinical Tests: Sterol or or steroid abnormalities using GC-MS, increased urinary pregnenolone and progesterone metabolites Molecular Tests: POR sequence variants Disease Mechanism: Disorder of steroid and cholesterol synthesis due to cytochrome P450 reductase deficiency Treatment/Prognosis: Airway management, hydrocortisone replacement, stress dose steroids, surgical correction of genital abnormalities, VP shunt for significant hydrocephalus, PT to minimize joint contracture 97 MULTIPLE CONGENITAL ANOMALIES ANTLEY-BIXLER SYNDROME Antley-Bixler Facial Features Frontal bossing Severe midface hypoplasia Short bulbous nose Depressed nasal bridge Small mouth Dysplastic ears that may be low set http://pediatricneuro.com/alfonso/newa10l.jpg 98 MULTIPLE CONGENITAL ANOMALIES BARDET-BIEDL SYNDROME Responsible genes: BBS1, BBS10 (multiple additional genes id’ed) Proteins: BBS1 protein, BBS10 protein Cytogenetic loci: 11q13, 12q21.2 Inheritance: AR (though 10% BBS thought to be tri-allelic) Clinical Features and Diagnostic Criteria: cone-rod dystrophy, truncal obesity, postaxial polydactyly, cognitive impairment, male hypogonadotrophic hypogonadism, complex female genitourinary malformations, and renal dysfunction. Night blindness by age 7-8 yrs, legally blind by age 15.5 yrs. A majority have significant learning difficulties, only a minority have severe impairment. Renal disease is a major cause of morbidity and mortality. Clinical Tests: atypical pigmentary retinal dystrophy with early macular involvement, renal anomalies on US Molecular Tests: Targeted mutation analysis: p.M390R BBS1 (18%-32% of BBS) and C91fsX95 BBS10 (10% of BBS). Disease Mechanism: Defects in cilia or intraflagellar transport (IFT) Treatment/Prognosis: visual aids and educational programs for the visually impaired; diet, exercise, and behavioral therapies for obesity; surgery to remove accessory digits; surgical repair of hydrocolpos, vaginal atresia, or hypospadias; HRT for hypogonadism. 99 583 MULTIPLE CONGENITAL ANOMALIES BARDET-BIEDL SYNDROME Beales P L et al. J Med Genet 1999;36:437-446 100 MULTIPLE CONGENITAL ANOMALIES BRANCHIO-OTO-RENAL SYNDROME Responsible gene: EYA1, SIX1, SIX5 Proteins: Eyes absent homolog 1, Homeobox protein SIX1 and SIX5 Cytogenetic loci: 8q13.3, 14q23, 19q13.32 Inheritance: AD Clinical Features and Diagnostic Criteria: malformations of the outer, middle, and inner ear associated with conductive, sensorineural, or mixed hearing impairment; branchial fistulae and cysts; and renal malformations, ranging from mild renal hypoplasia to bilateral renal agenesis Clinical Tests: Temporal bone CT, hearing test, renal US Molecular Tests: Mutation scanning (30%), Dupl/del testing (10%) Disease Mechanism: EYA1 encodes products important for inner-ear, kidney, and branchialarch development. Some mutations encode proteins that are rapidly degraded. Expression of SIX1 is necessary for normal development of the inner ear, nose, thymus, kidney, and skeletal muscle Treatment/Prognosis: excision of branchial cleft cysts/fistulae, fitting with appropriate aural habilitation, hearing impaired education programs. End-stage renal disease may require dialysis or renal transplantation. Surveillance includes semiannual examination for hearing impairment and annual audiometry to assess stability of hearing loss and semiannual/annual examination by a nephrologist if indicated 101 MULTIPLE CONGENITAL ANOMALIES BRANCHIO-OTO-RENAL SYNDROME Branchial fistula Trummer T et al. J Med Genet 2002;39:71-73 102 584 MULTIPLE CONGENITAL ANOMALIES CHARGE SYNDROME Responsible gene: CHD7 Protein: Chromodomain-helicase-DNA-binding protein 7 Cytogenetic locus: 8q12.1 Inheritance: AD Clinical Features and Diagnostic Criteria: 4/7: eye coloboma, heart anomaly (conotruncal defects, arch abnormalities), choanal atresia, growth and mental retardation, genitourinary malformations (microphallus), ear anomalies (ossicular malformations, Mondini defect of the cochlea) and/or deafness. Facial palsy, cleft palate, TE fistula, and dysphagia are commonly associated. 20-25% mortality in the first year Clinical Tests: echocardiogram, audiology evaluation, temporal bone CT, renal ultrasound Molecular Tests: CHD7 sequencing (60-65%) Disease Mechanism: Haploinsufficiency. This class of proteins is thought to have pivotal roles in early embryonic development by affecting chromatin structure and gene expression Treatment/Prognosis: Assess for airway compromise, swallowing problems, typical surgical correction of heart and GI malformations MULTIPLE CONGENITAL ANOMALIES CHARGE SYNDROME www.ncbi.nlm.nih.gov/.../bin/chargeFig1.jpg 104 MULTIPLE CONGENITAL ANOMALIES COFFIN-LOWRY SYNDROME Responsible gene: RPS6KA3 Protein: Ribosomal protein S6 kinase alpha-3 Cytogenetic locus: Xp22.2-p22.1 Inheritance: XLD Clinical Features and Diagnostic Criteria: severe to profound ID in males, short, soft fleshy hands, tapering fingers with small terminal phalanges, males <3% in height, microcephaly, stimulus induced drop episodes, kyphoscoliosis, characteristic facial features in older males, normal to profound ID in females. Clinical Tests: x-ray: thickened skull, anterior vertebrae beaking, metacarpal pseudoepiphyses Molecular Tests: RPS6KA3 sequencing (35-40%) Disease Mechanism: unclear, RPS6KA3 is a member of the Ras signaling cascade and participates in cellular events such as proliferation and differentiation Treatment/Prognosis: Medication for drop episodes, Rispieridone for self-injurious behavior, annual cardiac exam with echo every 5-10 years. 105 585 COFFIN-LOWRY SYNDROME MULTIPLE CONGENITAL ANOMALIES Coffin Lowry Facial Features Prominent forehead and eyebrows Full supraorbital ridges Marked ocular hypertelorism with downslanting palpebrae Low nasal bridge, blunt tip, and thick alae nasi and septum Large mouth, usually held open Patulous lips with everted lower lip Prominent ears http://clsf.info/Images/2005_July.JPG 106 MULTIPLE CONGENITAL ANOMALIES CORNELIA DE LANGE SYNDROME Responsible gene: NIPBL, SMC1A, SMC3, HDAC8, RAD21 Protein: Nipped-B-like protein, Structural maintenance of chromosomes protein 1A and 3, histone deacetylase 8, RAD21 Cytogenetic loci: 5p13.1, Xp11.22-p11.21, 10q25.2, Xq13.1, 8q24.11 Inheritance: AD (NIPBL and SMC3), XLR (SMC1L1) Clinical Features and Diagnostic Criteria: pre/postnatal growth retardation, low anterior hairline and synophrys, diaphragmatic hernia, upper limb anomalies (hypoplastic middle phalanx of the index finger, hypoplastic thenar eminence), ptosis, nystagmus, mod-severe ID, pulmonary valve stenosis and/or VSD Clinical Tests: non are diagnostic Molecular Tests: NIPBL sequencing (~50%), SMC1L1 sequencing (4%), SMC3 (<1%) Disease Mechanism: Unknown, the majority of mutations are truncating, likely leading to protein haploinsufficiency Treatment/Prognosis: Treat individual medical and developmental issues 107 MULTIPLE CONGENITAL ANOMALIES CORNELIA DE LANGE SYNDROME Facial Features Microbrachycephaly Synophrys, arched eyebrows Long, thick eyelashes Low-set posteriorly rotated and/or hirsute ears with thickened helices Depressed or broad nasal bridge, upturned nasal tip with anteverted nares, and prominence of the lateral aspects Long smooth philtrum, thin vermillion border of the upper lip with a midline "drip" appearance, downturned corners of the mouth High and arched palate with clefts Small widely-spaced teeth Micrognathia Short neck http://www.emedicine.com/ped/images/289503 108 586 MULTIPLE CONGENITAL ANOMALIES CRI-DU-CHAT (5p MINUS SYNDROME) Responsible gene(s): RPS14?, microRNA 145 and 146a? Protein(s): Cytogenetic locus: 5p15.2 Inheritance: 12% due to unequal segregation of a translocation or recombination involving a pericentric inversion in one of the parents, 85% sporadic de novo deletions (80% are on the paternal chromosome) Clinical Features and Diagnostic Criteria: Cat-like cry (abnormal laryngeal development), slow growth, microcephaly, ID, hypotonia, strabismus, characteristic facial features. Cat-like cry only when deletion limited to band 5p15.32 Molecular Tests: Most are visible, a few are submicroscopic and diagnosed by FISH for the critical region. Disease Mechanism: A study of 50 patients with deletions ranging from 5p15.2 to 5p13 and found no correlation with size of deletion and degree of mental impairment Treatment/Prognosis: Supportive care 109 MULTIPLE CONGENITAL ANOMALIES CRI-DU-CHAT (5p MINUS SYNDROME) Facial Features Microcephaly Round face Hypertelorism Micrognathia Epicanthal folds Low-set ears www.specialchild.com/archives/poster-child023 110 MULTIPLE CONGENITAL ANOMALIES FRYNS SYNDROME Responsible gene(s): unknown Protein(s): unknown Cytogenetic locus (loci): unknown Inheritance: AR Clinical Features and Diagnostic Criteria: LGA, coarse face, CL/CP, diaphragmatic defect, distal digital hypoplasia, ID in survivors, agenesis of the CC, optic and olfactory tract hypoplasia, encephalocele, GU malformation Clinical Tests: symptomatic Molecular Tests: none Disease Mechanism: unknown Treatment/Prognosis: The majority are stillborn or die in early neonatal period, 14% survive 111 587 MULTIPLE CONGENITAL ANOMALIES FRYNS SYNDROME Hypoplastic nails Fronal encephalocele and lissencephaly 112 MULTIPLE CONGENITAL ANOMALIES GREIG CEPHALOPOLYSYNDACTYLY Responsible gene: GLI3 Protein: Zinc finger protein GLI3 Cytogenetic locus: 7p13 Inheritance: AD Clinical Features and Diagnostic Criteria: Major findings: macrocephaly, ocular hypertelorism, preaxial polydactyly, cutaneous syndactyly. Developmental delay, ID, or seizures (<10%)- more common in those with large (>300 kb) deletions including GLI3. Allelic with Pallister-Hall syndrome (caused by GLI3 frame shifting mutations). Clinical Tests: 500-600 band karyotype 7p13 translocation or interstitial deletion (5-10%) Molecular Tests: GLI3 sequence analysis (70%) Disease Mechanism: GLI proteins regulate genes distal to Sonic Hedgehog in the SHH pathway. Pathogenesis of GCPS is haploinsufficiency Treatment/Prognosis: Surgical correction of polydactyly and syndactyly as indicated. CNS imaging if HC increasing faster than normal to r/o hydrocephalus 113 MULTIPLE CONGENITAL ANOMALIES GREIG CEPHALOPOLYSYNDACTYLY Macrocephaly, hypertelorism, and polysyndactyly http://www.ojrd.com/content/figures/1750-1172-3-10-1.jpg 114 588 MULTIPLE CONGENITAL ANOMALIES JOUBERT SYNDROME Responsible genes: NPHP1, AHI1, CEP290, TMEM67 and others (19 genes) Proteins: Nephrocystin-1, Jouberin, Centrosomal protein Cep290, Meckelin Cytogenetic loci: 2q13, 6q23.3, 12q21.32, 8q21.1-q22.1 Inheritance: AR (19 genes with rare-4% prevalence) Clinical Features and Diagnostic Criteria: Hypotonia in infancy leading to ataxia later, DD/ID, alternating tachypnea and/or apnea), pigmentary retinopathy, oculomotor apraxia or difficulty in smooth visual pursuits and jerkiness in gaze tracking. M:F, 2:1. Renal disease seen in those with retinal involvement. Rarely hepatic fibrosis. Clinical Tests: Molar tooth sign (cerebellar vermis hypoplasia) on MRI, ERG, renal US, LFT’s Molecular Tests: NPHP1 FISH or deletion analysis (1-2%), Sequencing AHI1 (11%), CEP290 (10%), TMEM67 (10%) Disease Mechanism: The CEP290 protein product modulates ATF4, a transcription factor implicated in renal cyst formation. Meckelin localizes to the primary cilium and plasma membrane of renal and biliary epithelial cells and other ciliated cells Treatment/Prognosis: apnea monitoring, ST, G tube if severe dyspahgia, surgery as needed for eye disease, dialysis for nephronophthisis 115 MULTIPLE CONGENITAL ANOMALIES JOUBERT SYNDROME Molar tooth sign 116 MULTIPLE CONGENITAL ANOMALIES KABUKI SYNDROME Responsible genes: KMT2D (66%), KDM6A Proteins: MLL2, Lysine-specific demethylase 6A Cytogenetic loci: 12q12-q14, Xp11.3 Inheritance: AD, XLD Clinical Features and Diagnostic Criteria: unique facial features, fetal finger pads, IQ<80, joint laxity, high palate, hypotonia, short stature, CHD, CL/P, scoliosis, renal anomalies, hearing loss, speech delay Clinical Tests: echocardiogram, renal ultrasound, eye exam, neuropsychological testing Molecular Tests: MLL2 gene sequencing, KDM6A gene sequencing and deletion testing Disease Mechanism: MLL2 encodes a protein that is part of the SET family of proteins, important to the epigenetic control of active chromatin states. Mutations are predicted to truncate the polypeptide chain before translation of the SET domain. H3K4 methylation by MLL2 is linked to the demethylation of H3K27 by KDM6A. Treatment/Prognosis: Individual medical problems are treated as in the general population. GH for short stature if deficient. At risk for immunodeficiency. 117 589 MULTIPLE CONGENITAL ANOMALIES KABUKI SYNDROME Facial Features Elongated palpebral fissures Eversion of the lateral third of the lower eyelid Arched and broad eyebrows Short columella with depressed nasal tip Large, prominent, or cupped ears http://kabukisyndrome.com 118 MULTIPLE CONGENITAL ANOMALIES MONOSOMY 1p36 Responsible genes: unknown Proteins: unknown Cytogenetic locus: 1p36 Clinical Features and Diagnostic Criteria: The most common terminal deletion syndrome. Hypotonia, developmental delay, growth retardation, obesity, microcephaly, orofacial clefting, typical facial features. Also minor cardiac malformations, cardiomyopathy, seizures, ventricular dilation, SNHL Clinical Tests: Brain CT/MRI Molecular Tests: The deletion can be detected by HR karyotype, confirmatory FISH required in most cases. The majority are maternally derived. Disease Mechanism: contiguous gene deletion syndrome Treatment/Prognosis: symptomatic treatment 119 MULTIPLE CONGENITAL ANOMALIES MONOSOMY 1p36 Facial Features: Straight eyebrows Deep-set eyes Midface hypoplasia Broad and flat nasal root/bridge Long philtrum Pointed chin Microbrachycephal y Epicanthal folds Posteriorly rotated, low-set, abnormal ears. www.theanswers.com 120 590 MULTIPLE CONGENITAL ANOMALIES PRADER-WILLI SYNDROME Responsible genes: Paternally expressed genes within the imprinted locus on 15q11-13 (SNURF-SNRPN, MKRN3, MAGEL2, and NDN ) Cytogenetic locus: 15q11-13 Inheritance: autosomal, expressed from paternal Ch 15 Clinical Features and Diagnostic Criteria: Hypothalamic insufficiency, neonatal hypotonia, developmental delay, hyperphagia leading to obesity, short stature, small hands and feet, hypogonadism, ID Molecular Tests: 3-5 Mb deletion of 15q11.2-q13 (~70%), matUPD (15%), PWS imprinting center defect (1-2%) Disease Mechanism: unknown Treatment/Prognosis: Monitor for feeding problems in infancy, obesity, OCD, psychosis, scoliosis, obstructive sleep apnea, diabetes, osteopenia 121 MULTIPLE CONGENITAL ANOMALIES PRADER-WILLI SYNDROME http://www.bcpwsa.com/images/header.jpg Arch Dis Child 2008;93:341-345 doi:10.1136/adc.2007 122 MULTIPLE CONGENITAL ANOMALIES RUBENSTEIN-TAYBI SYNDROME Responsible gene: CREBBP, EP300 Protein: CREB-binding protein, histone acetyltransferase-p300 Cytogenetic locus: 16p13.3, 22q13 Inheritance: AD though only a few cases of affected parent and child Clinical Features and Diagnostic Criteria: microcephaly, beaked nose, broad thumbs and toes, cryptorchidism, growth delay, severe ID (35-50), congenital heart defect, strabismus, ptosis, sleep apnea, tumors (meningioma, pilomatrixoma, leukemia), behavior problems Clinical Tests: ERG, echocardiogram, deletion or translocation occasionally seen on karyotype Molecular Tests: FISH CREBBP (~10%), direct sequencing CREBBP (40-60%), EP300 (~3%) Disease Mechanism: Some CREBBP mutations lead to abnormal acetylation of histones, an important step in transcription activation Treatment/Prognosis: Standard care for vision, hearing loss, heart defects, feeding problems. Some require thumb/toe surgery, behavior modification programs 123 591 MULTIPLE CONGENITAL ANOMALIES RUBENSTEIN-TAYBI SYNDROME Broad, deviated thumbs and first toes European Journal of Human Genetics (2006) 14, 981–985 124 MULTIPLE CONGENITAL ANOMALIES SMITH-MAGENIS SYNDROME Responsible gene: RAI1 Protein: Retinoic acid-induced protein 1 Cytogenetic locus: 17p11.2 Inheritance: AD (sporadic unless secondary to a parental balanced translocation) Clinical Features and Diagnostic Criteria: mild-moderate infantile hypotonia, feeding problems and FTT, short stature, brachydactyly, ophthalmologic and ORL abnormalities, early speech delay with or without hearing loss, peripheral neuropathy, sleep problems, and stereotypic maladaptive behaviors (self-injurious behaviors, inattention+hyperactivity, impulsivity, disobedience, the “self-hug” and “lick and flip” page turning motion), mild-mod ID, coarsening face over time Clinical Tests: Renal US, echo, spine x-ray, FISH, CMA for 17p11.2 deletion (~90%) Molecular Tests: RAI1 sequencing (5-10%) Disease Mechanism: The RAI1 gene product is thought to function in transcriptional regulation Treatment/Prognosis: ST, sensory integration, psychotropic meds for attention issues, behavioral therapies, melatonin may help with sleep, monitoring for hypercholesterolemia. Annual team eval, TFTs, fasting lipid profile, UA, scoliosis check, eye exam 125 MULTIPLE CONGENITAL ANOMALIES SMITH-MAGENIS SYNDROME Facial Features: Brachycephaly Midface retrusion Relative prognathism with age Broad, square-shaped face Everted, "tented“ vermilion of the upper lip Deep-set, close-spaced eyes http://www.nature.com/ejhg/journal/v16/n4/images/5202009f2.jpg 126 592 MULTIPLE CONGENITAL ANOMALIES TRIPLOIDY Cytogenetic abnormality: 69,XXY>69,XXX (69,XYY very rare) Inheritance: Sporadic without inc risk of recurrence Clinical Features and Diagnostic Criteria: >99% lost in first trimester, accounts for 6-10% of all SAb’s and 16-20% of all chromosomally abnormal SAb’s. Dysplastic calvaria with large posterior fontanelle, ¾ finger syndactyly, ASD, VSD, hydrocephalus, holoprosencephaly. Parent of origin effect: If Maternal: small placenta, severe asymmetric IUGR with a large head If Paternal: hydropic large placenta, well grown to mod symmetric IUGR, nl or microcephalic head Clinical Tests: Prenatal US, maternal serum hCG low Molecular Tests: Karyotype Disease Mechanism: Gynogenic triploidy (digyny): NDJ producing diploid oocyte, fertilization of ovulated primary oocyte, or polar body retention. Androgenic triploidy (Diandry) NDJ producing a diplod sperm or dispermy (most common) Treatment/Prognosis: Very poor prognosis, may be better if triploid mosaic 127 MULTIPLE CONGENITAL ANOMALIES TRIPLOIDY Classic 3/4 finger syndactyly of triploidy library.med.utah.edu 128 MULTIPLE CONGENITAL ANOMALIES TRISOMY 13, PATAU SYNDROME Inheritance: 20% due to a translocation Clinical Features and Diagnostic Criteria: The least common of the live born trisomy disorders. Holoprosencephaly, polydactyly, seizures, HL, microcephaly, midline CL/P, omphalocele, cardiac and renal anomalies, ID. Mosaic Tri 13: very broad phenotype from typical features of full trisomy to more mild ID and physical features and longer survival. Clinical Tests: Brain MRI, EEG, audiogram, echo, renal US Molecular Tests: Karyotype is diagnostic Disease Mechanism: 75% are due to maternal nondysjunction, 20% to a translocation, and 5% to mosaicism. Defect in fusion of the midline prechordial mesoderm in the first three weeks of gestation cause the major midline dysmorphic features. Treatment/Prognosis: 44% die in the first month, >70% die within one year. Severe ID exists in all survivors. 129 593 MULTIPLE CONGENITAL ANOMALIES TRISOMY 13, PATAU SYNDROME Cutis Aplasia www.prenatalpartnersforlife.org 130 MULTIPLE CONGENITAL ANOMALIES TRISOMY 18, EDWARDS SYNDROME Inheritance: Less than 1% due to a translocation Clinical Features and Diagnostic Criteria: clenched hand, fingers 2/5 overlap 3/4, IUGR, rocker bottom feet, micrognathia, prominent occiput, microphthalmia, VSD, ASD, PDA, generalized muscle spasm, renal anomalies, ID. Mosaic Tri 18 has variable but usually somewhat milder expression. Clinical Tests: Echo, abdominal US. Maternal serum screen: low AFP, hCG, and UE3. Molecular Tests: karytype is diagnostic Disease Mechanism: Maternal nondysjunction (90%), mosaicism (10%) Treatment/Prognosis: 50% die in first week, 90% die by one year 131 MULTIPLE CONGENITAL ANOMALIES TRISOMY 18, EDWARDS SYNDROME Typical digit 2 over 3 and 5 over 4 of Trisomy 18 Typical rocker bottom foot of Trisomy 18 www.gfmer.ch/.../gendis 132 594 MULTIPLE CONGENITAL ANOMALIES TRISOMY 21, DOWN SYNDROME Cytogenetic locus (loci): (21.22.1-22.2 has been called the DS critical region though there have been cases of duplication outside of this region who manifest DS Inheritance: 95% de novo, 5% due to Robertsonian translocation or isochromosome 21 Clinical Features and Diagnostic Criteria: mild-mod ID, hypotonia, growth delay, strabismus, adult cataracts, myopia, conductive HL, macroglossia, hypodontia, joint hyperflexibility, hypogenitalism, congenital heart defect, duodenal atresia, hirschprung, thyroid disease, early onset Alzheimers, transient myeloproliferation, ALL Clinical Tests: prenatal US abnormalities detected in 50%, maternal serum screen: high free beta HCG, low PAPP-A, Molecular Tests: maternal fetal free DNA testing, karyotype is diagnostic Disease Mechanism: 90% due to maternal meiosis nondisjunction (¾ MI error, ¼ MII error) Treatment/Prognosis: Supportive care, overall life expectancy is reduced 133 MULTIPLE CONGENITAL ANOMALIES TRISOMY 21, DOWN SYNDROME www.fetalcenter.com/images/Trisomy_21 134 MULTIPLE CONGENITAL ANOMALIES VACTERL (VATER) ASSOCIATION Responsible genes: unknown (HOXD13 21 bp deletions: 1 case report), FGF8?, PTF1A? Proteins: unknown Cytogenetic locus: unknown Inheritance: Isolated Clinical Features and Diagnostic Criteria: Vertebral anomalies, Anal atresia, Cardiac malformations (VSD, PDA, TOF, TOV), Treacheoesophageal fistula, Esophageal atresia, Renal anomalies, and Limb anomalies (polydactyly, humeral hypoplasia, radial aplasia, proximally placed thumb). Diagnosis requires 3 of 7 features and it is a diagnosis of exclusion. A variant is VACTERL with hydrocephalus which can be AR or XL. Clinical Tests: echo, spinal x-ray, limb x-ray, and renal US Molecular Tests: There isn’t a molecular test but rule out aneuploidy with karyotype, Fanconi anemia with DEB testing, and consider SALL1 sequencing to rule out TownesBrocks syndrome. Disease Mechanism: unknown Treatment/Prognosis: Severe cardiac malformation, anal atresia, TE fistula, and EA require surgical repair in the neonatal period 135 595 MULTIPLE CONGENITAL ANOMALIES VACTERL (VATER) ASSOCIATION Approximate days post-conception during which anatomic structures in VACTERL form (Stevenson, Mol Syndromol 2013;4:7–15 ) 136 MULTIPLE CONGENITAL ANOMALIES WOLF-HIRSCHORN SYNDROME (4p minus, Monosomy 4p) Responsible genes: 4p deletion, critical region includes two genes, WHSC1 and WHSC2 of unknown significance Protein: unknown Cytogenetic locus: 4p; critical region: 165-kb region between markers D4S166 and D4S3327 Inheritance: 87% de novo, 13% due to unbalanced translocation from a balanced parent Clinical Features and Diagnostic Criteria: “greek warrior helmet appearance”, microcephaly, pre and postnatal growth deficiency, ID of variable degree, seizures, facial asymmetry, ptosis, IgA deficiency, structural brain anomalies, CL/P, CHD (ASD>PVS>VSD>PDA>AI>TOF), renal US Clinical Tests: Distinctive EEG, Brain MRI, echo, plasma IgA level Molecular Tests: HR karyotype for 4p16.3 deletion (60-70%), FISH/array CGH for critical region deletion (>95%) Disease Mechanism: The function of WHSC1, WHSC2, and LETM1 in normal development and in WHS patients is not known Treatment/Prognosis: 2/3 develop valproate responsive atypical absence seizures, standard treatment of other medical problems 137 MULTIPLE CONGENITAL ANOMALIES WOLF-HIRSCHHORN SYNDROME (4p minus, Monosomy 4p) Facial Features: 'Greek warrior helmet appearance' of the nose (the broad bridge of the nose continuing to the forehead) Microcephaly High forehead with prominent glabella Ocular hypertelorism Epicanthus Highly arched eyebrows Short philtrum Downturned mouth Micrognathia Poorly formed ears with pits/tags medgen.genetics.utah.edu 138 596 NEUROLOGIC DISORDERS X-LINKED ADRENOLEUKODYSTROPHY Responsible gene: ABCD1 Protein: ATP-binding cassette sub-family D member 1 Cytogenetic locus: Xq28 Inheritance: X-LR Clinical Features and Diagnostic Criteria: a. Childhood cerebral: ADHD->total disability within 2 yrs b. Adrenomyeloneuropathy: late 20’s progressive paraparesis, sphincter disturbance, adrenocortical dysfunction c. Adrenocortical insufficiency (only); majority by age 7.5 (seen in 20% carrier females Clinical Tests: Brain MRI, VLCFA (not reliably abnl in carrier females) Molecular Tests: ABCD1 seq (92%); ABCD1 del/dup (6%) Disease Mechanism: Peroxisomal disorder, accumulation of saturated VLCFA Treatment/Prognosis: Corticosteroid replacement, BMT if diagnosed after changes visible on brain MRI but before significant neuropsych problems develop (Lorenzo’s Oil) 139 X-LINKED ADRENOLEUKODYSTROPHY NEUROLOGIC DISORDERS Clinical Features of Various Phenotypes of X-linked ALD *POW = parieto-occipital white matter. (Kim and Kim, RadioGraphics, 2005) 140 NEUROLOGIC DISORDERS EARLY ONSET FAMILIAL ALZHEIMER DISEASE Responsible genes: PSEN1, APP, PSEN2 Proteins: Presenelin-1, Amyloid beta A4, Presenilin-2 Cytogenetic loci: 14q24.3, 21q21, 1q31-q42 Inheritance: AD Clinical Features and Diagnostic Criteria: Dementia, confusion, poor judgment, language disturbance, agitation, withdrawal, and hallucinations. Early onset: <age 60 Clinical Tests: Gross cerebral cortical atrophy. Post mortem neuropath: A beta-amyloid neuritic plaques, intraneuronal neurofibrillary tangles, and amyloid angiopathy Molecular Tests: Seq.: PSEN1 (20-70%), APP (10-15%), PSEN2 (rare) Disease Mechanism: ?chromosomal instability and breakage at nonrandom sites? Triple dose of APP may explain Alzheimer’s in Tri 21. Treatment/Prognosis: Death from general inninition, malnutrition, and pneumonia. Clinical duration 8-10 yrs (range 1-25 yrs) EOFAD 1-6% OF ALL Alzheimer’s, 60% of which is familial, and 13% inherited in an AD manner. (<2% of all Alzheimer’s) LOFAD: Appears to be an assoc with APOE e4 but not sensitive or specific- supports the dx. APOE e2 may be protective. 597 NEUROLOGIC DISORDERS EARLY ONSET FAMILIAL ALZHEIMER DISEASE Beta-amyloid plaques: senile plaques appear as small collections of dark, irregular, thread-like structures often with a brownish material in the center. The central core is represented by amyloid and the irregular, beaded linear structures represent abnormal neurites (small dendrites and axons with degenerative changes). Nature. 1999 Jul 8;400(6740):173-7. 142 NEUROLOGIC DISORDERS ANGELMAN SYNDROME Responsible gene: UBE3A Protein: Ubiquitin protein ligase E3A Cytogenetic locus: 15q11-q13 Inheritance: loss of the maternally imprinted contribution in the 15q11.2-q13 (AS/PWS) region Clinical Features and Diagnostic Criteria: severe developmental delay or ID, severe speech impairment, gait ataxia and/or tremulousness of the limbs, and an inappropriate happy demeanor that includes frequent laughing, smiling, and excitability, microcephaly and seizures Clinical Tests: acquired microcephaly by age two years, Seizures before age three, abnl EEG: large amp. slow-spike waves Molecular Tests: 4-6 Mb del (65-75%), UBE3A mutation (11%), imprinting defect (2.5%), unbal chrom transloc (<1%), Pat UPD 15 (<1%), del of imprinting center (0.5%) Disease Mechanism: Disruption of E6AP ultimately causes an abnormality in the ubiquitin protein degradation pathway, but no clear AS-causing target protein yet identified Treatment/Prognosis: Typical care for medical issues, PT, OT, ST, and individualized education and behavior program. 143 NEUROLOGIC DISORDERS ANGELMAN SYNDROME Facial features: Protruding tongue Prognathia Wide mouth Widely spaced teeth Strabismus Light hair and eye color http://www.psychnet-uk.com/dsm_iv/pictures/angel.jpg 144 598 NEUROLOGIC DISORDERS CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) Responsible gene: NOTCH3 Protein: Neurogenic locus notch homolog protein 3 Cytogenetic locus: 19p13.2-p13.1 Inheritance: AD Clinical Features and Diagnostic Criteria: Stroke-like episodes before age 60, cognitive disturbance, behavioral abnormalities, migraine with aura Clinical Tests: Skin Bx EM: e- dense granules in media of arterioles. Brain MRI: T2 signal abnormalities in the WM of the temporal pole and external capsule, subcortical lacunar lesions (groups of rounded lesions at the junction of GM and WM. WM changes seen as early as age 21 yrs. Molecular Tests: NOTCH3 sequencing (>90%) Disease Mechanism: NOTCH genes encode transmembrane receptors involved in cell fate specification during development. The functional consequences of NOTCH3 mutations in the abnormal protein are not known. Treatment/Prognosis: supportive care, angiography and anticoagulants may precipitate CVA, smoking increases risk of stroke. Mean age to walk with asst.: 60yrs, bedridden by 64yrs, med. age of death 68 yrs. 145 NEUROLOGIC DISORDERS CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) Natural history of the main clinical manifestations of CADASIL 146 (Chabriat, The Lancet Neurology, 2009) NEUROLOGIC DISORDERS CANAVAN DISEASE Responsible gene: ASPA Protein: Aspartoacylase Cytogenetic locus: 17pter-p13 Inheritance: AR Clinical Features and Diagnostic Criteria: Macrocephaly, lack of head control, developmental delays by age 3-5 mos, severe hypotonia, never sit, walk, or speak. Hypotonia evolves to spasticity. Clinical Tests: High urine N-acetyl aspartic acid (NAA) Molecular Tests: 3 common mutations account for 99% of disease-causing alleles in Ash. Jewish, 50-55% in non-jewish. Disease Mechanism: Absence of aspartoacylase leads to build up of NAA in the brain leading to demyelination Treatment/Prognosis: Supportive care: nutrition, hydration, managing infectious disease, protecting airway. Life expectancy to the teens. 147 599 NEUROLOGIC DISORDERS CANAVAN DISEASE Macrocephaly and Hypotonia www.canavanresearch.org 148 NEUROLOGIC DISORDERS FAMILIAL DYSAUTONOMIA Responsible gene: IKBKAP Protein: IkappaB kinase complex-associated protein Cytogenetic locus: 9q31 Inheritance: AR Clinical Features and Diagnostic Criteria: Progressive, GI dysfunction, vomiting crises, recurrent pneumonia, altered sensitivity to pain and temperature, CV instability, autonomic crises, hypotonia, broad based ataxic gate deteriorates, decreased life expectancy. Dec taste and absence of fungiform papillae of the tongue, dec or absent DTR’s, absence of overflow tears with crying Clinical Tests: Pupillary hypersensitivity to parasympathetic agents, absence of axon flare response after intradermal histamine injection Molecular Tests: IVS20 (+6T>C); R696P in IKBKAP (>99% Ashkenazi Jewish population) Disease Mechanism: Abnormal development and survival of sensory, sympathetic and parasympathetic neurons Treatment/Prognosis: Aspiration precautions, hydration and elastic stockings for orthostatic hypotension, protect cornea with artificial tears, PT for contracture 149 NEUROLOGIC DISORDERS FAMILIAL DYSAUTONOMIA Smooth tongue without typical vascularized fungiform papillae (Axelrod and Gold-von Simson Orphanet Journal of Rare Diseases 2007) 150 600 NEUROLOGIC DISORDERS FRAGILE X Responsible gene: FMR-1 Protein: FMRP (Fragile X Mental Retardation Protein) Cytogenetic locus: Xq27.3 Inheritance: X-linked triplet repeat Clinical Features and Diagnostic Criteria: Delayed motor and verbal development, ID (mod-severe in boys, milder in girls), prominent jaw and forehead, high activity, autistic features. Carrier females: anxiety, OCD, depression, 20% have POF. Carrier Males: (>30% of males >50y), progressive intention tremor, ataxia, parkinsonism, and autonomic dysfunction. Two other loci: FraXE: only ID, FraXF: no phenotype Clinical Tests: None Molecular Tests: CGG triplet repeat detection. Southern Blot: good for small or large expansions, doesn’t give repeat #. PCR: Better quantification of repeat number, subject to allele dropout with large expansions. NL: 5-44 repeats, Intermediate: 45-58 repeats (gray zone), Pre-mutation: 59-200 repeats, Mutation: >200 repeats Disease Mechanism: >200 repeats leads to silencing by methylation. POF and ataxia thought to be due to toxic gain of function. Treatment/Prognosis: No specific treatment. 151 NEUROLOGIC DISORDERS FRAGILE X Facial Features: Long face, Prominent forehead Large ears Prominent jaw 152 (suzannebalvanz.blogspot.com/2007_07_01_archive.html) NEUROLOGIC DISORDERS HUNTINGTON DISEASE Responsible gene: HD Protein: Huntington Cytogenetic locus: 4p16.3 Inheritance: AD Clinical Features and Diagnostic Criteria: progressive motor disability involving both involuntary and voluntary movement (chorea, dysarthria, dysphagia progress to bradykinesia, rigidity, and dystonia) , cognitive decline (problems with planning or organization), psychiatric disturbances (personality change, affective psychosis, or schizophrenic psychosis. Mean age of onset 35-44 yrs (juvenile onset <20yrs ~10%). Clinical Tests: CT or MRI: characteristic atrophy of caudate and putamen. PET scan: dec uptake and metab. of glucose in the caudate nucleus (often abnl before MRI or CT). Molecular Tests: Targeted mut. analysis: trinucleotide CAG repeat expansion >36. 27-35: no symptoms but, if male, risk of expansion in children (6-10% risk of expansion with 35 repeats). 36-39: reduced penetrance, may never develop symptoms. >40: fully penetrant. >60 repeats: juvenile onset. Disease Mechanism: Unknown Treatment/Prognosis: Tx is symptomatic: neurolepetics, benzo’s, psychotropics, Median survival time: 15-18 yrs after onset, average age of death is 55 yrs. Suicide in 12%. 153 601 NEUROLOGIC DISORDERS HUNTINGTON DISEASE www.Nature.com 154 NEUROLOGIC DISORDERS KRABBE DISEASE Responsible gene: GALC Protein: Galactocerebrocidase Cytogenetic locus: 14q31 Inheritance: AR Clinical Features and Diagnostic Criteria: Infantile form: irritability to sensory stimuli, muscle hypertonicity, progressive neurologic deterioration, peripheral neuropathy, white matter disease, elevated CSF protein. Later onset (6 mos to 5th decade): weakness, vision loss, intellectual regression. Clinical Tests: CT: nonspecific- diffuse cerebral atrophy of grey and white matter. MRI: demyelination of the brainstem and cerebellum. Dec GALC enzyme activity (0-5% of normal activity). Abnl EEG, low nerve conduction velocity, Molecular Tests: GALC targeted mutation analysis: GALC 30-kb deletion (45% of Europeans, 35% of Mexicans); 809G>A mutation (50% of late onset Krabbe). GALC sequencing (virtually 100%) Disease Mechanism: Missense mutations result in unstable protein that is rapidly degraded Treatment/Prognosis: Hematopoietic stem cell transplant decreases morbidity and mortality when given to infants before they show symptoms. Supportive care to control irritability and spasticity if diagnosed when symptomatic. Infantile form: average age of death is 13 mos due to infections or resp failure. 155 NEUROLOGIC DISORDERS KRABBE DISEASE (http://disorders.eyes.arizona.edu/disorders/krabbe-disease) 156 602 NEUROLOGIC DISORDERS NEUROFIBROMATOSIS TYPE I Responsible gene: NF1 Protein: Neurofibromin Cytogenetic locus: 17q11 Inheritance: AD Clinical Features and Diagnostic Criteria: 2 or more of: 6x5mm (prepubertal) or 6x15mm (postpubertal) café au lait, 2 or more neurofibromas, one plexiform neurofibroma, axillary or inguinal freckling, optic glioma, 2 or more Lisch nodules, sphenoid dysplasia or thickening of long bone cortex, 1st degree relative with NF-1 Clinical Tests: x-ray, eye exam, brain MRI Molecular Tests: >500 mutations reported, usually unique to a particular family Disease Mechanism: Loss of function mutations impair ras GTPase mediated cellular proliferation and tumor suppression Treatment/Prognosis: The majority live normal lifespan. Surgery for bone malformations or painful or disfiguring tumors; clinical trials and use of MEK inhibitors for plexiform neurofibromas. Risk of malignant peripheral nerve sheath tumors in adolescence and young adulthood. 157 NEUROLOGIC DISORDERS NEUROFIBROMATOSIS TYPE I runkle-science.wikispaces.com 158 NEUROLOGIC DISORDERS PARKINSON DISEASE Responsible gene: Multiple, main gene PARK2 Protein: Parkin Cytogenetic locus: 6q25.2-q27 Inheritance: AD, AR, multifactorial Clinical Features and Diagnostic Criteria: bradykinesia, rigidity, and tremor, asymmetric limb involvement. Juvenile Onset AR PARK2 mutations, typical features, onset 20-40yrs. Clinical Tests: Good response to L-Dopa Molecular Tests: PARK2 sequencing Disease Mechanism: Unclear but thought to be due to loss of function by absent protein or protein inactivation Treatment/Prognosis: Dopamine therapy, PT, OT, ST. Some patients may benefit from palliodotomy or deep brain stimulation of the subthalamic nucleus. 159 603 NEUROLOGIC DISORDERS PARKINSON DISEASE 160 http://www.holisticonline.com/images/PD-ama-schematic1.GIF NEUROLOGIC DISORDERS RETT SYNDROME Responsible genes: MECP2 Proteins: MECP2 Cytogenetic loci: Xq28 Inheritance: XLD Clinical Features and Diagnostic Criteria: ID, developmental regression (especially language and hand use), acquired microcephaly, stereotypical wringing hand movements, hyperventilation, bruxism, paroxysmal laughing, prolonged QT, scoliosis Clinical Tests: EEG (nonspecific for Rett), ECG Molecular Tests: MECP2 sequencing (>80%), Need to test parents if a novel variant found. MECP2 MLPA or quantitative PCR testing for deletion (~16%). Disease Mechanism: Decreased function of loss-of-function of MECP2. Normally MECP2 binds methylated CpG islands. Treatment/Prognosis: Seizures are often difficult to manage, SSRI’s for agitation, monitor for scoliosis, periodic ECG to monitor for long QT Small subset have CDKL5 mutations and present atypically with early onset seizures 161 RETT SYNDROME NEUROLOGIC DISORDERS Genetic testing strategy www.lbl.gov 162 (Williamson and Christodoulou EJHG 2006) 604 NEUROLOGIC DISORDERS WILSON DISEASE Responsible gene: ATP7B Protein: Copper-transporting ATPase 2 Cytogenetic locus: 13q14.3-q21.1 Inheritance: AR Clinical Features and Diagnostic Criteria: Can present age 3-50 yrs. Liver disease: jaundice, self-limited hepatitis-like illness, autoimmune hepatitis, hepatic failure, chronic liver disease. Neurologic presentation: movement disorder, disorganization of personality Clinical Tests: Kayser-Fleisher rings on corneal exam, low serum Cu and ceruloplasmin, inc urinary copper excretion. Liver bx: inc copper storage. Molecular Tests: ATP7B sequencing (98%). H1069Q (35-45% Europeans), R779L (57% Asians), H714Q and delC2337 (40% Russians). Disease Mechanism: Loss of ATP7b function impairs holoceruloplasmin biosynthesis and biliary copper excretion with resultant copper-mediated oxidative damage, activation of cell death pathways, leakage of copper into plasma and eventual tissue copper overload. Treatment/Prognosis: Chelating agents, liver transplant 163 NEUROLOGIC DISORDERS WILSON DISEASE Kayser-Fleisher ring www.kellogg.umich.edu 164 NEUROMUSCULAR DISORDERS AMYOTROPHIC LATERAL SCLEROSIS Responsible genes: SOD1 (rare: SETX, VAPB, BSCL2, VCP, ALS2, SPG20, others) Protein: Superoxide dismutase Cytogenetic locus: 21q22 Inheritance: AD (AR ALS2 and SPG20) Clinical Features and Diagnostic Criteria: UMN: hyperreflexia, extensor plantar response, inc muscle tone, and weakness. LMN: weakness, muscle wasting, hyporeflexia, muscle cramps, and fasciculations. Frontotemporal dimentia Clinical Tests: EMG; Path: (1) degeneration and loss of the motor neurons in the anterior horns and in the motor nuclei of cranial nerves VII, X, and XI and most commonly the hypoglossal nucleus; and (2) axonal loss with decreased myelin staining in the lateral and anterior corticospinal tracts Molecular Tests: SOD1 mutation (20% familial, 3% sporadic ALS- 50% have the A4V Exon 1 mutation) Disease Mechanism: Toxic gain of function, not enzyme deficiency (SOD1 prevents oxidative damage to cells) Treatment/Prognosis: Primarily palliative, Riluzole (glutamate inhibitor) FDA-approved drug. Mean age of onset: 46 yrs if familial, 56 yrs if sporadic. Death usually caused by resp. muscle compromise. 165 605 NEUROMUSCULAR DISORDERS AMYOTROPHIC LATERAL SCLEROSIS http://www.esperanzapeptide.net/images/treatment-mnd.jpg 166 NEUROMUSCULAR DISORDERS CHARCOT MARIE TOOTH DISEASE CMT1: Abnormal myelin, AD, 50% of all CMT, PMP22 (17p11.2), MPZ (1q22), LITAF (16p13.1-p12.3), EGR2 (10q21.1-q22.1), NEFL (8p21) CMT2: Axonopathy, AD, 20-40% of all CMT, KIF1B and MFN2 (1p36.2), RAB7 (3q21), LMNA (1q21.2), GARS (7p15), NEFL (8p21), HSPB1 (7q), MPZ (1q22), GDAP1 (8q12-q21.1) CMT Intermediate Form: Combination of myelinopathy and axonopathy, AD, rare cause of CMT, DNM2 (19p12-p13.2), YARS (1p34-p35) CMT 4: Either myelinopathy or axonopathy, AR, rare cause of CMT, GDAP1 (8q13-q21.1), MTMR2 (11q22), CMT4B2 (11p15), SH3TC2 (5q32), NDRG1 (8q24.3), EGR2 (10q21.1-q22.1), PRX (19q13.1q13.2 CMTX: Axonopathy with secondary myelin changes, XLD, 10-20% of all CMT, GJB1 (Xq13.1). Clinical Features and Diagnostic Criteria: slowly progressive weakness and atrophy of distal muscles in the feet and/or hands beginning in the 1st-3rd decade; hearing loss; pes cavus foot deformity, hip dysplasia. Clinical Tests: nerve conduction studies, nerve biopsy Molecular Tests: Gene sequencing, deletion/duplication analysis Disease Mechanism: Abnormal peripheral myelination Treatment/Prognosis: orthopedic surgery, TCA’s, carbamazepine, or gabapentin for neuropathic pain. 167 NEUROMUSCULAR DISORDERS CHARCOT MARIE TOOTH DISEASE Pes cavus foot deformity Finger contracture 168 http://findmeacure.com/2011/03/22/charcot-marie-tooth-diseasecmt/ ACMG Genetics and Genomics Review Course June 18-21, 2015 606 NEUROMUSCULAR DISORDERS DUCHENNE AND BECKER MUSCULAR DYSTROPHY Responsible gene: DMD Protein: Dystrophin Cytogenetic locus: Xp21.2 Inheritance: XLR Clinical Features and Diagnostic Criteria: DMD: Symptoms present before age 5, progressive symmetrical muscular weakness, proximal>distal, calf hypertrophy, dilated cardiomyopathy (DCM). BMD: Later onset, less severe, weakness of quadriceps may be only sign, activity induced cramping. Preservation of neck flexor muscles (unlike DMD). DCM can occur in isolation Clinical Tests: CK 10x nl in DMD, 5x nl in BMD. Unreliable test for carrier females, tends to decrease with age. Molecular Tests: Multiplex PCR: DMD gene deletion (65% DMD, 85% BMD). Southern or quantitative PCR for gene duplication (6% DMD), DMD sequencing for small del/ins or point mutations (30% DMD) Disease Mechanism: Dystrophin binds actin and other membrane proteins. Mutations that lead to lack of dystrophin expression: DMD, those that lead to abnormal quality or quantity of dystrophin: BMD. Treatment/Prognosis: Supportive therapy, steroids may prolong walking 2-3 yrs. DMD: wheelchair dependent by age 13, ventilator by age 20, survival into 20’s. BMiDs: Wheelchair after age 16 (if at all), survival 40-50’s. Carrier females at risk for DCM. Exon skipping therapies, stop mutation readthrough under investigation. 169 NEUROMUSCULAR DISORDERS DUCHENNE AND BECKER MUSCULAR DYSTROPHY http://img.orthobullets.com/Pediatrics/Neuromuscular%20problems/Duchenes%20Muscular%20Dystroph y/Images/dystrophin_stains.jpg 170 NEUROMUSCULAR DISORDERS FRIEDREICH ATAXIA Responsible gene: FRDA Protein: Frataxin Cytogenetic locus: 9q13 Inheritance: AR Clinical Features and Diagnostic Criteria: Progressive degeneration of the dorsal root ganglia, posterior columns, corticospinal tracts, and the dorsal spinocerebellar tracts of the spinal cord and cerebellum. There is progressive limb and gait ataxia before age 25 yrs, absent tendon reflexes in the lower extremities. Within 5 years of disease onset: dysarthria, arefelxia, pyrimidal weakness of the legs, extensor plantar responses and distal loss of joint position and vibration sense. Also, scoliosis, pes cavus, optic nerve atrophy, hypertrophic cardiomyopathy, DM or glucose intolerance Clinical Tests: electrophysiologic evidence of axonal sensory neuropathy Molecular Tests: GAA triplet repeat expansion in FRDA intron 1 (96% homozygous) Normal 5-33, premutation 34-65, and disease causing: 66-1700 repeats. Disease Mechanism: It is believed that GAA expansion forms a stable DNA structure that interferes with transcription Treatment/Prognosis: Treatment is supportive: psychological, prostheses, walking aids, wheelchairs, PT, and ST 171 607 NEUROMUSCULAR DISORDERS FRIEDREICH ATAXIA Model of alteration in iron uptake in Friedreich's ataxia. Richardson D R et al. PNAS 2010;107:1077510782 172 NEUROMUSCULAR DISORDERS HEREDITARY NEUROPATHY WITH LIABILITY TO PRESSURE PALSIES Responsible gene: PMP22 Protein: Peripheral myelin protein 22 Cytogenetic locus: 17p11.2 Inheritance: AD Clinical Features and Diagnostic Criteria: adult with recurrent focal pressure palsies, mild polyneuropathy, absent ankle reflexes, reduced DTRs, mild-mod pes cavus deformity Clinical Tests: Prolongation of distal nerve conduction latencies (virtually 100%), normal general motor nerve conduction velocities, demyelination and tomaculous (focal nerve enlargement) on sural nerve biopsy Molecular Tests: PMP22 sequencing (20%), 1.5-Mb PMP22 deletion (80%) Disease Mechanism: HNPP is associated with decreased mRNA message for PMP22 and decreased peripheral myelin protein 22 in peripheral nerve. Treatment/Prognosis: Bracing, AFO for foot drop, unclear if surgical nerve decompression is helpful, avoid risk factors for pressure palsy: prolonged sitting with legs crossed, repetitive wrist movements, prolonged leaning on elbows, and rapid weight loss. 173 NEUROMUSCULAR DISORDERS LIMB-GIRDLE MUSCULAR DYSTROPHY Responsible gene (protein, cytogenetic locus): CAPN3 (Calpain 3, 15q15.1-q21.1), FKRP (Fukutin related protein, 19q13.1), LMNA (Lamin-A/C, 1q21.2), SGCA (alpha sarcoglycan, 17q12), SGCB beta sarcoglycan, 4q12), SGCD (delta-sarcoglycan, 5q33), SGCG (gammasarcoglycan, 13q12), DYSF (Dysferlin, 2p13.3) Inheritance: most AR, some rare AD subtypes Clinical Features and Diagnostic Criteria: AR Sarcoglycan LGMD: proximal limb weakness, difficulty running and walking, calf hypertrophy, onset age 3-15 (68% of childhood onset, 10% adult onset) Calpain AR LGMD proximal limb weakness, difficulty running and walking, calf atrophy, onset 2-40 yrs (10-30% AR LGMD). Dysferlin AR LGMD problems running and walking, foot drop, distal and/or pelvic weakness, transient calf hypertrophy, onset 17-23 yrs Clinical Tests: Inc serum CPK, dystrophic changes on muscle biopsy, sarcoglycan protein staining Molecular Tests: Gene sequencing (80-99%) Disease Mechanism: Sarcoglycanopathies disrupt dystrophin-dystroglycan complex, calpainopathy: unknown, dysferlinopathy: may be die to abnl membrane fusion Treatment/Prognosis: Supportive care to promote mobility and ambulation. Monitor for respiratory and orthopedic complications and for cardiomyopathy 174 608 NEUROMUSCULAR DISORDERS MYOTONIC DYSTROPHY TYPE 1 Responsible gene: DMPK Protein: Myotonin-protein kinase Cytogenetic locus: 19q13.32 Inheritance: AD Clinical Features and Diagnostic Criteria: Multisystem disorder of skeletal and smooth muscle, eyes, heart, endocrine system, and CNS. MILD cataract and mild myotonia (50-150 repeats) Classic muscle weakness and wasting, myotonia, cataract, and arrhythmia (100-1000 repeats). Have grip myotonia (sustained muscle contraction leads to inability to quickly release a hand grip) Congenital hypotonia and severe generalized weakness at birth often with resp. insufficiency and early death, MR is common (>2000 repeats) Clinical Tests: EMG, serum CK, muscle biopsy (internal nuclei, ring fibers, sarcoplasmic masses, type I fiber atrophy, inc # intrafusal muscle fibers), slitlamp exam Molecular Tests: CTG triplet repeat at the 3’-UTR of the DMPK (100%). PCR: detect repeats up to ~100, southern blot (detect repeats>100) Disease Mechanism: Cause thought to be due to gain of function RNA mechanism- the CUG repeats alter alternative splicing of other genes, including a CL- channel, resulting in myotonia Treatment/Prognosis: Symptomatic only 175 NEUROMUSCULAR DISORDERS MYOTONIC DYSTROPHY TYPE 1 Three ring fibers (one marked), atrophic myofibers, and central nuclei http://neuropathology.neoucom.edu/chapter13/chapter13cDystrophy.html 176 NEUROMUSCULAR DISORDERS NEMALINE MYOPATHY Gene (protein, chromosomal locus): ACTA1 (Actin, alpha skeletal muscle, 1q42.1), NEB (Nebulin, 2q22), TNNT1 (Troponin T, slow skeletal muscle, 19q13.4), TPM2 (Tropomyosin beta chain, 9p13.2-p13.1), TPM3 (Tropomyosin alpha-3 chain, 1q22-q23), RARE: CFL2 (Cofilin-2, 14q13.1), KBTBD13 (Kelch repeat and BTB domain containing protein 13, 15q22.31), KHLH40 (Kelch-like protein 40, 3p22.1), and KHLH41 (Kelch-like protein 41, 2q31.1) Inheritance: AR or AD Clinical Features and Diagnostic Criteria: weakness, hypotonia, and depressed or absent DTR’s. Weakness is usually most severe in the face, neck flexors, and proximal limb muscles. Age of onset: congenital, childhood, or adulthood. Clinical Tests: Muscle biopsy: the diagnostic hallmark is the presence of rod-like inclusions, nemaline bodies, in the sarcoplasm of skeletal muscle fibers with trichrome staining. Molecular Tests: ACTA sequencing: 15-25% of NM, ACTA Del/dup analysis: Exon 55. Disease Mechanism: NM is a disorder of thin filament anchoring proteins Treatment/Prognosis: No definitive correlation between # of rods and severity of the myopathy. Walking prior to 18 months is predictive of survival. 177 609 NEMALINE MYOPATHY NEUROMUSCULAR DISORDERS Nemaline inclusion www.pathology.vcu.edu 178 NEUROMUSCULAR DISORDERS SPINAL MUSCULAR ATROPHY Responsible genes: SMN1, SMN2 Proteins: survival motor neuron protein 1 and 2 Cytogenetic loci: 5q12.2-q13.3 Inheritance: AR Clinical Features and Diagnostic Criteria: arthrogryposis multiplex congenita, peripheral nerve hypomyelination. SMA I onset 0-6mo, muscle weakness, tongue fasiculations, absent DTRs SMA II muscle weakness onset after 6 months, finger trembling, low tone, absent DTRs, SMA III Weakness leads to frequent falls or trouble with stairs, onset 2-3yrs, proximal weakness (legs>arms), SMA IV adult onset Clinical Tests: EMG: denervation and diminished motor action potential amplitude. Muscle Bx: atrophy of type 1 and type 2 fibers Molecular Tests: Targeted mutation analysis: deletion of SMN1 exon 7 deletion (95-98%), SMN1 sequencing (2-5%). Carriers who have two copies of SMN1 in cis (~4% of the population) will be misdiagnosed as non-carriers. SMN2 copy # modifies the severity. 2 copies SMN2- SMA I, 3 copies- SMA II, 4-8 copies- SMA III. Absence of both SMN genes: lethal Disease Mechanism: Mutant SMN lacks the splicing-regeneration activity of wild type. Treatment/Prognosis: Optimize feeding and nutrition, PFT’s, sleep study for OSA, treat contractures, dislocations, and scoliosis; Nusinersen treatment 179 NEUROMUSCULAR DISORDERS SPINAL MUSCULAR ATROPHY Diagnostic algorithm for SMA (Lunn and Wang, The Lancet, 2008) 180 610 NEUROMUSCULAR DISORDERS SYNDROMIC CONGENITAL MUSCULAR DYSTROPHY (Fukuyama (FCMD), Muscle-Eye-Brain (MEB), Walker-Warburg (WWS), Congenital Muscular Dystrophy Type 1D (MDC1D) Responsible gene (protein, cytogenetic locus): FCMD; FCMD (Fukutin, 9q31); MEB: POMGNT1 (protein Omannosidase beta-1,2-N-acetylglucosaminyltransferase, 1p34-p33); WWS: POMT1 and POMT2 (Protein Omannosyltransferase 1 and 2, 9q34.1, and 14q24.3); MDC1D (LARGE, glycosyltransferase-like protein LARGE, 22q12.3-q13.1 Inheritance: AR Clinical Features and Diagnostic Criteria: Muscle weakness present at birth. Hypotonia and weakness. Joint contracture (MEB and WWS: elbow, FMD: hip, knee, ankle elbow). Clinical Tests: Muscle bx: dystrophic or myopathic pattern; inc serum CK; Muscle Bx: immunostaining; Brain MRI: Cobblestone complex (enlarged lat ventricles, flat brainstem, cerebellar hypoplasia) Molecular Tests: Disease Mechanism: Disruption of alpha dystroglycan (an integral component of the dystrophin-glycoprotein complex) Treatment/Prognosis: Weight control, PT, assist devices for ambulation, surgical correction of orthopaedic problems, monitoring of respiratory function 181 NEUROMUSCULAR DISORDERS SYNDROMIC CONGENITAL MUSCULAR DYSTROPHY Head lag due to hypotonia http://neuromuscular.wustl.edu/syncm.html NEUROMUSCULAR DISORDERS TAY-SACHS DISEASE Responsible gene: HEXA Protein: Hexosaminidase A Cytogenetic locus: 15q23-q24 Inheritance: AR Clinical Features and Diagnostic Criteria: Infantile weakness starts at 6 mo, exaggerated startle, seizures and vision loss by the end of the first year, neurodegeneration continuesdeaf, cannot swallow, weakening of muscles, and eventual paralysis, death in toddler years. Juvenile muscle coordination problems, seizures, and vision problems starting as young children. Chronic and adult onset start later, progress more slowly, more rare. Clinical Tests: HEXA enzyme activity, cherry red spot on eye exam Molecular Tests: Follow enzyme testing with DNA testing (some with a positive enzyme assay have a pseudodeficiency allele that does not cause Tay Sachs). HEXA 6 common mutation panel: 92% of Ashkenazi Jewish Disease Mechanism: Accumulation of GM2 gangliosides in the brain Treatment/Prognosis: Supportive only 183 611 NEUROMUSCULAR DISORDERS TAY-SACHS DISEASE Cherry red spot of the macula http://themedicalbiochemistrypage.org/images/cherryredspot.jpg 184 ONCOLOGIC DISORDERS BRCA1 and BRCA2 Hereditary Breast/Ovarian Cancer Responsible genes: BRCA1 and BRCA2 Proteins: Breast cancer type 1 and 2 susceptibility protein Cytogenetic loci: 17q21, 13q12.3 Inheritance: AD Clinical Features and Diagnostic Criteria: BRCA 1 and 2: Br, ovarian, prostate cancer. BRCA2: pancreatic Clinical Tests: mammography, MRI, BRCA1-related breast tumors show an excess of medullary histopathology, are of higher histological grade, and are more likely to be estrogen receptor-negative and progesterone receptor-negative. BRCA1-related ovarian cancer: excess of serous adenocarcinomas Molecular Tests: Full gene sequencing and deletion analysis (3-5%). Overall, about 3-5% of reports have variants of uncertain clinical significance). Ashkenzi founder 187delAG and 5382insC (BRCA1), and 6174delT (BRCA2) mutations are found in 20-30% of Jewish women with early breast cancer and in 45-60% of Jewish women diagnosed with ovarian cancer. Dutch women with early br or ovarian ca: often one of 3 large BRCA1 deletions. BRCA2 999del5 occurs in 7.7% of women and 40% of men with breast cancer from Iceland. Three Ashkenazi founders found in 2.4% of individuals of Ashkenazi Jewish descent. Disease Mechanism: BRCA1 and 2 are tumor suppressor genes Treatment/Prognosis: Prophylactic salpingo-oopherectomy, breast MRI, chemoprevention trials, and options for prophylactic surgery. 85% will develop Br ca by age 70 yrs. 185 ONCOLOGIC DISORDERS BRCA1 and BRCA2 Hereditary Breast/Ovarian Cancer 186 612 ONCOLOGIC DISORDERS FAMILIAL ADENOMATOUS POLYPOSIS Responsible gene: APC Protein: Adenomatous polyposis coli protein Cytogenetic locus: 5q21-22 Inheritance: AD (15-30% new mutation) Clinical Features and Diagnostic Criteria: adenomatous colonic polyps (100-1000) in childhood to adolescence, abdominal desmoid tumors, jaw osteoma, absent/supernumerary/malformed teeth, hepatoblastoma, thyroid cancer, epidermoid cysts. Attenuated FAP: fewer polyps, more proximal in the colon. Gardner syndrome: colonic adenomatous polyposis, osteomas, and soft tissue tumors. Turcot syndrome: colon cancer and CNS tumors (medulloblastoma more common in FAP) Clinical Tests: Clinical findings on colonoscopy Molecular Tests: APC sequence analysis abnormal (~90%), deletion (~5%) Disease Mechanism: Decrease in APC protein results in lack of b-catenin proteasome degradation and high levels of nuclear b-catenin protiein, binds to a transcription factor Tcf-4 or Lef-1 (T cell factor-lymphoid enhancer factor), and may activate the oncogenes c-Myc and cyclin D1 Treatment/Prognosis: Without colectomy, colon cancer is inevitable, and prophylactic colectomy is recommended in late teen-age years. The mean age of cancer in untreated individuals is 39 years. 187 ONCOLOGIC DISORDERS FAMILIAL ADENOMATOUS POLYPOSIS www.oncolink.com Colon with multiple adenomas 188 HEREDITARY NONPOLYPOSIS COLON CANCER (LYNCH SYNDROME) ONCOLOGIC DISORDERS Responsible gene (protein and cytogenetic locus): MLH1 (3p21.3, DNA mismatch repair protein MLH1), MSH2 (2p22-p21, DNA mismatch repair protein Msh2), MSH6 (2p16, DNA mismatch repair protein MSH6), and PMS2 (7p22, PMS1 protein homolog 2) Inheritance: AD Clinical Features and Diagnostic Criteria: HNPCC-related tumors: colon, endometrium, stomach, ovary, hepatobiliary tract, urinary tract, small bowel, brain/CNS. Amsterdam II Criteria: 3 or more family members (at least one 1st degree of the other 2) with HNPCC related cancers; 2 successive affected generations; 1 or more of the HNPCC-related cancers diagnosed before age 50; exclusion of FAP. Bethesda 2004: CRC diagnosed under age 50yrs, 2 HNPCC related tumors at once, CRC with high MSI in someone <age 60yrs, CRC in one or more 1st degree relatives with and HNPCC related tumor with 1 cancer diagnosed before age 50yrs, or CRC diagnosed in 2 or more 1st or 2nd degree relatives (any age).. Clinical Tests: Microsatellite instability (MSI) of tumor tissue, immuno-histochemistry of tumor tissue for the presence or absence of DNA mismatch repair proteins MLH1, MSH2, MSH6 and PMS2. Molecular Tests: Sequencing of all genes MLH1 (90-95%), MSH2 (50-80%). Deletion analysis MLH1 (5-10%), MSH2 (10-20%), MSH6 and PMS2 (less than 10% of Amsterdam criteria families combined). EPCAM deletion. Disease Mechanism: These proteins work in a recessive manner at the cellular level- LOH leads to absence of any functional protein and dysfunctional mismatch repair. Treatment/Prognosis: 70-80% lifetime risk of CRC. Colonoscopy every 1-2yrs by age 20-25. Current guidelines do not recommend endometrial sampling unless symptomatic. If CRC present, full colectomy with ileorectal anastomosis considered. Tumors respond better to immune checkpoint inhibitors. Recessive form – note rare AR constitutional mismatch repair deficiency syndrome with childhood cancer risk. 189 613 ONCOLOGIC DISORDERS HEREDITARY NONPOLYPOSIS COLON CANCER (LYNCH SYNDROME) www.mdanderson.org/images/hnpcc 190 ONCOLOGIC DISORDERS LI-FRAUMENI SYNDROME Responsible genes: TP53 Proteins: Cellular tumor antigen P53 Cytogenetic locus: 17p13 Inheritance: AD Clinical Features and Diagnostic Criteria: Proband with sarcoma <age 45 yrs, 1st deg relative with cancer <45 yrs, and 1st or 2nd deg relative with any cancer <45yrs or a sarcoma at any age. Increased risk of multiple primary tumors: bone, cartilage, and soft tissue sarcoma; early onset breast cancer; brain tumors (including choroid plexus carcinoma), childhood adrenocortical tumors, also GI malignancies, lung cancer and neuroblastoma. Clinical Tests: Pathology Molecular Tests: TP53 sequencing and deletion analysis Disease Mechanism: Abnormal DNA repair and genomic instability: P53 protein plays a role in determining whether cells undergo arrest for DNA repair or apoptosis Treatment/Prognosis: Toronto protocol (extensive screening with whole body MRI, brain MRI, breast MRI and abdominal U/S, biochemical screening for adrenal tumors) shown to improve mortality. Avoid or minimize exposure to radiation. 191 ONCOLOGIC DISORDERS LI-FRAUMENI SYNDROME Journal of Clinical Oncology, Vol 27, No 26 (September 10), 2009: pp e108-e109 192 614 ONCOLOGIC DISORDERS MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 Responsible gene: MEN1 Protein: Menin Cytogenetic locus: 11q13 Inheritance: AD Clinical Features and Diagnostic Criteria: MEN1= tumor in 2 of: parathyroid, enteropancreatic endocrine tissue, or anterior pituitary OR Tumor in one and 1st degree relative with MEN1. Facial angiofibroma, collagenoma, café au lait, lipoma Clinical Tests: Parathyroid function studies, anterior pituitary hormone abnormalities, Brain MRI Molecular Tests: MEN1 sequencing (70-90% familial, 65% sporadic), Dup/del testing (1-3%) Disease Mechanism: MEN1 is a tumor suppressor gene by regulating transcription of proteins involved in the regulation of cell proliferation and development Treatment/Prognosis: biochemical testing of serum concentrations of calcium (from age 8 yrs), gastrin (from age 20 yrs), pancreatic polypeptide (from age 10 yrs), prolactin (from age 5 yrs), abdominal CT or MRI (from age 20 yrs) and head MRI (from age 5 yrs). 193 ONCOLOGIC DISORDERS MULTIPLE ENDOCRINE NEOPLASIA TYPE 1 my.clevelandclinic.org/disorders/familial_multiple_endocrine_neoplasia 194 ONCOLOGIC DISORDERS MULTIPLE ENDOCRINE NEOPLASIA Type 2 Responsible gene: RET Protein: proto-oncogene tyrosine-protein kinase receptor ret Cytogenetic locus: 10q11.2 Inheritance: AD Clinical Features and Diagnostic Criteria: MEN2A two or more of medullary thyroid carcinoma, pheochromocytoma, or parathyroid adenoma/hyperplasia in a single person or close relatives. MEN2B mucosal neuromas of the lips and tongue, medullated corneal nerve fibers, Marfanoid habitus, and medullary thyroid carcinoma Clinical Tests: Calcitonin, catecholamines, catecholamine metabolites, Ca, PTH Molecular Tests: RET sequencing: Exon 10 and 11 (95% MEN2A), Exon 16 (95% MEN2B) Disease Mechanism: Gain of function mutations in RET lead to constitutive activation of tyrosine kinase Treatment/Prognosis: Prophylactic thyroidectomy (age dependent on specific mutations – by age 1 for MEN2B, by age 5 for most of MEN2A, screen for pheochromocytoma annually and prior to any surgery, annual calcitonin stim test, annual PTH screening. 195 615 ONCOLOGIC DISORDERS NEUROFIBROMATOSIS Type 2 Responsible gene: NF2 Protein: Neurofibromin-2 (Merlin) Cytogenetic locus: 22q12.2 Inheritance: AD Clinical Features and Diagnostic Criteria: Benign nerve tumors (schwannomas, meningiomas, ependymonas, astrocytoma). Hallmark is bilateral acoustic schwannoma, onset age 18-24 yrs, hearing loss, tinnitus, balance problems. Also cataracts, mononeuropathy, café-au-lait (fewer than in NF1). Clinical Tests: MRI/CT, BAER, audiology evaluation, eye exam Molecular Tests: NF2 sequencing (75%), dupl/del testing (10-15%) Disease Mechanism: NF2 is a tumor suppressor, 2nd hit leads to complete loss of function when one germline mutation present Treatment/Prognosis: Symptomatic tumors removed surgically (XRT may induce tumor formation). Bevacizumab may shrink vestibular tumors and stabilize hearing in some patients. 196 ONCOLOGIC DISORDERS NEUROFIBROMATOSIS Type 2 www.nfcalifornia.org/DiagnosticNF2 197 ONCOLOGIC DISORDERS PTEN HAMARTOMA TUMOR SYNDROME Responsible gene: PTEN Protein: Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase Cytogenetic locus: 10q23 Inheritance: AD Clinical Features and Diagnostic Criteria: Cowden: Presents 2nd/3rd decade: mucocutaneous facial and oral papules, gingival cobblestoning, acral keratosis; dystrophic and adenomatous multinodular goiter, GI polyps, adenosas and fibrocystic breast lesions, macrocephaly, dolichocephaly, lipomas, GU anom. High risk for breast, thyroid, and endometrial cancer. Bannayan-Riley-Ruvalcaba (BRR) macrocephaly, polyposis, lipomas, pigmented macules of the glans penis. Clinical Tests: Lesion pathology, MRA/MRI, CT Molecular Tests: PTEN seq (80%), promoter region mutations (10%) Disease Mechanism: Wild-type protein is a major lipid phosphatase that downregulates the PI3K/Akt pathway to cause G1 arrest and apoptosis Treatment/Prognosis: Annual derm exam, annual breast exam, annual breast MRI and mammography starting age 30, annual thyroid US starting age 14, Proteus syndrome: Distinct disorder - CT nevi, disprop. overgrowth, dysregulated adipose tissue, vascular malformation, risk of ovarian or parotid tumor in 2nd decade – somatic variants in AKT1 198 616 PTEN HAMARTOMA TUMOR SYNDROME ONCOLOGIC DISORDERS Cowden Syndrome Proteus syndrome www.uveitis.org/images/Image1.jpg http://www.childrenshospital.org/az/Site1965 199 ONCOLOGIC DISORDERS TUBEROUS SCLEROSIS COMPLEX Responsible genes: TSC1 and TSC2 Proteins: Hamartin and Tuberin Cytogenetic loci: 9q34, 16p13 Inheritance: AD (2/3 de novo) Clinical Features and Diagnostic Criteria: Skin: hypomelanotic macules, facial angiofibroma, shagreen patch, ungual fibromata. CNS: subependymal glial nodules, cortical tubers, giant cell astrocytoma, seizures. Renal: angiomyolipomas, epithelial cysts, <1% malignant transformation Heart: cardiac rhabdomyoma, tend to regress in infancy without intervention. Lung: lymphangiomatosis (TSC2, women aged 20-40 yrs) Eye: hamartomas or achromic patches. There is a TSC2/PCKD contiguous gene deletion syndrome with features of TS and PKD. Clinical Tests: brain MRI, echo, renal ultrasound, Wood’s lamp exam, eye exam, EEG Molecular Tests: TSC1 sequencing (30% familial, 15% sporadic) and TSC2 sequencing (50% familial and 60-70% sporadic) Disease Mechanism: Abnormal tumor suppressor activity Treatment/Prognosis: Renal US q1-3 yrs, renal CT/MRI if numerous lesions on US, semiannual renal US if angiomyolipomas <3.5-4.0 cm, chest CT if pulmonary symptoms; everolimus for renal angiomyolipoma or subependymal giant cell astrocytoma. mTOR inhibitors, everolimus and others, used for TSC-associated tumors. 200 ONCOLOGIC DISORDERS TUBEROUS SCLEROSIS COMPLEX www.uwo.ca/.../pictures/tuberousclinical.jpg 201 617 ONCOLOGIC DISORDERS VON HIPPEL-LINDAU SYNDROME Responsible gene: VHL Protein: Von Hippel –Lindau disease tumor suppressor Cytogenetic locus: 3p25 Inheritance: AD Clinical Features and Diagnostic Criteria: Hemangioblastoma (cerebellum, retina, spinal cord), pheochromocytoma (hypertension), renal cell carcinoma (40%), endolymphatic sac tumor. Somatic VHL mutations frequently seen in sporadic VHL associated tumors. Clinical Tests: Dilated eye exam, CT or MRI, urine catecholamine metabolites, renal US Molecular Tests: VHL sequencing (72%), Partial or complete gene deletion (28%) Disease Mechanism: Abnormal tumor suppressor function. Truncating or missense mutations that grossly disrupt protein folding lead to VHL Type I: low risk for pheo. Other missense mutations lead to VHL Type II: high risk of pheo. Reduced risk of renal cancer in those with complete gene deletion. Treatment/Prognosis: Annual eye exam from early childhood, Starting at age 5: fractionated metanephrines, BP. Starting age 15 every other year abdominal US, every 2-3 years brain and spine MRI. Temporal bone MRI if documented hearing loss or tinnutus. 202 ONCOLOGIC DISORDERS VON HIPPEL-LINDAU SYNDROME Lancet. 2003 Jun 14;361(9374):2059-67 203 XERODERMA PIGMENTOSUM Responsible genes: Most common subtypes: XPA, XPC, ERCC2, POLH Proteins: DNA-repair protein complementing XP-A cells, DNA-repair protein complementing XP-C cells, ONCOLOGIC DISORDERS TFIIH basal transcription factor complex helicase subunit, DNA polymerase theta Cytogenetic loci: 9q22.3, 3p25, 19q13.2-q13.3, 6p21.1-p12 Inheritance: AR Clinical Features and Diagnostic Criteria: severe sun sensitivity, UV exposure to conjunctiva, cornea, and lids-> severe keratitis, progressive neurologic deterioration: acquired microcephaly, dec/absent DTR’s, prog. SNHL, cognitive impairment. > 1000x inc. risk of skin and eye neoplasms Clinical Tests: Cellular UV hypersensitivity (a post UV exposure cellular survival plot reflecting capacity for DNA repair. Molecular Tests: Research only direct DNA testing of XPA (25%), XPC (25%), ERCC2 (15%), POLH (21%) Disease Mechanism: Impaired ability to sense, excise, and repair UV-induced DNA damage Treatment/Prognosis: Regular detailed skin and eye exam, regular audiometry, protection of all body surfaces from UV light, UV meter to detect unexpected sources of high levels of UV light (eg halogen lamps). 204 618 ONCOLOGIC DISORDERS XERODERMA PIGMENTOSUM http://phobos.ramapo.edu/~pbagga/xp.JPG 205 BECKWITH-WIEDEMANN SYNDROME OVERGROWTH DISORDERS Responsible genes: CDKN1C, H19, KCNQ1OT1 Proteins: cyclin-dep kinase inhib 1C, H19 maternally expressed untranslated mRNA, potassium voltage-gated channel, KQT-like subfamily, member 1 Cytogenetic locus: 11p15.5 Inheritance: AD (15%) Clinical Features and Diagnostic Criteria: hemihyperplasia, macrosomia, macroglossia, visceromegaly, embryonal tumors (e.g., Wilms tumor, hepatoblastoma, neuroblastoma, rhabdomyosarcoma), omphalocele, neonatal hypoglycemia, ear creases/pits, adrenocortical cytomegaly, and renal abnormalities Clinical Tests: AFP, abdominal CT Molecular Tests: Cytogenetically detectable abnormalities of 11p15 (<1%); loss of methylation at DMR2 (50%); gain of methylation at DMR1 (2% -7%); pat. UPD for 11p15 (10-20%); mutations in the CDKN1C (40% of familial cases and 5-10% of sporadic cases) Disease Mechanism: imprinted genes including growth factors and tumor suppressor genes in the 11p15.5 region Treatment/Prognosis: Screening for embryonal tumors: abdominal US every three months until eight years. Serum AFP concentration is monitored in the first few years of life for hepatoblastoma. 206 OVERGROWTH DISORDERS http://imaging.cmpmedica.com/consultantlive/images/photo_clinic BECKWITH-WIEDEMANN SYNDROME Facial features: Anterior linear ear lobe creases Posterior helical ear pits Macroglossia Hemihyperplasia Facial nevus flammeus Midface hypoplasia Infraorbital creases 207 619 OVERGROWTH DISORDERS SOTOS SYNDROME Responsible gene: NSD1 Protein: Histone-lysine N-methyltransferase, H3 lysine-36 and H4 lysine-20 specific Cytogenetic locus: 5q35 Inheritance: AD Clinical Features and Diagnostic Criteria: classic: macrocephaly, pointed chin, tall stature and increased body mass, delayed motor skills, delayed cognitive, verbal, and social development, advanced BA. Less common: phobias, aggression, OCD, ADD, abnormal EEG and seizure, chronic OM and constipation, congenital heart defects, strabismus, hyper/hypothyroidism, possible inc risk of tumors (saccrococcygeal teratoma and neuroblastoma). Clinical Tests: Bone age. Brain MRI or CT may show inc ventricles Molecular Tests: MLPA or FISH for 5q35 microdeletion including NSD1: ~15% (70% in Japanese). NSD1 sequencing: 27-93% (12% in Japanese) Disease Mechanism: Haploinsufficiency of NSD1. May be related to genes affecting growth. Treatment/Prognosis: Supportive treatment, most end up of ave adult Ht, IQ ranges from normal to ID. Cancer screening is not rec. (risk ~1%) 208 OVERGROWTH DISORDERS SOTOS SYNDROME www.gfmer.ch Facial features: malar flushing, sparse frontotemporal hair, high bossed forehead, downslanting palpebral fissures, a long narrow face, and prominent narrow jaw; the head is said to resemble an inverted pear 209 CONNECTIVE PREMATURE AGING TISSUESYNDROMES DISORDERS ATAXIA WITH OCULOMOTER APRAXIA TYPE 1 and TYPE 2 Responsible genes: APTX, SETX Proteins: Aprataxin, Probable Helicase Senataxin Cytogenetic loci: 9p13.3, 9q34 Inheritance: AR Clinical Features and Diagnostic Criteria: childhood onset of slowly progressive cerebellar ataxia, followed by oculomotor apraxia and a severe primary motor peripheral axonal motor neuropathy. Oculomotor apraxia progresses to external ophthalmoplegia. Clinical Tests: Cerebellar atrophy, axonal neuropathy on EMG and biopsy, low serum albumin, high cholesterol. Type 2: Inc AFP Molecular Tests: sequencing APTX (Inc incidence in Portugal and Japan) and SETX. Mutation detection rate unknown. Disease Mechanism: There is direct involvement of aprataxin in the DNA single-strand break repair mechanisms; mutations in the APTX gene destabilize aprataxin and cells from individuals with AOA1 are characterized by enhanced sensitivity to agents that cause singlestrand breaks in DNA Treatment/Prognosis: PT, wheelchair by age 15-20 yrs, educational support, high protein low cholesterol diet 210 620 CONNECTIVE PREMATURE AGING TISSUESYNDROMES DISORDERS ATAXIA WITH OCULOMOTER APRAXIA TYPE 1 and TYPE 2 Sagittal T2-weighted section showing severe cerebellar atrophy predominantly in the vermis (Le Ber et al, Brain, 2003) 211 CONNECTIVE PREMATURE AGING TISSUESYNDROMES DISORDERS COCKAYNE SYNDROME Responsible genes: ERCC6, ERCC8 Proteins: DNA excision repair protein ERCC-6 and ERCC-8 Cytogenetic loci: 10q11, Chromosome 5 Inheritance: AR Clinical Features and Diagnostic Criteria: CS Type I: normal prenatal growth, severe FTT in first 2 years, progressive deterioration of vision, hearing, CNS, and peripheral nervous syndrome. Type II: growth failure at birth, little or no postnatal neurological development, kyphosis, scoliosis, joint contracture. Type III: normal growth and development or late onset. Xeroderma Pigmentosum-CS: facial freckling, early skin cancer, ID, spasticity, short stature, hypogonadism (no demyelination). Clinical Tests: Brain MRI: leukodystrophy. Eye exam: pigmentary retinopathy, cataracts, demyelinating peripheral neuropathy. Abnormal DNA repair on skin fibroblasts Molecular Tests: Gene sequencing ERCC6 (75%), ERCC8 (25%) Disease Mechanism: Abnormal transcription-coupled nucleotide excision repair (preferential removal of UV-induced pyrimidine dimers and other transcription blocking lesions) Treatment/Prognosis: PT, dental exams, skin exams, sunscreen if photosensitive. Death in 1st-2nd decade Type I, by age 7 yrs Type II. 212 CONNECTIVE PREMATURE AGING TISSUESYNDROMES DISORDERS COCKAYNE SYNDROME www.ufowijzer.nl 213 621 CONNECTIVE PREMATURE AGING TISSUESYNDROMES DISORDERS HUTCHINSON-GILFORD PROGERIA SYNDROME Responsible gene: LMNA Protein: Lamin-A/C Cytogenetic locus: 1q21.2 Inheritance: AD (all de novo, paternal age effect) Clinical Features and Diagnostic Criteria: short stature, wt<<ht, head large for face, diminished sc fat, prominent scalp veins, generalized alopecia, delayed and crowded teeth, delayed fontanelle closure, pear shaped thorax, small chin, thin limbs, tight joints, wide based shuffling gate. Sclerodermatous skin changes over lower abdomen and thighs Clinical Tests: Elevated urine hyaluronic acid (unreliable for Dx). ECG, echo, and carotid duplex scans for stenosis. X-ray for clavicular absorption, acroosteolysis, coxa valga Molecular Tests: LMNA G608G Exon 11 (100%) Disease Mechanism: G608G leads to abnl splicing and the mutant form of prelamin A that results is thought have a dominant negative effect leading to progressive defects in nuclear architecture Treatment/Prognosis: Optimize nutrition, age appropriate schooling, PT, aspirin. Annual ECG, echo, carotid duplex, lipid profiles, dental exam and x-ray. Hip x-rays every few yrs to evaluate for avascular necrosis of the femoral head. Severe atherosclerosis develops even with nl lipid profiles, usually die of MI or CVA (ave lifespan 13 yrs); clinical trials 214 CONNECTIVE PREMATURE AGING TISSUESYNDROMES DISORDERS HUTCHINSON-GILFORD PROGERIA SYNDROME seattlepi.nwsource.com 215 PULMONARY SYSTEM ALPHA-1-ANTITRYPSIN DEFICIENCY Responsible gene: SERPINA1 Protein: AAT Cytogenetic locus: 14q32.1 Inheritance: AR Clinical Features and Diagnostic Criteria: Adult COPD, childhood and adult liver disease (obstructive jaundice and raised transaminases in kids, cirrhosis and fibrosis in adults). Age of onset 40-50y if a smoker, 60’s if not. Clinical Tests: Low plasma AAT (also low in other resp d/o inc CF), Demonstration of deficient variant of the AAT protein by protease inhibitor typing Molecular Tests: Targeted mutation testing of SERPINA (95% E42K) Disease Mechanism: Loss of sufficient protease inhibition by AAT Treatment/Prognosis: Liver transplant is a cure (donor liver produces AAT). Intravenous augmentation therapy 216 622 PULMONARY SYSTEM ALPHA-1-ANTITRYPSIN DEFICIENCY (commons.wikimedia.org/wiki/File:Conditions_associated_with_Alpha-1_Antitrypsin_Deficiency.png) 217 PULMONARY SYSTEM CFTR-RELATED DISORDERS Responsible gene: CFTR Protein: cystic fibrosis transmembrane conductance regulator Cytogenetic locus: 7q31.2 Inheritance: AR Clinical Features and Diagnostic Criteria: Cystic fibrosis (CF): chronic airway infection, chronic sinusitis, meconium ileus, malabsorption due to pancreatic insufficiency, male infertility due to azoospermia. Progression to end stage lung disease. Congenital bilateral absence of the vas deferens (CBAVD) occurs in men without pulm. or GI Sx of CF. Clinical Tests: sweat test, decreased semen volume with low pH, high [citric acid], high [acid phosphatase], low [fructose] Molecular Tests: Common mutation testing or full gene sequencing. Intron 8 5T variant: variably penetrant, test for if R117H mutation. 5T with 12 or 13 TG tract (just 5’ of 5T) has the strongest adverse effect on proper intron 8 splicing. deltaF508: 30-80% of mutant alleles depending upon ethnic group. Disease Mechanism: CFTR forms a regulated cell membrane chloride channel. 4 mutation classes: I. reduced or absent synthesis, II. block in protein processing, III. block in regulation of CFTR chloride channel, IV. altered conductance of CFTR chloride channel. Treatment/Prognosis: antibiotics, bronchodilators, steroids, mucolytics, chest PT, lung transplant, pancreatic enzymes, fat soluble vitamins, microscopic sperm aspiration; some examples of mutationspecific therapies. 218 PULMONARY SYSTEM CFTR-RELATED DISORDERS Cysticfibrosis.com 219 623 RENAL DISORDERS ALPORT SYNDROME AND THIN BM NEPHROPATHY Responsible genes: XL: COL4A5, AR: COL4A3 and COL4A4, AD: COL4A3 and COL4A4 Proteins: Collagen alpha-3(IV) chain, Collagen alpha-4(IV) chain, Collagen alpha-5(IV) chain Cytogenetic loci: 2q36-q37 (COL4A3 and COL4A4), Xq22.3 (COL4A5) Inheritance: 80% X linked, 15% AR, 5% AD Clinical Features and Diagnostic Criteria: Spectrum from progressive renal disease with cochlear and ocular abnormalities (Alport) to isolated hematuria with a benign course (thin BM nephropathy). Clinical Tests: Microhematuria, eventually proteinuria. Anterior lenticonus virtually pathognomonic, EM on renal biopsy Molecular Tests: Sequencing and deletion testing COL4A3, COL4A4, COL4A5 (80-100%) Disease Mechanism: Type IV Collagen is found ubiquitously and is the major collagen component of BMs. Alport due to abnl secretion of collagen alpha 3,4,and 5 (IV) chains Treatment/Prognosis: ESRD: 60% by 30 yrs and 90% by 40 yrs and Deafness: 80-90% SN deafness by age 40 in males, later in life in females with XL Alport. Renal progression and deafness is slower in AD Alport and ocular lesions uncommon. Juvenile onset HL in AR Alport. 220 RENAL DISORDERS ALPORT SYNDROME AND THIN BM NEPHROPATHY www.ncbi.nlm.nih.gov/books/NBK22265/ 221 RENAL DISORDERS POLYCYSTIC KIDNEY DISEASE Responsible genes: PKD1, PKD2 and PKHD1 Proteins: Polycystin-1, Polycystin-2, Fibrocystin Cytogenetic loci: 16p13.1, 4q21, and 6p21.1-p12 Inheritance: AD (PKD1, PKD2) AR (PKHD1) Clinical Features and Diagnostic Criteria: AD PKD Enlargement of both kidneys, renal cysts, hematuria, polyuria, flank pain, renal stones, urinary infection. Cysts in liver, pancreas, and intestine; heart valve defects, intracranial aneurysm. AR PKD Fetal or neonatal death, impaired lung formation, pulmonary hypoplasia due to oligohydramnios, renal failure, hepatic fibrosis. Most present prenatally or early infancy Clinical Tests: Abdominal US, prenatal US, MRI Molecular Tests: PKD1 and PKD2 sequence analysis (85%). Large deletion including PKD1 and TSC2: manifestations off PKD and tuberous sclerosis Disease Mechanism: Unclear, decreased amount of functional protein? Treatment/Prognosis: PKD2 mutations show later onset and slower rate of progression. ESRD age 60 yrs 222 624 RENAL DISORDERS POLYCYSTIC KIDNEY DISEASE Liver and kidney cysts www.learningradiology.com 223 SKELETAL DYSPLASIA ACHONDROPLASIA Responsible gene: FGFR3 Protein: Fibroblast growth factor recepter 3 Cytogenetic locus: 4p16.3 Inheritance: AD; 80% de novo Clinical Features and Diagnostic Criteria: short stature, rhizomelic shortening, trident hand, frontal bossing, midface hypoplasia, macrocephaly, OSA, spinal cord compression Clinical Tests: Narrowing of interpediculate distance, caudal spine; notch-like sacroiliac groove, circumflex or chevron seat on the metaphysis Molecular Tests: 98% FGFR3 G1138A; ~1% FGFR3 G1138C Disease Mechanism: Constitutive activation of FGF R (GOF mutations)activation of negative growth control Treatment/Prognosis: achondroplasia growth curves, surgery or CPAP for OSA, role of GH unclear, leg lengthening, suboccipital decompression, spinal fusion, LPA support group 224 ACHONDROPLASIA SKELETAL DYSPLASIA Rhizomelic shortening, frontal bossing, midface hypoplasia, macrocephaly myweb.lsbu.ac.uk medgen.genetics.utah.edu Genu varum 225 625 SKELETAL DYSPLASIA CLEIDOCRANIAL DYSPLASIA Responsible gene: RUNX2 Protein: Runt-related transcription factor 2 Cytogenetic locus: 6p21 Inheritance: AD (high proportion de novo) Clinical Features and Diagnostic Criteria: delayed closure of the cranial sutures, hypoplastic or aplastic clavicles, multiple dental abnormalities. Abnormally large wide open anterior fontanel, midface hypoplasia, brachydactyly, recurrent OM, hearing loss, normal intellect. Clinical Tests: X-ray: clavicular hypoplasia, open sutures, wormian bones, poor or absent sinus pneumatization, hypoplastic scapulae, wide symphysis pubis and sacroiliac joints, large femoral neck and epiphyses, pseudoepiphyses of the metacarpals and metatarsals, deformed and short middle phalanges, osteopenia. Molecular Tests: RUNX2 sequencing and array for microdeletions (60-70%). Disease Mechanism: Independently mediates DNA binding and protein heterodimerization; mutations abolish DNA binding Treatment/Prognosis: Hearing test, dental referral, ear tubes, helmets if large skull defects 226 SKELETAL DYSPLASIA CLEIDOCRANIAL DYSPLASIA Hypoplastic/ absent clavicles allow opposition of the shoulders anteriorly Large skull defects (www.lab3d.odont.ku.dk) (www.pediatriconcall.com) 227 SKELETAL DYSPLASIA DIASTROPHIC DYSPLASIA Responsible gene: SLC26A2 Protein: Sulfate transporter Cytogenetic locus: 5q32-q33.1 Inheritance: AR Clinical Features and Diagnostic Criteria: limb shortening, normal-sized skull, hitchhiker thumbs, small chest, large joint contracture, cleft palate, cystic ear swelling, ulnar deviation of fingers, clubfoot, low tone, normal IQ Clinical Tests: x-ray: cervical kyphosis, incomplete thoracic vertebrae ossification, coronal clefting of lower thoracic and lumbar vertebrae, narrowed interpedicular distance L1 to L5, distal humerous can be bifid or v shaped, rounded distal femur, advanced bone age. Cartilage histopathology: paucity of sulfated proteoglycans in cartilage matrix. Abnormal incorporation of sulfate into macromolecules in cultured chondrocytes Molecular Tests: SLC26A2 targeted mutation analysis (65% one of 5 mutations), SLC26A2 sequencing(>90%) Disease Mechanism: Undersulfation of proteoglycans affects the composition of the extracellular matrix and leads to impaired proteoglycan deposition which is necessary for proper enchondral bone formation Treatment/Prognosis: Maintain joint positioning and mobility as much as possible, clubfoot deformities tend to recur after surgical correction, scoliosis surgery best if postponed until after puberty. Joint contractures and spine deformity worsen with age. Total arthroplasy may diminish joint pain. 228 626 SKELETAL DYSPLASIA DIASTROPHIC DYSPLASIA Limb shortening Normal-sized skull Hitchhiker thumbs Small chest Large joint contracture Ulnar deviation of fingers Clubfoot www.thefetus.net 229 FGFR-RELATED CRANIOSYNOSTOSIS SKELETAL DYSPLASIA (Pfeiffer, Apert, Crouzon, Beare-Stevenson, FGFR2-related Isolated Coronal Synostosis, Jackson-Weiss, Crouzon with Acanthosis Nigricans, and Muenke) Responsible genes: FGFR1, FGFR2, FGFR3 Proteins: Basic fibroblast growth factor receptor 1, 2, and 3 Cytogenetic loci: 8p11.2-p11.1, 10q26, 4p16.3 Inheritance: AD Clinical Features and Diagnostic Criteria: All but Muenke and FGFR2-related Isolated Coronal craniosynostosis are associated with bicoronal craniosynostosis or cloverleaf skull, distinctive facial features, and variable hand and foot anomalies (broad and/or syndactylous). Developmental delay/ID, hearing loss, and visual impairment common. Clinical Tests: Brain CT or MRI for hydrocephalus, spinal x-rays for vertebral anomalies Molecular Tests: FGFR1 sequencing (5% Pfeiffer 1); FGFR2 sequencing (100% Crouzon, JacksonWeiss, Apert, Pfeiffer 2 and 3, and FGFR2-related isolated coronal synostosis); FGFR3 sequencing (100% Crouzon with Acanthosis Nigricans); FGFR3 targeted mutation analysis (100% Muenke) Disease Mechanism: Mutations cause increased R affinity thought to promote excessive receptor down-regulation. Treatment/Prognosis: Coordinated neurosurgical, ORL, and dental care, follow for scoliosis, limb anomalies rarely benefit from surgery 230 FGFR-RELATED CRANIOSYNOSTOSIS SKELETAL DYSPLASIA (Pfeiffer, Apert, Crouzon, Beare-Stevenson, FGFR2-related Isolated Coronal Synostosis, Jackson-Weiss, Crouzon with Acanthosis Nigricans, and Muenke) http://www.ida.org.in/Infor mation/newimages/cranios ynostosis.1.jpg http://webspace.webring.com/people/jc/crouzonsyndrom e/family2007c.jpg http://static.guim.co.uk/sys-images 231 627 SKELETAL DYSPLASIA HEREDITARY MULTIPLE OSTEOCHONDROMAS SYNDROME Responsible genes: EXT1, EXT2 Proteins: Exostosin-1, Exostosin-2 Cytogenetic loci: 8q24.11, 11p11.2 Inheritance: AD Clinical Features and Diagnostic Criteria: Exostoses (benign cartilage-capped bony growths) arising from the growth plate of the long bones or from the surface of flat bones (scapula). Limb length inequity and bowed long bones can develop. Short metacarpals. Can have mass effect compression of nerves and blood vessels. Clinical Tests: x-ray may detect mildly affected individuals Molecular Tests: EXT1 and EXT2 sequencing: >70% detection rate, del/dup studies: 20% Disease Mechanism: EXT1/2 encode glycosyltransferases, mutations lead to actin accumulation and cytoskeltal abnormalities Treatment/Prognosis: Growth ceases after skeletal maturation. 0.5-2% of cases degenerate to chondrosarcoma. Treatment is surgical resection. 232 SKELETAL DYSPLASIA HEREDITARY MULTIPLE OSTEOCHONDROMAS SYNDROME Exostoses www.learningradiology.com 233 SKELETAL DYSPLASIA HYPOCHONDROPLASIA Responsible gene: FGFR3 Protein: Fibroblast growth factor receptor 3 Cytogenetic locus: 4p16.3 Inheritance: AD Clinical Features and Diagnostic Criteria: Short stature, stocky build, rhizo- or mesomelia, limited elbow extension, brachydactyly, mild joint laxity, macrocephaly, scoliosis, genu varum, lumbar lordosis, mild-mod ID, LD, adult onset osteoarthritis Clinical Tests: x-ray: elongated distal fibula, short lumbar pedicles, short distal ulna, chevron deformity of distal femur metaphysis, flattened acetabular roof Molecular Tests: Targeted mutation analysis: N540K (C1620A) (49%), N540K (C1620G) (21%). Exon 9, 10, 13, or 15 sequencing (80%) Disease Mechanism: unknown but mouse models suggest FGFR3 is a negative regulator of bone growth Treatment/Prognosis: Monitor for S/Sx spinal cord compression (MRI or CT foramen magnum), sleep study id history c/w OSA, ortho eval if severe genu varum impairs walking. 234 628 SKELETAL DYSPLASIA HYPOCHONDROPLASIA Short stature Stocky build Rhizo- or mesomelia Limited elbow extension Brachydactyly Macrocephaly Scoliosis Genu varum Lumbar lordosis (www.nature.com/.../n12/fig_ta b/5201700f4.html) 235 SKELETAL DYSPLASIA OSTEOGENESIS IMPERFECTA Responsible genes: COL1A1 and COL1A2 Proteins: Collagen alpha 1(I) chain, Collagen alpha 2(I) chain Cytogenetic loci: 17q21.33, 7q21.3 Inheritance: AD and rare AR Clinical Features and Diagnostic Criteria: Fractures with little or no trauma, relative short stature, blue sclera, dentinogenesis imperfecta, post-pubertal HL, ligamentous laxity, easy bruising. OI Type II: perinatal lethal, palpable callus formation on ribs, hips in “frog-leg” position, short bowed extremities. OI Type III: severe, skull descends on cervical spinebrainstem compression, obstructive hydrocephalus, syringomyelia Clinical Tests: x-ray: fractures of varying ages, spinal compression fracture, wormian bones, protrusio acetabuli, osteopenia. Cultured fibroblasts (98% Type II, 87% all others) Molecular Tests: COL1A1 and COL2A1 sequencing: ~100% Type I, 98% Type II, 60-70% Type III, 0-80% Type IV Disease Mechanism: Type I: premature stop codon->unstable mRNA->dec amount type I collagen. Types II, III, IV: mutations alter collagen structure Treatment/Prognosis: Bisphosphonate to decrease bone resorption, GH to increase linear growth and bone formation 236 SKELETAL DYSPLASIA OSTEOGENESIS IMPERFECTA Bowing of the femur folding.stanford.edu 237 629 SKELETAL DYSPLASIA SAETHRE-CHOTZEN SYNDROME Responsible gene: TWIST1 Protein: Twist-related protein 1 Cytogenetic locus: 7p21 Inheritance: AD Clinical Features and Diagnostic Criteria: coronal synostosis, facial asymmetry, ptosis, 2/3 hand syndactyly, mild-moderate developmental delay in a minority, short stature, parietal foramina, vertebral fusions, radioulnar synostosis, cleft palate, maxillary hypoplasia, congenital heart defect Clinical Tests: echo, x-ray for vertebral abnormalities, audiologic testing, and karyotype: translocations, inversions, or ring chromsome 7 have been reported Molecular Tests: TWIST1 sequencing: >50%, del/dup testing: complete deletion of the TWIST1 gene 11-28% Disease Mechanism: haploinsufficiency by gene deletion, rapid degradation of abnormal protein, or altered subcellular localization of abnormal protein Treatment/Prognosis: endocrine eval if plateau in growth, craniofacial team management, surgical repair of cleft palate and craniosynostosis, eye exams to monitor for evidence of increase ICP 238 SKELETAL DYSPLASIA SAETHRE-CHOTZEN SYNDROME Facial Features: Coronal synostosis Facial asymmetry Ptosis Maxillary hypoplasia (The Cleft Palate-Craniofacial Journal: May 2010, Vol. 47, No. 3, pp. 318-321) 239 630 Writing Exam Questions WRITING EXAM QUESTIONS Debra Schindler, PhD Senior Education Specialist, Office of Curricular Affairs Saint Louis University School of Medicine 633 634 Designing Single Best Answer Multiple Choice Questions This is a short summary of the main points to consider in writing single best answer multiple choice examination questions. It is intended as a guide and checklist to facilitate what is a very difficult task: crafting a good examination that assesses your students’ knowledge and problem-solving abilities, and not their test-taking skills. Most of the material here comes straight from Case and Swanson’s Constructing Written Test Questions For the Basic and Clinical Sciences.1 There are three basic questions that you should be able to answer by the time you finish reading these materials. x How do I construct the question? x How do I construct the answer choices? x How do I construct an exam with a balance of difficulty levels? Only with practice, however, will you be able to apply the basic principles of question writing described here. A well-crafted exam question is usually the product of several iterations of less “ideal” exam questions, and testing the item with students. How to Use This Guide First Steps. The section titled First Steps is the place to start. Use the three steps on this page to review and revise each of your current exam questions. If you find that your questions and your exam meet the criteria listed under First Steps, then your exam is probably in good shape. If you don’t read anything else, read this section. The Structure of Questions and Answers / Question Difficulty. These two sections provide a more detailed explanation of the “how” and “why” of the First Steps. Second Steps- Polishing Vignettes. When you are ready to add new questions, modify existing questions to include more patient-based problems, or improve your existing vignettes, refer to this section. Case and Swanson’s Item Review. This is a more detailed checklist to use in reviewing your test items. If your test item meets the “bare bones” criteria of the First Steps, then use this checklist to check for any additional areas of difficulty. Content Integration. The last page of this document contains an example of how one patient vignette can be used in four different disciplines, providing a means of integrating knowledge across course and discipline boundaries. 1 Susan M. Case and David B. Swanson, Constructing Written Test Questions For The Basic and Clinical Sciences (Philadelphia: National Board of Medical Examiners, 1998). Prepared by the Office of Curricular Affairs, St. Louis University School of Medicine, September 28, 2004. Debra L. Schindler, Ph.D., Evaluation Coordinator 635 First Steps Writing good test questions is hard and takes time. Here are some first steps to take in reviewing and revising your existing test questions. 1. Cover the options. Can you still answer the question? If not, rewrite the question to make it complete. Why? Ideally, you would like your students to answer a question without having any hints (i.e. answer choices)- ideally, they should be able to generate the answer from the knowledge they have inside their heads. So- reinforce the cognitive structure of their medical knowledge by asking a question that requires accessing the knowledge base. HINT: Don’t use the phrase “Which of the following…” if possible. Use the full range of question words available in English: who, what, when, where, why, how, which. Using these question words almost always forces you to write a complete question. 2. Order the options. Make sure that you can order your options (including the correct answer) from o Best to Worst o First to Last o Most Likely to Least Likely o Etc. Why? When your students generate answers in their heads, you would like those possible answers to be logically structured. We must help students build the scaffolding required to organize all of the knowledge and skills they need to acquire, and then teach them, through question and answer, how to retrieve and apply that knowledge in a structured way. A clearly focused question, with options that can be ordered logically, reinforces both the structure of knowledge that students are building, and the thought process needed to retrieve that knowledge. If your options cannot be ordered in any logical manner, they probably aren’t homogenous, and your question probably isn’t clearly focused. 3. Provide a balanced level of difficulty and challenge your students. They work hard and want to demonstrate their knowledge and skills. On every exam, try to provide a balance between questions that ask students to recall facts, questions that ask students to apply those facts, and questions that ask students to generate new solutions. Using questions that assess different levels of cognitive ability can produce important feedback for both students and faculty. Students can identify the types of questions (and thought processes) that they have the most difficulty with, and focus their efforts in those areas. Faculty can identify areas of teaching and/or content that may need strengthening or may actually need less time in class. 2 Prepared by the Office of Curricular Affairs 636 The Structure of Questions and Answers2 Single best answer style questions should have the following structure: Component A 32-year old man has a 4-day history of progressive weakness in his extremities. He has been healthy except for an upper respiratory tract infection 10 days ago. His temperature is 37.8 C (100 F), blood pressure is 130/80 mm Hg, pulse is 94/min, and respirations are 42/min and shallow. He has symmetric weakness of both sides of the face and the proximal and distal muscles of the extremities. Sensation is intact. No deep tendon reflexes can be elicited; the plantar responses are flexor. Stem (e.g., a clinical or laboratory case presentation). This need not be a long presentation, and may in fact, elicit simple recall. What is the most likely diagnosis? Lead-in question A. B. C. D. E. One correct answer + four distractors Acute disseminated encephalomyelitis Guillain-Barré syndrome Myasthenia gravis Poliomyelitis Polymyositis The Question/Stem The stem and lead in question should provide enough information to allow the student to answer the question without looking at the options. More information on creating a vignette is provided on pages 10-11. Case and Swanson provide many examples of leadin questions for basic sciences on pages 39-40, and examples for clinical science items Health and Health Maintenance, Mechanisms of Disease, Diagnosis, and Management) on pages 61-65. 2 Material on writing questions and answers is borrowed and adapted from Susan M. Case and David B. Swanson, Constructing Written Test Questions For The Basic and Clinical Sciences (Philadelphia: National Board of Medical Examiners, 1998). Prepared by the Office of Curricular Affairs 637 3 The Answer/Options Note that the incorrect options in the example above are not totally wrong. The options can be diagrammed as follows: D Least Correct C A E B Most Correct Even though the incorrect answers are not completely wrong, they are less correct than the "keyed answer." The examinee is instructed to select the "most likely diagnosis"; experts would agree that the most likely diagnosis is B; they would also agree that the other diagnoses are somewhat likely, but less likely than B. Using a continuum provides a more realistic context for deriving the correct answer. Physicians must choose, for example, the best drug from among several possible choices: under different circumstances each of the choices may be the ‘best.” The circumstances of the case that you provide to students, however, should dictate the “best” choice, and thus reflect the correct decision-making process. As long as the options can be laid out on a single continuum, in this case from "Most Likely Diagnosis" to "Least Likely Diagnosis," options in single best answer questions do not have to be totally wrong. Other option sets can include next steps, tests, treatments, prognoses, etc., as long as all of the options in a set are homogeneous. The next example illustrates the most common type of flaw in single best answer questions: non-homogeneous option sets. Component Which of the following is true about pseudogout? Stem A. B. C. D. E. One correct answer + four distractors It occurs frequently in women It is seldom associated with acute pain in a joint. It may be associated with a finding of chondrocalcinosis It is clearly hereditary in most cases It responds well to treatment with allopurinol After reading the stem, the examinee has only the vaguest idea what the question is about. In an attempt to determine the "best" answer, the examinee has to decide whether "it occurs frequently in women" is more or less true than "it is seldom associated with acute pain in a joint." This is a comparison of apples and oranges. In order to rank-order the relative correctness of options, the options must differ on a single dimension or else all options must be absolutely 100% true or false. 4 Prepared by the Office of Curricular Affairs 638 The options for this test item can be diagrammed as follows: Gender (A) Rx (E) Inheritance (D) False (B) Associations (C) True The options are heterogenous and deal with miscellaneous facts; they cannot be rankordered from least to most true along a single dimension. Although this question appears to assess knowledge of several different points, its inherent flaws preclude this. The question by itself is not clear; the item cannot be answered without looking at the options. Prepared by the Office of Curricular Affairs 639 5 Question Difficulty The three questions below are examples of the evolution of questions on the USMLE Step 1 exam, beginning with question type 1 from the 1980s, question type 2 from the 1990s, and ending with question type 3, a format that has been added to the currently administered Step 1 exam.3 Each question type represents a different level of difficulty and requires a different thought process to arrive at the correct answer. As faculty in Phase 1 and 2 courses build banks of test questions, questions at all three levels should be constructed and questions from all three levels should be included on every exam. Exams may contain very few type 3 questions at the highest level of cognitive ability, but such questions can help identify students who are truly at the top of their class (as measured by the exam only). Distinguishing between types of questions can also help identify students who are comfortable with repeating directly from the course material, but who have trouble with integrating information and forming independent conclusions. Just as the USMLE examination contains all three types of questions, so should SLUSOM course examinations. Type 1: Comprehension, Knowledge Parkinson’s disease is caused by loss of neurons in which of the following areas of the brain? A) Caudate nucleus B) Cerebral cortex C) Mamillary bodies D) Substantia nigra E) Subthalamic nucleus Type 2: Application A 72-year-old man presents complaining of shaking in his right hand and trouble starting movements. On physical examination, the patient has a resting tremor of the right hand that decreases with active movement. The man’s face is expressionless, and his voice is very soft. Ratchet-like resistance to passive movement is noted in both arms. He also has a slightly stooped posture, and a slow, shuffling gait. Which of the following areas of the brain is most likely affected in this patient? A) Caudate nucleus B) Cerebral cortex C) Mamillary bodies D) Substantia nigra E) Subthalamic nucleus Type 3: Analysis, Synthesis, Evaluation A 72-year-old man presents complaining of shaking in his right hand and trouble starting movements. On physical examination, the patient has a resting tremor of the right hand that decreases with active movement. The man’s face is expressionless, and his voice is very soft. Ratchet-like resistance to passive movement is noted in both arms. He also has a slightly stooped posture, and a slow, shuffling gait. Which of the following areas of the labeled brain below is most likely affected in this patient? A) A B) B C) C D) D E) E 3 The examples in this table have been reproduced from a Kaplan Medical Power Point presentation made November 21, 2002 at Saint Louis University School of Medicine, with permission from Kaplan, Inc. It may not be reproduced or altered without their permission. 6 Prepared by the Office of Curricular Affairs 640 The table below provides several additional ways to think about question item construction and difficulty. Test Question Level of Difficulty 1 Easy to Moderate 2 Moderate to Difficult 3 Very Difficult Analysis Synthesis Evaluation Bloom's Taxomony Comprehension Knowledge Walvoord and JohnsonAnderson's criteria for assigning difficulty4 The information in the question is directly from the course presentation, although it may have changes in wording or phraseology. Course materials provide all of the background information needed to answer the question. There is a direct, visible connection between the course material and the question. Course materials provide all of the background information needed to answer the question, although there is no clear, visible connection between the course material and the question. Type 1:1980s style questions Type 2: 1990s style questions Type 3: Current style of questions USMLE Step 1 Question types (as illustrated on page 7) Application 4 Walvoord, B.E, and Johnson-Anderson, V. (1998) Effective Grading. A Tool for Learning and Assessment. San Francisco: Jossey-Bass. Prepared by the Office of Curricular Affairs 641 7 Second Steps- Polishing Vignettes As you work on your existing questions, you may want to write new questions, and refine your old questions to include more patient-centered problems, such as patient, clinical, and laboratory vignettes. Case and Swanson recommend using templates to help generate questions. Their explanations are summarized below, and references are provided to guide to the specific sections of their book which contain more information. Basic Science Questions Template: 5 A (patient description) is unable to (functional disability). Which (structure) is most likely to have been injured? Example: A 65-year-old man has difficulty rising from a seated position and straightening his trunk, but he has no difficulty flexing his leg. Which muscle is most likely to have been injured? A. B. C. D. E. Gluteus maximus Gluteus minimus Hamstrings Lliopsoas Obturator internus Patient vignettes for basic science questions may include some or all of the following information: age, gender, site of care, presenting complaint, duration, patient history (with or without family history), physical findings, +/- results of diagnostic studies, +/initial treatment, subsequent findings, etc. Clinical Science Questions Clinical vignettes should begin with the presenting problem, history, physical findings, results of diagnostic studies, initial treatment, subsequent findings, etc. The vignette may include all or only part of this information, but the information should be presented in this order. There should be one clearly formulated problem, which the student should be able to answer without looking at the options. Example: A 52-year-old man has had increasing dyspnea and cough productive of purulent sputum for 2 days. He has smoked one pack of cigarettes daily for 30 years. His temperature is 37.2 C (99 F). Breath sounds are distant with a few rhonchi and wheezes. His leukocyte count is 9000/mm3 with a normal differential. Gram’s stain of sputum shows numerous neutrophils and gram-negative diplococci. X-ray films of the chest show hyperinflation. What is the most likely diagnosis? 5 Page 39 in the Case and Swanson book list many other templates appropriate for basic science questions. 8 Prepared by the Office of Curricular Affairs 642 Tips: x Don’t use “real patients”—they are too complicated, their findings may contain “red herrings,” and they sometimes lie. Use incidental findings when appropriatestudents should know how to filter information for what is most relevant in any given situation. A paper and pencil test can’t capture the visual and verbal cues that a physician gets from a patient, so avoid trying to write those cues into the vignette. For example, rather than writing “The patient claims to smoke one pack of cigarettes per day,” write “the patient smokes one pack of cigarettes per day.” Tailor the patient and his or her vignette to include accurate information, focused on the problem-solving or decision-making behavior that you want students to demonstrate. x Provide reference materials (e.g,. a table of normal laboratory values), if in real life, the physician would use such reference materials to obtain information or help make a decision. Prepared by the Office of Curricular Affairs 643 9 Case and Swanson’s Item Review Susan Case and David Swanson recommend the following guidelines for writing these types of test questions in the context of medical school assessment. 6 Subject each question to the five “tests” implied by rules below. If a question passes all five, it is probably well phrased and focused on an appropriate topic. The Basic Rules for One-Best Answer Items Each item should focus on an important concept, typically a common or potentially catastrophic problem. Don’t waste testing time with questions assessing knowledge of trivial facts. Focus on problems that would be encountered in real life. Avoid trivial, “tricky,” or overly complex questions. Each item should assess application of knowledge, not recall of an isolated fact. The item stems may be relatively long; the options should be short. Vignettes provide a good basis for a question. For medical students, patient vignettes are useful, even for beginning students with little exposure to clinical problems. These vignettes should begin with the presenting problem of a patient, followed by the history (including duration of signs and symptoms), physical findings, results of diagnostic studies, initial treatment, subsequent findings, etc. Vignettes may include only a subset of this information, but the information should be provided in this specified order. For some students in some topic areas, vignettes may be very brief; in other areas, longer, more complicated vignettes are more appropriate. The stem of the item must pose a clear question, and it should be possible to arrive at an answer with the options covered. To determine if the question is focused, cover up the options and see if the question is clear and if the examinees can pose an answer based only on the stem. Rewrite the stem and/or options if they could not. All distractors (i.e., incorrect options) should be homogeneous. They should fall into the same category as the correct answer (e.g., all diagnoses, all tests, all treatments, all prognoses, all “next steps in solving the problem”). Rewrite any dissimilar distractors. Avoid using “double options” (e.g., do W and Y; do Y because of Z) unless the correct answer and all distractors are double options. Rewrite double options to focus on a single point. All distractors should be plausible, grammatically consistent, logically compatible, and of the same (relative) length as the correct answer. Order the options in logical order (e.g. numeric), or in alphabetical order. Avoid technical item flaws that provide special benefit to testwise examinees or that pose irrelevant difficulty. Do NOT write any questions of the form “Which of the following statements are correct?” or “Each of the following statements is correct EXCEPT.” These questions are unfocused and have heterogeneous options. 6 Susan M. Case and David B. Swanson, Constructing Written Test Questions For The Basic and Clinical Sciences (Philadelphia: National Board of Medical Examiners, 1998) 33. 10 Prepared by the Office of Curricular Affairs 644 Content Integration7 To lighten everyone’s burden in writing exams, consider sharing good vignettes across courses, and maybe working in some curricular integration at the same time. This example illustrates how a single vignette can be used in combination with questions from multiple disciplines, allowing integration of content from across the curriculum in a single examination. The questions posed after the vignette are application type questions (using Bloom's taxonomy), requiring two steps in the thought process needed to arrive at an answer: students must first draw a conclusion, and then apply knowledge of an associated fact. 1. A 72-year-old man presents complaining of shaking in his right hand and trouble starting movements. On physical examination, the patient has a resting tremor of the right hand that decreases with active movement. The man’s face is expressionless, and his voice is very soft. Ratchet-like resistance to passive movement is noted in both arms. He also has a slightly stooped posture and a slow, shuffling gait. Pharmacology Which of the following treatments for this disease acts by inhibiting monoamine oxidase B? Pathology Which of the following pathologic findings is characteristic of this disease? (A) Amantadine (B) Benztropine (C) Bromocriptine (D) Levodopa (E) Selegiline (A) Amyloid plaques (B) Howell-Jolly bodies (C) Kaiser-Fleischer rings (D) Lewy bodies Behavioral Science The neurotransmitter that is deficient in this disease is thought to be involved in the pathogenesis of which of the following psychiatric disorders? Biochemistry The neurotransmitter involved in the pathogenesis of this disease is derived from which of the following amino acids? (A) Bipolar disorder (B) Borderline personality disorder (C) Major depressive disorder (D) Obsessive-compulsive disorder (E) Schizophrenia (A) Arginine (B) Glycine (C) Histidine (D) Tryptophan (E) Tyrosine 7 The example in this table has been reproduced from a Kaplan Medical Power Point presentation made November 21, 2002 at Saint Louis University School of Medicine, with permission from Kaplan, Inc. It may not be reproduced or altered without their permission. Prepared by the Office of Curricular Affairs 645 11 646 Sample Exam Questions & Answers SAMPLE EXAM QUESTIONS & ANSWERS 649 650 2017 Genetics and Genomics Review Course Sample Questions & Answers This document contains a consolidation of sample questions with the answers following each section. All questions have been organized by category for ease of review. Section Title A Genetic Transmission B Biochemical Genetics C Clinical Genetics D Neurogenetics E Molecular Genetics F Genetic Counseling G Cancer Genetics H Cytogenetics I Prenatal/Reproductive Genetics J Mendelian Genetics K Developmental Genetics L. Genomic Medicine 651 A. Genetic Transmission (Quantitative Genetics) Questions A1-A35 A1. Assume that 500,000 serial newborns were examined for new mutation cases of a dominant disorder with 100% penetrance. Ten affected infants were found with unaffected parents. Which of the following represents the calculated mutation rate? A. B. C. D. E. A2. A couple are both carriers for an autosomal recessive disorder. They have four children. What is the probability that at least one child is affected? A. B. C. D. E. A3. 40/256 81/256 160/256 175/256 216/256 What is the coefficient of inbreeding (F) for a mating of half-siblings? A. B. C. D. E. A4. 1 x 10-6 2 x 10-6 1 x 10-5 2 x 10-5 4 x 10-5 1/4 1/8 1/16 1/32 1/64 A couple requests genetic testing for their newborn child (arrow) who is at risk of a sex-linked recessive disorder that affects the child’s brother and uncle. A linkage study is performed using a marker closely linked to the disease locus. There are two alleles at the marker site: A and B, and the distance between the marker locus and the disease locus is estimated to be 5 cM. What is the risk of this child developing signs of the disorder, assuming complete penetrance? A. B. C. D. E. 0.0475 0.0500 0.0950 0.0975 0.1000 652 A5. I-1 was affected with Hemophilia (Factor VIII deficiency). What is the probability that the pregnancy (IV-4) will result in a boy affected with Hemophilia? A. B. C. D. E. 1/4 1/8 1/9 1/18 1/36 I II 1 2 1 2 III 2 1 IV 1 2 3 P 4 A6. A couple presents for prenatal counseling and you obtain the family history documented in the pedigree below. They have two healthy sons and she is pregnant again. What is the approximate probability that her fetus will be severely affected with hydrocephalus? A. B. C. D. E. A7. 1/5. 1/8. 1/10. 1/20. 1/32. Assume a three allele autosomal DNA polymorphism with gene frequencies of 0.50, 0.25, and 0.25. What proportion of the population will be heterozygous for these polymorphisms? A. B. C. D. E. 4/16 (0.25) 6/16 (0.375) 8/16 (0.5) 10/16 (0.625) 12/16 (0.75) 653 A8. A woman has a child with an autosomal recessive disorder that affects 1:40,000 in the population. She remarries and the couple requests recurrence risk counseling. What is their approximate risk of having an affected child? A. B. C. D. E. A9. What is the probability that a couple who are both heterozygous for the (F508 mutation for CF will have two affected and two unaffected with CF if they have four children? A. B. C. D. E. A10. 9% 18% 27% 36% 45% Assume you discover a new X-linked platelet antigen detected by a monoclonal antibody. Heterozygous and homozygous positive females are antigen positive. You find 20% of males to be antigen positive and 80% to be antigen negative. Assuming Hardy-Weinberg equilibrium, what percent of females would you predict to be antigen positive? A. B. C. D. E. A12. 9/32 9/64 9/128 27/128 27/256 Assume the following frequencies for the ABO blood group. The A allele = 0.30; the B allele = 0.10; and the O allele = 0.60. What percent of people should be blood group A? A. B. C. D. E. A11. 1/500 1/400 1/200 1/100 1/50 16% 20% 32% 36% 40% A recessive condition has a population frequency of 1:90,000. Choose the number below that is closest to the number of generations required to reduce the allele frequency by half, assuming that all homozygous individuals are unable to reproduce? A. B. C. D. E. 50 75 150 300 600 654 A13. There is a three allele DNA polymorphism. A 10 kb fragment has an allele frequency of 0.10; a 7 kb fragment has an allele frequency of 0.10; and a 5 kb fragment has an allele frequency of 0.80. Assuming Hardy-Weinberg equilibrium, what percent of individuals should be heterozygous for this polymorphism? A. B. C. D. E. A14. A GT repeat polymorphism has five alleles all with a frequency of 0.20. What proportion of people should be heterozygous (informative)? A. B. C. D. E. A15. 0.10 0.20 0.50 0.80 0.90 You find an autosomal (two allele) RFLP and 25% of people are homozygotes for the upper band. What should be the allele frequency for that band? A. B. C. D. E. A16. 34% 36% 50% 64% 66% 0.05 0.10 0.125 0.25 0.50 A couple seeks counseling regarding their risk of having a child with an autosomal recessive condition that affects the brother of one partner (see pedigree). The population frequency of the disorder is 1:400. What is the risk to one of their offspring of being affected? A. 1/40 B. 1/60 C. 1/80 D. 1/100 E. 1/120 655 A17. What is the coefficient of inbreeding (F; equal to the chance of homozygosity by descent) for the child of the mating shown below? A. B. C. D. E. A18. A couple comes to you for genetic counseling. The wife had two siblings that died from WerdnigHoffman syndrome (or spinal muscular atrophy 1, SMA1), an autosomal recessive disease with a prevalence of approximately 1 in 20,000 livebirths. The husband reports a negative family history. What is the probability that this couple will have a child with SMA1? A. B. C. D. E. A19. 1 in 70 1 in 120 1 in 240 1 in 360 1 in 440 A 35-year old man presents for genetic counseling because his mother recently died from an autosomal dominant late-onset progressive neuromuscular disorder. By age 35 years, 75% of individuals carrying the mutant gene have begun to show symptoms of muscle weakness and slurred speech. A thorough evaluation by a neurologist did not detect any signs of this condition. The gene for this condition is unknown and there are no available carrier tests. What is the likelihood that this man inherited the mutant gene from his mother? A. B. C. D. E. A20. 1/4 1/8 1/16 1/32 1/64 1 in 5 1 in 10 1 in 20 1 in 30 1 in 50 Which of the following numbers below is the closest estimate of the mutation rate for an autosomal dominant disorder with a population frequency of 1:5,000 in which reproductive fitness is approximately 0.3? A. 0.0001 B. 0.0002 C. 0.0003 D. 0.0004 E. 0.0005 656 A21. Linkage is a violation of which of the following genetic concepts? A. B. C. D. E. A22. The presence of an allele or genotype at one locus being necessary for the expression of a genotype at a second locus is described by which of the following genetic concepts? A. B. C. D. E. A23. Hardy-Weinberg equilibrium Mendel’s law of independent assortment Mendel’s law of segregation Recombination Variability of expression pleiotropy penetrance linkage disequilibrium variable expression epistasis A couple has a son with a multifactorial disorder in which it is more common for females to be affected. Which of the following statements about recurrence risk is true? A. Recurrence is higher for a next child than if a daughter had been affected, and would be higher for a daughter than a son B. Recurrence is higher for a next child than if a daughter had been affected, and would be higher for a son than a daughter C. Recurrence is lower for a next child than if a daughter had been affected, and would be higher for a daughter than a son D. Recurrence is lower for a next child than if a daughter had been affected, and would be higher for a son than a daughter E. Recurrence is 50% since a son is affected. A24. Mutations in the CFTR gene can affect multiple organ systems, including respiratory, gastrointestinal, and reproductive systems. This is an example of which of the following genetic concepts? A. B. C. D. E. A25. pleiotropy penetrance linkage disequilibrium variable expression epistasis Males with two mutations in the CFTR gene can have classic CF (lung involvement, pancreatic insufficiency, male infertility) or only infertility. This is an example of which of the following genetic concepts? A. B. C. D. E. pleiotropy penetrance linkage disequilibrium variable expression epistasis 657 A26. A GeneReviews article notes the incidence of an autosomal recessive disease is 1 in 10,000. A man has an affected child and has remarried and his new wife is pregnant. His mutation is known and is found to be transmitted to the fetus. Carrier detection for the new wife is impractical. What is the chance the fetus is affected? A. B. C. D. E. A27. A woman has a son with an X-linked recessive lethal disorder and no other family history of the condition. Her daughter has one healthy son. What is the daughter’s risk of being a carrier? A. B. C. D. E. A28. 1/2 1/3 1/4 1/6 1/12 What is the approximate odds ratio of a disorder in a person who carries an allele that is seen in 30% of those with the disorder but only 20% of controls? A. B. C. D. E. A30. 0.10 0.20 0.33 0.50 0.66 A couple’s first child is a boy with a very severe X-linked disease. There is no carrier testing for this disease and the incidence is 1 in 100,000 boys. Neither partner has a family history of this disease. The probability that the couple will have another affected child is about? A. B. C. D. E. A29. 1 in 25 1 in 50 1 in 64 1 in 100 1 in 200 1.5 0.8 1.7 1.9 2.4 Elizabeth’s maternal grandfather has hemophilia due to a factor VIII deficiency. Elizabeth has married an unaffected man and has four healthy sons. Elizabeth is pregnant again with a male fetus. What is the approximate probability that this fetus will develop hemophilia? A. 0 B. 1/4 C. 1/25 D. 1/33 E. 1/50 658 A31. Leevi, a 32-year-old man of Finnish descent had a sibling who died at the age of 10 from cystic fibrosis (CF). Leevi, who does not himself have CF, has recently married and would like to start a family, but is concerned about the risk of passing CF to his children. DNA samples are not available from his brother or from his parents, who are both deceased. He undergoes DNA testing for the 70 most common CFTR mutations – a panel that detects 90% of the mutations found among individuals of northern European descent. He tests negative for all 70 mutations. Given this information, what is the probability that he is a heterozygous carrier for CF? A. B. C. D. E. A32. 2/3 1/4 1/6 1/15 1/25 Rob's father has Huntington's disease. Rob's mother has no family history of Huntington's disease. Although Rob does not want to know if he has inherited the mutation, he is concerned about the potential risks for his children. Rob's wife, who has no family history of Huntington's disease, is pregnant. Prenatal testing is performed, using the alleles of an STR marker linked (q = 0.0) to the Huntington gene. The allele results are shown below on the pedigree of Rob's family. Using this information, what is the approximate chance that the fetus has inherited the mutated (expanded) copy of the Huntington gene? A. 0 B. 1/4 C. 1/2 D. 3/4 E. 1 A33. A 22-year-old man is at 50% risk of having inherited a mutation for a dominantly inherited disorder. He has no signs of the disorder, but the condition displays age-dependent penetrance, so that 20% of individuals at age 22 would expect to be affected. What is his approximate risk of having inherited the mutation? A. B. C. D. E. 0.86 0.52 0.44 0.24 0.20 659 A34. Mr. and Mrs. Smith come to see you in your clinic very distressed. At 38 years old, Mrs. Smith is pregnant with her fifth child. Her firstborn died from complications of cystic fibrosis. The couple’s three other children are not affected with this condition, but Mr. and Mrs. Smith are concerned the fetus may have CF. They have come to you for prenatal diagnosis. You screen the Smiths for the 25 most common CF mutations, but they do not have any of these mutations. You conclude they are carriers of rare CF mutations. Fortunately, there is a microsatellite marker very tightly linked (theta = 0.00) to the CF-causing gene. You obtain DNA from all the surviving family members and the fetus (via CVS) and genotype them with respect to this linked marker. The corresponding alleles are shown below: 9,3 9,4 ? 9,9 3,4 3,9 9 9,4 4 Based on these results, what is the chance that the fetus has CF? A. 0 B. 1/4 C. 1/2 D. 3/4 E. 1 A35. What is the approximate risk that the woman indicated by the arrow in the pedigree below will have a son with the X-linked recessive lethal disorder that affects her brother? A. B. C. D. E. 0.50 0.25 0.10 0.05 0.01 660 A36. A case control study is done in which a specific allele is tested in affected and unaffected individuals. The allele is found in 40% of affected individuals but only 10% of controls. Which of the following is the best estimate of the odds ratio of disease given the presence of the allele compared with the allele not being present? A. B. C. D. E. A37. 2 4 6 8 10 A couple seek counseling regarding their risk of having a child with a specific recessive disorder that occurs in 1:40,000 births in the mother’s ancestry and 1:10,000 in the father’s. Which of the following is the chance that they would have an affected child? A. B. C. D. E. 1:5,000 1:10,000 1:20,000 1:400,000,000 1:1,600,000,000 A38. 37. You are evaluating a large family depicted in the pedigree below. Which of the following modes of inheritance is most compatible with this family history? A. B. C. D. E. Autosomal recessive Digenic Maternal X-linked dominant X-linked recessive 661 Answers to Genetic Transmission (Quantitative Genetics) Questions A1-A38 A1. For the direct method of estimating the mutation rate, Number of sporadic cases with condition ȝ [numbers of individuals examined x #sporadic cases The correct answer is C A2. The easiest approach is to calculate the probability that none of the children is affected which is (3/4)4. This is 81/256. The difference (175/256) is the probability that at least one child will be affected. It is not as easy to calculate the probability, for example, that two children would be affected and two children would be unaffected. This would involve using the binomial expansion or making very careful calculations to consider all the possible birth orders and multiplying these out. The correct answer is D. A3. Expect a question on coefficient of inbreeding. You can memorize the usual numbers, calculate the probability that the offspring of the mating will be homozygote by descent at a given locus in simple cases, or use the method of path coefficients as described below: Using Sewall Wright’s path coefficient method, the coefficient of relationship of the parents is (1/2)n where n is the number of links through common ancestors on a path through the pedigree. Here n = 2 for the path through from the father through his mother to his half-sister (mother of child). The coefficient of relationship of the parents is therefore 1/4. The coefficient of inbreeding for the child is ½ the coefficient of relationship for the parents or 1/8. The correct answer is B. A4. Based on the affected sibling, the mother most likely has the A allele in coupling with the disorder. The risk to the child is therefore 2T – 2T2 = 0.0950. The correct Answer is C. A5. Use Bayesian calculation that III-1 is a carrier. Prior Conditional Joint Posterior Carrier 1/2 1/2x1/2x1/2 1/16 1/9 Carrier risk x 1/4 = risk of affected boy = 1/36. The correct answer is E. 662 Non-Carrier 1/2 1 8/16 8/9 A6. Aqueductal stenosis can be X-linked and appears to be so in this family. The probability she is a carrier is a Bayesian calculation. Carrier 1/2 1/2x1/2 1/8 1/5 Prior Conditional Joint Posterior Non-Carrier 1/2 1 4/8 4/5 The chance she is a carrier (1/5) times the chance of an affected male (1/4) = 1/20. The correct answer is D. A7. It is easiest to calculate the frequency of each of the homozygotes which is simply the sum of the square of the gene frequencies. Thus the frequency of homozygotes would be 1/4 + 1/16 + 1/16 = 6/16. Thus the proportion of the population which would be heterozygous would be 10/16. The correct answer is D. A8. Explanation: q 1 40,000 1 2q = 1/100 = risk partner is a carrier 200 Risk = 1(1/100)(1/4) = 1/400 The correct Answer is B. A9. The probability for two of each is 1/4 x 1/4 x 3/4 x 3/4 but this can occur in any of six orders. The affecteds may be 1 + 2, 1 + 3, 1 + 4, 2 + 3, 2 + 4, or 3 + 4. Therefore the answer is 6(1/4)2 (3/4)2 = 27/128. You could use binomial expansion. The answer is D. A10. Individuals of genotype AA would be 0.3 X 0.3 = 0.09. Individuals of genotype AO would be 2 X 0.3 X 0.6 = 0.36. Adding these together gives 0.45. The correct answer is E. A11. The male data gives a gene frequency for antigen negative of p = 0.8. Antigen positive homozygotes would be 0.2 X 0.2 = 0.04. Heterozygous females would be 2 X 0.2 X 0.8 = 0.32. Adding these two together gives the correct answer, D. A12. Explanation: q 1 90, 000 1 300 n = 1/q = 300 The correct Answer is D. A13. It may be easier to calculate the frequency of homozygotes which would be each allele frequency squared. The sum of each allele frequency squared is 0.66. This would be the frequency of homozygotes. The frequency of heterozygotes would be 0.34 = 34%. The correct answer is A. 663 A14. Similarly for this question, the frequency of homozygotes would be each gene frequency squared. Since they are all same, one can calculate 5 X 0.2 X 0.2 = 0.2 for the frequency of homozygotes. Thus the heterozygotes would be 0.80. The correct answer is D. A15. The allele frequency would be the square root of the frequency of the homozygotes which would be 0.5. The correct answer is E. A16. The male in the couple has a 2/3 risk. The frequency of the allele is 1/20 (square root of 1/400), so the carrier frequency is approximately 1/10. Hence their risk of having an affected child is (2/3)(1/10)(1/4)= 1/60 The correct Answer is B. A17. Using Sewall Wright’s path coefficient method, the coefficient of relationship of the parents is (1/2)n where n is the number of links through common ancestors on a path through the pedigree. Here n = 5 for the path through from the father through his great-grandfather to the mother and also n = 5 from the father through his great-grandmother to the mother. The coefficient of relationship of the parents is therefore 1/32+1/32 = 1/16. The coefficient of inbreeding for the child is ½ the coefficient of relationship for the parents or 1/32. The correct answer is D. A18. The wife has a 2/3 risk of being a carrier. The allele frequency is the square root of 1/20,000 = 1/141. The carrier frequency is twice that, about 1/70. Hence the risk to a child is 2/3 x 1/70 x ¼ = 1/420. E is closest to this value. A19. This is best handled as a Bayesian calculation: Prior Conditional Joint Posterior Inherited Gene 1/2 1/4 1/8 1/5 Did Not Inherit Gene 1/2 1 4/8 The correct answer is A. A20. Explanation: 2p = 1/5,000 p = 1/10,000 ľűŴġľġĩĲİĲıĭıııĪĩıįĸĪġľġıįııııĸġſġıįıııĲġĩųŦŮŦŮţŦųġŵũŢŵġŴġľġĲ-F) The correct Answer is A. A21. The correct answer is B. A22. The correct answer is E. 664 A23. This is an example of the Carter effect. Since the disorder is more common in daughters, the fact that a son was affected says that the liability in the couple must be higher than if a daughter had been affected. This makes the recurrence risk higher for the next child, but recurrence for this trait is always higher for a daughter. The correct Answer is A. A24. The correct answer is A. A25. The correct answer is D. A26. We are told essentially that q2 equals 1/10,000 (q=1/100). The chance that the fetus will be affected is the chance that the mother is a carrier (essentially 2q) times the ½ chance that she will pass on the altered gene. Therefore 1/100 x 2 x 1/2 = 1/100. The correct answer is D. A27. Explanation: The The mother has a 2/3 risk of being a carrier. For the daughter: Prior Conditional Joint Posterior Is carrier 1/3 1/2 1/6 1/5 not carrier 2/3 1 2/3 4/5 correct Answer is B. A28. The correct answer is D. Answer: 2/3 chance of being a carrier and 1/4 chance of having any affected child = 1/6. A29. Explanation: The correct Answer is C. A30. The correct answer is D. – Begin with the prior risk that Elizabeth is a carrier for hemophilia. Her mother is an obligate carrier and Elizabeth’s carrier risk is ½. Then use Bayesian analysis to calculate the risk given Elizabeth has four unaffected sons. Prior Conditional Joint Posterior Carrier 1/2 1/2x1/2x1/2x1/2 1/32 1/17 Non-Carrier 1/2 1 16/32 16/17 So, given that she had four unaffected sons, Elizabeth’s new risk of being a carrier is 1/17. She is pregnant with a male fetus so the chance she passes along the mutant chromosome becomes 1/17 x ½ = 1/34 ~ 1/33 probability. 665 A31. The correct answer is C. – Begin with the prior risk that Leevi is a carrier for CF. Because he is not affected with the disorder, the risk he is a carrier is 2/3. Then use Bayesian analysis to calculate the risk given his negative testing results. Prior Conditional Joint Posterior Carrier 2/3 1/10 2/30 1/6 Non-Carrier 1/3 1 10/30 5/6 Conditional (results of negative screening test) 1/10 (the chance he has a mutation not detected by the panel) 1 (if he is not a carrier, you expect him to test negative) So, given that he tested negative for the CF panel, his new risk of being a carrier for CF is 1/6. A32. The correct answer is A. – Although Huntington's disease can be tested for directly, Rob does not want to know if he has inherited the mutation. By testing the fetus for a linked marker, this can be avoided. The goal of this type of approach is to determine whether the fetus has inherited the copy of Huntington gene that originated in Rob's father. Note that we do not know if this is the expanded or normal copy. In the example given above, the fetus inherits the copy of Huntington gene that originated in Rob's mother, which is of normal size. This puts the chance of Huntington's disease closest to zero for this fetus. A33. Explanation: prior conditional joint posterior 0.5 0.8 0.4 0.4/0.9 0.5 1 0.5 The correct Answer is C. A34. The correct answer is E. – CF is an autosomal recessive disease and since the first child was affected with CF, you know both parents are carriers. The common mutation panel doesn’t identify either parent’s mutation, but you can use the linked marker in a diagnostic manner. The alleles shown are the alleles present at the linked marker. A priori you do not know which allele of the marker is on the same chromosome as the copy of the CF gene with the mutation. You can look at the alleles present in the unaffected children however, to get an idea of the combinations that do not result in CF. Thus by process of elimination, you can deduce that “9,9”, “3,4”, “3,9” are not associated with homozygosity for the mutation. That leaves only “9,4” as the combination that represents both copies of the mutation and the probability that the fetus will have CF is closest to 1. Note that it is the “9” allele from Dad and the “4” allele from Mom. (Mom has a “9,4” combination, but in her family, the mutation is associated with the “4” allele, not the “9” allele). 666 A35. Explanation: Mother: prior conditional joint posterior 4P 1/16 P 1-4Pγ 1 P P 1/2 0.05 0.05/0.95 = 0.0526 0.90 1 0.90 Daughter: prior conditional joint posterior The correct Answer is D. A36. CORRECT ANSWER: C SOURCE OF ITEM TOPIC: Lecture/Syllabus KEYWORDS: odds ratio, multifactorial EXPLANATION: The odds of disease given the allele are 40/10 = 4; the odds of disease given not having the allele = 60/90 = 2/3; the odds ratio is 4 divided by 2/3 = 6 A37. CORRECT ANSWER: C SOURCE OF ITEM TOPIC: Lecture/Syllabus KEYWORDS: population genetics, risk assessment EXPLANATION: The allele frequency in the mother’s population is 1/200, so her risk of being a carrier is 1/100. The allele frequency in the father’s population is 1/100, so his risk of being a carrier is 1/50. Their risk of having an affected child is 1/100 x 1/50 x 1/4 = 1/20,000. Remember to calculate the carrier frequency, not the allele frequency. A38. CORRECT ANSWER: D SOURCE OF ITEM TOPIC: Lecture/Slides KEYWORDS: Mendelian inheritance EXPLANATION: The founding male transmits to all his daughters but not to his son; his daughters transmit to half their offspring. It is possible for autosomal dominant to explain this pedigree, but that choice is not provided. It is not autosomal recessive, as it is transmitted from generation to generation; it is not maternal, as the founding transmitting individual is male; it is not likely X-linked recessive, since many females are affected. Digenic inheritance is unlikely given the many couples who have had affected offspring. 667 B. Biochemical Genetics/Newborn Screening Questions B1-B72 The vignette for the next two questions is the same: B1. New parents bring their 1-week-old baby boy to the pediatrician's office and report that he is not feeding as well over the last 24 hours and that his diapers have an odor like sweaty feet. Which of the following disorders is the most likely cause of this child's problems? A. B. C. D. E. Isovaleric acidemia Maple syrup urine disease. Methylmalonic acidemia. Phenylketonuria Propionic acidemia B2. Which of the following laboratory tests is most likely to provide a definitive diagnosis? A. B. C. D. E. Comprehensive Metabolic Panel Plasma amino acids Plasma ammonium level Urine amino acids Urine organic acids The vignette and response options for the next 2 items are the same. Select one answer for each item in the set. For each description, select the associated glycogen storage disorder (A-D). . Glycogen storage diseases result from several different enzyme deficiences, a few of which are listed below. Match the clinical description below with the appropriate diagnosis from this list. You may use an answer once, more than once, or not at all. A. B. C. D. Type la is glucose-6-phosphatase deficiency or von Gierke. Type II is (alpha-glucosidase (acid maltase) deficiency or Pompe. Type III is debrancher deficiency or Forbes. Type V is muscle phosphorylase deficiency or McArdle B3. A six-month-old boy presents with hepatomegaly, renomegaly, hypoglycemia and lactic acidosis. B4. A six-month-old boy presents with severe hypotonia, massive cardiomegaly, progressive weakness and markedly elevated CPK. 668 B5. A four-day-old infant boy is brought to the emergency room lethargic and no longer taking his formula. A metabolic profile reveals metabolic acidosis and his ammonium level is normal. Which of the following diagnoses is most likely to be found with subsequent metabolic screening studies? A. Citrullinemia B. Maple syrup urine disease C. Methylmalonic acidemia D. Ornithine transcarbamylase deficiency E. Proprionic acidemia B6. A 6 year old girl is referred to genetics following an episode of pancreatitis. During her hospitalization, she received hyperalimentation and became obtunded. An ammonia level during this episode was 200 meq/L. Following discontinuation of the hyperalimentation and treatment of the pancreatitis, the girl was discharged home on a low fat diet. She has not had any further episodes of pancreatitis or lethargy. You suspect she has a specific metabolic disorder. Which of the following laboratory evaluations is the BEST way to obtain a specific diagnosis? A. B. C. D. E. B7. You are seeing a couple whose first child is a Duarte/classical Galactosemia genetic compound heterozygote. His parents (who have not been tested) ask about the risk for their next child to have classical galactosemia (Gal/Gal). Knowing that the frequencies of the Duarte (1/27) and Gal (1/278) alleles, which of the following do you think is their risk to have a Gal/Gal child? A. B. C. D. E. B8. Gene sequencing Plasma amino acids Plasma ammonia level Protein challenge Urine orotic acid 1/278 1/278 x 2/3 1/278 x ½ 1/278 x 2/3 x ½ 1/278 x ½ x ½ You are notified by the state newborn screening laboratory that a seven-day-old neonate, born at 33-weeks gestation and is receiving antibiotics for possible sepsis and total parenteral nutrition (TPN). He was found to have a phenylalanine level (Phe) of 4.8 mg/dL (291 (M). Which of the following interventions is the most appropriate next step in evaluating or managing this child's care? A. B. C. D. E. Stop (TPN) briefly and remeasure Phe on glucose and IV fluids. Modify antibiotic coverage patient is probably receiving Monitor the phenylalanine level weekly over next month. Restrict the infant's phenylalanine intake Obtain urine for assessment of biopterin metabolite levels. 669 B9. A 3 year old boy is referred to genetics clinic for evaluation of hypotonia, seizures, and developmental delay. His older brother, age 8, was also hypotonic and has been diagnosed with autism. You suspect a disorder of creatine metabolism. Which of the following studies is the best option to confirm a diagnosis for this child? A. B. C. D. E. B10. A nine- month-old infant contracts a viral illness and is unable to take her usual amount of formula over the previous 24 hours. She is found dead in her bed the following morning. Which of the following disorders accounts for approximately 5% of cases of sudden infant death syndrome (SIDS) and is the most likely cause of this unfortunate infant’s death? A. B. C. D. E. B11. Glutaric aciduria Type I Glycogen storage disease Type III MCAD deficiency OTC deficiency Propionic academia A couple comes for genetic counseling because the husband’s sister lost a child to Tay-Sachs (TSD) disease. The wife has a negative family history for TSD, is not Jewish, and is currently 6 weeks pregnant. A. B. C. D. E. B12. Measure plasma guanidinoacetoacetate Measure plasma creatine to creatinine ratio Perform a brain MRI Perform plasma amino acids to measure an ornithine level Sequence the SLC6A8 creatine transporter gene Perform molecular TSD analysis on the wife. Perform molecular TSD analysis on both the husband and the wife. Perform no testing since the risk is low for having a child with TSD Perform serum HexA and molecular TSD analysis on the husband. Perform serum HexA testing on the wife A 9-year-old boy is referred by a neurologist for evaluation of a 6-month history of decreasing intellectual performance. An MRI shows prominent white matter changes. Serum biochemistries reveal evidence of mild adrenal dysfunction. Which of the following biochemical pathway components is most likely to be encoded by the gene for this disorder? A. B. C. D. E. Lysosomal enzyme Mitochondrial enzyme involved in fatty acid oxidation Peroxisomal enzyme involved in fatty acid oxidation Peroxisomal membrane protein Transcription factor 670 B13. You are asked to evaluate a one month old asymptomatic girl who had an elevated tyrosine on newborn screening. Succinylacetone testing was subsequently positive. Which of the following interventions is the best initial course of action for this infant? A. B. C. D. E. B14. You are asked to evaluate a newborn baby in the neonatal intensive care unit who is noted to have proximal shortening of the humeri (and to a smaller degree the femurs) along with punctate calcifications in cartilage, coronal clefting of the several vertebrae and congenital cataracts. You suspect a peroxisomal disorder as the likely etiology. Which of the following biochemical abnormalities is most likely to be found when appropriate peroxisomal studies are performed? A. B. C. D. E. B15. Low plasmalogen levels High very long chain fatty acid levels High 7-dehydrocholesterol levels Low phytanic acid levels Low very long chain fatty acid levels Hyperammonemia is a cardinal finding in the newborn period of several metabolic disorders, however late onset hyperammonemia can also occur. Which of the following disorders is most likely to present outside the newborn period with progressive neurologic findings and mild hyperammonemia? A. B. C. D. E. B16. Place the infant on a low phenyalanine diet Place the infant on NTBC and a low tyrosine diet Reassure the family that the infant had transient tyrosinemia of the newborn Refer the infant for evaluation for a liver transplant Repeat tyrosine levels at monthly intervals Arginase deficiency Citrullinemia OTC deficiency Propionic academia Sepsis Pycnodysostosis, a skeletal dysplasia, is caused by mutations in which of the following proteins? A. B. C. D. E. Cartilage-specific parathyroid related protein Cathepsin K, a lysosomal enzyme Collagen Type IX Dystosin, a fibrillar structural protein FGFR4 671 B17. You are asked to evaluate a 10 month old boy with macrocephaly, seizures, and a leukodystrophy pattern on MRI. You suspect Canavan disease. Which of the following analyses is the best screening test to confirm your assumption? A. B. C. D. E. B18. Acute intermittent prophyria, a defect in heme biosynthesis, is characterized by which of the following inheritance patterns? A. B. C. D. E. B19. Non-paternity Pseudodeficiency Unreliability of the assay for carrier testing Decline of enzyme activity with age His being affected with the disease but asymptomatic Specific diagnosis of Glycogen Storage Disease Type IA is available by which of the follwoing sets of laboratory analyses? A. B. C. D. E. B21. A digenic disorder in 2 sequential enzymes in the heme biosynthesis As an autosomal dominant disorder As an autosomal recessive disorder As an X-linked disorder affecting only males Maternally since the enzyme is encoded by the mitochondrial genome A 15 month old boy with hypotonia, optic atrophy, and neurologic regression is found to have metachromatic leukodystrophy with 0.3 nmol/hr/mg protein Arylsulfatase A activity in white blood cells. Activity in the mother’s and father’s white blood cells is 8.6 nmol/hr/mg and 1.2 nmol/hr/mg protein, respectively, with a normal control range of 11-25 nmol/hr/mg protein. The low activity in the father is most likely the result of which of the following genetic issues? A. B. C. D. E. B20. Acyl carnitine profile Lysosomal enzyme screening on white blood cells Plasma amino acids Urine organic acids Very long chain fatty acids Enzyme assay of liver or muscle Enzyme assay of skin fibroblasts or liver Enzyme assay of liver or molecular analysis of the glucose-6-phosphatase gene Molecular analysis of the glucose-6-translocase gene only Western blotting of muscle with specific antibody to the protein A new autosomal recessive disorder of N-linked glycosylation and sterol metabolism has recently been described and involves a defect in an enzyme involved in the synthesis of the dolichol, a polyprenol required for the synthesis and transfer of dolichol-linked monosaccharides to proteins. Which of the following screening tests is most likely to detect this new disorder? A. B. C. D. E. Enzyme assay of the new protein Plasma cholesterol Plasma sterol analysis Serum transferrin isoelectric focusing Urine oligosaccharide chromatography 672 B22. The metabolic defect in Fabry disease is a deficiency of which of the following enzymes? A. Į-galactosidase B. Į-glucosidase C. E-galactosidase D. E-glucosidase E. cerebroside E-galactosidase B23. Which of the following is a major biochemical abnormality in X-linked adrenoleukodystrophy? A. B. C. D. E. B24. Malignancy is a major risk in which of the following metabolic disorders? A. B. C. D. E. B25. Cystinosis Hurler syndrome Maple syrup urine disease Propionic acidemia Tyrosinemia A 35 year woman is referred for evaluation of ptosis, limitation of eye gaze, and a cardiac arrhythmia. You suspect a mitochondrial disorder. Which of the following mutations would be most compatible with her clinical features? A. B. C. D. E. B26. cerebroside E-galactosidase deficiency dicarboxylic aciduria pipecolic acidemia plasmalogen deficiency very long chain fatty acid excess A de novo dominant mutation in a nuclear encoded gene for an OXPHOS subunit A heteroplasmic deletion involving a portion of the mitochondrial genome A homoplasmic missense mutation in a mitochondrial gene for an OXPHOS subunit A heteroplasmic single base change in a mitochondrial rRNA gene A heteroplasmic single base change in a mitochondrial tRNA gene The best diagnostic approach for screening for Zellweger syndrome in an infant with organomegaly and dysmorphic features is which of the following tests? A. B. C. D. E. Chromosome analysis CT scan of the brain Plasma carnitine levels Plasma very long chain fatty acids levels Radiographs of entire infant skeleton 673 B27. Which of the following abnormalities is the most likely diagnosis in a 3-day-old infant who develops lethargy, vomiting, respiratory alkalosis and hyperammonemia with undetectable plasma citrulline and massive orotic aciduria? A. B. C. D. E. B28. An asymptomatic infant who is found to have hyperphenylalaninemia on newborn screening should be tested for which of the following associated problems? A. B. C. D. E. B29. D1-antitrypsin deficiency hepatitis C hepatorenal tyrosinemia tyrosine aminotransferase deficiency acetyl CoA-carboxylase deficiency A 37 year old woman is referred for a history of unexplained stroke. During her evaluation she was found to have proteinuria. Her 42 year old brother has been on renal dialysis for 2 years and is awaiting a renal transplant. Which of the following is the most likley diagnosis in this family? A. B. C. D. E. B31. biopterin synthetic defects catechol-o-methyltransferase liver disease microdeletion deficiency of chromosome 12 porphyria cutanea tarda Which of the following disorders is the most likely diagnosis in an infant with progressive liver failure, elevated serum alpha-fetoprotein and succinylacetone in the urine? A. B. C. D. E. B30. argininosuccinate synthetase deficiency carbamylphosphate synthase I deficiency nonketotic hyperglycinemia ornithine transcarbamylase deficiency propionic acidemia Alport syndrome Fabry disease MELAS syndrome Von Hippel Lindau syndrome X-linked Adrenoleukodystrophy Which of the following abnormalities is shared by the hyperornithinemia-hyperammonemiahomocitrullinuria (HHH) syndrome and gyrate atrophy of the choroid and retina? A. B. C. D. E. hyperammonemia hyperglutaminemia hyperornithinemia orotic aciduria progressive retinal degeneration 674 B32. Intermittent treatment with metronidazole is recommended in patients with propionic academia or methylmalonic acidemia for which of the following therapeutic effects? A. B. C. D. E. B33. Neonatal hypotonia, seizures, apnea and hiccups are features of which of the following inborn errors of metabolism? A. B. C. D. E. B34. Cobalamin complementation studies on the patient’s fibroblasts Measurement of serum B12 level and total homocysteine in the mother Measurement of serum B12 level and total homocysteine in the sister Measurement of transcobalamin II levels in the patient Sequencing the methylmalonyl CoA mutase gene in the patient Very long branch-chain fatty acids undergo E-oxidation in which of the cellular organelles? A. B. C. D. E. B36. citrullinemia galactosemia isovaleric acidemia maple syrup urine disease nonketotic hyperglycinemia A six month old girl is referred for megaloblastic anemia. She is otherwise healthy and has been exclusively breast fed. Metabolic screening reveals mildly elevated methylmalonic acid in her urine and a plasma total homocysteine level of 27 uM (nl 5-8). The mother states that her older daughter, now age 3, who is the patient’s maternal half-sister, had similar anemia as an infant, but is “now fine.” Which of the following assessments is the best course of action in the evaluation of this patient? A. B. C. D. E. B35. gut flora produce substantial amounts of propionate it promotes normal bowel function it provides a source of reducing equivalents it reduces the incidence of sepsis reduction of enteric bacteria synthesis of valine and isoleucine endoplasmic reticulum Golgi apparatus mitochondria nuclear envelope peroxisomes Transferrin isoelectric focusing is useful in diagnosing which of the following disorders?: A. B. C. D. E. Acute Intermittent Porphyria Congenital disorders of glycosylation Hemophilia B Iron-deficiency anemia Marfan syndrome 675 B37. Which of the following mucopolysaccharidoses includes preserved cognitive function? A. B. C. D. E. B38. A seven month old boy is referred for coarse facial features and hepatomegaly. His x-rays demonstrate beaking of the lumbar vertebrae, a J-shaped sella tursica, and ribs that widen anteriorly. Lysosomal enzyme screening from blood reveals elevated levels oIDU\OVXOIDWDVH$DQGȕ-glucuronidase. Whichof the following diagnoses is most likely to explain this infant’s clinical findings? A. B. C. D. E. B39. Androgen receptor deficiency Aromatase deficiency Cholesterol desmolase deficiency 5-Į-Reductase deficiency. 21-Hydroxylase deficiency Thrombosis and strokes are a frequent complication of which one of the following diseases? A. B. C. D. E. B41. Hurler syndrome I-cell disease Metachomatic leukodystrophy MPS VII, Sly disease Pseudo-Hurler polydystrophy Metabolic defects that cause Congenital Adrenal Hyperplasia usually involve which of the following enzyme deficiencies? A. B. C. D. E. B40. Hunter syndrome (MPS II) Hurler syndrome (MPS IH) Maroteaux-Lamy syndrome (MPS VI) Sanfilipo syndrome (MPS III) Sly disease (MPSVII) Congenital Adrenal Hyperplasia Galactosemia Homocystinuria Medium Chain AcylCoA Dehydrogenase Deficiency Phenylketonuria Which of the following NBS acylcarnitine profiles is most suggestive of MCAD deficiency? A. B. C. D. E. Increased C3 and C4 Increased C5-OH Increased C6, C8 and C8/ C10 ratio Increased C14-OH, C16-OH, C18-OH and C18:1-OH Increased C14:1 and C14:1/ C12:1 ratio 676 B42. The MS/MS result of newborn screening sample number 1257 (shown at the bottom) is most consistent with which of the following disorders? d3-Leu 100 % Ala Leu d4-Ala Phe Normal d5-Phe Tyr d6-Tyr d -Met Met 3 0 Phe 100 Elevated Phenylalanine PKU 1257 Sample % 0 140 160 180 200 220 240 260 m/z 280 A. d5 Phe deficiency B. Hyperphenylalaninemia C. Maple Syrup Urine Disease D. Normal result E. Tyrosine deficiency B43. A 20 month old girl is referred for hypotonia, developmental delay and difficult to control seizures. As part of her evaluation you recommend a lumbar puncture to be sent for special biochemical analyses, including neurotransmitter studies. You wish to exclude a disorder where a medical treatment can result in a dramatic improvement in symptoms. Which of the following disorders would meet this criterion and be applicable in this patient? A. B. C. D. E. adenylosuccinate lyase deficiency Cerebral folate deficiency GLUT1 deficiency Menkes disease Nonketotic hyperglycinemia 677 B44. The results of BH4 challenge tests are shown below for individuals A-D who have MHP, Mild PKU, or PKU. Which individuals appear to respond to BH4? B A C D Blau N.PKU & BH4 SPS Verlagsgesellschaft mbH, Heilbronn 2006; p 409. A. B. C. D. E B45. Individuals A, and B but not C and D appear to respond . Individuals A, and D but not B and C appear to respond. Individuals A, B, C and D appear to respond. Individuals A, B and C but not D appear to respond Individuals C, and D but not A and B appear to respond A neonate, Sam, was referred for an elevated Gal-1-P and E Coli sepsis at 8 days of age. Sam is four-weeks-old and he is on Prosobee (lactose free) formula. Which of the following laboratory tests is most likely to yield information that will guide dietary recommendations for Sam? A. B. C. D. E. Detection of urinary carbohydrate by Clinitest Galactose-1-P uridyltransferase activity level N1314D and -119GTCA deletion genotyping Q188R genotyping Total galactose level in his blood 678 B46. Sarah is a five-week-old girl who is brought to clinic by her foster parents. She is on regular formula. Her foster parents just received Sarah’s newborn screening results that show Sarah had phenylalanine levels of 660 and 1220 μmol/L at 3 and 32 days of age, respectively. After obtaining plasma amino acids, which of the following interventions is the best management plan in this situation? A. B. C. D. E. B47. Your state Newborn Screening (NBS) Laboratory sends you a batch report of five abnormal NBS results on neonates discharged from your facility two days ago. Which of the following abnormal NBS results warrants your immediate attention for finding the patient and performing definitive testing? A. B. C. D. E. B48. Biotinidase enzyme level was 1.2 times the normal cutoff C6 and C8 levels were 1.2 times the normal cutoff GALT enzyme level was 1.2 times the normal cutoff Immunoreactive Trypsinogen level 0.8 times the normal cutoff Phenylalanine level was 1.2 times the normal cutoff You are asked to evaluate a 34-week fetal demise with fetal hydrops. Congenital heart defects, arrhthymias, and immune causes have been excluded. Which of the following inborn errors of metabolism is most likely to present this way? A. B. C. D. E. B49. Start Sarah on a phenylalanine-free formula Start Sarah on large neutral amino acids Start Sarah on PEGylated phenylalanine ammonium lyase injections Obtain urine pterins and start Sarah on a phenylalanine-free formula Obtain urine pterins and start Sarah on tetrahydrobiopterin (Sapropterin) Maple syrup urine disease OTC deficiency Sialidase deficiency Very long chain acyl CoA dehydrogenase deficiency Zellweger syndrome You are treating a one year-old with propionic acidemia diagnosed by newborn screening. Despite optimal management, the infant is not growing well, requires G-tube feeds, and has frequent hospitalizations with intercurrent illnesses and mild hyperammonemia. You decide to treat the infant with carglumic acid (N-carbamylglutamate) at the beginning of the next illness to try to prevent the hyperammonemia. Which of the following mechanisms best describes the action of carglumic acid in preventing elevated NH3? A. B. C. D. E. Acting as a synthetic required cofactor for carbamyl phosphate synthetase I Increasing the renal excretion of urea Inhibiting N-acetylglutamate synthase Inhibiting the production of organic acids that inhibit the urea cycle Removal of nitrogen by an alternate pathway 679 B50. You are asked to perform a consult for an internist regarding a 46-year-old man with significant liver disease and early cirrhosis. He has a 10-year history of significant alcohol consumption and darkened complexion in skin-exposed areas. You suspect porphyria cutanea tarda. Which of the following patterns of laboratory porphyrins or porphyrin precursors would be consistent with this diagnosis? A. B. C. D. Elevated red blood cell (RBC) free protoporphyrin and stool protoporphyrin Elevated RBC free and Zinc (Zn) protoporphyrin and stool protoporphyrin Elevated urine aminolevulinic acid (ALA), porphobilinogen (PBG) and uroporphyrin Elevated urine ALA, PBG, and coproporphyrin III and stool coproporphyrin III and protoprphyrin E. Elevated urine uroporphyrin and stool isocoproporphyrin B51. You are asked to consult about a 10-month-old girl with hypotonia and developmental delay. This is the couple’s first child, and there is no known consanguinity in the family. Her plasma lactate is elevated (6 mM/L, normal <2). Her MRI shows basal ganglia lesions and white matter changes. You suspect Leigh syndrome. Which of the following mutations would be most compatible with this diagnosis? A. B. C. D. E. B52. A 2½-year-old toddler has neurologic regression, seizures, and a very low level of serum hexosaminidase A activity. On ophthalmologic exam, which of the following clinical findings would you expect to find? A. B. C. D. E. B53. A de novo autosomal dominant mutation in a nuclear-encoded gene for an OXPHOS subunit A heteroplasmic deletion involving a portion of the mitochondrial genome A heteroplasmic single base change in a mitochondrial tRNA gene A homoplasmic missense mutation in a mitochondrial gene for an OXPHOS subunit Compound heterozygous mutations in a nuclear encoded gene for an OXPHOS subunit Cherry red spot in the macula Corneal clouding on slit lamp examination Optic atrophy consistent with white matter disease Raised intraocular pressure Retinitis pigmentosa at the periphery of the retina You are asked to consult about a 2-month-old girl with hypotonia, seizures, and an elevated plasma lactate (8 mM/L, normal <2). Brain MRI shows a thin corpus callosum but no other abnormalities. You suspect pyruvate dehydrogenase deficiency. Which of the following is the most likely mode of inheritance in this infant? A. B. C. D. E. Autosomal dominant Autosomal recessive Mitochondrial Sporadic X-linked 680 B54. A 6-month-old infant presents with FTT, enlarged liver, hypotonia, and developmental delay. You diagnose Carbohydrate Deficient Glycoprotein syndrome Type Ib based on transferrin glycosylation and gene sequencing. Which of the following interventions is the most appropriate treatment for this disorder? A. B. C. D. E. B55. You are asked to evaluate a 2-year-old boy with coarse facies, developmental delay, and hepatomegaly. You note that the corneas are clear; there is mixed sensorineural and conductive hearing loss; and dysostosis multiplex on skeletal survey. Which of the following mucopolysaccharidoses (MPS) is the most likely diagnosis? A. B. C. D. E. B56. Development of sensorineural or conductive hearing loss Lack of development of antibodies in treated patients Loss of visual acuity and retinal detachment Normal gross motor skills after 12 months of therapy Progressive cardiac enlargement A couple is 9 weeks pregnant. A previous child died shortly after birth with a severe presentation of Smith-Lemli-Opitz disease. Which of the following clinical/laboratory findings is most likely to be found with an affected fetus in the current pregnancy? A. B. C. D. E. B58. Hunter syndrome (MPS II) Hurler syndrome (MPS I) Maroteaux-Lamy syndrome (MPS VI) Morquio syndrome (MPS Type IVA) Sanfilippo syndrome (MPS III) Early enzyme replacement therapy has dramatically altered the prognosis for infantile Pompe disease. Which of the following represents an unexpected clinical finding in long-term treated survivors? A. B. C. D. E. B57. Enzyme replacement therapy Oral mannose supplementation Supportive care only Bone marrow transplant Low carbohydrate elemental formula Abnormal microarray with small deletion on chromosome 11q Abnormal nuchal translucency at 10 weeks on prenatal ultrasound High maternal serum alpha-fetoprotein level Low maternal serum estriol level Polyhydramnios on prenatal ultrasound at 24 weeks You evaluate a 6-day-old term girl in the emergency room with an unremarkable prenatal and family history. She was discharged home at 2 days of age on regular infant formula. For 24 hours she has been eating poorly (<1 ounce per feed) and seems “sleepy.” She has vomited once. Her CBC is normal, her electrolytes show a metabolic acidosis and the ammonia level is 500 mg/dl. Which of the following metabolic laboratory results are you the most likely to find? A. B. C. D. E. Elevated plasma glycine with 3-OH-propionate, methylcitrate present on urine organics acids Low citrulline on plasma amino acids along with high orotic acid in urine Markedly elevated citrulline on plasma amino acids and normal urine organic acids Markedly elevated plasma glycine with normal urine organic acids Normal plasma amino acids and large suberylglycine peak on urine organic acids 681 B59. An 8-year-old boy and his family recently moved to the United States from Russia. His parents report their son has a genetic disorder and needs a special diet. Newborn screening was never performed. On physical examination you note macrocephaly and choreoathetotic movements. Height and weight are 25-30th centile for age. The parents note he has some mild cognitive deficits, but attended regular school in Russia. Which of the following metabolic disorders is the most likely diagnosis? A. B. C. D. E. B60. A couple, who had a previous child with classic Hurler syndrome who died of disease complications, comes to you for pre-conception counseling. Each parent has been molecularly confirmed to carry a known pathogenic mutation. Which of the following interventions will likely provide the best outcome for a future affected pregnancy? A. B. C. D. E. B61. Canavan disease Glutaric academia Type I Isovaleric acidemia Medium chain acyl-CoA dehydrogenase deficiency Phenylketonuria HLA matched (66%; 4/6) unrelated donor bone marrow transplant in the early postnatal period HLA matched liver transplant when the infant reaches a weight of 10 kilograms Institution of chaperone therapy as early as possible in the postnatal period Institution of enzyme replacement therapy at the first sign of neurologic regression Unrelated donor, HLA matched (100%; 6/6) cord blood transplant in the early postnatal period A 15-year-old healthy girl is running long distances as a member of her high school track team. The morning after a 10-mile run on a hot day, she notices a red color to her urine. She is referred to you to exclude an inborn error of metabolism. Her creatine kinase is markedly elevated at 15,000 U/L and urine myoglobin is positive. Which of the following disorders of fatty acid oxidation is most compatible with this presentation? A. B. C. D. E. Carnitine palmitoyltransferase I (CPT I) deficiency Carnitine palmitoyltransferase II (CPT II) deficiency Long chain 3-OH Acyl-CoA dehydrogenase deficiency Medium chain acyl-CoA dehydrogenase deficiency Short chain acyl-CoA dehydrogenase deficiency NEWBORN SCREENING B62. You are seeing a neonate, Katie, who has a positive Newborn Screening (NBS) test result. The NBS test has a sensitivity of 0.95, a specificity of .90 and a positive predictive value of 0.10. What is the probability that Katie actually has the disorder? A. B. C. D. E. B63. 95% 90% 10% 9.5% 5% A Newborn Screening (NBS) test of 1000 neonates yielded 100 who tested positive. Subsequent diagnostic testing showed that only 10 of the 100 who tested positive and none of those who tested negative on NBS actually had the disease. Which of the following is the best estimate of the sensitivity of the NBS test? A. B. C. D. E. 90/990 10/100 100/990 900/990 10/10 682 B64. A neonate, Billy, failed his newborn hearing screening test and was also found to have a 17 hydroxyprogesterone (17OHP) level that is 1.5 times the upper range of normal. Which of the following disorders do you think Billy is most likely to have? A. B. C. D. E. B65 ACTH Deficiency Congenital Adrenal Hyperplasia Pendred Syndrome Primary Congenital Hypothyroidism Secondary Congenital Hypothryroidism A 6-day-old neonate, Sammy, presents with acidosis, ketonuria, hyperammonemia, neutropenia and thrombocytopenia. Sammy’s Newborn Screening (NBS) test shows an isolated elevation of C5. Which of the following disorders is Sammy most likely to have? A. B. C. D. E. Biotinidase Deficiency Holocarboxylase Deficiency Isovaleric Acidemia Methylmalonic Acidemia Propionic Acidemia Source: Newborn Screening Slides 42-43 and 49 Keywords: acidosis, ketonuria, hyperammonemia, neutropenia, thrombocytopenia, C5 Explanation: Isovaleric Acidemia presents with acidosis, ketonuria, hyperammonemia, neutropenia, thrombocytopenia, an elevated C5 and the odor of sweaty feet. Biotinidase Deficiency presents with alopecia, rash and elevated C5-OH. Holocarboxylase Deficiency presents with elevated C5-OH. Methylmalonic Acidemia and Propionic Acidemia present with elevated C3. B66. A newborn infant is diagnosed with Medium Chain AcylCoA Dehydrogenase Deficiency (MCADD) based on newborn screening (NBS) results. While awaiting the molecular analysis to confirm the NBS result, which of the following complications should you warn the parents about during their initial visit to your medical genetics clinic? A. B. C. D. E. B67. Hyperammonemia Hypoglycemia Ketosis Neutropenia Thrombocytopenia You are adjusting the cutoffs for a Newborn Screening (NBS) test for a metabolic condition. Your adjustments increase the number of true positive results and reduce the number of false positive results. Both of these changes will increase which of the following NBS test parameters? A. B. C. D. E. False negative rate False positive rate Positive predictive value Sensitivity of the test Specificity of the test 683 B68. As you evaluate a 4-day-old boy with a blood sugar of 37, an anion gap of 21, 4+ urinary ketones and an ammonia level of 197, the State Newborn Screening (NBS) Lab contacts you to report an emergency NBS result. The emergency result is most likely to reveal an increase in which of the following levels? A. B. C. D. E. B69. You are seeing a 4-day-old girl with abnormal Newborn Screening (NBS) result. She has a 2-day history of emesis and diarrhea. On physical examination she is quite icteric with hepatomegaly. Which of the following abnormalities is most likely to be found on the NBS report? A. B. C. D. E. B70. Cystic Fibrosis Galactosemia Homocystinuria Pendred Syndrome Phenylketonuria You evaluate a 6-day-old girl whose phenylalanine (Phe) level was increased on her Newborn Screening (NBS) test obtained at 36 hrs of age. Which of the following factors decreases the positive predictive value of her results? A. B. C. D. E. B72. C0 acylcarnitine level is decreased C5DC level is increased Citrulline level is decreased GALT level is decreased Phenylalanine level is increased You evaluate a 3-day-old boy with an abnormal Newborn Screening (NBS) result. He has a 2-day history of poor feeding, emesis and diarrhea. On physical examination he is somewhat icteric and has hepatomegaly. Which of the following disorders is most likely to be implicated by the abnormal NBS result? A. B. C. D. E. B71. Biotinidase enzyme activity C3 acylcarnitine C6, C8 & C8/C10 GALT enzyme activity Succinylacetone Early rise of Phe level Fasting before heel stick Increased Phe/Tyr ratio Rapid rise of Phe level Total Parenteral Nutrition A new screening (NBS) test was evaluated on stored NBS blood spots from 200 individuals confirmed to have Pompe Disease and 200 normal controls. Of these 400 samples 160 tested positive and 240 tested negative. Which of the following is the maximum sensitivity of the new screening test for Pompe Disease? A. 160/400 or 0.40 B. 240/400 or 0.60 C. 160/200 or 0.80 D. 40/200 or 0.20 E. 160/240 or 0.67 684 Answers to Biochemical Genetics/Newborn Screening B1-B72 B1. The odor of sweaty feet is classically associated with isovaleric acidemia. The odor of untreated PKU is sometimes described as having a mousy odor. The odor of untreated maple syrup urine disease is as suggested by the name of the disease. Odor is not a prominent feature for propionic or methymalonic. The correct answer is A. B2. Urine organic acids will provide a characteric profile that allows for clinical diagnosis. The correct answer is E B3. Patient A has the typical phenotype of type la GSD. The correct answer is A B4 Patient B has a typical phenotype of Type II GSD. The correct answer is B B5. High ammonia would be a feature of all of these disorders except maple syrup urine disease. The correct answer is B. B6. The clinical vignette is suspicious for heterozygous OTC deficiency. Female carriers can become symptomatic when ill or if they receive a high protein load (such as hyperalimentation). In the past, a protein challenge or allopurinol loading test followed by measurement of urine orotic acid was often used to make a diagnosis. Today gene sequencing is the best method. Urine orotic acid alone or ammonia level will not be abnormal unless the patient is in the midst of an acute crisis. The correct answer is A B7. One parent must be a Gal carrier and the other a Duarte carrier. There is a possibility that the parent who is a Duarte carrier is also a Gal carrier. Such individuals have about 25% of normal gal activity, which would not produce clinical signs of galactosemia. The risk that the other allele in this parent is Gal is 1/278. In this case the risk to a child would be ¼. Therefore the overall risk is 1/278 x ¼. The correct answer is E. B8. There are two reasons why this infant may have an elevated phe on newborn screening – immaturity of the HPPD enzyme and TPN. The best way to exclude an inherited disorder in phe metabolism would be to stop the TPN briefly (~4-6 hr) and provide calories with glucose and recheck the phe off the TPN. The correct answer is A B9. The history is fairly classic for X-linked creatine transporter deficiency which can account for up to ~1% of X-linked MR. Other features include hypotonia, seizures, and autism. Screening can be performed by urine guanidinoacetoacetate (GAA) which is elevated or by finding a low creatine peak on brain MRI with spectroscopy. It is not specific on plain MRI. While the disorder is technically a defect in ornithine metabolism, ornithine levels are normal. Plasma creatine/creatinine ratio is used to detect the 2 other disorders of creatine metabolism (AGAT and GAMT deficiency). The correct answer is E 685 B10. MCAD presents with hypoketotic hypoglycemia and can resemble Reye syndrome or SIDs. Although initially it was thought that a higher percentage of cases of SIDS were likely due to defects in fatty acid oxidation, prospective and retrospective studies now demonstrate that this accounts for ~5% of SIDs. While OTC and propionic acidemia patients can occasionally die unexpectedly, there are usually some warning signs – vomiting, lethargy, irritability. GA I presents with MR, a movement disorder, and macrocephaly. GA II could be a rare cause of sudden death. The correct answer is C. B11. Depending on how rapidly one could obtain results, the BEST answer might be serum HexA on husband (+/- molecular) and WBC hexA on the wife. The major points of this question are (1) serum hexA is not accurate during pregnancy and one needs to do WBC hexA determinations and (2) molecular testing is not helpful in the non-Ashkenazi Jewish population since most mutations will not be detected. The correct answer is D. B12. The clinical features here are strongly suggestive of X-linked adrenoleukodystrophy. Although biochemically this disorder is characterized by accumulation of VLCFA, the gene defect is NOT an enzyme involved in fatty acid metabolism. Rather, it is a peroxisomal membrane protein which may be involved in transport of the enzyme. The correct answer is D. B13. The elevated tyr + succinylacetone is diagnostic for Type I tyrosinemia. Current standard treatment is NTBC + low tyr diet (tyrex formula). The advent of newborn screening and NTBC has resulted in a markedly reduced need for liver transplantation. The correct answer is B B14. Rhizomelic chondrodysplasia patients have normal VLCFA and normal numbers of peroxisomes. Most patients have elevated phytanic acid levels from impaired phytanic acid oxidase acitivity. The correct answer is A. B15. Arginase deficiency does not present with crises in the newborn period. It presents typically with spastic paraplegia and MR although later episodes of mild hyperammonemia can occur. The correct answer is A B16. Pycnodysostosis is actually a lysosomal storage disease caused by Cathepsin K deficiency. The correct answer is B. B17. While gene sequencing would be a definitive test, screening can be performed by urine organic acids which will detect elevated N-acetylaspartic acid (NAA). An elevated NAA peak could also be seen on MRI spectroscopy. The correct answer is D. B18. AIP is an autosomal dominant enzyme deficiency. Most patients are asymptomatic. The correct answer is B B19. Pseudodeficiency, with 5-15% of normal enzyme activity, is found in ~2% of European Caucasian alleles. It results from 2 single base “polymorphisms” in the Arylsulfatase A gene and can complicate carrier testing and prenatal diagnosis. For families where this is found, molecular testing or use of sulfatide loading as a natural substrate is the preferred assay. The slightly low level of activity in the mother would be typical for an obligate carrier. The correct answer is B. 686 B20. The glucose-6-phosphatase enzyme is expressed only in liver. Prior to isolation of the gene, prenatal diagnosis was not possible. Translocase deficiency causes GSD Ib. The correct answer is C. B21. Since this involves a defect in N-linked glycosylation, serum transferrin isoelectric focusing should produce an abnormal pattern. This would be the most efficient screening test. Cholesterol and sterol patterns may be normal; insufficient information is provided concerning their utility. Urine oligosaccharide chromatography is used for lysosomal storage disorders such as mannosidosis and fucosidosis. The correct answer is D. B22. Expect some simple questions about basic metabolic defects in the general exam. The defect in Fabry disease was referred to as Į-galactosidase A in the past. The correct answer is A. B23. Pipecolic acidemia and plasmalogen deficiency can occur in various peroxisomal disorders. Cerebrosidase E-galactosidase deficiency is the defect in Krabbe disease. Dicarboxylic aciduria is characteristic of some of the defects of fatty acid oxidation. The hallmark metabolic abnormality in adrenoleukodystrophy is accumulation of very long chain fatty acids, although this abnormality also occurs in other peroxisomal disorders. The correct answer is E. B24. Some autosomal recessive disorders with substantial risk of malignancy are glycogen storage disease type I, tyrosinemia, hemochromatosis, and others less prominently. Immunodeficiency disorders and DNA repair disorders also relevant. The correct answer is E. B25. The features are classic for Kearn-Sayre syndrome which involves heteroplasmic deletions or duplications of portions of the mitochondrial genome. The correct answer is B. B26. Chromosome analysis and plasma carnitine levels would not be helpful in any specific way to make the diagnosis of Zellweger syndrome. Skeletal X-rays might detect stippling and CT scan of the brain might show abnormalities of gyral formation. However, plasma very long chain fatty acid analysis would be by far the best choice In this test and would be abnormal in the vast majority of cases. The correct answer is D. B27. The clinical features are classic for a urea cycle disorder. Levels of citrulline on amino acids are key to deciding which disorder is present. Undetectable citrulline is found in males with OTC and CPS deficiencies. In OTC, carbamyl phosphate is shunted to pyrimidine synthesis resulting in high orotic acid. In CPS deficiency, no carbamyl phosphate is made and so there is no elevation in orotic acid. (see pathway) The correct answer is D. B28. Tetrahydrobiopterin is a cofactor for phenylalanine hydroxylase (along with oxygen) that converts phe to tyr ~2% of infants with inherited hnyperphenylalaninemia have a defect in the synthesis or recycling of the biopterin cofactor. All infants with confirmed hyperphe should be tested for a biopterin defect by blood and urine pterin screening. The correct answer is A. 687 B29. Hepatorenal tyrosinemia is also called tyrosinemia type I and results from a defect in last step of tyrosine degradation fumarylacetoacetate hydrolase (FAH). The correct answer is C. B30. In the past several years, it has been recognized that most female Fabry carriers develop symptoms. Proteinuria develops in late childhood although only ~10% develop frank renal failure. Most affected males (disorder is X-linked) develop renal failure if untreated, with onset of symptoms around 30 years of age. Fabry accounts for ~5% cryptogenic strokes and can be seen. Alport is often X-linked and has renal disease (nephritis) and hearing loss, but not strokes. MELAS has strokes but not this pattern of renal disease. In Von Hippel Lindau, vascular malformatons are present in multiple tissues and there can also be renal cysts. The correct answer is B. B31. Gyrate atrophy of the retina is caused by deficiency of ornithine aminotransferase (OAT). It is diagnosed by finding elevated ornithine on plasma amino acids. The correct answer is C. B32. Metronidazole is an antibiotic used against anaerobic bacteria and protozoa. Some gut flora can generate propionate (which can be converted to MMA). Thus, some doctors advocate using medtronidazole to reduce gut flora and propionate production in PA or MMA. The correct answer is A. B33. These are classic findings in nonketotic hyperglycinemia, particularly the seizures and hiccups, which may occur prenatally. The correct answer is E. B34. This is likely a transient B12 deficiency in the patient and her sister secondary to B12 deficiency in the mother. The fact that the sisters have different fathers makes a defect in cobalamin metabolism itself in the patient or her sister much less likely. While transcobalamin II deficiency can produce megaloblastic anemia in infants (often with FTT and other sx), this is very rare and is also an autosomal recessive disorder. The reason for B12 deficiency in the mother is often undiagnosed pernicious anemia, but it can also occur in strict vegans. The correct answer is B. B35. The correct answer is E. B36. The correct answer is B. B37. Intellectual function is usually normal in MPSVI. It is also preserved in MPS IVA (Morquio) and the Scheie variant of Hurler (MPS IS). The correct answer is C. B38. The features are suggestive of a mucopolysaccharidosis or storage disorder with significant somatic involvement. Elevation of multiple lysosomal enzymes occurs in I-cell disease (ML II) and pseudoHurler polydystrophy (MLIII) because the enzymes are not targeted to the lysosome properly and are, instead, secreted. The two disorders are allelic with I-cell being more severe and presenting earlier. The defect is in a sugar phosphotransferase found in the Golgi that properly targets enzymes to the lysosome. The correct answer is B. 688 B39. 21-Hydroxylase deficiency causes ~95% of cases of CAH. Aromatase deficiency causes inability to synthesize estrogen and affected females have ambiguous genetalia and primary amenorrhea. 5-Į-Reductase deficiency converts testosterone into the more potent dihydrotestosterone. Affected males can have pseudovaginal perineoscrotal hypospadias Androgen receptor deficiency causes androgen insensitivity and feminization of affected males. The correct answer is E. B40. The correct answer is C. Thromboembolic events cause the death of 50% of individuals affected by Homocystinuria by 20 yrs. B41. The correct answer is C. Increased C6, C8 and C8/ C10 ratio is the profile for MCAD and these are derived from medium chain fatty acids. Increased C14-OH, C16-OH, C18-OH and C18:1-OH is the profile for LCHAD Deficiency. Increased C14:1 and C14:1/ C12:1 ratio is the profile for VLCAD deficiency. Increased C3 and C4 These are seen in Propionic academia and SCAD deficiency, respectively. B42. The peak for phe is markedly elevated. The correct answer is B B43. Cerebral folate deficiency is an autosomal recessive disorder due to mutations in a folate receptor, FOLR1. It results from brain-specific folate deficiency, and, if recognized, can be treated with high dose oral folinic acid. This often leads to a dramatic decrease in seizures and marked improvement in tone and development. There is no good treatment for the other disorders although some therapies are available (Menkes, NKH). Menkes is also X-linked, and these symptoms would not be expected in a female carrier. The correct answer is B. B44. Both A and B show a decrease in phe levels following administraton of BH4. The other two graphs do not (C,D). The correct answer is A B45. Sam’s GPUT (GALT) enzyme level should clarify if he has classical galactosemia which is associated with both an elevated Gal-1-P and E Coli sepsis. A. and E.— Since Sam may have been on a lactose free formula for several weeks his blood galactose and urinary carbohydrate level by Clinitest are likely to be normal and will not clarify if he has classical galactosemia. C. and D.— Determining if he has Duarte (N1314D and -119GTCA deletion) or Gal (Q188R) genotypes alone would only clarify dietary recommentations for Sam if he should he be found to be homozygous for the Q188R mutation. The correct answer is B. B46. Sarah’s Phenylalanine level of 1220 μmol/L strongly suggests that she has Phenylketonuria (PKU). You should obtain a confirmatory test (plasma amino acid levels of Phe & Tyr), exclude BH4 disorders (urine pterins) and start a Phe free formula while waiting for the test results. A. Starting a Phenylalanine free formula doesn’t exclude BH4 disorders. B. or C. Similarly starting her on large neutral amino acids or PEGylated Phenylalanine Ammonium Lyase rather than a Phe free formula are also not standard care. E. Starting her on BH4 (Sapropterin) at 20 mgm/kg/day rather than a Phe free formula is not standard care. The correct answer is D. 689 B47. C6 and C8 levels at 1.2 times the normal cutoffs suggests that the baby may have MCAD Deficiency which can cause lethal hypoketotic hypoglycemia following fasting, gastroenteritis or poor feeding. This baby should be evaluated as soon as possible, the NBS repeated, the parents be given instructions for what to do if the baby has poor feeding or lethargy, and diagnostic tests for MCAD considered. A. Increased biotinidase enzyme levels do not indicate biotinidase deficiency. C. Increased GALT enzyme levels do not indicate galactosemia. D. Decreased Immunoreactive Trypsinogen levels do not indicate cystic fibrosis. E. A mildly increased Phe level should be repeated but PKU is not a life-threatening disorder like MCAD. The correct answer is B. B48. The correct answer is C. Keywords: fetal hydrops, inborn error of metabolism Several lysosomal storage disorders have severe forms with prenatal onset, including sialidase deficiency. B49. The correct answer is A. Keywords: propionic acidemia, carglumic acid Carglumic acid is a synthetic analog of N-acetylglutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI). In organic acidemias, NAG synthase or CPSI reactions are compromised, probably by the organic intermediates that build up during acute illnesses. While designed to treat NAG synthase deficiency, carglumic acid has been found to be effective in reducing hyperammonemia in organic acid disorders, such as PA, MMA, IVA, by increasing CPSI activity acting as a cofactor. B50. The correct answer is E. Keywords: cirrhosis, porphyria cutanea tarda, porphyrins Different porphyrias have different sites and patterns of porphyrin metabolite and/or product accumulation. All of the erythropoietic cutaneous porphyrias have some accumulation in RBCs. The only hepatic porphyria with RBC accumulation is ADP (ALA-dehydratase-deficient porphyria) which is extremely rare. Other hepatic porphyrias accumulate metabolites in urine or urine + stool. AIP, the most common has early metabolites (ALA, PBG) in urine only (answer C). PCT is occasionally an inherited or, more often, acquired deficiency of URO-decarboxylase and accumulation of uroporphyrin is found in urine. The presence of increased stool isocoproporphyrin is diagnostic. B51. The correct answer is E. Keywords: lactate, Leigh syndrome Most mitochondrial disease in infants affect nuclear genes. Leigh syndrome has multiple etiologies including nuclear genes in complexes IV or I and nuclear SURF1 mutations that encode a protein involved in the assembly of Complex IV. These variants of Leigh syndrome are typically inherited in an autosomal recessive fashion. B52. The correct answer is A. Keywords: hexosaminidase A, cherry red spot Answer: A. Hex A is missing in Tay-Sachs disease. The classic eye finding is a cherry red spot which is present by the time neurologic symptoms begin. The macula is normal and the cherry red spot is due to lipid accumulation in the surrounding neural retina. 690 B53. The correct answer is E. Keywords: Leigh syndrome, lactate X-OLQNHG0XWDWLRQVLQWKH(Įsubunit are the most common. This gene is on the X chromosome and severe cases are usually lethal in hemizygous males. B54. The correct answer is B. CDG Type Ib is the only type currently known with a specific treatment. The defect is mannose-6-phosphate isomerase. B55. The correct answer is A. Keywords: Hunter syndrome, coarse facies, hepatomegaly MPSII is X-linked and found mostly in males. Features include coarse facies, hepatomegaly and dysostosis multiplex like Hurler syndrome, but there is hearing loss and usually no corneal clouding (may occasionally see by slit lamp but not clinically significant.) B56. The correct answer is A. Keywords: Pompe disease, hearing loss Many patients have hearing loss which was not appreciated previously because of their early death. Osteopenia is also found. Cardiac disease usually improves quickly. Motor skills improve but residual weakness is typical. >90% of patients develop IgG antibodies but this does not cause inhibitory activity against the recombinant enzyme. B57. The correct answer is D. Keywords: Smith-Lemli-Opitz, prenatal Cholesterol is a precursor for the synthesis of steroid hormones and many fetuses affected with SLOS are associated with low maternal serum estriol. B58. The correct answer is A. This should be an organic acid disorder because of acidosis, type and timing of presentation, and high ammonia. Option A is consistent with propionic academia. Option B would be consistent with OTC and one would not expect heterozygous females to present in neonatal period. Option C would be typical of urea cycle and expect normal pH or mild alkalosis early in course. Option D would be consistent with nonketotic hyperglycinemia which has early seizures and normal routine labs including chemistries and ammonia. Option E is diagnostic for MCAD, which almost never presents in first week of life. B59. The correct answer is B. The macrocephaly and movement disorder with relatively intact development is typical of treated GAI. 691 B60. The correct answer is E. Early matched umbilical cord blood or matched bone marrow transplant has best outcome. B61. The correct answer is B. Late onset CPTII deficiency typically presents as intermittent rhabomyolysis with strenuous exercise. Option A - CPTI presents with liver disease. Option C - LCHAD has liver, cardiac and acute muscle symptoms, usually presenting earlier. Option D - MCAD does not have muscle involvement. Option E - SCAD is usually not disease-associated and if pathologic does not have skeletal muscle involvement. B62. NEWBORN SCREENING The correct answer is C. Source: Newborn Screening Slides 7-10 Key Words: Sensitivity, specificity, positive predictive value Explanation: Positive Predictive Value (PPV) is the fraction of those who have a + screen results who are affected = True Positive/ (True Positive +False Positive). If the PPV= 0.10, then 10% of those with a + screening result will be affected. B63. The correct answer is E. Source: Newborn Screening Slides 7-8 Key Words: Screening, sensitivity Explanation: Sensitivity is the fraction of affecteds who screen positive = True Positive/ (True Positive +False Negative). In this case there were 10 TPs (affecteds who screen +) and none of those who were affected screened negative. Since none of the remaining 90 who screened + were affected, there were no FNs. Thus sensitivity = 10/ (10+0) = 10/10. B64. The correct answer is B. Source: Newborn Screening Slides 52 and 61-63 Keywords: newborn hearing screening, 17OHP Explanation: Of the choices only Congenital Adrenal Hyperplasia (CAH) causes an elevation in 17OHP. CAH is potentially life threatening and an elevated 17OHP on NBS is an emergency. ACTH is elevated rather than low in CAH and low ACTH doesn’t cause an elevated 17OHP. Pended Syndrome does cause deafness in newborns but does not cause either an elevated 17OHP or congenital hypothyroidism. It does cause euthyroid goiter in adolescence. B65. The correct answer is C. Source: Newborn Screening Slides 42-43 and 49 Keywords: acidosis, ketonuria, hyperammonemia, neutropenia, thrombocytopenia, C5 Explanation: Isovaleric Acidemia presents with acidosis, ketonuria, hyperammonemia, neutropenia, thrombocytopenia, an elevated C5 and the odor of sweaty feet. Biotinidase Deficiency presents with alopecia, rash and elevated C5-OH. Holocarboxylase Deficiency presents with elevated C5-OH. Methylmalonic Acidemia and Propionic Acidemia present with elevated C3. 692 B66. The correct answer is B. Source: Newborn Screening Slides 39-41 Keywords: MCADD, NBS Explanation: Children with MCADD who are fasted or have poor po intake are prone to develop hypoketotic hypoglycemia. Before NBS for MCADD the first crisis was fatal in 25% of affected children. The other answers are not typical for MCADD. B67. The correct answer is C. Positive predictive value is the fraction with a positive screen that are affected. Since the PPV = TP/ (TP+FP) both increasing the TP and reducing the FP will increase the PPV. A. False negative rate (FNR) is the fraction of affecteds that test negative. Since the FNR = FN/ (FN+TP) increasing TP will decrease the FNR. B. False positive rate (FPR) is the fraction of unaffected that test positive. Since the FPR = FP/ (FP+TN) decreasing the FP will decrease the FPR. D. Sensitivity of the test is the fraction of affecteds who screen positive. Since Sensitivity = TP/ (TP+FN) increasing TP can increase the sensitivity but reducing FP has no additional effect as it does on the PPV. E. Specificity of the test is the fraction of unaffected who screen negative. Specificity = TN/ (TN+FP) reducing the FP can increase the sensitivity but increasing the TP has no additional effect as it does on the PPV. B68. The correct answer is B. This neonate has hypoglycemia, ketoacidosis and hyperammonemia. These are all consistent with an organic acidemia. C3 acylcarnitine is elevated in methylmalonic acidemia and propionic acidemia. A. Deficiencies rather than increases in Biotinidase enzyme activity cause disease. C. Increases in these analytes are seen in MCAD deficiency which is usually not associated with ketoacidosis. D. Deficiencies rather than increases in GALT enzyme activity cause galactosemia. E. Increases in Succinylacetone are seen in tyrosinemia type 1 which usually doesn’t present this early. B69. The correct answer is D. This neonate has emesis, diarrhea, icterus and hepatomegaly. The early onset and all of these signs and symptoms are seen in galactosemia which is caused by GALT deficiency. A. C0 acylcarnitine level is decreased in carnitine transporter deficiency that can have hepatomegaly due to cardiac failure but usually not emesis and diarrhea. B. C5DC level is increased in GA1 which presents with macrocephaly and hypotonia. C. Citrulline level is decreased in OTC which is X linked and doesn’t present with above. E. Phenylalanine level is increased in PKU which doesn’t present with above. B70. The correct answer is B. This neonate has poor feeding, emesis, diarrhea, icterus and hepatomegaly. The early onset and all of these signs and symptoms are seen in galactosemia. A. Cystic Fibrosis can present with diarrhea but not usually with poor feeding, emesis or hepatomegaly. C. Homocystinuria is usually asymptomatic in neonates. D. Pendred Syndrome can present with hearing loss but not usually with poor feeding, emesis or hepatomegaly. E. Phenylketonuria is usually asymptomatic in neonates. B71 The correct answer is E. TPN can cause Phe elevations which represent false positives that decrease the PPV=TP/ (TP+FP). A. Early rise of Phe level is seen in PKU and it increases TP and the PPV. B. Fasting before heel stick decreases FP and increases the PPV. C. Increased Phe/Tyr ratio increases TP and the PPV. D. Rapid rise of Phe level is seen in PKU and it increases TP and the PPV. 693 B72. The correct answer is C. Source: Lecutre/Slides Keywords: Sensitivity, True positive, False negative Explanation: Sensitivity is the ratio of True positive/True positive + False negative results. There were 200 confirmed cases and 200 normal control samples. If all 160 positives were from the 200 confirmed cases then the maximum sensivity was 160/200 or 0.80. A. 160/400 or 0.40 is the ratio of Positives/True positives + False negatives + True Negatives B. 240/400 or 0.60 is the ratio of False negatives + True negatives / True positives +False negatives + True Negatives C. 160/200 or 0.80 is the maximum ratio of True positives / True positives + False negatives D. 40/200 or 0.20 is the ratio False negatives / True positives + False negatives E. 160/240 or 0.67 is the ratio of Positives / Negatives 694 C. Clinical Genetics Questions C1-C98 C1. A concerned mother brings her 18-month-old daughter to the pediatrician's office because she has been intermittently constipated. Her mother became alarmed while changing her diaper after straining to have a stool, she found some red, tissue bulging from the rectum. Which of the following procedures or laboratory studies is most like to identify the appropriate diagnosis? A. B. C. D. E. C2. Guanine nucleotide binding proteins play regulatory role in many signal transduction pathways within the cell. Abnormalities of guanine nucleotide binding protein have been associated with several clinical disorders. Which of the following disorders results from inactivating mutations of the guanine nucleotide binding protein Gs? A. B. C. D. E. C3. Albright hereditary osteodystrophy insulin unresponsiveness McCune-Albright syndrome nephrogenic diabetes insipidus type II diabetes mellitus An infant dies at ten days of age, and autopsy reveals clinical features that include: agyria, cerebellar hypoplasia, Dandy-Walker cyst, microphthalmia, and retinal detachment with retinal dysplasia. Which of the following syndromes if the most likely diagnosis? A. B. C. D. E. C4. Colonoscopy for Crohn disease. Molecular analysis for Duchenne muscular dystrophy Rectal biopsy Hirschsprung disease Renal ultrasound for Beckwith-Wiedemann syndrome Sweat chloride test for cystic fibrosis Meckel-Gruber syndrome Miller-Dieker syndrome Neu-Laxova syndrome Pallister-Hall syndrome Warburg syndrome Prader-Willi is a genetic syndrome characterized by failure to thrive during the first year of life, developmental delay in most patients, small hands and feet (more noticeable in late childhood), crytorchidism in males, a facial gestalt that is recognizable in many patients and foodseeking behaviour after age two. Even so clinical diagnosis can be difficult because of the variability in the phenotype and laboratory studies have become the mainstay of diagnosis. Which of the following laboratory analyses is most likely to reveal a positive finding in a young child where the constellation of clinical features above is not readily identifiable? A. B. C. D. E. FISH analysis for missing SNRPN locus High-resolution chromosome analysis Imprinting assessment with DNA methylation assay Routine chromosome analysis and karyotype Subtelomeric probe analysis by FISH studies 695 C5. A newborn infant has the Robin sequence. The mother was diagnosed with arthritis in childhood and has had a retinal detachment. Which of the following is the most likely syndromic diagnosis? A. B. C. D. E. C6. Radial ray anomalies distinguished by the presence or absence of thumbs can provide a clue toward an appropriate diagnosis. In which of the following syndromes are thumbs consistently present? A. B. C. D. E. C7. Thrombocytopenia and absent radius syndrome Fanconi anemia Holt-Oram syndrome VATER association de Lange syndrome Soft cystic masses in the auricle which develop into hypertrophic cartilage are a typical feature of which of the following skeletal dysplasias? A. B. C. D. E. C8. Acromesomelic dysplasia Rieger syndrome Stickler syndrome Treacher Collins syndrome Warburg syndrome Achondroplasia Camptomelic dysplasia Chondroectrodermal dysplasia Diastrophic dwarfism Larsen syndrome A 28-year-old woman underwent amniocentesis for chromosomal analysis when her fetus was found to have limbs that were short for the assigned dates. The karyotype was 46,XY. At birth the baby had female appearing genitalia. These clinical findings are most consistent with which of the following syndromes ? A. B. C. D. E. Achondrogenesis type IA Achondrogenesis type II Campomelic dysplasia Jeune thoracic dystrophy Thanatophoric dysplasia 696 C9. A 1-year-old boy presents with an episode of fever on a 90 degree day in July and there is no evidence of an infection. His parents note that he never seems to sweat in the extreme heat of the summer. He has extremely sparse hair. Skull x-rays show absence of most of the primary tooth buds. Which of the following statements reflects the most likely inheritance pattern for this disorder? A. B. C. D. E. C10. A newborn infant has extreme hypotelorism, microcephaly, midline cleft, poor temperature regulation, seizures, and no anomalies below the neck. Which of the following syndromes is best described by these findings? A. B. C. D. E. C11. Ellis van Creveld syndrome Holoprosencephaly Meckel-Gruber syndrome Trisomy 13 Trisomy 18 A newborn infant has cleft lip and palate, postaxial polydactyly, microphthalmos, and congenital heart disease. Which of the following syndromes is best described by these findings? A. B. C. D. E. C12. He likely has an autosomal dominant disorder. He likely has an autosomal recessive disorder. He likely has a sporadically occurring disorder. He likely has an X-linked dominant disorder. He likely has an X-linked recessive disorder. Ellis van Creveld syndrome Holoprosencephaly Meckel-Gruber syndrome Trisomy 13 Trisomy 18 Haploinsufficiency is the mechanism underlying the genotype/phenotype relationship for which of the following disorders? A. B. C. D. E. achondroplasia acute intermittent porphyria Huntington disease multiple endocrine neoplasia, type 2A transthyretin amyloidosis 697 C13. Reproductive fitness is a measure of an individual’s likelihood of being able to have offspring where a fitness level of 1 is equivalent to the normal population and a fitness of 0 represents an inability to reproduce. In the relationships outlined below the disorders are abbreviated as follows: Huntington disease (HD), tuberous sclerosis (TS), neurofibromatosis type 1 (NF), and osteogenesis imperfecta type II (OI). Which of the following relationships best describes the reproductive fitness of these disorders from greatest to least? A. B. C. D. E. C14. A six-year-old girl presents to clinic with a history of cleft palate, tetralogy of Fallot, height at the 3rd centile, and mental retardation. Which of the following syndromes is the most likely diagnosis? A. B. C. D. E. C15. Cardiofaciocutaneous syndrome Down syndrome Otopalatodigital syndrome Stickler syndrome Velocardiofacial syndrome A 7-year-old boy with cleft palate, height at the 3rd centile, myopia, and a family history of retinal detachment presents to orthopedic clinic with hip pain and a referring diagnosis of Legg-Perthes disease. Which of the following sysndromes is most consistent with this patient’s clinical presentation? A. B. C. D. E. C16. HD>NF>TS>OI HD>TS>NF>OI NF>HD>OI>TS NF>OI >HD>TS TS >NF>OI>HD Coincidental Legg-Perthes disease in a child with nonsyndromic cleft palate Otopalatodigital syndrome Stickler syndrome Struger’s multiple epiphyseal dysplasia with cleft palate syndrome Velocardiofacial syndrome A newborn baby is found to have hydrocephalus, spina bifida, and clubbed feet. Which of the follwoing genetic etiologies provides the best description of this baby’s clinical features? A. B. C. D. E. A multiple congenital anomalies syndrome A single primary malformation A probable teratogenic syndrome A probable chromosome abnormality An association ] 698 C17. You are evaluating a newborn with bilateral absence of the radii, with intact thumbs. Which of the following clinical findings is most likley to be associated with this baby’s presentation? A. B. C. D. E. C18. A child with hypoplastic left heart syndrome is being evaluated prior to consideration of a heart transplant. Which of the following karyotypes is most likely to be found on chromosomal analysis? A. B. C. D. E. C19. Aarskog Noonan Robinow Pfeiffer Velocardiofacial A man with a right-sided triphalangeal thumb and left sided absent thumb has a child with bilateral absence of the radius and an atrioventriculoseptal defect. Which of the following genetic concepts accounts for this clinical history? A. B. C. D. E. C21. 45,X 46,XX 46,XX,del(22)(q11.2q11.2) 46,XY.ish del(7)(q11.23q11.23)(ELN-) 47,XXY You are asked to evaluate a 6-year-old boy with mild developmental delay and a distinctive dysmorphic facial appearance. In taking the history, you are told by his mother that his father has the exact same appearance. Which of the following syndromes are you most likely to remove from your differential diagnosis based on this clinical history? A. B. C. D. E. C20. Cytogenetic testing would show an extra copy of an E group chromosome. Hypoplasia of the mammary tissue is a common associated finding. Thrombocytopenia is a common complication. Tracheo-esophageal fistula is a common associated finding. When DEB is added to the media, increased chromosomal breakage is seen. Genetic heterogeneity Multifactorial inheritance Phenotypic pleiotropy Variable expression with complete penetrance Variable penetrance with incomplete expressivity A newborn boy manifests macrosomia (birth weight 5.2 Kg), coarse facies, and post-axial polydactyly. Which of the following clinical findings are you most likely to encounter when caring for this child? A. B. C. D. E. Cardiac conduction defects Delayed bone age Father to son transmission Late onset retinitis pigmentosa Uniparental disomy for chromosome 11p. 699 C22. A four-year-old girl is found to have relative macrocephaly, a broad forehead, a large open anterior fontanelle, and Wormian bones on X-ray. Her parents were told that she suffered bilateral clavicular fractures at birth, and they have never healed. Her father has a broad forehead, wide set eyes, and a history of dental problems, including supernumerary and ectopic teeth. Which of the following clinical findings has been associated with the underlying disorder? A. B. C. D. E. C23. Cytogenetic testing is needed to confirm the diagnosis. Most affected individuals have an adult height under 5 feet. Retinal detachment is a long-term risk. There is a lifetime increase risk for fractures. There is an increase rate of Caesarian section for affected women. A 3-year-old girl has hypodontia, alopecia, and mild developmental delay. Examination reveals hyperpigmented hyperkeratotic streaks. Her sister has severe developmental delay with seizures. Her mother has partial adontia and atrophic scalp hair. Which of the following clinical findings is most commonly associated with this disorder? A. B. C. D. E. Affected females can only have unaffected males or affected females. Chromosome analysis of fibroblasts reveal an abnormality in 90% of cases. Germline mosaicism is a common etiology. One sees fewer than expected males in affected families. 67% of cases represent new mutations C24. A 3-year-old boy manifests short stature, “Hitchhiker” thumbs, and “cauliflower” ears. What is the recurrence risk for his unaffected parents of having a subsequent child with the same condition? A. <1% B. 5-7% C. 12.5% D. 25% E. 50% C25. Which of the following genes is most likely to be mutated in an individual with a hypercoagulable state? A. B. C. D. E. C26. Which of the follwoing symptoms is the most common presenting complaint in individuals with hemochromatosis? A. B. C. D. E. C27. Factor VIII Factor IX Prothrombin Thrombin von Willebrand factor joint pain fatigue change in skin pigmentation nausea and vomiting decreased libido Hereditary angioedema is due to deficiency in which of the following systems? 700 A. B. C. D. E. C28. Which of the following is responsible for a phase I reaction in drug metabolism? A. B. C. D. E. C29. Allergy Arrhythmia Hair loss Hepatitis Seizure You are asked to evaluate a well infant girl with apparently isolated Pierre Robin sequence. Which of the following diagnostic tests is most likely to yield a useful clinical finding? A. B. C. D. E. C33. Asthma Chronic myelogenous leukemia Coronary artery disease Diabetes Hypertension A sodium channel polymorphism can be associated with which of the following side effects of medication? A. B. C. D. E. C32. Hypertension Asthma Tuberculosis Leukemia Diabetes Imatinib is used in the treatment of which of the following disorders? A. B. C. D. E. C31. ABC transporter Acetylase Glucuronyl transferase P450 enzyme Transcarbamylase The thiopurine methyltransferase polymorphism is important in treatment of which of the following disorders? A. B. C. D. E. C30. Antibodies B-cells Complement Macrophages T-cells Chromosome analysis Electrocardiogram (ECG) Eye exam FISH for del22q11.2 Skeletal survey Chromosome analysis would be most helpful in diagnosis of which of the following syndromes? 701 A. B. C. D. E. C34. Which of the following would be most useful in differentiating non-accidental injury (NAI) from osteogenesis imperfecta in a 5-month-old girl with unexplained fractures? A. B. C. D. E. C35. Blue sclerae Healing fractures of different ages Low socioeconomic status of the parents Retinal hemorrhages Type I collagen analysis A 3 year old girl has severe developmental delay, microcephaly, no speech, grand mal seizures, and ataxic limb movements. Chromosome analysis and FISH testing for a 15q11 deletion are normal. Which of the following genetic tests would be most appropriate to order in trying to determine the underlying etiology? A. B. C. D. E. C36. Marfan syndrome Neurofibromatosis 1 Smith-Magenis syndrome Pfeiffer syndrome Williams syndrome Cholesterol/7-delhydrocholesterol ratio FMR1 analysis MECP2 sequence analysis Methylation studies of chromosome 11p15 Uniparental disomy for chromosome 7 Assuming complete penetrance for a point mutation, this pedigree is most consistent with A. autosomal dominant inheritance of a gene imprinted (not expressed) for the maternal allele B. autosomal dominant inheritance of a gene imprinted (not expressed) for the paternal allele C. mitochondrial inheritance D. X-linked dominant inheritance E. X-linked recessive inheritance C37. Which of the following is associated with increased sensitivity to treatment of small cell lung cancers with gefitinb? A. B. C. D. E. C38. BCR-ABL translocation EGF receptor mutation N-myc amplification RAS mutation RET mutation Which of the following hematologic disorders is commonly associated with a gene inversion? 702 A. B. C. D. E. C39. Which of the following immune deficiency disorders is associated with thrombocytopenia? A. B. C. D. E. C40. Hemophilia A Hemophilia B Protein S deficiency Thalassemia Von Willebrand disease Adenosine deaminase deficiency Ataxia-telangiectasia Severe combined immune deficiency Velocardiofacial syndrome Wiscott-Aldrich syndrome Which of the following mutations is the most common mutation associated with hemochromatosis? A. B. C. D. E. C282Y H63D I105T Q283P R330M C41 – C43: For each consultand described below, choose the chance that the consultand might have a child with a neural tube defect (in the US) from the percentages listed in A-E. You may choose an answer once, more than once, or not at all. Percentages A. < 0.1% B. 0.1% to 1.9% C. 2% to 4% D. 5% to 10% E. >10% C41. A man with asymptomatic L-5 spina bifida occulta discovered on X-ray following a car accident C42. A woman whose maternal aunt's daughter had a baby with anencephaly C43. A normal couple whose first two children have isolated spina bifida 703 C44 – C46: For each situation described below, choose the coping response that best describes the consultands behavior, from those listed in A-E. You may choose an answer once, more than once, or not at all. A. B. C. D. E. Anger Denial Intellectualization Projection Reaction formation C44. A father who is preoccupied with learning all about the embryology of his newborn son's cleft lip C45. A mother who insists that her 4 year old daughter with spina bifida is not walking because of her many hospitalizations C46. A mother who ascribes her son's fetal alcohol syndrome to her obstetrician's negligence C47. Which of the following proteins represents the defective protein responsible for familial hypercholesterolemia? A. B. C. D. E. C48. ApoB-100 ApoE ApoB-48 Lipoprotein lipase LDL receptor Which of the following skeletal disorders has been associated with defective Wnt signaling? A. B. C. D. E. Osteogenesis imperfecta Osteopetrosis Pseudohypoparathyroidism Achondroplasia Osteoporosis DEVELOPMENTAL GENETICS C49. During development, specification and determination involve the stepwise acquisition of a stable cellular phenotype of gene expression specific to the particular fate of each cell, and regulation of gene expression depends on epigenetic changes. Which of the following is NOT an epigenetic change? A. B. C. D. E. Transcription complex stabilization Transposon insertion Modification of histones in chromatin DNA methylation Alternative promoter usage 704 C50. You are asked to consult on a newborn girl in the NICU. She was born with shortened fingers on her right hand with syndactyly of the 3rd and 4th right fingers. On examination, you find an absence of the right pectoral muscle. Which of the following mechanism is the most likely cause of these anomalies? A. B. C. D. E. C51. A 4-month-old boy presented with new onset seizures. His physician ordered an MRI, and he is found to have agyria. The patient is referred to you. On examination, the child has no dysmorphic features. Family history is remarkable for a seizure disorder in his mother, who is a stay at home mother who finished high school. Which of the following molecular alterations is the most likely reason for these findings in this patient? A. B. C. D. E. C52. Deformation Disruption Dysplasia Malformation Migration ARX mutation DCX mutation Deletion of the terminal end of 17p EMX2 mutation LIS1 mutation A newborn male infant in the NICU is found to display hypotelorism, small head circumference, thin nose and his mother has a single maxillary incisor. Which of the following genetic defects is the most likely cause of this newborn’s phenotype? A. B. C. D. E. Chromosomal anomaly DNA repair enzyme mutation Fibroblast growth factor receptor mutation RAS pathway component mutation Sonic hedgehog mutation 705 C53. You are asked to evaluate a small child with asymmetric head shape, midface hypoplasia and digital anomalies. You obtain a head CT with 3-D reconstruction that demonstrates the finding below. Which of the following genetic defects is the most common cause of the radiographic finding seen on this 3-D CT image? A. B. C. D. E. C54 Chromosomal anomaly DNA repair enzyme mutation Fibroblast growth factor receptor mutation RAS pathway component mutation Sonic hedgehog mutation The correct answer is E A nasal sample was sent on a patient that was seen in clinic who you suspect has a problem with ciliary function. The results found abnormalities in the dynein arms of the cilia. Which of the following diagnoses is most consistent with this finding? A. Bardet-Biedl syndrome B. Grieg cephalopolysyndactyly syndrome C. Holoprosencephaly D. Joubert syndrome E. Situs inversus MENDELIAN TRANSMISSION C55. Consider a disorder inherited as an autosomal dominant with complete penetrance in which reproductive fitness is 0.8 and the disease frequency is 1/50,000. What is the rate of new mutation? A. 1/1,000,000 B. 1/500,000 C. 1/250,000 D. 8/500,000 E. 8/1,000,000 706 C56. A man with hemochromatosis is married to a woman who is not affected, but is of the same ethnicity as he. Before doing genetic testing, what is closest to the risk of their having a child homozygous for a hemochromatosis mutation? Assume a population frequency of homozygosity to be 1/400. A. 1/5 B. 1/10 C. 1/20 D. 1/40 E. 1/80 C57. A woman has retinitis pigmentosa (RP) and is found to be heterozygous for two unlinked genes, each of which has been implicated in RP. Her partner does not have RP and is unrelated. The chance for a child of this couple to have RP is? A. 0 B. 1/8 C. 1/4 D. 1/2 E. 1 C58. What is the risk that a person with phenylketonuria (PKU) would have a child with PKU, assuming his partner is not related but is of the same ethnicity? Assume a population frequency of 1/10,000. A. 1/1,000 B. 1/500 C. 1/200 D. 1/100 E. 1/50 C59. A couple who are first cousins request counseling regarding their risk of having a child with alpha-1-antitrypsin deficiency, a rare autosomal recessive trait. Their grandfather is affected with the disorder. What is the risk to their child of being homozygous for a mutation for the condition? A. 1/4 B. 1/8 C. 1/16 D. 1/32 E. 1/64 707 C60. A couple who are first cousins ask about their risk of having a child with a rare autosomal recessive disorder that affected the sister of their common grandmother. The grandmother was not affected by this condition that exhibits complete penetrance. What is the risk to their child? A. B. C. D. E. C61. You are counseling a couple where both parents are affected with NF1. They have just had a baby, who at birth has no signs of the disorder. You explain that although NF1 follows is autosomal dominant disorder, features such as café-au-lait spots may not appear at birth, so their child still needs to be followed clinically, and that genetic testing is possible. You also note that homozygotes for NF1 mutation do not survive in utero. The couple asks you to estimate the chance that the baby has inherited an NF1 gene mutation. You quote the family which of the following risks? A. B. C. D. E. C62. 1/128 1/96 1/32 1/24 1/6 1/4 1/3 1/2 2/3 1 A genetic trait is associated with a fitness of 0.8 and is found in 1 in 10,000 individuals in the population. Assuming mutation/selection balance, what is the mutation rate? A. B. C. D. E. 0.00001 0.00002 0.00008 0.002 0.008 PHARMACOGENETICS C63. A 70 year old woman has had a heart valve replacement and needs to be treated with a drug to reduce platelet function. You are considering starting her on clopidigrel. Which of the following genetic tests would be appropriate to inform this decision? A. CYP2C19 B. CYP2D6 C. N-acetyl transferase D. Thiopurine methyltransferase E. VKORC1 708 C64. A patient you are following learns of a clinical trial for the disorder. It is a phase I trial. What should the patient expect if he participates? A. The trial will determine whether the treatment is effective. B. The trial will determine whether the treatment is more effective than alternatives. C. The trial will determine the maximum tolerated dose of drug and pharmakokinetics. D. The trial will monitor for long-term side effects of treatment. E. The trial will determine whether the drug affects fertility. C65. The parent of a child with cystic fibrosis has read about a new medication, ivacaftor, which can help some with the disorder. Which of the following mutations would need to be present in the child to justify treatment? A. 3849+10kbC>T B. G551D C. G542X D. phe508del E. R117H C66. A case-control study reveals an allele in 700/1000 cases and 300/1000 controls. What is closest to the odds ratio of a carrier of the allele having the disease compared with a noncarrier having the disease? A. 1 B. 3 C. 5 D. 7 E. 10 C67. A 60 year old man develops severe respiratory depression after a dose of codeine. Which of the following genes is most appropriate to test in an effort to find an explanation? A. CYP2C19 B. CYP2D6 C. N-acetyl transferase D. Thiopurine methyltransferase E. VKORC1 709 SYSTEM-BASED SINGLE GENE DISORDERS C68. A boy is seen with Hirschsprung disease. No apparent syndrome is found. Which of the following genes is most likely to be mutated if a genetic test is done? A. AKT1 B. APC C. PTEN D. RAS E. RET C69. A child with autism spectrum disorder is found to have a deletion at 15q13.3. For which of the following clinical problems is the child at greatest risk? A. congenital heart defect B. deafness C. epilepsy D. obesity E. retinitis pigmentosum C70 Exome sequencing reveals a heterozygous pathogenic mutation in MYO7A. Which of the following phenotypes is most likely? A. B. C. D. E. C71. AD deafness AR deafness Congenital myopathy Ectodermal dysplasia Usher syndrome A 2 year old girl is seen with streaky patches of hyperpigmentation. She had had seizures in the newborn period, which subsequently resolved. She had multiple skin lesions in the early weeks of life that began with vesicular lesions and later evolved. A genetic test is done. Which of the following types of mutation is most likely to be found? A. Amino acid substitution B. Deletion C. Frameshift mutation D. Mosaic trisomy E. Stop mutation C72. A newborn has failed her hearing screen. An electrocardiogram is ordered and is found to be abnormal. Which of the following findings is most likely? A. Elevated P wave B. Elevated R wave C. Elongated PR interval D. Elongated QT interval E. Right bundle branch block 710 C73. A man has 3 schwannomas, no vestibular schwannomas, and a normal exam. His schwannomas came to attention because of pain. Which of the conditions best explains this presentation? A. NF1 B. NF2 C. schwannomatosis D. tuberous sclerosis complex E. von Hippel Lindau syndrome C74. A newborn is having multiple seizures and you learn that there is a family history of the same in multiple relatives on the father’s side. Which of the following type of gene is most likely to be responsible for this history? A. chloride channel encoding gene B. gaba receptor encoding gene C. nicotinic acid receptor encoding gene D. potassium channel encoding gene E. sodium channel encoding gene C75. 33. A 64-year-old man is seen with tremor, unsteady gait, and memory loss. Which of the following genetic tests would be most likey to be informative? A. ApoE B. ATM C. DYT1 D. FMR1 E. LRRK2 C76. 40. A 40 year old man with tuberous sclerosis complex has a progressively enlarging renal angiomyolipoma. Which of the following is the most appropriate approach to therapy? A. treatment with bevacizumab B. treatment with everolimus C. treatment with imatinib D. nephrectomy E. treatment with rapamycin 711 C77. 45. A newborn with severe weakness is evaluated in the nursery. He is intubated and moves very little. Exam reveals little movement but weakly elicitable deep tendon reflexes. On taking a family history it is noted that his mother has visible mild facial weakness. What is the most likely diagnosis for the infant? A. Charcot-Marie-Tooth disease B. congenital muscular dystrophy B. Duchenne muscular dystrophy C. myotonic dystrophy D. spinal muscular atrophy C78. You are seeing Ms Andrews who is a 36-year-old woman who recently developed dyspnea and fatigue. She was found to have a mean pulmonary arterial pressure of 33 mm Hg on an otherwise normal echocardiogram. Her father is healthy. Her mother was also healthy until she developed right heart failure during pregnancy. Ms Andrews’ pulmonologist asks you “Which of the following genes is most likely to bear a mutation that is the cause of her condition?” A. B. C. D. E. C79. ACVRL1 BMPR1B BMPR2 EAG SMAD9 You are seeing Joey, a child who had an elevated Immunoreactive Trypsinogen newborn screening result. Since Joey was found to have a Phe508del/G551D CFTR genotype which of the following medications is likely to potentiate his CFTR function? A. B. C. D. E. Gleevec (Imatinib) Kalydeco (ivacaftor) Mucomyst (acetylcysteine) Pulmozyme (dornase) Tobi (tobramycin) 712 C80. You are seeing Seth who is a 4-month-old boy who has had recurring respiratory infections, diarrhea and failure to thrive since one month of age. Seth had two maternal uncles who died at 4 and 7 months of recurring bacterial infections. Which of the following genes would you test first to find the cause of Seth’s problems? A. B. C. D. E. C81. Sammy is a 12-year-old boy is referred to you for genetic testing to determine the cause of his Common Variable Immune Deficiency (CVID). His was recently diagnosed because of recurring Streptococcal, then Klebsiella pneumonia which was complicated by meningitis at 6 years of age. An abnormality in which of the following genes is the one that is most likely to cause Sammy’s CVID? A. B. C. D. E. C82. ADA IL2RG STAT3 TNFR5R13B ZAP70 You are seeing a 32-year-old man, Todd, who has recently been found to have an area of aortic dilation. Which of the following disorders is least likely to be the cause of Todd’s aortic aneurysm? A. B. C. D. E. C83. ADA FOXP3 IL2RG STAT3 ZAP70 Ehlers Danlos Syndrome Familial Thoracic Aortic Aneurysm and Aortic Dissection (TAAD) Loeys-Dietz Syndrome Marfan Syndrome Pseudoxanthoma Elasticum You are seeing a brother and sister (Will and Cindy) who are 15 and 19 years of age. Both have recently been found to have multiple renal cysts. You are asked to determine the genetic cause of their renal cysts. Which of the following genes is most likely to show a mutation on molecular testing for this disorder? A. B. C. D. E. PKD1 PKD2 PKHD1 OCRL TSC2 713 C84. You are evaluating Sarah, a 13-month-old girl, who has microcytic anemia (MCV=56) with 5% Hb Bart. Which of the following hemoglobinopathies do you think is the most likely cause of Sarah’s findings? A. B. C. D. E. C85. You are evaluating Paul who is a 5-day-old boy. Paul has a large hematoma at his Vitamin K injection site and he continues to ooze after initial bleeding from his circumcision three days ago. He has a normal prothrombin time, von Willebrand and F9 factor levels and no family history of coagulation problems. You suspect hemophilia A. Which of the following mutations in the F8 do you predict is most likely to explain Paul’s problems? A. B. C. D. E. C86. Deletion/duplication IVS1 inversion IVS22 inversion Missense 51 promoter A 4-year-old girl has sparse hair, abnormal teeth, and absence of sweating. Which of the following genes is most likely to be responsible for this disorder? A. B. C. D. E. C87. Į-Thalassemia rait ȕ-Thalassemia trait Hb Bart syndrome Hb E disease Hb H disease EDA1 EDAR GJB6 MSX1 SLC45A2 A skin biopsy is done on a young woman with severe scarring due to epidermolysis bullosa. Which of the following sites is most likely to demonstrate skin separation in this biopsy? A. B. C. D. E. Below basement membrane Epidermis Multiple layers in skin Sub-dermis Within basement membrane 714 C88. A 30-year-old man develops congestive heart failure and has left ventricular hypertrophy by echocardiogram. If this clinical finding is genetically determined, which of the following is the most likely mode of inheritance? A. B. C. D. E. C89. Autosomal dominant Autosomal recessive Mitochondrial X-linked dominant X-linked recessive You evaluate a 2-year-old boy, Ethan, with multiple fractures after mild trauma. The family pedigree (below) reveals additional family members with a similar clinical phenotype. Ethan’s two sisters have no history of fractures. The most likely cause of fractures in this family is a mutation in which of the following genes? A. B. C. D. E. C90. COL1A1/2 IFITM5 LEPRE1 RPIB SERPINF1 You evaluate an 8-year-old girl with eczema since 3 months of age, recurring boils and cyst forming pneumonia since 1 year of age but no significant problems with diarrhea. Which of the following disorders is the most likely cause of her problems? A. B. C. D. E. AR Severe Combined Immune Deficiency Common Variable Immune Deficiency Hyper IgE Syndrome Hyper IgM Syndrome XL Severe Combined Immune Deficiency 715 C91. You evaluate an 8-year-old boy with “very soft skin” and myopia. On physical examination you find his skin is quite soft, especially on his face; he has bilateral inguinal hernias, and normal joint mobility. Which of the following disorders is the most likely diagnosis? A. B. C. D. E. C92. You evaluate a 19-year-old man (Ralph) with chronic hip and knee pain. His mother had hip and knee replacements at 42 years of age. On your physical examination you find Ralph’s height is at the 10th percentile, he has decreased range of motion of both hips and knees, but no scoliosis. Which of the following disorders is the most likely diagnosis? A. B. C. D. E. C93. Ehlers Danlos Syndrome Hypochondroplasia Marfan Syndrome Multiple Epiphyseal Dysplasia Osteogenesis Imperfecta You are seeing a child who presents with small teeth and dystrophic nails. Her hair is normal, she has normal skin, and she is able to sweat normally. Which of the following genes is the most likely to explain her disorder? A. B. C. D. E. C94. Cutis laxa Syndrome Classic Ehlers-Danlos Syndrome Loeys Dietz Syndrome Marfan Syndrome Pseudoxanthoma Elasticum EDA EDAR GJB6 MSX1 WNT10A You are evaluating a 17-year-old girl, whose primary care physician (PCP) reports has primary amenorrhea but is otherwise normal. The primary care physician obtained a pelvic ultrasound which showed a “blind vagina and no uterus”. Laboratory results showed low 17-OHP but high testosterone levels. Which of the following diagnoses provides the best explanation of her clinical findings? A. B. C. D. E. Congenital adrenal hyperplasia Complete androgen insensitivity Luteinizing hormone deficiency Turner syndrome 46, XY sex reversal, SRY related 716 C95. You are seeing a 6-month-old girl who has short stature, rachitic skeletal changes and serum biochemistries which included: sodium 138, potassium 4.6, chloride 105, bicarbonate 22, BUN 8, creatinine 0.3, glucose 87, calcium 10.4, ionized calcium 4.4, magnesium 2.2, phosphorus 6.4 mg/dL (Nl: 3.0 – 6.0 mg/dL) alkaline phosphatase 44 U/L (Nl: 110 – 320 U/L) and vitamin B6 (pyridoxal 5’-phosphate) level 2449 nmoles/L (Nl: 10 – 110 nmoles/L). Which of the following clinical findings is associated with her disorder? A. B. C. D. E. C96. A woman is referred for genetic testing because of a severe disorder of skin blistering and scarring. There is no known prior family history of the condition. Biopsy reveals scarring in the dermis below the basement membrane of the skin. Which of the following genes is the most likely to explain her condition? A. B. C. D. E. C97. COL7A1 EXPH5 KRT14 LAMB3 TGM5 You are seeing an eight-year-old boy with microcephaly, telecanthus, coarse facies, genital anomalies, hypotonia, intellectual disability & mild anemia thought to be due to thalassemia. Which of the following genes is the best candidate to explain his findings? A. B. C. D. E. C98. Craniosynostosis Hypocalemia Hypocalciuria Osteopetrosis Tall stature ATRX HBA1 HBA2 HBB HBG1 You are evaluating a young boy with proportionate short stature, relative macrocephaly, and hypospadias. His bone age is delayed. You arrange for testing of imprinting defects on chromosome 11. Which of the following changes is the most likely to explain his features? A. B. C. D. E. CDKN1C mutation Gain of methylation of the maternal IC1 site Hypomethylation of the paternal IC1 site Microdeletion of the maternal IC1 site Paternal uniparental disomy 717 Answers to Clinical Genetics Questions C1-C98 C1. Answer: E. Rectal prolapse is a frequent feature of CF and should raise concern about that diagnosis. C2. Answer: A. Mutations causing deficiency of G proteins occur in Albright osteodystrophy while activating mutations in the same gene cause McCune-Albright. C3. Answer: E. This is a typical description for a patient with Warburg syndrome. A patient wlth Meckel-Gruber syndrome would more likely have encephalocele, polydactyly, and polycystic kidney disease. The Pallister-Hall syndrome is characterized by hypothalamic hamartoblastoma, hypopitutarism, imperforate anus, and postaxial polydactyly. The Neu-Laxova syndrome is characterized by microcephaly or lisssencephaly, elfin-facies with exophthalmos, and syndactyly with subcutaneous edema. The Miller-Dieker syndrome is characterized by lissencephaly. C4. Answer C. Consensus clinical diagnostic criteria are accurate, but the mainstay of diagnosis is DNA-based methylation testing to detect abnormal parent-specific imprinting within the Prader-Willi critical region (PWCR) on chromosome 15; this testing determines whether the region is maternally inherited only (i. e., the paternally contributed region is absent) and detects more than 99% of affected individuals. Methylation-specific testing is important to confirm the diagnosis of PWS in all individuals, but especially those who have atypical findings or are too young to manifest sufficient features to make the diagnosis on clinical grounds. (www.genereviews.org) C5. Answer: C. This would be a reasonably good story for Stickler syndrome. The arthropathy can simulate juvenile rheumatoid arthritis. Retinal detachment is a feature. Robin sequence can occur in childhood. The other disorders would all be quite different. C6. Answer: A. The thumbs may be absent in any of these disorders except TAR where they are consistently present. C7. Answer: D. This description of the changes in the ear is typical of diastrophic dwarfism. It is a very distinctive change but is usually not present in the neonatal period. C8. Answer: C. Female appearing genitalia in an XY infant is a common feature of campomelic dysplasia, but is not a feature of the other disorders. C9. Answer: E. This clinical description is very suggestive of anhidrotic ectodermal dysplasia, which is usually X-linked. Many other forms with different inheritance occur. This story is typical for the X-linked disorder but is not diagnostic of that pattern of inheritance. C10. Answer: B. This is a typical description of holoprosencephaly. The trisomies and Meckel-Gruber syndrome would likely be associated with additional features below the neck. The CNS abnormalities would not be a feature of Ellis van Creveld although cardiac defect and limb abnormalities would be present. C11. Answer: D. This is a typical clinical description for Trisomy 13. 718 C12. Answer: B. All the conditions listed are autosomal dominant disorders. Missense mutations with some type of aberrant function are involved in MEN2A and achondroplasia. The mechanism is not clear for Huntington disease, but there is no evidence that deletion of this region causes the disease, and the evidence favors some type of gain of function. A harmful effect of the mutant protein is involved in amyloidosis. Many of the mutations in acute intermittent porphyria and in some of the other porphyries are obvious loss of function mutations indicating that haploinsufficiency is the mechanism of dominance for AIP. C13 Answer: A. Reproductive fitness is relatively normal in HD. It is somewhat reduced, but not severely so in NF. Fitness is substantially reduced, but not to 0, in TSC. OI type II is lethal neonatal OI and reproductive fitness is 0. Keep in mind that a reproductive fitness of 1 is equal to the normal population and a reproductive fitness of 0 is no ability to reproduce. C14. Answer: E. Conotruncal defects are most common in Down syndrome and clefting would be less typical. It is unlikely a child with Trisomy 13 or 18 would survive to age 6 years. Congenital heart defect would not be expected with otopalatodigital syndrome and it is much less common than VCFS. Clefting and TOF are not features of CFC. All of the features listed are common in VCFS. C15. Answer: C. The retinal detachment is the unique feature in this question- it is only found in Stickler syndrome. C16. Answer: B. This is a single primary malformation because the other malformations are a direct result of the first. A MCA syndrome includes anomalies that arise independently, not causatively. An association is a group of anomalies that frequently arise together (e.g. VACTERL) without an apparent relationship to each other or a known single gene cause. The vast majority of neural tube defects are isolated and multifactorial in origin, not chromosomal or teratogenic. C17. Answer: C. Absent radii is associated with absent or abnormal thumbs except in one syndromeThrombocytopenia-Absent Radius syndrome. These children have a high rate (as the name suggests) of developing thrombocytopenia. The other answers suggest other syndromes that commonly manifest radial hypoplasia, but in all the thumbs are also absent: Trisomy 18 (answer B), Fanconi syndrome (answer D), and VATER (answer E). Answer C suggests ulnar mammary syndrome, in which ulnar ray (not radial ray) defects are associated with mammary hypoplasia. C18. Answer: B. The most common cytogenetic finding seen with hypoplastic left heart (HLH) is a normal karyotype. Turner syndrome (45,X) is among the most common cytogenetic abnormalities seen with HLH, but is still not as common as a normal karyotype. Other syndromes seen with HLH include Down syndrome, and Smith Lemli Opitz.. HLH is not seen at an increased frequency in Williams syndrome [46,XY.ish del(7)(q11.23q11.23)(ELN-)], VCFS/DiGeorge [46,XX,del(22)(q11.2q11.2)], or Klinefelter syndrome [47,XXY]. C19. Answer: A. While all the syndromes have a distinctive facial appearance, Aarskog syndrome is an X-linked disorder, so father-to-son transmission would not be seen. The other syndromes are autosomal dominant. C20. Answer: D. The description fits Holt-Oram syndrome, and represents variable expression with complete penetrance. 719 C21. Answer: A. The child in this scenario has Simpson-Golabi-Behmel syndrome, an X-linked overgrowth syndrome caused by mutations in Glypican 3 on Xq26. There is no parent of origin effect, and male to male transmission is not seen. These children have a high rate of cardiac conduction defects, which may account for the observation of unexplained death. Bone age is advanced initially, then becomes normal. Retinitis pigmentosa is not a common long-term complication. C22. Answer: E. This scenario describes a child with Cleidocranial dysplasia (CCD). Women with CCD have an increased rate of needing a Caesarian section in childbirth. They do not have an increased fracture rate. Mild short stature can be seen, but adult heights are usually at the low end of the normal range. Retinal detachment is not a manifestation. Cytogenetic testing is not helpful. Some children have a microdeletion on 6p21, but it is not cytogenetically visible. These children usually have developmental delay/mental retardation in addition to the usual CCDS findings. C23. Answer: D. The condition describes is Incontinentia Pigmenti (IP), an X-linked male lethal trait. In such conditions one sees fewer than expected males in a given pedigree, as ½ of male fetuses (those affected) are lost. Germline mosaicism is not common in IP. For an X-linked lethal trait, approximately 1/3rd of cases are new mutations. There is no cytogenetic abnormality associated with IP. Chromosome analysis of fibroblasts demonstrates an abnormality in approximately 40% of cases of Hypomelanosis of Ito, a related but clinically distinct disorder. Affected females can have unaffected males and affected and unaffected females. C24. Answer: D. This child’s description is classic for Diastrophic Dysplasia, which is an autosomal recessive trait caused by mutations in the DDST gene. C25. Answer: C. The prothrombin G20210A mutation is associated with the hypercoagulable state. Mutation of any of the other listed proteins would lead to bleeding tendency, not increased tendency to form blood clots. C26. Answer: B. Fatigue is the most common presenting complaint, although all of the listed items are features of hemochromatosis. C27. Answer: C. Hereditary angioedema is due to C1q inhibitor deficiency in the complement system. C28. Answer: D. Phase I reactions are oxidations, reductions, and hydrolysis reactions; one of the major enzyme systems involved in phase I reactions is the P450 system. C29. Answer: D. The TPMT polymorphism affects metabolism of drugs such as 6-mercaptopurine, 6thioguanine, etc., which have a role in treatment of leukemia and autoimmune disorders. C30. Answer: B. Imatinib targets the BCR-ABL kinase that results from the Philadelphia chromosome, which is associated with chronic myelogenous leukemia. C31. Answer: B. The sodium channel polymorphism can lead to arrhythmia upon exposure to specific drugs. 720 C32. Answer: C. In approximately 50% of cases, Pierre Robin sequence (PRS) is part of a genetic syndrome, and half of that is Stickler syndrome (SS). The majority of those with SS have eye involvement (only those cases caused by COL11A2 mutations do not). The eye findings of SS are usually evident in the newborn period. Chromosome abnormalities are unlikely in an otherwise normal baby with PRS. Del22q11.2 is the next most common syndromic cause of PRS, so it should be ordered in every baby with PRS. However, del22q11.2 is only about half as common as SS as the cause of PRS. Cardiac conduction abnormalities are unlikely in this setting. C33. Answer: C. SMS is caused by deletions of 17p11.2 visible with routine chromosome analysis (>550 bands), although deletions may be missed on first inspection. There is evidence that the majority of the manifestations may be attributable to haploinsufficiency of the RAI1 gene. While a small percentage of Neurofibromatosis 1 (NF1) is caused by deletions of the NF1 gene, the majority of cases are caused by point mutations in the NF1 gene. Furthermore, a cytogenetically visible deletion is very rare. Williams syndrome is caused by submicroscopic deletions of 7q11.2, encompassing the elastin gene and others as well. The Pfeiffer syndrome (PS) phenotype can be caused by a variety of different point mutations in FGFR1 (mild type 1 PS), FGFR2 (more severe type 1 as well as types 2 and 3 PS), and FGFR3 (some cases of FGFR3-related craniosynostosis, or Muenke syndrome, can look like PS). Marfan syndrome is generally caused by point mutations in fibrillin-1 on chromosome 15q21. C34. Answer: D. Blue sclerae are seen in OI types 1, 2 and 3, but are also seen in most healthy babies as a normal variant. Healing fractures of different ages can be seen in OI as well as NAI. Type I collagen analysis is considered the ‘gold standard’ for the diagnosis of OI, but it can be normal in over 10% of OI, so a negative analysis does not rule out OI. NAI can be seen in families of any level of socioeconomic status of the parents. Retinal hemorrhages are not a feature of OI, but do suggest a diagnosis of NAI. C35. Answer: C. Mutations in MECP2 cause Rett syndrome, but also can cause an Angelman syndrome-like phenotype, as described above. Uniparental disomy (UPD) 7 causes idiopathic short stature and a small percentage of cases of Russell-Silver syndrome. FMR1 is the gene for Fragile X syndrome, and does not typically present with these findings. The ratio of cholesterol/7dehydrocholesterol is used to diagnose Smith-Lemli-Opitz syndrome. C36. Answer: B. The disorder was not expressed in the mother of the two affected children, since these women inherited the gene from their father. Their sons each inherited the mutation from a female, though, and therefore are affected. C37. Answer: B. EGF receptor mutations are found in small cell lung cancers that are likely to respond to gefitinib. C38. Answer: A. Hemophilia A is commonly associated with an inversion of the factor VIII gene. C39. Answer: E. C40. Answer: A. 721 C41. The correct answer is B. Although this man probably does not have an increased risk to have a child with an NTD, the question requires you to recognize that there is a "background" incidence of NTDs in the population and to know that the incidence is about 1 per 1000 in the U.S. Although this man's risk is not increased, it would not be lower than the population incidence, the correct answer is (B) C42. The correct answer is B. In the U.S. and Canada, data on risk to first cousins of an affected range from 0.3 to 0.9%, but a second cousin (the relationship between this woman’s fetus daughter and her cousin’s baby with anencephaly) don’t have a risk significantly above background. There does appear to be a higher risk when the proband is related through the mother rather than through the father. Even though this consultand may have a slightly higher risk than the first man, the correct answer is still (B). C43. The correct answer is D. The data for risk following two affecteds differ between the U.S. and the U.K., reflecting the lower population incidence here. However, in both places, the risk is at least twice what it would be after only 1 affected. Iin the U.S., this is about 6.4%; in the U.K., at least 10%. Since the question asks about U.S. figures, the correct answer is (D). C44. The correct answer is C. The father in (J7) is trying to achieve some mastery over a situation over which he has no control by intellectualizing. C45. The correct answer is B. The mother in (J8) is denying the reality of her daughter's disability by attributing it to a cause that, at age 2 or 3 years old might still have been legitimate. C46. The correct answer is D. The mother in J9, who presumably has been counseled regarding her son's diagnosis and it's cause, is projecting her own guilt onto the obstetrician. C47. The correct answer is E. C48. Answer: E. A mutation in LDL receptor protein (LRP6) has been found in a family with a number of conditions, including osteoporosis; and an LRP5 mutation in association with pseudogloma osteoporosis syndrome. Both are involved in the Wnt signaling pathway. DEVELOPMENTAL GENETICS C49. The correct answer is B. Keywords: Epigenetic change, transposon insertion Transposon insertion is a genomic change, not an epigenetic change. Epigenetic changes are not inherited, and are not permanent changes in DNA. The other choices are all examples of epigenetic changes. C50. The correct answer is B. Keywords: Poland anomaly, syndactyly Explanation: The clinical vignette describes a child with Poland anomaly. Poland anomaly is believed to result from a disruption of blood flow during development before birth. This disruption is thought to occur at about the sixth week of embryonic development and affect blood vessels that will become the subclavian and vertebral arteries. 722 C51. The correct answer is B. Keywords: Agyria, lissencephaly, Agyria is lissencephaly. Mutations in ARX, DCX and LIS1, as well as terminal 17p deletions cause lissencephaly. EMX2 mutations cause schizencephaly, ruling our D. Since there are no dysmorphic features, 17p deletions (C, which cause Miller-Dieker syndrome with craniofacial dysmorphisms) can be ruled out. Similarly, since the patient has normal genitalia, that rules out ARX mutations (A). Isolated lissencephaly can be caused by LIS1 or DCX mutations. Since the mother has seizures, but clearly no lissencephaly, and the patient is male, the most likely explanation is (B) DCX mutation, not (E) LIS1 mutation. C52. The correct answer is E. Keywords: holoprosencephaly, sonic hedgehog The clinical case described a child with the outward appearance of holoproencephaly. This is supported by the single maxillary incisor in his mother. The most common cause of holoprosencephaly are mutations in sonic hedgehog C53. The correct answer is C. Keywords: Craniosynostosis, fibroblast growth factor receptor (FGFR) The CT scan displays craniosynostosis. The most common causes of craniosynostosis are mutations in FGFR1, 2 and 3. C54 The correct answer is E Bardet-Biedl syndrome (BBS genes), Grieg cephalopolysyndactyly syndrome (GLI3), Holoprosencephaly (Sonic hedgehog and other genes) and Joubert syndrome (multiple genes) are caused by defects in signal transduction in the cilia, but they do not display structural cilia defects. Situs inversus is often caused by mutations in ciliary dyneins, which can be detected in electron micrographs as structural defects in the dynein arms. MENDELIAN TRANSMISSION C55. The correct answer is B Source: Genetic Transmission lecture Keywords: population genetics, dominant, selection, mutation Explanation: P = ps 2p = 1/50,000, hence p = 1/100,000; s = 0.2 (1-fitness); therefore m = (1/100,000)(0.2) = 1/500,000 C56. The correct answer is C. Source: Genetic Transmission lecture Keywords: autosomal recessive, pseudodominance, risk assessment Explanation: The carrier frequency is 2(1/20) = 1/10; the risk to a child is 1/20; we are ignoring the possibility that the mother is a homozygote, which is possible, but adds little to the risk. 723 C57. The correct answer is C. Source: Genetic Transmission Lecture Keywords: genetic transmission, Mendelian, digenic Explanation: This is an example of digenic inheritance. The woman has a 1/2 chance of transmitting each of the mutant alleles, so a 1/4 chance of transmitting both to an offspring. C58. The correct answer is D. Source: Genetic Transmission Lecture Keywords: autosomal recessive, Hardy-Weinberg, genetic counseling, risk assessment Explanation: The frequency of the gene is the square root of 1/10,000 = 1/100. The carrier frequency is therefore 1/50. The chance of having an affected child is 1(1/50)(1/2) = 1/100 C59. The correct answer is C. Source: Genetic Transmission Lecture Keywords: consanguinity, risk assessment, autosomal recessive Explanation: Both of the parents of the couple are obligate carriers. Each partner therefore has a risk of ½ of having inherited a mutation. Each in turn would have a ½ chance of passing the mutation on to a child. The risk to a child is therefore (1/2)(1/2)(1/2)(1/2) = 1/16. C60. The correct answer is B. Keywords: inbreeding; population genetics; genetic risk assessment Explanation: The grandmother has a 2/3 risk of being a carrier. There is a 1/4 chance that she passed the mutation on to either parent and hence a 1/16 chance that she passed it to both. If both are carriers, they have a 1/4 chance of having an affected child. Hence the risk here is (2/3)(1/16)(1/4) = 1/96 C61. The correct answer is D. Keywords: genetic transmission, Mendelian genetics, genetic risk Explanation: The parents are both heterozygous for an NF1 mutation. There is a one in four chance that a fetus will be homozygous and therefore will die in utero, a one-in four chance that an offspring will be homozygous for the wild type gene, and a one in two chance for being heterozygous for either one of the parent’s mutations. Since the homozygous mutation embryo will not survive to birth, a live born child has a 2/3 risk of being affected with NF1. C62. The correct answer is B. Keywords: population genetics; Hardy-Weinberg; mutation rate Explanation: P = sq2; s = 1 – 0.8 = 0.2; hence P = (1/10,000)(0.2) = 0.0001(0.2) = 0.00002 724 PHARMACOGENETICS C63. The correct answer is A. Source: Genomic Medicine lecture Keywords: pharmacogenetics Explanation: CYP2C19 is required to activate clopidigrel, so would be appropriate to test. C64. The correct answer is C. Source: Genomic Medicine lecture Keywords: clinical trials Explanation: A phase I trial looks at maximum tolerate dose and side effects. A phase II trial looks at efficacy and a phase III trial compares the treatment to alternatives. Longterm effects are monitored in phase IV. C65. The correct answer is B. Source: Genomic Medicine lecture Keywords: treatment, cytic fibrosis Explanation: Ivacaftor targets the mutant protein due to the G551D mutation increasing Cl- flow through the mutant CFTR. C66. The correct answer is C. Source: Genomic Medicine lecture Keywords: genetic association, case-control study Explanation: The odds of a carrier having disease are 700/300 = 7/3; the odds of a noncarrier having disease are 300/700 = 3/7. The odds ratio is (7/3)/(3/7) = 49/9§5 C67. The correct answer is B. Source: Genomic Medicine lecture Keywords: pharmacogenetics Explanation: Codeine is converted to morphine by the CYP2D6 enzyme; a rapid metabolizer would produce too much morphine at a standard dose and might have this reaction SYSTEM-BASED SINGLE GENE DISORDERS C68. The correct answer is E. Source: Systems I lecture Keywords: Hirschsprung disease, genetic testing, RET gene Explanation: RET mutations are the most common associated with non-syndrome Hirschsprung disease. C69. The correct answer is C. Source: Systems I lecture Keywords: autism, chromosome 15, deletion Explanation: Those with deletions at 15q13.3 are at risk for epilepsy, in addition to autism. 725 C70. The correct answer is A. Source: Systems I lecture Keywords: deafness, Usher syndrome, genetic heterogeneity, exome sequencing Explanation: MYO7A mutation can lead to AD deafness, AR deafness, or Usher syndrome; the fact that heterozygous mutation was found here suggests an AD mode of inheritance, and hence deafness. C71. The correct answer is B. Source: Systems I lecture Keywords: mutation, incontinentia pigmenti Explanation: The clinical history and exam suggest incontinenti pigmenti. This is associated with IKBKG mutation, which in 80% of cases is a deletion. C72. The correct answer is D. Source: Systems I lecture Keywords: Long QT, deafness, Jervell and Lange-Nielsen syndrome Explanation: Jervell and Lange-Nielsen syndrome is associated with congenital deafness and long QT interval. C73. The correct answer is C. Source: Neurogenetics lecture Keywords: neurofibromatosis, phakomatosis Explanation: multiple schwannomas plus pain without vestibular tumor is characteristic of schwannomatosis; mosaicism for NF2 would be a less likely possibility. None of the other disorders is likely to present with multiple schwannomas. C74. The correct answer is D. Source: Neurogenetics lecture Keywords: epilepsy, genetic testing Explanation: The history is most compatible with benign neonatal convulsions, which are associated with mutations in a potassium channel encoding gene. C75. The correct answer is D. Source: Neurogenetics lecture Keywords: FXTAS, dementia, tremor Explanation: The clinical history is most compatible with fragile X ataxia tremor syndrome. C76. The correct answer is B. Source: Neurogenetics lecture Keywords: tuberous sclerosis complex, treatment, angiomyolipoma Explanation: everolimus is FDA approved for treatment of progressive angiomyolipoma associated with tuberous sclerosis complex 726 C77. The correct answer is D. Source: Neurogenetics lecture Keywords: neuromuscular, anticipation Explanation: The weakness, preserved reflexes, and occurrence of milder weakness in the mother are all suggestive of myotonic dystrophy. Charcot-Marie-Tooth would not present at this age and deep tendon reflexes would be absent in spinal muscular atrophy. Congenital muscular dystrophy would not explain the mother’s weakness. C78. The correct answer is C. Source: Systems Based Disorders II slides 7- 8 Key Words: pulmonary arterial pressure, heart failure Explanation: pulmonary arterial pressure of 33 mmHg and otherwise normal echocardiogram indicates pulmonary arterial hypertension (PAH). Maternal history of right heart failure during pregnancy suggests Heritable PAH. Slide 8 states that ~75% of HPAH cases are due to BMPR2 mutations and that ACVRL1, BMPR1B, EAG and SMAD9 are all rare causes of HPAH. C79. The correct answer is B Source: Systems Based Disorders II slide 6 and Newborn Screening slide 50 Key Words: CFTR, G551D, potentiates Explanation: Kalydeco (ivacaftor) is an allele specific drug that potentiates CFTR function in individuals who have G551D CFTR mutations. C80. The correct answer is C. Source: Systems Based Disorders II slides 10, 12 and 14-16 Key Words: recurring infections, onset 1-3 months, maternal uncle Explanation: Recurring infections beginning at 1 month, FTT, and maternal uncle who died of recurring infections all suggest XL SCID. IL2RG mutations are found in > 99% of males with SCID. ADA is AR, FOXP3 causes IPEX syndrome (XL but very different phenotype), STAT3 causes Hyper IgE (AD) and ZAP70 causes AR SCID. C81. The correct answer is D. Source: Systems Based Disorders II slides 10-12 and 15-16 Key Words: Recurring pneumonia, complicating meningitis, 12 years Explanation: Sammy’s onset of recurring Streptococcal, then Klebsiella pneumonia complicated by meningitis after 2 years of age is classic for CVID. TNFRSF13B (TAC1) is currently the gene that is most often found to bear mutations causing CVIB. TAC1 mutations are found in 10-15% and ICOS mutations in 1% of CVIB cases. ADA causes ADA Deficiency, IL2RG causes XL SCID, STAT3 causes Hyper IgE (AD) and ZAP70 causes AR SCID. 727 C82. The correct answer is E. Source: Systems Based Disorders II slides 41, 44-45 and 47 Key Words: Aortic root dilation Explanation: Pseudoxanthoma Elasticum frequently causes mineralization of the internal elastic lamina resulting in arterial narrowing but not aortic dilation. All the other answers are associated with aortic dilation. C83. The correct answer is A. Source: Systems Based Disorders II slides 50-53 Keywords: Multiple renal cysts, teenage Explanation: AD Polycystic Kidney Disease (PKD) is characterized by 3 or more (unilateral or bilateral renal cysts) in an individual aged 15-39 years. PKD1 mutations cause 85% of cases and PKD2 causes 15%. PKHD1 causes AR Polycystic Kidney Disease which presents in neonatal period. OCRL causes Lowe Syndrome which does not have renal cysts and is XL. TSC2 can be deleted with PKD1 resulting in PKD in utero & Tuberous Sclerosis but such contiguous TSC2- PKD1 deletions are rare. C84. The correct answer is E. Source: Systems Based Disorders II slides 22-23 Key Words: microcytic anemia, MCV=56, 5% Hb Bart. ([SODQDWLRQ+E+'LVHDVHLVXVXDOO\FDXVHGE\GHOHWLRQRIĮJORELQJHQHVDQGLWFDXVHV PLFURF\WLFDQHPLDRIWKLVVHYHULW\ZLWKa+E%DUWĮDQGȕWKDOWUDLWDQG+E(GRQRW cause Hb Bart. Hb Bart Syndrome is usually lethal before or just after birth and is associated with ~90% Hb Bart. C85. The correct answer is C. Source: Systems Based Disorders II slides 18 and 20 Key Words: Hematoma, oozing, prothrombin, von Willebrand, F9 Explanation: Prolonged oozing after trauma, normal prothrombin; and normal von Willebrand and F9 factor levels leave F8 deficiency or Hemophilia A as most likely problem. All the answers are known F8 mutations but IVS22 inversions are found in 48% of severe cases and often as new mutations. C86. The correct answer is B Keywords: ectodermal dysplasia Explanation: This is most likely to be a form of non-X-linked hypohydrotic ectodermal dysplasia, which could be associated with EDAR mutation. EDA1 is associated with the X-linked form; GJB6 with hydrotic ectodermal dysplasia; MSX1 with Witkop syndrome; SLC45A2 with oculocutaneous albinism. C87. The correct answer is A. Keywords: epidermolysis bullosa Explanation: Separation occurs below the basement membrane in dystrophic EB; it occurs in the epidermis in simplex EB, within the basement membrane in junctional EB, and at multiple layers in Kindler syndrome. 728 C88. The correct answer is A. Keywords: cardiomyopathy Explanation: Hypertrophic cardiomyopathy is usually autosomal dominant if it is due to a monogenic cause. C89. The correct answer is A. Ethan, and his brother, mother and maternal grandfather all had many fractures after mild trauma. This is consistent with and AD form of OI which is most likely OI Type 1 which is caused by mutations in COL1A1/2 in >90% of cases. IFITM5, LEPRE1, RPIB, and SERPINF1 are genes that cause AR forms of OI. C90. The correct answer is C This 8-year-old girl has had eczema since infancy and recurring boils and cyst forming pneumonia since 1 year of age but no significant problems with diarrhea. The triad of recurrent skin boils, cyst forming pneumonia and high serum IgE is classic for Hyper IgE syndrome. A. AR Severe Combined Immune Deficiency presents with recurrent infections, diarrhea, and Pneumocystis, but not cyst forming pneumonia. B. Common Variable Immune Deficiency presents with recurrent sinopulmonary infections and diarrhea, but not cyst forming pneumonia. D. Hyper IgM Syndrome presents with recurrent infections, diarrhea and neutropenia but not cyst forming pneumonia. E. XL Severe Combined Immune Deficiency presents with candidiasis, absent tonsils and persistent infections. It almost always only affects males. C91. The correct answer is A. He has “very soft and loose skin”, especially on his face, myopia, bilateral inguinal hernias, but normal joint mobility. This is most consistent with Cutis Laxa. B. Classic Ehlers Danlos Syndrome has hyper mobile joints. C. Loeys Dietz Syndrome doesn’t have soft skin or hernias. D. Marfan Syndrome doesn’t have soft skin or normal joint mobility. E. Pseudoxanthoma Elasticum does not have myopia or lax skin and has emphysema. C92. The correct answer is D. Ralph has chronic hip and knee pain and decreased ROM of his hips and knees. His mother had early hip and knee replacements. This is most consistent with Multiple Epiphyseal Dysplasia. A. Ehlers-Danlos syndrome has increased ROM of joints. B. Hypochondroplasia has short stature. C. Marfan Syndrome has scoliosis and infrequently requires hip or knee replacements. E. Osteogenesis Imperfecta presents with recurring fractures rather than chronic pain. C93. The correct answer is D. SOURCE: Lecture/Slides KEYWORDS: Ectodermal dysplasia 729 EXPLANATION: The child’s abnormal teeth and nails, with normal skin, hair, and sweating, are compatible with Witkop syndrome, associated with MSX1 mutations. EDA and EDAR are both associated with hypohidrotic ectodermal dysplasia. GJB6 is associated with Clouston syndrome, which leads to hyperkeratosis and abnormal hair in addition to abnormal nails. WNT10A is associated with Odonto-Onycho-Dermal Dysplasia, which also leads to hair and skin abnormalities. C94. CORRECT ANSWER: A SOURCE OF ITEM TOPIC: Lecture/Slides KEYWORDS: Amenorrhea, blind vagina and testosterone. EXPLANATION: Complete androgen insensitivity is associated with 46, XY; absent or rudimentary müllerian structures, normal or elevated testosterone , dihydrotestosterone & luteinizing hormone and it is XL due to AR variants. CAH would have high 17-OHP levels, LH deficiency causes low testosterone levels, Turner syndrome and 46, XY sex reversal females, SRY related should not have a blind vagina or absent uterus. C95. CORRECT ANSWER: A SOURCE: Lecture/Slides KEYWORDS: Hypophosphatasia EXPLANATION: The child’s short stature, rachitic skeletal changes and a low serum ALP are all typical of Hypophosphatasia and craniosynostosis is often found in the perinatal or infantile form. Hypercalcemia, hypercalciuria, low bone density and short stature are typical rather than hypocalcemia, hypocalciuria, osteoporosis and tall stature. C96. CORRECT ANSWER: A SOURCE: Lecture/Slides KEYWORDS: epidermolysis bullosum EXPLANATION: Dystrophic epidermolysis bullosum with scarring in the dermis can be AD or AR and is associated with COL7A1 mutations. LAMB3 is associated with junctional EB, where scarring is at the basement membrane, and the others with EB simplex, with scarring in the basal layer. C97. CORRECT ANSWER: A SOURCE: Lecture/Slides KEYWORDS: Microcephaly, telecanthus, coarse facies, genital anomalies, hypotonia, intellectual disability & thalassemia. EXPLANATION: ATRX pathogenic variants cause Alpha Thalassemia XL Intellectual Disability Syndrome. The other genes encode alpha, beta or gamma globin. C98. CORRECT ANSWER: C SOURCE OF ITEM TOPIC: Lecture/Slides KEYWORDS: Russell-Silver syndrome, imprinting EXPLANATION: Hypomethylation of the paternal IC1 site explains about 45% of cases of Russell-Silver syndrome. Hypomethlation leads to blockage of expression of the IGF2 locus. The other mechanisms are all associated with Beckwith-Wiedemann syndrome. 730 D. Neurological Genetics Questions D1-D40 D1. A 10-year-old boy visits an ophthalmologist for the first time after complaining to his parents that he is having trouble seeing the interactive whiteboard at school. The ophthalmologist finds a mild refractive error, but is most concerned about finding Lisch nodules. The presence of iris Lisch nodules is a helpful diagnostic feature of which of the following syndromes? A. B. C. D. E. D2. A young girl with new onset seizure disorder is examined by a neurologist and found to have non-traumatic periungual and subungual fibromas. These clinical features are a primary diagnostic feature of which of the following syndromes? A. B. C. D. E. D3. Gardner syndrome Neurofibromatosis type 1 Sturge-Weber syndrome Tuberous sclerosis complex von Hippel-Lindau disease A 30-year-old woman with a complicated medical history dies of renal cell carcinoma. Renal cell carcinoma is a primary cause of death in which of the following syndromes? A. B. C. D. E. D4. Gardner syndrome Neurofibromatosis type 1 Sturge-Weber syndrome Tuberous sclerosis complex von Hippel-Lindau disease Gardner syndrome Neurofibromatosis type 1 Sturge-Weber syndrome Tuberous sclerosis von Hippel-Lindau disease Two sisters are affected with cataracts in infancy, intellectual impairment, microcephaly, nystagmus, and moderate growth deficiency. Which of the following syndrome is the most likely diagnosis? A. B. C. D. E. Abetalipoproteinemia Ataxia telangiectasia Friedreich ataxia Marinesco-Sjogren syndrome Type 3 Gaucher disease 731 D5. A 40-year-old man presents with pain in his leg associated with a palpable mass. Biopsy reveals a schwannoma. He previously had a schwannoma removed from his arm. An MRI of the brain shows no abnormality and an ophthalmological examination is normal. There is no known family history of similar problems. Molecular analysis of which one of the following genes would be most likely to reveal a mutation that might explain his phenotype? A. B. C. D. E. D6. A 45 year old presents with ringing in the ears and hearing loss. He is found to have a vestibular schwannoma. Concerned that he might have NF2, you arrange an eye examination. Which of the following would be supportive of this diagnosis? A. B. C. D. E. D7. maternal inheritance of the CAG repeat expansion maternal uniparental disomy of the CAG repeat expansion new mutation of the CAG repeat expansion paternal inheritance of the CAG repeat expansion paternal uniparental disomy of the CAG repeat expansion Which of the following disorders is associated with duplication of gene on chromosome 17? A. B. C. D. E. D9. Lisch nodules optic atrophy papilledema posterior subcapsular cataract thickened corneal nerve An eight-year-old girl develops signs and symptoms of Huntington disease, presenting with rigidity. Early onset is most likely the result of which of the following genetic abnormalities? A. B. C. D. E. D8. NF1 NF2 SPRED1 INI1 PTPN11 Charcot-Marie-Tooth disease Hereditary liability to pressure palsies Miller-Dieker syndrome NF1 Spinal muscular atrophy Which of the following is the most common mutation responsible for infantile spinal muscular atrophy? A. B. C. D. E. gene conversion of SMNT to SMNC deletion of NAIP gene deletion of SMNT gene point mutation of SMNT gene duplication of SMNC gene 732 D10. Which of the following genes includes a polymorphism that is associated with risk of Alzheimer disease? A. B. C. D. E. D11. You are seeing a child with tuberous sclerosis and examine both parents. The child's mother is found to also have hypopigmented macules. Genetic testing is performed and reveals a TSC2 mutation in both mother and child. The mother reports being in good health, with no history of seizures, normal development, and normal activity. Which of the following tests is most likely to be normal even if she does have tuberous sclerosis complex? A. B. C. D. E. D12. Basal cell nevus syndrome NF1 NF2 TSC1 TSC2 The most common autosomal dystonia 1 (DYT1) mutation is found in which ethnic group? A. B. C. D. E. D14. brain MRI pulmonary CT ophthalmological examination echocardiogram renal ultrasound Which of the following disorders is most likely to be associated with renal cysts? A. B. C. D. E. D13. amyloid precursor protein apoB-48 apoE presenilin prion protein African-Americans Ashkenazi Jews Finns Mennonites Southeast Asians Pantothenate kinase associated neurodegeneration is associated with basal ganglia accumulation of which of the following metals? A. B. C. D. E. Calcium Copper Iron Magnesium Zinc 733 D15. The triplet repeat expansion responsible for Huntington disease is located in which of the following areas of the Huntingtin gene? A. B. C. D. E. . D16. Individuals with CADASIL are at risk for which of the following complications? A. B. C. D. E. D17. abnormal methylation chromosomal abnormality gene conversion single base pair mutation triplet repeat expansion The CTG repeat expansion responsible for myotonic dystrophy affects which of the following molecular processes for the DMPK gene or gene product? A. B. C. D. E. D19. Berry aneurysm cavernous hemangiomas hemangioblastomas multiple strokes stroke-like episodes A newborn has weakness, hypotonia, absent reflexes, and tongue fasciculations. Which of the following genetic mechanisms is most likely responsible for this clinical presentation? A. B. C. D. E. D18. at an intron-exon border in an exon in an intron in the 3’ untranslated region near the promoter Level of transcription protein degradation in the proteosome RNA binding protein RNA editing RNA splicing A 55-year-old man is seen in Neurology Clinic because of unsteady gait. On examination he is found to have ataxia and a tremor. Brain MRI reveals white matter lesions in the basal ganglia. The patient has no children, but he has a sister who has a son with impaired cognitive development. Genetic testing of the patient is most likely to reveal which of the following findings in the FMR1 gene? A. B. C. D. E. 37 CAG repeats 45 CAG repeats 60 GAA repeats 75 CGG repeats 250 CGG repeats 734 D20. The most likely mutation in an individual with hereditary neuropathy with predisposition to pressure palsies is the result of which of the following molecular mechanisms? A. B. C. D. E. D21. A 17-year-old man is evaluated for weakness in the lower extremities that has been gradually getting worse over the past 3 years. He is found to have pes cavus and his knee and ankle jerk reflexes cannot be elicited. His exam is otherwise normal. Family history reveals that his father was affected similarly. Which of the following diagnoses is most likely to explain his clinical findings? A. B. C. D. E. D22. A cleft in the brain A double band of cortex Lack of cerebral gyri Large numbers of small cerebral gyri Small numbers of large cerebral gyri Mutations in the D-synuclein gene are most likely to result in which of the following neurologic findings? A. B. C. D. E. D24. Charcot-Marie-Tooth disease Friedreich ataxia Hereditary amyotrophic lateral sclerosis Limb-girdle muscular dystrophy Myotonic dystrophy Lissencephaly is characterized by which of the following anatomic abnormalities? A. B. C. D. E. D23. Single base change Deletion Duplication Inversion Imprinting change Ataxia Dementia Spasticity Tremor Weakness A 60-year-old man presents to neurology clinic with a history of late-onset, progressive cerebellar ataxia and intention tremor. A family history reveals his daughter has a son with fragile X syndrome. Which of the following alterations in the FMR1 gene is most likely to be found in this patient? A. B. C. D. E. Point mutation 30 CGG repeats 80 CGG repeats 250 CGG repeats Deletion of the FMR1 gene 735 D25. You see a couple for genetic counseling because of a family history of Duchenne muscular dystrophy. See pedigree below. She is now 30 weeks pregnant and ultrasound reveals a male; she asks whether it will be possible to test her son at birth for the disorder. Which of the following is the best way to establish whether the child is affected at birth? A. B. C. D. E. D26. Which of the following neuromuscular diseases is associated with deletion of a repeated sequence in the chromosome 4q subtelomeric region? A. B. C. D. E. D27. physical examination testing the dystrophin gene for intragenic deletions sequencing the dystrophin gene muscle biopsy serum creatine phosphokinase testing Charcot-Marie-Tooth Disease Congenital fiber type disproportion Duchenne muscular dystrophy Facio-scapulo-humeral muscular dystrophy Limb girdle muscular dystrophy Patients with basal cell nevus syndrome are most likely to develop which of the following brain tumors? A. B. C. D. E. Glioblastoma Hemangioblastoma Medulloblastoma Optic glioma Pilocytic astrocytoma 736 D28. Which of the following disorders results from a triplet repeat expansion in the 3’ untranslated region of the causative gene? A. B. C. D. E. D29. An individual at risk for Huntington disease is found to have 40 CAG repeats. Which of the following allele types best describes the implications of this result? A. B. C. D. E. D30. Fragile X syndrome Friedreich ataxia Huntington disease Myotonic dystrophy Spinocerebellary ataxia Disease allele Intermediate allele Normal allele Reduced penetrance allele Polymorphic allele Which of the following is associated with mutation in a GTPase activating protein? A. B. C. D. E. NF1 NF2 TSC1 TSC2 Schwannomatosis D31. Which of the following movement disorders is associated with a GAG deletion? A. B. C. D. E. D32. Parkinson disease Huntington disease Spinocerebellar ataxia Tourette syndrome Torsion dystonia Notch3 gene mutations cause which of the following neurologic complications? A. B. C. D. E. Ataxia Deafness Dystonia Seizures Strokes 737 D33. First-time parents report that their 8-month-old infant son has recurrent vomiting episodes, feeding problems and does not appear to shed tears when upset and crying. Which of the following neurologic disorders is the most likely diagnosis? A. B. C. D. E. D34. Myasthenia gravis affects which of the following components of the neuromuscular system? A. B. C. D. E. D35. Central core myopathy Duchenne muscular dystrophy Myotonic dystrophy Nemaline myopathy Spinal muscular atrophy A 7-year-old with NF1 develops an acute left hemiparesis. Which of the following clinical findings is the most likely cause of acute stroke in NF1? A. B. C. D. E. D37. Cardiac muscle Extracellular matrix surrounding muscle Muscle cytoskeleton Neuromuscular junction Sarcomere A massively elevated CK (creatine kinase) level is found in a 4-year-old boy with progressively worsening gross motor delay. This finding is typical for which of the following neuromuscular disorders? A. B. C. D. E. D36. Charcot-Marie Tooth disease Familial dysautonomia Friedreich ataxia Hereditary dystonia Von Hippel Lindau syndrome Blood clotting abnormality Cardiac rhabdomyoma Embolus from cardiac arrhythmia Moyamoya syndrome Optic glioma You are following a 30-year-old pregnant woman with von Hippel-Lindau syndrome. Which of the following findings is the most important to monitor her for during the pregnancy? A. B. C. D. E. Endolymphatic sac tumor Pheochromocytoma Renal Cancer Retinal hemangioblastoma Vestibular schwannoma 738 D38. A 50 year old man presents with pain and is found to have multiple schwannomas. A brain MRI shows no evidence for vestibular schwannoma. Which pair of genes would be appropriate to test to determine whether he might have schwannomatosis? A. B. C. D. E. D39. A 50-year-old woman has a direct-to-consumer genetic test that reveals she is homozygous for the E4 allele of ApoE. Which of the following conditions has the highest relative risk of occurring given this result? A. B. C. D. E. D40. NF1 and NF2 NF2 and TSC2 NF1 and TSC2 TSC2 and LZTR1 SMARCB1 and LZTR1 Alzheimer disease Ataxia telangiectasia Hereditary spastic paraplegia Parkinson disease von Hippel-Lindau syndrome After his younger sister (III-2) was diagnosed as having Gaucher disease, a 25-year-old man (III-I) is found to be carrier for Gaucher disease as seen in the pedigree below. Which of the following conditions has the highest relative risk given his carrier status? A. Alzheimer disease B. Charcot-Marie Tooth Disease C. Myotonic Dystrophy D. Parkinson Disease E. PKAN Associated Neurodegeneration D41. A 40-year-old man presents with ataxia and MRI reveals a cerebellar hemangioblastoma. He is also found to have a retinal hemangioblastoma by ophthalmological exam and sensorineural hearing loss by audiology. Which of the following lesions is the most likely to be responsible for his hearing loss? A. B. C. D. E. brainstem hemangioblastoma endolymphatic sac tumor glioma meningioma vestibular schwannoma 739 Answers to Neurological Genetics Questions D1-D40 D1. Answer: B. Lisch nodules are a very helpful clinical feature of neurofibromatosis type 1 and are an important part of family evaluations. D2. Answer: D. The primary diagnostic criteria for tuberous sclerosis include cortical hamartomas, multiple retinal hamartomas, and angiofibromas or periungual fibromas. Other criteria include infantile spasms, hypomelanotic patches, single retinal hamartoma, subependymal or cortical calcifications, multiple renal tumors, cardiac rhabdomyoma, and first-degree relative with TS. D3. Answer: E. The von Hippel-Lindau disease has the most significant association with renal cell carcinoma. Retinal angiomas, angiomas of the cerebellum, and pheochromocytomas are other features. D4. Answer: D. This is a typical clinical description for Marinesco-Sjogren syndrome. Ataxia telangiectasia would be characterized by the telangiectasia and the absence of cataracts. The onset would be later with less mental impairment early on. Friedreich ataxia would have a later onset and not be associated with many of the findings listed here. Type 3 Gaucher disease would not be associated with cataracts or early growth deficiency and would likely have associated splenomegaly. Abetalipoproteinemia would have a quite different presentation with none of these dysmorphic features. D5. Answer D. The occurrence of an multiple schwannomas presenting with pain is most typical of schwannomatosis, associated with INI1 mutation. Schwannomas could also be associated with NF2, but one would expect bilateral vestibular schwannomas and cataract in individuals with NF2. D6. Answer: D. Posterior subcapsular cataract is characteristic of NF2; other features listed are typical of NF1 (papilledema is optic nerve swelling in the setting of increased intracranial pressure). D7. Answer: D. Paternal inheritance in some cases is associated with the juvenile presentation of Huntington disease, with rigidity. D8. Answer: A. Chariot-Marie Tooth disease is associated with PMP22 duplication; hereditary neuropathy with liability to pressure palsies is associated with deletion of this gene. 740 D9. Answer: C. Deletion of the SMNT gene is associated with SMA; sometimes the mutation consists of gene conversion of SMTC to SMNT D10. Answer: C. D11. Answer D. Adults with tuberous sclerosis complex are unlikely to have cardiac rhabdomyoma, since this lesion tends to regress with time. The other studies are indicated for screening, even in the absence of signs or symptoms. D12. Answer: E. Renal cysts occur when a contiguous deletion occurs between the TSC2 and PKD1 genes. D13. Answer: B. D14. Answer: C. D15. Answer: B. The triplet repeat is within an exon and encodes a polyglutamine tract in the protein. D16. Answer: D. D17. Answer: C. This is a description of spinal muscular atrophy, which in some cases is due to a gene conversion event of SMNC to SMNT D18. Answer: C. D19. Answer D. The patient has signs of FXTAS (fragile X tremor-ataxia syndrome), associated with premutation expansion of the CGG repeat in the FMR1 gene. D20. Answer: B. Hereditary neuropathy with predisposition to pressure palsies is due to PMP22 deletion; Charcot-Marie-Tooth disease occurs when the gene is duplicated. D21. Answer A. Pes cavus and lack of deep tendon reflexes is suggestive of peripheral neuropathy, and the family history suggests dominant inheritance. Friedreich ataxia would produce other signs and is recessive; the exam is not suggestive of the other diagnoses. D22. Answer: C. Lissencephaly is characterized by a smooth brain surface without gyri. Polymicrogyria is the occurrence of a large number of small gyri; pachygyria is the occurrence of a small number of large gyri; schizencephaly is the occurrence of a cleft in the brain. 741 D23. Answer: D. D-synuclein mutations result in an inherited form of Parkinson disease, which is characterized by tremor, rigidity, and bradykinesia. D24. Answer: C. Fragile X tremor/ataxia syndrome occurs in males with FMR1 premutation alleles. D25. Answer E. Creatine phosphokinase levels will be elevated at birth for a boy with Duchenne muscular dystropy, well before muscle weakness will be apparent. Genetic testing could be done, but the family mutation is not known, so a negative test will be difficult to interpret, and should not be necessary. Physical exam will be normal at this age and muscle biopsy, besides being invasive, will likely miss pathology this early in life D26. Answer: D. FSH dystrophy is due to deletions of a repeat region in the 4q subtelomere. D27. Answer: C. Optic gliomas (which are pilocytic astrocytomas) are characteristic of NF1; hemangioblastomas are seen in von Hippel Lindau syndrome. D28. Answer: D. Myotonic dystrophy is due to a CTG repeat expansion in the 3’ untranslated region of the DMPK gene. The expansion for fragile X occurs in the promoter region; for Friedreich ataxia in an intron; for Huntington disease and spinocerebellar ataxia in exons. D29. Answer: A. Normal alleles are 10-26 repeats; intermediate alleles 27-35 repeats; disease alleles are >36 repeats with reduced penetrance from 36-39. D 30. Answer: A. The NF1 gene product, neurofibromin, is a GTPase activating protein for Ras. D31. Answer E. Torsion dystonia is most commonly associated with GAG deletion in the DYT1 gene. D32. Answer: E. Notch3 mutations are associated with CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). D33. Answer B. Familial dysautonomia includes absent tearing, autonomic neuropathy, episodic vomiting, feeding disorder, and absent fungiform papillae. D34. Answser: D. Myasthenia gravis is a disorder of the neuromuscular junction; usually it is autoimmune, but there are rare inherited forms. D35. Answer: B. Duchenne and Becker dystrophies are associated with major CPK elevations. The other disorders either do not cause elevation, or the elevation is typically minor. D36. Answer: D. Keywords: NF1, stroke, vascular Explanation: Individuals with NF1 are at increased risk of internal carotid occlusion and associated moyamoya syndrome. 742 D37. Answer B. Keywords: von Hippel-Lindau syndrome Explanation: Women with VHL should be followed during pregnancy for development of pheochromocytoma. The other matters are not specifically critical during pregnancy, and vestibular schwannoma is not a feature of VHL. D38. Answer E. Keywords: neurofibromatosis, schwannomoatosis Explanation: SMARCB1 and LZTR1 are both associated with schwannomatosis. Schwannomas can be found in NF2 patients, but usually in association with vestibular schwannomas and other features. TSC2 is associated with tuberous sclerosis complex, not schwannomatosis. D39. CORRECT ANSWER: A SOURCE OF ITEM TOPIC: Lecture/Syllabus KEYWORDS: Alzheimer disease, ApoE EXPLANATION: The ApoE E4 allele is associated with an increased risk of Alzheimer disease. None of the other conditions is associated with the ApoE4 genotype. D40. CORRECT ANSWER: D SOURCE OF ITEM TOPIC: Lecture/Slides KEYWORDS: Gaucher, glucocerebrosidase, Parkinson disease EXPLANATION: Having one N370S glucocerebrosidase allele has been indicated as a risk factor for Parkinson disease. None of the other conditions is associated with carrier status for glucocerebrosidase. D41. CORRECT ANSWER: B SOURCE OF ITEM TOPIC: Lecture/Slides KEYWORDS: von Hippel-Lindau, hearing loss EXPLANATION: The presence of cerebellar and retinal hemangioblastoma is indicative of von Hippel-Lindau syndrome, which is also associated with endolymphatic sac tumor, that can cause hearing loss. A brainstem hemangioblastoma is not likely to cause hearing loss, though it could occur in VHL. The other tumors are not associated with VHL; vestibular schwannoma is typical for NF2. 743 E. Molecular Genetics Questions E1-E79 E1. You have cloned a candidate gene for an autosomal recessive disorder. You have identified a dinucleotide repeat polymorphism in intron 1 of the gene. Solid symbol is affected. Three alleles for the repeat are found in this family. Which of the following assessments are you most likely to provide regarding the likelihood that the gene you identified is the disease gene in this family? A. B. C. D. E. Very encouraging Slightly encouraging No evidence either way Slightly discouraging Virtually rule it out 1 2 3 E2. The diagram below represents linkage analysis for an autosomal dominant disorder where the grandfather is deceased. A dinucleotide repeat with a marker within the disease gene is shown with three alleles. What is the probability that the fetus (P) is affected by this disease? A. B. C. D. E. 1.0 0.75 0.50 0.25 0.0 P 1 2 3 E3. For this CF family, DNA results are as indicated. Assume analysis for 20 mutations with detection of 90% CF mutant chromosomes. Neg = negative for 20 mutations. Assume carrier frequency of 1 in 25. Which of the following probabilities represents the likelihood that individual #6 is a carrier based on the data shown? A. B. C. D. E. 1 1in 7 1 in 11 1 in 16 1 in 20 1 in 24 5 2 6 Neg 744 3 4 7 E4. For this CF family, DNA results are as indicated. Assume analysis for 20 mutations with GHWHFWLRQRI&)PXWDQWFKURPRVRPHVǻ) ǻ)1HJ QHJDWLYHIRUPXWDWLRQV8. = unknown (untested) CF allele, Nl = presumptive normal allele. Assume carrier frequency of 1 in 25. What is the probability that individual #6 is a carrier based on the data shown? A. B. C. D. E. 1 1 in 2 1 in 4 1 in 9 1 in 10 1 in 11 5 2 3 6 Neg 4 7 'F/Nl UK/Nl 'F/UK E5. A dinucleotide repeat analysis for a polymorphism within a gene is analyzed for an autosomal recessive disorder as shown below. What is the probability that the pregnancy (P) is affected? A. B. C. D. E. 1.0 0.75 0.50 0.25 0.0 P 1 2 3 E6 A 20-year-old male presents with proximal muscle weakness but is ambulatory, has frequent falls, and has significant fatigue during mild exercise. SMN1 deletion analysis is ordered, and testing is done using quantitative PCR analysis. The results indicate that the patient has one SMN1 allele detected. Which of the following rationales provides the best plan of action in response to this result? A. The patient has a both a gene deletion and a point mutation (or small deletion/insertion) and should have SMN1 gene sequence analysis. B. The patient has juvenile amyotrophic lateral sclerosis and would benefit by testing ALS2 gene. C. The patient has X-linked spinal muscular atrophy and should be tested for a mutation in UBA1 gene. D. The patient is deleted for both copies of SMN2 and should be tested by MLPA to more accurately assess number of gene copies. E. The patient is deleted for both SMN1 genes but has a false negative test due to interference of multiple copies of the pseudogene SMN2 an no additional testing is warranted. 745 E7. Fred is an 8-year-old boy referred because his physician was concerned he might have Fragile X Syndrome (FXS). The lab report of Fred’s FXS study says that his DNA yielded hybridizing fragments of 5.2 and 2.8 kbs indicating methylated and unmethylated FMR1 alleles. Which of the following explanations accounts for these test results? A. B. C. D. E. E8. DNAs from the patient and another individual were swapped The patient has a normal FXS test result The patient has Klinefelter Syndrome The patient is mosaic for Fragile X Syndrome The restriction endonuclease digestion was incomplete A DNA analysis using a dinucleotide repeat within a gene for an autosomal dominant disorder is presented below. The father and deceased grandfather are affected. . What is the probability that the pregnancy (P) is affected? A. B. C. D. E. 1.0 0.75 0.5 0.25 0.0 P 1 2 3 E9. This is a family with X-Iinked Duchenne muscular dystrophy. A Southern blot is depicted using a fragment of the cDNA for the gene as a probe. Bands indicate presence or absence with no attempt to distinguish intensity (dosage). Assume no gonadal mosaicism. Which of the following conclusions can be drawn about this family? A. B. C. D. E. It is uncertain if the mother is a carrier or not The mother and only the mother is a carrier The mother and the grandmother are carriers The mother is definitely not a carrier The mother, the grandmother and the aunt are all carriers. Normal male and female pattern 746 E10. Protein truncation testing is best suited for detecting which of the following types of mutations? A. B. C. D. E. E11. triplet repeat expansions. promoter mutations. missense mutations. large deletions. frameshift mutations. A researcher has a candidate gene for an autosomal recessive disorder and studies a dinucleotide repeat within intron 1 in the family depicted below. What are the odds favoring that this is the disease gene relative to the chances of a result such as this favoring linkage occurring by chance? A. B. C. D. E. 16 to 1 32 to 1 64 to 1 128 to 1 256 to 1 AB AC E12. AC AC AC The R117H mutation (Arg to His at position 117) in the cystic fibrosis gene is associated with considerable phenotypic heterogeneity. This is explained at least in part by which of the following types of polymorphism? A. B. C. D. E. E13. AC CD A polymorphism involving a splice site. A polymorphism involving the polyadenylation site. A polymorphism involving the promoter. A polymorphism involving the signal sequence. A polymorphism mapping to a locus other than CFTR. Mary is 35-year-old and has a 4-year-old boy with developmental delay and large ears. She has 6year-old daughter with attention deficit hyperactivity disorder. Mary is being seen by her OB for ovarian insufficiency. Her father at age 61 has recently developed tremors. Which of the following explanations accounts for the clinical information found in this family history? A. Her father and children have a point mutation in the FMR1 gene that shows variable expressivity. B. Her father and children have unrelated disorders, Parkinson disease and nonspecific developmental delay. C. Her father and her daughter have small CTG expansions in the premutation range, while her son has a full CTG expansion, demonstrating anticipation. D. Her father carries a premutation of >80 CGG repeats and has FXTAS, while the son has inherited an expansion of >200 CGG repeats and has Fragile X. E. Her father carries a reduced penetrance CAG repeat expansion, explaining his milder symptoms and later age of onset. 747 E14. A patient’s malignant melanoma biopsy specimen is tested for the BRAF V600E mutation to guide chemotherapy. Which of the following findings would be the most likely cause of a false negative result? A. B. C. D. E. E15. The genetic length of the human genome is best represented by which of the following pairings? A. B C D. E. E16. 8800 cM for male map and 2700 for female map. 8800 cM for male map and 5400 for female map. 8800 cM for female map and 5400 for male map. 4400 cM for male map and 2700 for female map. 4400 cM for female map and 2700 for male map. A woman has a brother and son affected with Duchenne dystrophy. No deletion could be found, so linkage analysis was used to test her current pregnancy (P) which is a male fetus. An STR at the 5' end of the gene and another at the 3' end of the gene were tested as shown below. What is the best estimate of the probability that the fetus is affected? A. B. C. D. E. E17. A chromosomal deletion A chromosomal duplication A low level of somatic mosaicism A sample mix up A trinucleotide repeat expansion close to 0% close to 25% close to 50% close to 75% close to 100% P 5’STR alleles 3’STR alleles 1 5 1/2 5/6 1 5 1 6 Mutations can affect the function of a gene in a number of ways. Which of the following is the term used for a mutation that causes substitution of a single amino acid? A. B. C. D. E. frameshift in-frame deletion missense nonsense splicing 748 E18. ȕ-thalassemia and cystic fibrosis are autosomal recessive disorders in which a large number of mutations have been discovered. Which of the following characteristics of a gene is most useful in designing a sensitive mutation assay? A. B. C. D. E. E19. Indirect detection of an abnormal gene can be used for diagnosis in situations where the diseaseproducing mutation is unknown. Which of the following characteristics of a DNA marker is most important for accuracy in linkage studies? A. B. C. D. E. E20. can be detected using PCR highly polymorphic low recombination rate with disease locus physical distance from disease gene is known polymorphisms are absent at the marker site Which of the following investigative assays would be the most appropriate method to use for testing 50 variants including point mutations, deletions and insertions in 10 different genes as a screening test for the reproductive population? A. B. C. D. E. E21. distribution frequency of mutations among different ethnic/racial groups distribution frequency of mutations among exons frequency of missense, nonsense, frameshift and splicing mutations frequency of single nucleotide polymorphisms with a gene. frequency of structural rearrangements (e.g.insertions/deletions) Fluorescence Resonance Energy Transfer Liquid Bead Array Multiplex Ligation-dependent Probe Amplification Next generation sequencing Sanger sequencing You have clinically diagnosed achondroplasia in an infant whose parents are unaffected. You submit the infant’s DNA for molecular analysis. The laboratory reports an FGFR3 GLY380ARG (GGG to AGG) substitution due to an 1138G-A change. This change was reported in 96% of 17 sporadic and 6 familial cases of achondroplasia studied. Which of the following mechanisms is most likely to have caused this change in the DNA? A. B. C. D. E. A trinucleotide repeat expansion Gene conversion Methylation of a CG Recombination Slipped misspairing 749 E22. In order to test for the homozygous deletion in patients affected with SMA which of the following procedures must be accomplished initially? A. B. C. D. E. E23. Which of the following is the most accurate test for the diagnosis of Fragile X syndrome? A. B. C. D. E. E24. Allele specific amplification Chemical cleavage Protein truncation Reverse dot blotting SSCP Which of the following characteristics is the most likely mechanism of action of the triplet repeat expansion in Huntington disease? A. B. C. D. E. E26. Allele specific oligonucleotides Allele specific PCR Protein truncation Quantitative PCR Southern blotting Which of the following molecular methods is a non-gel based assay for the detection of point mutations? A. B. C. D. E. E25. perform multiplex PCR for SMN exons 1-4 distinguish the telomeric (SMN1) SMN gene from the centromeric SMN (SMN2) gene perform a dosage assay on the SMN1 gene use Southern blotting to identify the common SMN1 junction fragment determine a loss of heterozygosity using closely linked markers inside the SMN1 gene affects the overall chromatin configuration decreases gene methylation inhibits gene expression inhibits protein translation works by a gain of function Advantages of the microsatellite repeats compared to restriction fragment length polymorphisms for indirect testing include which of the following characteristics? A. B. C. D. E. Microsatellites repeats have negligible recombination errors Microsatellites repeats are intronic Microsatellites repeats are multi-allelic Microsatellites are invariable in length Microsatellites repeats are amenable to PCR detection 750 E27. A 50-year-old woman with aplastic anemia was found to have a nonsense and a silent mutation in the FANCA gene, confirming the diagnosis of Fanconi anemia. All of her siblings were tested for both mutations as potential bone marrow donors for transplant. Her asymptomatic older sister was found to have both mutations in her fibroblasts but only the silent mutation in her blood. Which of the following explanations provides the most likely reason for this finding? A. A gene conversion event has occurred in the asymptomatic sister’s fibroblast cells. B. The asymptomatic sister has had a reversion event in her bone marrow, explaining the difference in blood. C. The asymptomatic sister is not affected with Fanconi anemia and therefore no further monitoring is required. D. The diagnosis is incorrect, because the silent mutation does not change an amino acid in the protein and thus, it is really a benign polymorphism. E. There is a lab error in her sister’s blood test result because these are germline variants and all tissues within each individual will carry the same variants. E28. You submitted a DNA sample to determine if there is a mutation in an arginine residue “hotspot” in the NRAS gene. Which of the following alterations is most likely to result from a substitution that changes this arginine codon from AGG to ATG? A. B. C. D. E. E29. Which of the following codons each result the in termination of translation? A. B. C. D. E. E30. AUG & UAA AUG, UAA, UGA & UAG UUU, UAG, UGA & UAA UUU, UAA & UAG UGA, UAA & UAG Which of the following genes is commonly associated with intragenic inversion? A. B. C. D. E. E31. Creates a frameshift downstream Creates a nonsynonymous substitution Creates a start codon Creates a synonymous substitution Creates a termination codon Factor VIII deficiency Factor IX deficiency Factor XI deficiency Von Willebrand disease Factor V Leiden Which of the following events typically occurs when ultraviolet light damages DNA? A. B. C. D. E. Double strand breaks. Purines dimerize within a strand Purines dimerize between strands. Pyrimidines dimerize within a strand Single strand breaks. 751 E32. You have performed linkage analysis using restriction fragment length polymorphisms (RFLPs) to follow the inheritance pattern of defective beta-globin genes in a family with beta-thalassemia (shown below). Assuming that the RFLP does not show recombination with the beta-globin locus, what is the most likely diagnosis for the daughter (indicated by the open circle) who is at risk for this autosomal recessive disorder? A. B. C. D. E. E33. The patient has a fully penetrant HD allele. The patient has a nonpenetrant allele, ruling out the diagnosis of HD. The patient has a reduced penetrance allele, confirming the diagnosis of HD. The patient’s personality disorder is caused by another gene defect. This result is normal so another disorder must be responsible. Mrs. Soft has a history of multiple fractures, mild short stature and hearing loss. She brings her two-month-old son, Carl, to you for evaluation. Carl has no history of fractures. He has a normal physical exam. Which of the following evaluations is the most accurate way to determine if Carl has the same condition as his mother? A. B. C. D. E. E35. ? A 54-year-old woman with mild neurological symptoms of ataxia and personality disorder was found to have a 38 CAG repeat in the huntingtin (HTT ) gene. Which of the following explanations provides the most likely reason for this finding? A. B. C. D. E. E34. Daughter is heterozygous for beta thalassemia Daughter is heterozygous for a beta globin gene deletion Daughter is homozygous normal Pattern indicates non-paternity The results are not informative Determine if Carl has a mutation in his COL1A1 or COL1A2 genes Determine if Carl has an abnormal ABR or OAE test Determine if Carl’s skull radiographs demonstrate Wormian bones Determine if Mrs. Soft and Carl share a COL1A1/COL1A2 mutation Determine if radiographs of Carl’s extremity bones show fractures Core promoter elements that direct transcription include which of the following motifs? A. B. C. D. E. CAAT and Acceptor splice sequences CAAT box and Initiator sequence CAAT and TATA boxes and Poly-A addition signal TATA box and Branch point consensus TATA boxes and Donor splice sequences 752 E36. Trans-acting transcriptional regulatory sequences include which of the following DNA components? A. B. C. D. E. E37. PCR is most useful for which of the following DNA analyses? A. B. C. D. E. E38. 1:200 1:500 1:1000 1:1500 1:2000 A male is found to have 400 CGG repeats. Which of the following disorders is the most likely diagnosis? A. B. C. D. E. E40. RFLP analysis VNTR analysis Short Tandem Repeat (STR) analysis CGG full expansion analysis in males CTG full expansion analysis in females A disorder is recessive and has a carrier frequency of 1:100 in Ashkenazi Jews. The test includes one mutation and the test sensitivity is estimated at 90%. What is the probability that an Ashkenazi Jewish individual with a negative test result is a carrier for this disorder? A. B. C. D. E. E39. DNA methylation of CpG dinucleotides CAAT boxes Promoter enhancers Transcription factors Response elements Fragile X syndrome Friedreich ataxia FXTAS Huntington disease Myotonic dystrophy A male is found to have 46 CAG repeats. Which of the following disorders is the most likely diagnosis? A. B. C. D. E. Fragile X syndrome Friedreich ataxia FXTAS Huntington disease Myotonic dystrophy 753 E41. Your lab is testing a patient, with brain iron accumulation on MRI showing the “eye of the tiger” sign, for the PANK2 gene using sequence analysis and identified a missense variant in exon 3 along with a small insertion in exon 5. The PANK2 database shows the insertion to be pathogenic but has no information about the missense variant. Both Polyphen and SIFT predict the missense mutation to be possibly damaging. The predicted amino acid change is from serine to phenylalanine. dbSNP lists an rs number with unknown frequency for the missense variant. 1000 Genomes indicates a frequency of 0.001 for the missense variant. There are no deletions or duplications detected by MLPA. There are no other symptomatic individuals in the family. Which of the following interpretations would best explain these results? A. B. C. D. E. E42. A 50-year-old man is found to have 120 CGG repeats. Which of the following disorders is the most likely diagnosis? A. B. C. D. E. E43. Fragile X syndrome Friedreich ataxia FXTAS Huntington disease Myotonic dystrophy A male fetus is found to have 1600 CTG repeats. What is the most likely diagnosis? A. B. C. D. E. E44. The missense variant is benign. The missense variant is pathogenic. The missense variant is a VUS (variant of unknown significance). These findings confirm the diagnosis. These findings make the diagnosis unlikely. Fragile X syndrome Friedreich ataxia FXTAS Huntington disease Myotonic dystrophy An individual is found to have a 33 and a 300 GAA repeat. Which of the following results is the most likely interpretation? A. B. C. D. E. Affected with Huntington disease Affected with Friedreich ataxia Affected with myotonic dystrophy Carrier of Friedreich ataxia Female carrier of Fragile X with symptoms 754 E45. An 18-year-old young man is found to have thousands of colon polyps and his blood sample is sent for sequence analysis of the APC gene. The results are negative. Which of the following conclusions is the best explanation of his findings and the next step in his clinical management? A. B. C. D. E. E46. Which of the following findings would be interpreted as a positive test result for Spinal Muscular Atrophy? A. B. C. D. E. E47. Melting curve analysis by FRET. Multiplex ligation probe amplification. Oligonucleotide assay. Quantitative PCR. Sanger sequence analysis. In one form of familial isolated growth hormone (GH) deficiency, GH mutations cause skipping of exon 3. In heterozygotes, the GH protein product lacking exon 3 accumulates and kills the GH secreting cells to prevent secretion of normal GH molecules from the normal GH gene. This result is best described by which of the following genetic concepts? A. B. C. D. E. E49. A homozygous deletion of exon 7 in the cenSMN gene. A homozygous deletion of exon 7 in the telSMN gene. A homozygous duplication of exon 8 in the telSMN gene. A homozygous duplication of exon 8 in the cenSMN gene. A homozygous duplication of both exon 7 and exon 8 in the cenSMN gene. Which of the following molecular tests would be most useful method for diagnosis of a 5-yearold boy with progressive neuromuscular disease, a positive Gower sign, and elevated creatine phosphokinase values? A. B. C. D. E. E48. He definitely does not have FAP but he should undergo colectomy soon. He is a good candidate for MYH polyposis gene analysis and testing should be done He is a good candidate for HNPCC analysis and testing should be done He has a mutation in the regulatory region of the gene and testing should be done He may have a large gene deletion in the APC gene and dosage analysis should be done Dominant negative effect Gain of function mutation Haploinsufficiency Loss of function mutation Protein genocide Achondroplasia has a high mutation rate and is most likely the result of which of the following genetic concerns? A. B. C. D. E. Highly repetitive sequence Large gene size Maternal age effect Methylated CG dinucleotide Paternal age effect 755 E50. Which of these disorders is most likely due to loss of function of the protein product? A. B. C. D. E. E51. Which of the following disorders is most likely due to a gain of function in the protein product? A. B. C. D. E. E52. Fragile X Friedreich Ataxia FXTAS Myotonic dystrophy Spinobulbar Muscular Atrophy Which of the following disorders is most likely due to a gain of function toxic RNA? A. B. C. D. E. E53. Fragile X syndrome FXTAS Huntington Disease Myotonic Dystrophy Spinobulbar Muscular Atrophy Fragile X Friedreich Ataxia Huntington Disease Myotonic Dystrophy Spinalbulbar Muscular Atrophy The figure below represents a methylation Southern blot analysis performed on a symptomatic individual. The probe is SNRPN and methylation sensitive and insensitive enzymes were used. Which of the following is the best interpretation of the result for the individual in lane 2? 1 2 3 maternal paternal A. B. C. D. E. Angelman Syndrome due to the absence of the maternal allele Angelman Syndrome due to the absence of the paternal allele Normal individual based on the single targeted band in the blot. Prader Willi Syndrome due to absence of the maternal allele Prader Willi Syndrome due to the absence of the paternal allele 756 E54-E57. The following table should be used in answering questions E54-57: Disease-Positive 70 30 100 Test Positive Test Negative Total E54. 5% 30% 70% 95% 100% In the above 2x2 table, what is the false negative rate? A. B. C. D. E. E58. 5% 30% 70% 95% 100% In the above 2x2 table, what is the false positive rate? A. B. C. D. E. E57. 5% 30% 70% 95% 100% In the above 2x2 table, what is the test specificity? A B. C. D E. E56. Total 75 125 Given the 2x2 table, what is the test sensitivity? A. B. C. D. E. E55. Disease-Negative 5 95 100 5% 30% 70% 95% 100% A metastatic colorectal cancer patient is being tested to determine whether he is a good candidate for imatanib therapy. Which of the following test results would be most favorable for administering EGFR inhibitor therapy? A. B. C. D. E. A positive result for a mutation in BRAF. A negative result for a mutation in FLT3. A negative result for a mutation in KRAS. A positive result for a mutation in NPM. A positive result for a mutation in NRAS. 757 E59. Your lab is validating a new assay for an autosomal recessive disorder using 100 positive and 100 negative control samples. The assay detected 98 positive samples out of the positive controls and got 1 positive result in the negative controls. The targeted mutation panel of the assay is designed to detect 90% of mutations reported in the database of clinically affected individuals. Which of the following assessments provides the best interpretation of your data? A. B. C. D. E. . E60. What is the recommended nomenclature for the major mutation in the CFTR gene in Caucasian populations? (Pick the best answer) A. B. C. D. E. E61. 'F508 c.F508del deltaF508 p.F508del phe508del If you are screening a colorectal cancer population to detect HNPCC, which of the following evaluation plans is the most efficient strategy? A. B. C. D. E. E62. The analytical sensitivity is ~99%. The analytical specificity is ~98%. The clinical sensitivity is ~80%. The clinical sensitivity is ~90%. The negative predictive value is ~99%. Family history using Bethesda criteria followed by gene testing MLH1 & MSH2 Full gene sequencing for MLH1, MSH2, MSH6 in all patients Immunohistochemical analysis of MLH1, MSH2 & MSH6, followed by sequencing of the indicated gene Methylation analysis of the MLH1 promoter using blood specimens Microsatellite instability (MSI) followed by MLH1 & MSH2 sequencing According to a recent national survey of laboratory directors, which of the following laboratory descriptions was the strongest predictor of laboratory quality (and fewer errors)? A. B. C. D. E. ABMG Board certification of the laboratory director Academic setting in a medical school Laboratory certification by CAP Participation of the laboratory director in professional societies Participation of the laboratory in proficiency testing programs 758 E63. Mary’s sister’s 2-year-old son was recently diagnosed with Duchenne muscular dystrophy (DMD). He was tested by microarray analysis and found to have a deletion of exons 44-52. Mary is 6 weeks pregnant and wants to know her DMD carrier status. Her blood sample is sent to a different lab that uses MLPA for dystrophin carrier testing. The lab does not find the exon 44-52 deletion but instead finds an exon 3 deletion. Which of the following explanations most likely accounts for these results? A. B. C. D. E. E64. You see a couple with a history of several miscarriages in prenatal genetics clinic and you take a detailed medical history. You recommend molecular/cytogenetic analyses and after blood is drawn from each of them, you complete the appropriate requisition forms to accompany each of the specimens to the lab. Which of the following items listed on the requisition is most important to provide the laboratory staff enabling them to set up the appropriate study? A. B. C. D. E. E65. Mary’s DNA has a variant at the probe binding site causing a false positive result. Mary’s mother is a germline mosaic for two different dystrophin mutations. One of the labs had a sample mix up, misidentifying the sample. The exon 3 deletion represents a new mutation that occurred in Mary. The microarray analysis produced false positive results due to incomplete hybridization. Clinical indication for the molecular/cytogenetic analyses Documentation that patient fasted for at least 8 hours Patient’s family’s ethnic and ancestral origins Patient’s primary medical insurance provider Source of the tissue sample to be used in the study A clinical laboratory has received funding from their institution to implement next generation sequencing in their laboratory. The laboratory is tasked to evaluate the instruments and sequencing chemistries on the market and provide a justification for selection of a particular instrument and chemistry. Amongst the parameters being evaluated the laboratory is looking at Phred scores. A Q40 Phred Score reflects which of the following likelihoods that the base call is inaccurate. A. B. C. D. E. 1:40 1:4,000 1:100 1:1,000 1:10,000 759 E66. A clinical laboratory has developed next generation sequencing panel for a disorder, which has shown to be associated with mutations in several genes. The laboratory has completed test validation is preparing educational material for the test launch. Which of the following is not an accurate description of the gene panel? A. B. C. D. E. E67. 9. A clinical laboratory has been advised to develop next generation sequencing based tests using paired end sequencing instead of single read sequencing. Paired end sequencing can be helpful in increasing the ability to correctly map short sequence reads when which of the following molecular analysis issues is present? A. B. C. D. E. E68. The library fragment size is larger than the size of the repetitive region The library fragment size is smaller than the size of the repetitive region The library fragment size is equal to the size of the repetitive region Longer sequencing primers are used Longer molecular barcodes are used 10. Next Generation sequencing technology is being widely adopted in clinical laboratories. As a part of the guidance documents from CAP, CDC and ACMGG tests are evaluated for several parameters, including analytical specificity. The analytical specificity of a test is a reflection of which of the following statistical measures? A. B. C. D. E. E69. Detection of point mutation, small insertion or deletion in a gene Confirmation of an established clinical diagnosis when single gene sequencing is negative Detection of point mutation in promoters and introns Detection of intragenic copy number variation within the genes Perform mutation analysis in an affected individual with differential diagnosis False negative rate Positive prediction value False positive rate Negative prediction value Standard deviation Next Generation Sequencing technology is being widely adopted in clinical laboratories. As a part of the guidance documents from CAP, CDC and ACMGG tests are evaluated for several parameters, including analytical specificity.The analytical sensitivity of a test is a reflection of which of the following statistical measures? A. B. C. D. E. False negative rate Positive prediction value False positive rate Negative prediction value Standard deviation 760 E70. You provide a clinical description of a patient to the molecular cytogeneticist at your facility including: a prominent nasal root, bulbous nasal tip, hypocalcemia, immunodeficiency, and conotruncal heart abnormality. Which of the following laboratory techniques is best used to confirm the suspected diagnosis in this patient? A. B. C. D. E. E71 A family with an affected father and two affected siblings with an undiagnosed autosomal dominant condition is examined by a clinical geneticist. Since the clinical presentation is not indicative of a specific disorder the clinical geneticist decides to order whole exome sequencing. Whole exome sequencing interrogates approximately what fraction of the exome? A. B. C. D. E. E72. Next-generation sequencing Routine cytogenetic testing Southern Blot Fluorescence-in-situ hybridization (FISH) or DNA microarray Sanger sequencing of the TBX1 gene 100% 92% 70% 50% 20% Known pathogenic variants are found in a variety of genetic databases available electronically through the internet. In searching for the implications of a de novo variant found on exome sequencing of a patient with unexplained developmental delay and dysmorphic features you find the exact same variant reported in the Human Genetic Mutation Database (HGMD) associated with a milder phenotype. However, a search of the Online Mendelian Inheritance in Man (OMIM) database and LOVD (Locus Specific database) does not confirm this relationship. Which of the following is the best explanation for the differences in the information available in these databases? A. Cancer causing changes (somatic mutations) are not included in these databases. B. Each database has a unique way of identifying which variants to include and call pathogenic C. Mitochondrial mutation data is only included in some of these databases. D. Sources of identified variants are the same across these databases. E. Splice-site and regulatory regions of human nuclear genes are not included. 761 E73. A clinical laboratory is undergoing validation of next generation sequencing (NGS) technology using CAP molecular check list. They are validating gene panels and exome sequencing but will be outsourcing the “bench work” to a CLIA certified core sequencing facility and performing in house bioinformatics analysis. Which is most appropriate file to be returned to the laboratory for analysis? A. B. C. D. Sequence + images Short sequence reads aligned to the reference sequence Sequence + quality scores Variants + quality scores E. Variant calls and images E74. Next Generation Sequencing (NGS) technology is being widely adopted in clinical laboratories. As a part of the guidance documents from CAP, CDC and ACMGG tests are evaluated for several parameters which include ability to detect insertion and deletion. NGS based tests tend to have a reduced analytical sensitivity for small insertions and deletions (in/dels) as a result of which of the following characteristics of in/dels detection? A. B. C. D. E. E75. In/dels negatively influence cluster generation on the flow cell In/dels lead to altered chromatin structure In/dels tend to have strand bias and reads containing them are filtered out Short reads containing in/dels are more difficult to map unambiguously The polymerases used do not sequence efficiently through in/dels Based on the clinical presentation and nuclear study, a diagnosis of Hereditary Paraganglioma-Pheochromocytoma syndrome was considered in a 36-year-old woman. The eight exons of the succinate dehydrogenase complex, subunit B (SDHB) gene were sequenced and the variant c.434 C>T (p.R115X) was detected in the fourth exon of this gene. What is the most likely outcome of the mRNA transcribed from this allele? A. mRNA will be translated into truncated protein that has deleterious gain of function B. mRNA will be translated into truncated protein that has dominant negative effect C. mRNA will undergo nonsense mediated mRNA decay (NMD) D. mRNA will be translated into full-length protein E. This variant will inhibit the transcription of mRNA from this allele 762 E76. A 5-year-old boy was referred for delays in language development. He had no functional communication, but was able to repeat words and phrases. He displayed some repetitive behavior and did not play with toys in the typical manner. Testing for Autism Spectrum Disorder was recommended. Which of the following analyses is likely the first tier test to be performed on this child? A. Chromosomal analysis B. Expanded CGG repeat in FMR1 (Fragile X) C. Expanded CCG repeat in FMR2 (FRAXE) D. Non-syndromic autism gene panel E. Syndromic autism gene panel E77. American College of Medical Genetics and Genomics published new sequence interpretation guidelines in 2015. These guidelines are now widely used for interpretation of variants in genes associated with disease. The guidelines give five categories for variant classification (Pathogenic, Likely Pathogenic, Variant of uncertain Significance, Likely Benign and Benign) with rules to classify them. Which of the following are these rules a best fit for when interpreting a variant in a clinical setting? A. Genes without evidence as causing disease B. Genes with moderate to strong evidence as being causative of disease and a well-established reported variant spectrum C. Genes with only strong evidence of being causative of disease and a well-established reported variant spectrum D. Sequence variants of all types irrespective of the gene/ disease causality evidence E. Truncating variants only (nonsense, frameshift and splice site) E78. A new gene is reported in the medical literature in 2013, associated with an inherited autosomal dominant form of colon cancer. Multiple genes have been reported to be causative of inherited forms of colon cancer. Subsequent publications establish the causal relationship of gene: colon cancer occurs with evidence a loss of function effect from the variants of the gene. A founder pathogenic variant is reported in the Chinese population and most Chinese patients are found to have this pathogenic variant, with only two other pathogenic variants reported from a publication coming from China. A laboratory in China has made a decision to develop a test for this gene. Which of the following technical approaches is preferred for this gene in order to identify pathogenic variants causative of disease? A. Exome sequencing to detect variation in all ~22,000 genes in the human genome. B. Gene panel approach using next generation sequencing to detect variants in all genes known to causative of inherited form of colon cancer including this newly reported covering all exon and flanking intron/exon boundaries C. Next generation full sequencing single gene assay covering the entire genomic region of the gene to detect all types of variants 763 D. Sanger sequencing single gene assay covering all exons and flanking exon/intron boundaries to detect all types of variants E. Targeted pathogenic variant detection approach using methods such DHPLC, or PCR followed by restriction enzyme to detect the common Chinese pathogenic variant E79. A silent sequence variant was identified, in a Caucasian individual with no reported family history of skin disease, in a gene associated with an autosomal dominant form of skin disease. This silent variant occurred at the last base of the exon 3 (ATG start in exon 1) and has not been reported in the literature. ExAC, GnomAD and dbSNP, and 1000G have a collective allele frequency of 0.00005% for this variant. The laboratory reported the variant using the ACMG sequence interpretation guidelines as a Variant of Uncertain Significance (VUS). Which of the following steps should the laboratory take classify the variant as pathogenic or benign? A. Perform reanalysis of the variant, by obtaining new blood samples from patient and family along with performing functional analyses on patient skin biopsy fibroblasts. B. Recommend exome or genome sequencing to identify other possible causes of the skin d disease. C. Report the case as” negative” and conduct no further investigations into the nature of this silent variant. D. Sign up for Google Alerts to be notified when this specific variant is reported in the medical literature by a reputable research group. E. The patient’s physician should search the medical literature to try to find a research group working on this gene in order to help the family 764 Answers to Molecular Genetics E1-E79 E1. There are two noteworthy issues. The mother fails to give an allele to the affected and maybe she has a deletion for the locus. Second, the other child who fails to receive an allele from the mother supports that this phenomenon is real but gets the opposite allele from the father and is unaffected. The other unaffected sibs have genotypes different from the affected. This is very encouraging! The correct answer is A. E2. The phase cannot be deduced in the father of the pregnancy and the analysis is of no help. The risk for the fetus is 0.5. The correct answer is C. E3. This is best calculated on a computer program such as MLINK but can be estimated by a Bayesian calculation. The correct answer is B. Prior Conditional (Neg.) Joint Post. Carrier 5/10 1/10 5/100 5/55 = 1/11 Noncarrier 5/10 1 50/100 50/55 = 10/11 E4. Since the analysis definitely is not detecting the mutation on the paternal side, the risk is unchanged by DNA analysis and is 1 in 2. The correct answer is A. E5. This is an intercross result for an autosomal recessive disorder and the data on the grandparents are irrelevant. If the fetus were homozygous 11 or 22, it would be a definite carrier. Since it is 12, there is a 50% probability for affected and 50% for noncarrier. The correct answer is C. E6. Both copies of SMN1 are deleted in approximately 95% of patients. The remaining 5% of patients have one deleted gene and a nonsense, frameshift or missense mutation in the other gene and would require sequence analysis. All other options were distractors. The correct answer is A. E7. The hybridizing fragments of 5.2 and 2.8 kb represent methylated, non-expanded (inactive FMR1) and non-methylated, non-expanded (active FMR1) alleles, respectively. The most likely reason for a male to have both methylated and non-methylated, non-expanded FRM1 alleles is that he has an XXY Karyotype and Klinefelter syndrome. A) Swapping DNA samples is possible but this should have resulted in a sample from a female generating a male pattern. Also a repeat sample should be requested and its analysis will eliminate the possibility that his FXS sample was exchanged with that of an XX female. b) FXS result for a normal male would be 2.8 kb only. d) Mosaicism for FXS would yield 2.8 kb and a smear of methylated, expanded FRM1 alleles >~6.6 kb in size. e) An incomplete endonuclease digestion should have been noticed on exam of the digestion products after gel electrophoresis and is likely to not generate discrete hybridizing bands. The correct answer is C. 765 E8. The phase in the father can be deduced to be allele 2 with the disease allele since both had to come from the grandfather. The fetus receives allele 3 from the father and allele 2 from the mother and is unaffected. The correct answer is E. E9. There is an abnormal junction fragment in this blot. The affected child has the junction fragment. The mother also has the junction fragment and is definitely a carrier. None of the other female individuals has the abnormal fragment and therefore all are predicted to be noncarrier. The correct answer is B. E10. The protein truncation assay is based on the detection of truncated peptides due to any mutation that causes premature termination of translation. The correct answer is E. E11. The first of the five affected children in this sibship sets the phase. For each subsequent affected child, the chances of receiving this same dinucleotide genotype is 1 in 4. Therefore the probability that all of the subsequent affected children would inherit the same dinucloeotide genotype by chance is ¼ x ¼ x ¼ x ¼ = 1/256. Therefore the odds of obtaining a result favoring linkage by chance would be 1 in 256. The correct answer is E. E12. The phenotype associated with the R117H mutation is influenced by the 5T/7T/9T polymorphism in intron 8, which affects whether exon 9 is included in the transcript. The correct answer is A. E13. FXTAS is caused by large premutations in older individuals and the symptoms include tremors and ataxia, explaining the grandfather. The boy is affected with fragile X syndrome due to a CGG expansion in FMR1. The mother inherited the premutation from her father and passed an expanded full mutation to her son. All other options were distractors. The correct answer is D. E14. DNA for analysis is isolated from tissue blocks or slides that contain portions of tumor and possibly adjacent, non tumor tissue. If the portion of the sample selected for DNA isolation contains little tumor but abundant normal tissue, molecular analysis may be false negative. a) A Chromosomal deletion including BRAF would not cause a false negative result because the V600E mutated allele would remain if the deletion was trans and if the deletion is in cis, V600E is not there to detect. d) A sample mix up (exchange of samples) is possible but selection of sections of tumor for DNA isolation makes this unlikely. e) The BRAF V600E mutation is a missense mutation rather than a trinucleotide repeat expansion. The correct answer is C. E15. You should know that the female map is greater than the male map, and that the sex-average map is about 3700 cM. The correct answer is E. 766 E16. These data are consistent with a recombination event occurring between the two markers for the current pregnancy. This means that there is little ability to use the linkage data to predict the outcome of the pregnancy and the a priori risk of 50% would be the most appropriate. The correct answer is C. E17. The correct answer is C. E18 The correct answer is A. E19. The correct answer is C. E20. The correct answer is B. The liquid bead array can be highly multiplexed and is frequently used for large targeted assays. The FRET assay is not a multiplex and is well suited to a single target. MLPA is for detection of deletions and duplications, not point mutations. NGS and Sanger sequencing would not be suitable for large scale testing for a highly multiplexed panel. The correct answer is B. E21. The FGFR3 GLY380ARG (GGG to AGG) substitution that was reported to cause 96% of Achondroplasia cases changes the G of a CG dinucleotide to an A by methylation of the C on the complementary strand that is paired to the G of the forward strand. Methylation of Cs on the forward strand cause CG to TG transitions. Methylation of Cs on the complementary (reverse) strand causes CG to CA transitions on the forward strand. a) GGG to AGG is not a trinucleotide expansion. b) While a GGG to AGG substitution might be caused by gene conversion, CG transitions are much much more common. d or e) This is not an example of either recombination or slipped misspairing. The correct answer is C. E22. It is imperative to separate the SMN1 gene from the SMN2 gene before testing for the homozygous deletion. This is most commonly done by using a restriction enzyme site present in the SMN2 gene, but lacking in the SMN1 gene. The correct answer is B. E23. Southern blotting detects both the large CGG expansions and the methylation status The correct answer is E . E24. A major advantage of dot blotting techniques is that gels are not used which allows one to screen many samples. The correct answer is D. E25. The CAG repeats all seem to work by a gain of function mechanism. The correct answer is E. E26. Microsatellites repeats are multi-allelic and therefore are often highly informative. The correct answer is C. 767 E27. Reversion is a common event in the FANCA gene. Both blood and fibroblasts are tested for breakage analysis and then for DNA to identify the mutation. A number of patients show differences in the mutations identified in the blood and fibroblasts. The reversion event most likely occurs in bone marrow. Note that a silent mutation can be either benign or disease-causing. In this case the silent mutation leads to the creation of an alternate splice site. The correct answer is B. E28. Changing base 2 of a codon always produces a missense (nonsynonymous) substitution. Changing base 1 almost always does so, while changing base 3 causes a missense mutation much less frequently. a) AGG to ATG doesn’t create a frameshift. c) While AGG to ATG might create a start codon, remember this is an internal codon and it is unlikely to be embedded in a Kozak sequence (GCCPuCCAUGG). Without the surrounding Kozak sequence the ATG encodes an internal Methionine. d) Changing base 2 of a codon never produces a synonymous substitution. e) AGG to ATG doesn’t create a termination codon. The correct answer is B. E29. The correct answer is E. E30. Factor VIII deficiency is often due to inversion within the factor VIII gene. The correct answer is A. E31. The most common lesions in DNA caused by UV damage are pyrimidine dimers. The exact position of the new bonds can vary. These can be T-T, T-C, or C-C dimers. The dimers involve adjacent bases on the same strand. The correct answer is D. E32. The correct answer is B. E33. To answer this question you have to know the size categories for CAG repeats in HD. A 38 repeat allele is in the reduced penetrance category (36 to 39 repeats). A symptomatic individual with a 38 CAG repeat in HTT would be interpreted as the results are consistent with a diagnosis of HD. The correct answer is C. E34. Mrs. Soft most likely has Osteogenesis Imperfecta (OI) type 1 which is caused by missense mutations, small insertions or deletions, or exon-skipping mutations of COL1A1 or COL1A2. You should test Mrs. Soft to confirm that she has a detectable COL1A1 or COL1A2 OI causing mutation. You can then test her son for her mutation. If he is positive, then he is at great risk to develop findings of OI. If he is negative for the mutation, then he is at population risk to develop OI. a) His not having an OI causing COL1A1 or COL1A2 mutation could result from Mrs. Soft not having OI Type 1, he has her mutation but it is not detectable, or he does not have her mutation. Thus if he has a negative result you are in doubt. b, c and e) These options are all looking for pleiotropic effects of OI that can have reduced penetrance and thus could be negative at two months of age. The correct answer is D. E35. The correct answer is B. 768 E36. The correct answer is D. E37. The correct answer is C. RFLPs, while some can be detected by PCR, many cannot and require Southern analysis. VNTRs are large repeating units and require Southerns, as do full expansions for Fragile X and myotonic dystrophy. E38. The correct answer is C. This requires a Bayesian risk calculation. Prior Conditional Joint Posterior Carrier 1/100 10/100 10/10000 1/991 (~1/1000) Non-Carrier 99/100 1 99/100 E39. The correct answer is A. The only CGG expansion disorders in this list is Fragile X and FXTAS. A large expansion of this size would lead to FRAXA, while a premutation would cause FXTAS. The size of the CGG repeat is consistent with Fragile X symptoms, not FXTAS. Myotonic is CTG, FXTAS E40. The correct answer is D. HD is the only disorder in this list that has CAG expansions. E41. While the missense variant has not been reported, it is predicted to be pathogenic by multiple predictive algorithms and is found at a very low frequency in the population, and is seen in this affected individual along with a known pathogenic mutation. Thus, it may be pathogenic. However, clinically, it would probably be reported as a variant of unknown clinical significance until additional evidence, such as family studies or functional analysis provided evidence for reclassifying it. Caveats to the report may include that it is suspected pathogenic. The correct answer is C. E42. The correct answer is C. FXTAS is caused by premutations in FRAXA. E43. The correct answer is E. Myotonic dystrophy is caused by CTG expansions. Large expansions are found in congenital myotonic dystrophy and are maternally derived. E44. The correct answer is D. Only 4% of FRDA patients will have an expansion and a point mutation. Most FRDA carriers will have an expansion and a normal allele. If this were a symptomatic individual, one would recommend the point mutation analysis. For diagnostic testing, finding one expansion confirms that the individual is at least a carrier but does not confirm the diagnosis, although it is strongly suggestive. E45. The correct answer is E. All of these are possibilities, however, the best NEXT STEP that should be done after sequence analysis for APC is gene deletion analysis. Then, MYH gene testing would be indicated. At present clinical labs are not testing for mutations in the regulatory region of APC. If APC and MYH are ruled out, then he would most likely not have FAP. It is unlikely that he has HNPCC due to the thousands of polyps. 769 E46. The correct answer is B. The important concept is that there are two SMN genes, one centromeric and one telomeric. The pseudogene is centromeric, while the gene that causes SMA is telomeric. Both genes are very similar at the molecular level and can be distinguished by SNPs in exons 7 and 8. Clinical laboratory testing is based on finding a homozygous deletion of exon 7 in the SMNtel gene, so it is critical to be able to separate the SMNtel from the SMNcen. In most SMA cases, a homozygous deletion of both exons 7 and 8 is found. However, the homozygous absence of exon 7 is found in almost all SMA cases. E47. The correct answer is B. The patient clearly has DMD. The majority of mutations in the dystrophin gene are deletions and duplications. The only test listed which detects deletions and duplications is MLPA. E48. The correct answer is A E49. The correct answer is D (repeat question (see #21) without vignette and different distractors) E50. The correct answer is A. The only disorder listed that is due to loss of function of protein product is FRAXA. The function of the normal protein product is an RNA-binding protein. E51. The correct answer is E. SBMA is due to a gain of function protein that most likely affects transcription. E52. The correct answer is D. Myotonic dystrophy is most likely due to the gain of function of a toxic RNA that interferes with normal splicing in a number of RNAs leading to faulty protein products. E53. The correct answer is E. Lane 1 is a normal individual with both maternal and paternal contributions. Lane 2 is Prader Willi Syndrome patient lacking a paternal contribution; lane 3 is Angelman Syndrome. E54. The correct answer is C. The clinical test sensitivity is calculated by determining the number of individuals who have disease and are positive for the test divided by the total number of individuals with disease who were tested (both positive & negative). E55. The correct answer is D. The clinical test specificity is calculated by determining the number of individuals who do not have disease and are negative for the test divided by the total number of individuals without disease who were tested (positive and negative). E56. The correct answer is A. The false positive rate is determined by the number of individuals without disease who tested positive over the total number of individuals without disease who were tested. E57. The correct answer is B. The false negative rate is the number of individuals with disease who tested negative over the total number of individuals with disease who were tested. 770 E58. The correct answer is D. KRAS is downstream from EGFR. Auto-activating mutations that occur in KRAS result in unregulated growth, thus bypassing EGFR regulation by inhibitor drugs. Prior to prescribing EGFR inhibitors, patients are tested for KRAS mutations. Only in the absence of a KRAS mutation is the mCRC patient placed on the EGFR inhibitor drug. E59. The correct answer is C. The clinical sensitivity refers to the percent of patients that the assay will correctly identify. In this case, the disorder is autosomal recessive, indicating that two mutations would be found in each patient. The assay detects 90% of the variants found in the patient database. The clinical sensitivity is 0.9 x 0.9 = 0.81, or approximately 80%. E60. The correct answer is D. The p stands for protein, the F for phenylalanine, 508 is the codon, and del is the deletion. I did not give the cDNA nomenclature at the nucleotide level because that would not be the common name that most people would recognize on the general exam. It is recommended to give both proper and common nomenclature on reports. The proper nomenclature should always be used in describing a novel variant and in sequence analysis and data base reporting. The molecular folks should be able to name a mutation using current nomenclature standards, given the sequence information. E61. The correct answer is C. A combination of all of these testing strategies can used in HNPCC screening in a large colorectal cancer population. The only incorrect statement is that promoter methylation and BRAF analysis is done on the tumor, not in blood (germline). The most efficient strategy listed includes IHC followed by the indicated gene analysis. It is efficient in that it decreases the number of multiple gene analyses. E62. The correct answer is E. The strongest indicator of high quality and low error rate was laboratory participation in proficiency testing for all analytes tested. E63. The correct answer is A. The greatest technical concern about the MLPA is regarding SNPs at the probe site that would interfere with binding of the probe and ligation, which produce false positive results. Generally, this would result in only a single exon being apparently deleted. For this reason, single exon deletion results should be confirmed using another technique. While some of the distractors could also happen, by far, the most likely explanation is as described above. E64. The correct answer is A. Keywords: clinical indication, miscarriages Explanation: The clinical indication will allow the laboratory staff to select the appropriate first study to perform. The patient’s family’s ethnic/ancestral origins may impact what is targeted in some studies, but in the instance of multiple miscarriages, is less likely to play a significant role. Fasting is not an issue with molecular/cytogenetic analyses. The patient’s health insurance provider should not impact the decision of the most appropriate study to perform. The source of tissue while important for some studies, is obviously blood in this instance. 771 E65. The correct answer is E Keywords: Phred score, sequence chemistry Explanation: Phred scores are indicative of the quality of the sequence data and the higher the Phred score lower the error rate in the sequence data. A Phred score of Q40 is considered the highest and best Phred indicative of an error rate of 1:10,000 in the sequence data. E66. The correct answer is D. Keywords: mutations, clinical diagnosis Explanation: Gene panels can be easily developed using next generation sequencing (NGS) technology. These panels are useful to confirm the clinical diagnosis and also when the disorder is in the list of differential diagnosis. At this time NGS can detect only point mutations and small insertions and deletion in any regions of the gene. NGS cannot detect intragenic copy number variation. E67. The correct answer is B Keywords: paired end sequencing, single read sequencing Explanation: NGS technology is based on short read sequencing. Single read sequencing does not generate high fidelity data as it does not read from both ends of the sequence and therefore does not interrogate repetitive region effectively. Paired end sequencing can effectively address this issue. E68. The correct answer is C. Keywords: False positive rate, False negative rate Explanation: Analytical specificity is defined as the ability of an analytical method to measure only the sought-for analyte or measurand. Numerically characterized by determination of interferences and non-specific responses to other analytes or materials. E69 The correct answer is A. Keywords: False positive rate, False negative rate Explanation: Analytical sensitivity is defined as the ability of an analytical method to detect small quantities of the measured component. Numerically characterized by determination of detection limit. E70. The correct answer is D Keywords: clinical description, cytogenetics Explanation: The clinical description is specific and indicative of a targeted cytogenetic abnormality, which can be identified by FISH analysis. Methodologies such as next generation, routine cytogenetic testing are useful when a specific condition is not indicated from the clinical presentation. Southern blot analysis is labor intensive method to detect the abnormality whereas sequencing will not detect the common mutation in the indicated abnormality. E71 The correct answer is B. Keywords: Whole exome sequence, coverage Explanation: Certain regions of the exomes are still refractory to the exome analysis so whole exome sequencing has “Holes” in it. These holes contain regions not yet sequenced by the Human Genome project, difficult to sequence regions such as repeat regions and complex sequences. Still a large amount of the exome is covered by exome sequencing. 772 E72. The correct answer is B. Keywords: human variation, databases Explanation: The methods for identifying variants differs from database to database. Some databases rely on searching the medical literature for published reports of variants and others allow for investigators to submit the variants they have identified in an online report. Some databases include mitochondrial and/or somatic mutations. E73. The correct answer is C. Keywords: sequence analysis, bioinformatics Explanation: The file generated by NGS should contain all the relevant information required for analysis using NGS analysis pipeline. The image files are large and not needed for sequence analysis. The reference sequence should be a standard sequence using by a clinical laboratory to validate their analysis pipeline so it important the laboratory performing the analysis chooses the reference sequence. The laboratory should generate their own variant file using their reference sequence instead of a pre computed variant file which could lead to errors. E74. The correct answer is D. Keywords: in/del detection, analytical sensitivity Explanation: In contrast to the commonly used Sanger sequencing methodology which generates long sequence reads NGS generates short sequence reads. The short read sequencing technology is fast and allows generation of large amount of data using sophisticated sequence chemistries, which involve cluster generation in a flow, high fidelity polymerases. A drawback of the short sequence reads is that they can map nonspecific regions of the genome and thus cause errors in the in/del detection. E75. The correct answer is C. Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance mechanism that degrades transcripts with nonsense mutations in their open reading frame (ORF). Mutations that UHVLGHDWOHDVWQWƍWRDQH[RQMXQFWLRQGLUHFWWKHDIIHFWHG mRNA to rapid decay. In contrast, the nonsense mutations within the last exon do not activate NMD and yield a stable mRNA that directs the synthesis of C-terminally truncated polypeptides. E76. The correct answer is A. Chromosomal analysis as a cytogenetic abnormality accounts for ~15% of genetic causes related to ASD. 773 E77. CORRECT ANSWER B: SOURCE: Lecture/Syllabus KEYWORDS: sequence interpretation, variant classification EXPLANATION: Genes with moderate to strong evidence as being causative of disease and a well-established reported variant spectrum. The ACMG sequence variant guidelines clearly state that it is important to look into reported gene: disease causality with applying the sequence interpretation guidelines. This includes the reported evidence for gene: disease relationship, variant spectrum, effect of the pathogenic variant (eg Loss of function) and any contradictory evidence. E78. CORRECT ANSWER B: SOURCE: Lecture/Syllabus KEYWORDS: colon cancer, gene panel, founder, pathogenic variant EXPLANATION: A gene panel approach using next generation sequencing to detect variants in all genes known to causative of inherited form of colon cancer including this newly reported gene, covering all exon and flanking intron/ exon boundaries is the best answer. This approach will ensure all types of variation are detected in the newly reported gene (since two other pathogenic variants have been reported) and other previously known genes are also covered. A next generation sequencing approach is the most cost effective approach. E79. CORRECT ANSWER: A SOURCE OF ITEM TOPIC: Lecture/Syllabus KEYWORDS: silent variant, Variant of Uncertain Significance (VUS), splice site defect EXPLANATION: The laboratory should perform reanalysis of the variant, contact Physician/ Counselor to obtain additional family members and try to obtain a new blood samples/skin biopsy to perform functional analysis to investigate a possible splice site defect. The clinical report should clearly state the findings on the gene and recommended testing for additional family members to aid in interpretation of the variant. If possible, the laboratory should perform cDNA seq or collaborate with a research laboratory to resolve the variant. 774 F. Genetic Counseling Questions F1-F40 F1. Which of the following lists accurately describes the risks for an Ashkenazi Jewish couple to have a child affected with one of the following disorders? (CF = cystic fibrosis) A. CF > Tay Sachs = Gaucher B. Gaucher > CF > Tay Sachs C. Tay Sachs > Gaucher > CF D. Gaucher > Tay Sachs = CF E. Tay Sachs > CF > Gaucher F2. An uncle and his niece have consensual sex and she becomes pregnant. The uncle’s sister (maternal aunt of the niece) has a rare autosomal recessive disorder which is fully penetrant. Which of the following risks best describes the likelihood that the pregnancy shown is affected with the condition? A. 1 in 12 B. 1 in 16 C. 1 in 18 D. 1 in 24 E. 1 in 32 P F3. This family is affected with an X-linked disorder where penetrance is complete in hemizygous males, and heterozygous females are asymptomatic. What is the probability that individual III, 4 is a carrier. A. B. C. D. E. 1/2 or 0.50 1/4 or 0.25 1/5 or 0.20 1/8 or 0.125 1/10 or 0.10 I 1 II 2 1 3 2 3 4 1 2 3 III 775 4 F4. This family is affected with an autosomal recessive disorder. What is the probability of the disease in the current pregnancy? Two sisters married two brothers. A. B. C. D. E. 0.125 0.0625 0.052 0.039 0.035 P F5. An autosomal recessive disease can be diagnosed with molecular detection of mutations. The current test identifies 80% of carriers. This test identifies a man as a definite carrier of this disease. His spouse’s test is negative. Assuming a population carrier risk of 1 in 25, what is the probability that this man's spouse is a carrier? A. B. C. D. E. F6. Twin, adoption and family studies for pyloric stenosis suggest that genetic factors are important in the etiology of this birth defect, and it appears to follow a multifactorial pattern of inheritance. The incidence among male babies is 5/1000, and the incidence among female babies is 1/1000. Your patient, Dorothy, was born with pyloric stenosis, and she is currently pregnant. Another patient, Eleanor, is also pregnant and the father of the baby was born with pyloric stenosis. There is no other family history of pyloric stenosis in either family. Among the following individuals, who is at highest risk to have pyloric stenosis? A. B. C. D. E. F7. slightly greater than 0.032 exactly 0.032 slightly less than 0.032 slightly greater than 0.008 exactly 0.008 Dorothy’s daughter Dorothy’s son Eleanor’s daughter Eleanor’s son Each has the same risk Which of the following viral agents is the most common identifiable cause of congenital viral infections? A. B. C. D. E. Cytomegalovirus Herpes simplex Mumps Rubella Varicella 776 F8. Which of the following clinical findings is most likely in infants with congenital CMV infection? A. B. C. D. E. F9. A woman comes to clinic because she had a CT of the lower spine and then discovered she was 3-4 weeks pregnant at the time. The radiologist says the dose was less than 0.01 gray (Gy). Her risk for fetal malformation and/or mental retardation from the X-ray exposure is closest to which of the following estimates? A. B. C. D. E. F10. 1 in 10 or more 1 in 50 1 in 100 1 in 500 1 in 1000 or less Which of the following congenital heart defects carries the highest recurrence risk? A. B. C. D. E. F11. Cataracts Congenital heart disease Intracranial calcification Hydrocephalus Asymptomatic child Pulmonary atresia Truncus arteriosus Tricuspid atresia Ebstein's anomaly Endocardial fibroelastosis For the pedigree below, assume complete penetrance and that solid symbols identify individual who are affected with the disorder. The pedigree below is most consistent with which of the following type of inheritance? A. B. C. D. E. Imprinted gene expressed only from the maternal allele Imprinted gene expressed only from the paternal allele Sex limited autosomal dominant inheritance X-linked dominant inheritance X-linked recessive inheritance 777 F12. A 30 year-old woman elects to undergo maternal serum marker screening for aneuploidy. The test results indicate a 1 in 1,000 risk for Down syndrome. An ultrasound examination is subsequently performed at 18 weeks gestational age. There is a singleton fetus with adequate growth. Amniotic fluid volume is normal. All anatomic findings are normal. Which of the following choices would be the most appropriate management for this patient? A. B. C. D. E. F13. Amniocentesis Fetal echocardiogram No further testing Repeat serum marker testing Ultrasound to determine the Nuchal Thickening (NT) measurement A pedigree with two individuals affected with autosomal recessive polycystic kidney disease is depicted below. Two brothers married two sisters. The brothers had an affected deceased sister and one of the couples had an affected child. The other couple is pregnant (P). The probability that the fetus is AFFECTED is closest to which of the following fractional statements? A. B. C. D. E. 1 in 4 1 in 9 1 in 12 1 in 16 1 in 32 P F14. The results of prenatal study for an autosomal recessive disorder are depicted below. A dinucleotide repeat within the gene for the disease was analyzed in the parents, an affected child, an unaffected child, and the fetus. Which of the following percentages represents the probability that the fetus is AFFECTED? A. 100% B. 67% C. 50% D. 25% E. 0% P 778 F15. A woman has two brothers and a maternal uncle institutionalized with a rare form of severe X-linked mental retardation (X-MR). She has two normal sons and is pregnant again with a male fetus. What is the probability that the fetus is affected with the X-MR disorder? A. B. C. D. E. F16. 1 in 8 1 in 10 1 in 12 1 in 14 1 in 16 The chance that the mother (not retarded) of a boy with fragile X mental retardation will have both triplet repeats on her two X chromosomes in the normal size range is closest to which of the following percentages? A. B. C. D. E F17. The border between normal and expanded triplet repeat size for Huntington disease is closest to which of the following numbers of triplet repeats. A. B. C. D. E F18. 50% 25% 10% 5% 0% 35 repeats 45 repeats 55 repeats 65 repeats 75 repeats Older children and adults with normal hemoglobin typically have 97% Hemoglobin A, 2% Hemoglobin A2, and < 1% Hemoglobin F. Which of the following results would you expect to see on the quantitative hemoglobin electrophoresis for a beta-thalassemia carrier? A. B. C. D. E. F19. Ĺ+HPRJORELQ$Ļ+HPRJORELQ$Ļ+HPRJORELQ)+HPRJORELQ6 Ļ+HPRJORELQ$Ĺ+HPRJORELQ$Ĺ+HPRJORELQ)+HPRJORELQ6 Ļ+HPRJORELQ$Ļ+HPRJORELQ$Ĺ+HPRJORELQ)+HPRJORELQ6 Ĺ+HPRJORELQ$Ļ+HPRJORELQ$Ļ+HPRJORELQ)+HPRJORELQ6 Ļ+HPRJORELQ$Ļ+HPRJORELQ$Ļ+HPRJORELQ)+HPRJORELQ6 The recurrence risk is higher if the index case is female for which of the following disorders? A. B. C. D. E. cleft palate lethal osteogenesis imperfecta neural tube defect profound childhood deafness of unknown cause pyloric stenosis 779 F20. The risk of a severe abnormality or mortality related to a genetic disorder for the first child of a cousin mating is closest to which of the following percentages? A. B. C D. E. F21. first 1% 3% 6% 9% 12% Which of the following explanations is MOST likely to account for the great variation in severity that can occur between full siblings with cystic fibrosis? A. epistatic interaction with modifying genes B. linkage disequilibrium C. multiple loci for the cystic fibrosis gene D. multiple alleles for the cystic fibrosis gene E. uniparental disomy F22. A couple whose first son has isolated hydrocephalus with aqueductal stenosis asks about their risk to have another affected child. There are no other affected individuals in the family, and the boy has a normal karyotype. Which of the following percentages represents the approximate chance that their next child would be affected? A. B. C. D. E. F23. 1% 3% 6% 12% 25% A screening program has determined that a severe autosomal recessive condition occurs in 1/10,000 newborns in a population. A new test is devised to screen for carriers for this disease using tears collected on filter paper. For those testing positive, carrier status can reliably be confirmed with a serum enzyme assay. An experimental heterozygote screening program yields the following results: Total screened: 5,000; screen positive: 135; confirmed heterozygotes: 85 The sensitivity of this screening test is closest to which of the following percentages? A. 1% B. 2% C. 15% D. 85% E. 99% 780 F24. A couple is referred for prenatal counseling during the 18th week of pregnancy because of a positive 2nd trimester screen result, which revealed an increased risk for trisomy 18. They state at the start of the session that they are uncertain whether to proceed with amniocentesis. The pregnant patient later states that she wishes to have an amniocentesis, but that her husband is reluctant. Which of the following management activities would be the most appropriate next step in genetic counseling for this couple? A. Arrange for an amniocentesis appointment as soon as possible as long as the wife wants the procedure. B. Refer the couple for couples counseling so that they can resolve the issue with an impartial mediator. C. Suggest the couple defer making a decision about whether to have the procedure, and set up a return appointment for the following week. D. Explore with the couple the possible outcomes associated with deciding to have (or not have) the amniocentesis. E. Use confrontation to clarify for the couple the source of their disagreement so that their true feelings are known. F25. Which of the following reasons is the MOST important reason to screen newborns for sickle cell anemia? A. determine the incidence of the disease in various populations B. identify other at-risk family members C. prevent the subsequent birth of affected siblings D . provide genetic counseling to the parents E.. reduce morbidity and mortality in affected infants F26. A man has a child with an extremely rare autosomal recessive disorder. His first wife dies and he now marries the half sister of his first wife. The new couple is pregnant. The chance the fetus is affected with the condition is about A. B. C. D. E. 1 in 4 1 in 8 1 in 16 1 in 32 1 in 64 P 781 F27. You are scheduled to see a patient whose medical records indicate that he has mild, unilateral hearing loss, heterochromia, and a white, frontal streak in his hair. These findings would be most consistent with which of the following syndromes? A. B. C. D. E. F28. A normal-appearing infant boy who passed his initial newborn hearing screen but remained in the NICU for an infection develops severe hearing loss after treatment with streptomycin. The baby’s mother had hearing loss in her early 20s and the baby’s maternal grandmother became completely deaf by her late 40s. Molecular testing of which of the following genes is most likely to shed light on this family’s hearing loss? A. B. C. D. E. F29. Alport syndrome Pendred syndrome Treacher Collins syndrome Usher syndrome Waardenburg syndrome USH2A GBJ2 GNJ6 MT-RNR1 MT-TS1 Which of the following types of craniofacial clefting is associated with the highest sibling risk of recurrence (assuming non-syndromic)? A. B. C. D. E. bilateral cleft lip and palate cleft soft palate alone cleft hard palate alone unilateral cleft lip alone unilateral cleft lip and palate 782 F30. A 50-year old woman presents for genetic counseling because her father (age 75) has recently been diagnosed with Alzheimer’s disease. The woman is concerned about this family history because her 30-year old daughter is currently pregnant with her first child. She states that she wants to pursue genetic testing, in order to find out whether her daughter and grandchild are at risk. Which of the following interventions is the best response to this woman? A. Arrange genetic testing to determine the ApoE genotype for this 50-year old patient. B. Arrange genetic testing for the 75-year old father to determine whether he carries a ApoE genotype associated with increased risk, before offering testing to your patient. C. Arrange a genetic counseling appointment for the 30-year old daughter, since she is currently pregnant and it will be quickest to start with offering testing for her. D. Decline to arrange genetic testing in this family, because of the limited clinical validity and utility. E. Determine what this 50-year patient hopes to gain from having testing, and what the interpretation will be for each potential result. F31. In the U.S., when a woman with a singleton pregnancy has had a positive high MSAFP of 3.5 MoM between 15 - 20 weeks gestation which of the following outcomes for the baby is most likely to occur? A. B. C. D. E. F32. have an open neural tube defect have an abdominal wall defect miscarry or be stillborn be small-for-dates be normal at birth The likelihood that a child resulting from incest between two first-degree relatives will have a significant abnormality (e.g., birth defect, AR disorder, intellectual impairment) is estimated to be closest to which of the following percentages? A. B. C. D. E. 5% 10% 15% 25% 40% 783 F33 After a genetic counseling appointment with a pregnant patient, a genetic counselor recognizes that she is feeling angry towards her patient because the patient is continuing to use alcohol and marijuana during the pregnancy. The counselor seeks out a colleague to discuss the case, and realizes that her reaction stems from her own recent experience with miscarriage despite attempting to maintain a healthy lifestyle. Which of the following concepts best describes the counselor’s reaction to this prenatal patient? A. B. C. D. E. F34. Counter-transference Transference Confrontation Advanced empathy Self-reflection Your office receives a phone call from a woman who says that she wants to self-refer herself for genetic counseling and testing because of a family history of Huntington disease. She also states that she wants to know more about what protection there is against possible discrimination in insurance coverage. Which of the following statements represents the clearest description of the protection provided by the Genetic Information Nondiscrimination Act of 2008 (GINA)? A. GINA prohibits health insurers from making decisions about insurance coverage based on whether someone is affected and manifests a genetic condition. B. GINA prohibits health insurers from using the results of genetic testing to make decisions about insurance coverage. C. GINA prohibits health insurers from using the results of genetic testing to make decisions about insurance coverage, except for group policies at companies which employ fewer than 15 people. D. GINA prohibits insurance companies from using the results of genetic testing in decisions about insurance coverage. E. GINA requires insurance companies to provide coverage for genetic testing. F35. A woman has a son with an X-linked recessive lethal disorder, and her maternal half-brother had the same X-linked recessive lethal disorder. There is no other family history of the condition. Her daughter has one healthy son. What is the daughter’s chance to be a carrier? A. 1/4 B. 1/3 C. 1/2 D. 2/3 E. 5/6 784 F36. You are scheduled to see a woman whose family history is consistent with inheritance of an autosomal dominant RB1 mutation with reduced penetrance. Your patient’s brother, maternal uncle, and maternal grandmother all had bilateral retinoblastoma. However, your patient and her mother have both had a normal ophthalmologic exam. Review of the literature reveals that RB1 mutations with 90% penetrance have been documented. If an RB1 mutation with 90% penetrance is segregating in this family, what is the chance for your patient’s offspring to inherit the RB1 mutation? A. B. C. D. E. F37. 0 1/40 1/22 1/11 ½ You are scheduled to see a patient at 17 weeks gestation because of a positive second trimester maternal serum screening result which showed a maternal serum alphafetoprotein (AFP) level of 2.59 multiples of the median (MOM). During the initial contracting process, your patient tells you that she is certain that she wants to proceed with amniocentesis, because of the likelihood of fetal anomalies. Which of the following options is the most appropriate next step? A. Ask the patient to describe for you what she has been told about her positive screening result. B. Determine the patient’s primary sources of emotional support during her pregnancy. C. Educate the patient about the possible causes of an increased maternal serum AFP result. D. Explain the risks and limitations associated with amniocentesis. E. Provide the patient with the informed consent form to sign, indicating that she is choosing to proceed with amniocentesis. F38. A couple comes to you for genetic counseling because the husband had two siblings who died from cystic fibrosis (CF; an autosomal recessive disease with an incidence of approximately 1 in 2,500 livebirths). No molecular testing was done for either of his siblings. Both members of the couple choose to have carrier testing, using a mutation panel test which detects 90% of CF mutation carriers in their population. You are surprised to find that the wife receives a positive result, and is a carrier of the Phe508del mutation. However, the husband’s test is negative; no mutations are identified. What is probability that their first child will be affected? A. B. C. D. E. 1/2500 1/150 1/44 1/24 1/6 785 F39. You see a 4-year-old boy and his parents for a follow-up visit in genetics clinic. He was initially referred from craniofacial clinic for a sub-mucous cleft, delayed speech, a ventricular septal defect, and characteristic facial features. The parents were told these features may be consistent with a deletion of chromosome 22q11.2, and the syndrome was briefly described. The chromosomal microarray analysis you obtained following his initial visit revealed a 22q11.2 deletion and the family comes to discuss the results. After disclosing the confirmation of the diagnosis to the parents, which of the following actions is the most appropriate next step? A. Assess the parents’ reaction to this diagnosis, then ask if they would like to hear more information. B. Describe the 22q11.2 deletion syndrome and the clinical features associated with this diagnosis. C. Explain how 22q11.2 deletion syndrome is inherited and discuss testing for other family members. D. Inform the parents of the limitations and potential benefits of chromosomal microarray testing. E. Provide support and reassurance for the parents that everything will be okay. F40. Mr. and Mrs. B are referred for genetic counseling because of a family history of cystic fibrosis. Mr. B and Mrs. B are first cousins (their mothers are sisters), and Mrs. B’s brother was affected with cystic fibrosis. No molecular testing was done, and there is no further family history of CF. The chance that Mr. and Mrs. B’s first child will be affected with CF is closest to which of the following probabilities? A. B. C. D. E. F41. 1/32 1/24 1/18 1/8 1/6 A woman (II-4) had a half-brother who died of a lethal disorder in his early twenties. Her 15-year-old son (III-5) has inherited the same disorder. Her daughter (III-4) has one healthy son (IV-1). There is no other family history of the condition. Based on the most likely inheritance pattern consistent with the pedigree below, what is the daughter’s (III-4) chance to be a carrier? A. 1/4 B. 1/3 C. 1/2 D. 2/3 E. 5/6 786 Answers to Genetic Counseling Questions F1-F40 F1. Gaucher of the nonneuronopathic type is clearly the most frequent at about 1 in 600-1,000. Tay Sachs is generally quoted at about 1 in 3,600, and the incidence for CF is not well documented but is probably about the same or less frequent than Tay Sachs and definitely would not be more frequent than Tay Sachs. The correct answer is D. F2. The chance the man is a carrier is 2/3. The chance his niece (sexual partner) is a carrier is about 1/3. The chance of an affected is about 2/3 x 1/3 x 1/4 = 1/18. The correct answer is C. F3. This is a simple Bayesian calculation. Prior Conditional Joint Post 0.5 0.5 x 0.5 0.125 0.2 0.5 1.0 0.5 0.8 Thus the probability that individual II-4 is a carrier is 0.2 and the probability that individual III-4 is a carrier is 0.1. The correct answer is E. F4. The prior probability that the pregnant couple are both carriers is 0.25. Calculations to take into account that the two normal offspring are provided below. Prior Conditional Joint Post 0.25 0.75 x 0.75 0.14 0.157 0.75 1.0 0.75 0.843 The posterior probability that they are both carriers is 0.157. This number times 0.25 times the risk for an affected of 0.039. The correct answer is D. F5. One way to make this calculation is to use a Bayesian method. The point is that the correct answer is not exactly 0.008 which is the probability that a person is a carrier times the probability that a carrier will have a negative test. The proper calculation is shown below. The correct answer is D. Prior Conditional Joint Post 1/25 0.20 0.008 0.0083 787 24/25 1.0 0.96 0.9917 F6. The correct answer is B. F7. The most frequent identifiable congenital viral infection is with CMV, although the majority of these infants are asymptomatic. The correct answer is A. F8. As mentioned above, the majority of infants with congenital CMV are asymptomatic. The correct answer is E. F9. According to Harper p. 277, the risk would be 1 in 1000 or less with this exposure. The correct answer is E. F10. According to Harper p.219, the recurrence risk for all of these cardiac malforrmations is relatively low (1.0-1.3%).The recurrence risk for endocardial fibroelastosis is substantially higher at 3.8%. The correct answer is E. F11. This pedigree is not consistent with X-linked inheritance and complete penetrance because of the evidence of male to male transmission of the trait. Sex limited autosomal dominant inheritance would be a possibility except for the statement to assume complete penetrance and the transmission through an asymptomatic male. The pedigree would fit well for a mutation in an imprinted gene where the gene is expressed only from the maternal allele, and the phenotype occurs only when the mutant gene is inherited from the mother. The correct answer is A. F12. The correct answer is C. F13. The probability that the father of the current pregnancy would be a carrier is 2/3. The probability that the mother is a carrier is ½. If they are both carriers, the probability that the fetus would be affected is ¼. Thus, 2/3 x ½ x ¼ = 1/12. The correct answer is C. F14. This is an intercross data set for the affected child and the fetus. The linkage analysis indicates that the fetus is either homozygous affected or is a noncarrier. A heterozygous result for the disease would involve homozygosity for the upper or lower dinucleotide allele as is the case for the unaffected sib. There is a 50-50 probability that the fetus is affected. The correct answer is C. F15. This requires a Bayesian calculation. The woman’s mother is an obligate carrier of this disorder. Prior Conditional (2 nl sons) Joint Post Is carrier 1/2 1/4 1/8 1/5 Is not carrier 1/2 1 1/2 Hence she has a 1/5 risk of being a carrier, so the chance that her male fetus is affected is 1/5 x ½ = 1/10. The correct answer is B. 788 F16. All mothers of males with fragile X full mutations have at least a pre-mutation allele. Hence the correct answer is E, 0. F17. The correct answer is A. F18. The correct answer is B. F19. The correct answer is E. F20. The background risk for the general population is about 3%; the incremental risk for first cousins is another 3%. The correct answer is C. F21. The correct answer is A. F22. About ¼ of males with aqueductal stenosis have the X-linked form; the risk to the next child is P[XL] x P[male] x P[affected] = ¼ x ½ x ½ . The correct answer is C. F23. q2 = 1/10,000; q = 1/100, 2pq = 1/50. Therefore should have 100 carriers among 5,000 individuals; found 85, so 85% sensitivity. The correct answer is D. F24. The correct answer is D. F25. The correct answer is E. F26. The man is a definite carrier. There is a 1 in 2 chance that his double mother-in-law is a carrier and a 1 in 2 chance that she passed the altered gene to his new spouse ( ½ X ½). The chance that the father will pass on an altered gene is ½ . The chance that the mother will pass on an altered gene to the fetus is ½ X ½ X ½ . The chance the fetus is affected is about 1 in 16. The correct answer is C. F27. Patient has classic features of Waardenburg’s and, apart from deafness, no apparent signs of the other syndromes: Alport (e.g., glomerulonephritis, lenticonus), Usher (e.g., RP, vestibular dysfunction), Treacher-Collins (e.g., mandibulo-facial dysostosis; cleft palate; Pendred (e.g., goiter, vestibular dysfunction). The correct answer is E F28. Baby’s deafness has apparently arisen in reaction to aminoglycoside exposure. Later age of hearing loss in (presumably unexposed) mother and maternal grandmother is consistent both with maternal inheritance and effects of mtDNA mutation: A1555G in MT-RNR1; a) is one of the Usher genes and is AR, not accounting for the three-generation HL hx.; b) is connexin 26— usually AR, though sometimes AD, but not likely to have been associated with initially normal NB hearing screen with loss after streptomycin exposure; c) is connexin 30, presumably AR, and e) the other mtDNA mutation, is a heteroplasmic mutation and associated with progressive, childhood-onset SN HL with skin changes, but not with aminoglycoside toxicity. The correct answer is D 789 F29. The correct answer is A. a) = 5.7-8% b) = 3.8%, c) = 5.4%, d) = 1.6-2.5%, and e) = 3.3-4.2%.] F30. The correct answer is E. F31. With an MSAFP 3.5 MoM, 18% will have problem found with US or amnio; if US and amnio are normal, and additional 28.5 will have problem later in pregnancy, but the baby is still most likely to be normal at birth. At a 2.5 MoM cut-off, about 2-3% of women will have a positive high MSAFP. Of these, 0.1% of all women would be expected to have a fetus with a NTD, and about an equal percentage will have a fetus with ventral wall defect. For an MSAFP between 3 and 4.9 MoM, the overall risk for poor outcome is about 41%. With MSAFP >5 MoM, the risk for an adverse outcome is about 91%. The correct answer is E F32. The correct answer is E. 40% F33. The correct answer is A. F34. The correct answer is B GINA prohibits health insurers from requesting genetic information of an individual or the individual’s family members, or using it for decisions regarding coverage, rates, or preexisting conditions. Answer A is incorrect, because GINA does not does not prohibit the health insurer from determining eligibility or premium rates for an individual based on the manifestation of a disease or disorder in that individual. Answer C is incorrect; it is the employment provisions of GINA which do not apply if an employer has fewer than 15 employees. Answer D is incorrect, because the insurance provisions of GINA only cover health insurance; they do not extend to long-term care, life, or disability insurance. Answer E is incorrect, because GINA does not mandate coverage for any particular genetic test or treatment. F35. The correct answer is B Keywords: Bayesian calculation Explanation: The woman is herself an obligate carrier, since she has an affected son and an affected maternal half-brother. So the daughter’s prior (Mendelian) risk to be a carrier is 1/2. Use Bayesian calculation to calculate her chance to be a carrier given that she has a healthy son. Prior Condition (healthy son) Joint Posterior Carrier 1/2 1/2 1/4 1/3 Non-carrier 1/2 1 1/2 2/3 The probability that the daughter is a carrier is 1/3. 790 Ȉ F36. The correct answer is C Keywords: Retinablastoma, reduce penetrance Explanation: Your patient’s mother is an obligate carrier of the RB1 mutation in the family. Use a Bayesian calculation to determine the chance that your patient is a carrier, given that she has a normal ophthalmologic exam. Prior Condition (nl ophth. exam) Joint Posterior Carrier 1/2 1/10 Non-carrier 1/2 1 1/20 1/11 10/20 10/11 Ȉ Probability your patient is a carrier = 1/11. Probability your patient’s child will inherit the mutation = 1/22 F37. The correct answer is A Keywords: Maternal serum screen, genetic counseling Explanation: The most appropriate next step will be to assess the patient’s current understanding of the implications of the positive screening result. This will allow you to provide the further counseling information and support in a manner that will be most helpful to your patient. Clearly the session will also include providing information about the possible causes of a positive screen result (C) and her options for further testing (D); however, this should follow an assessment of her current understanding. During the session, you will also assess her sources of emotional support (B), but this would not be done during the initial contracting process. F38. The correct answer is D Keywords: Cystic fibrosis recurrence risk, Bayesian calculation Explanation: The probability that the wife is a carrier is 1. Use Bayesian calculation to calculate the probability that the husband is a carrier. Prior Condition (neg. carrier test) Joint Posterior Carrier 2/3 1/10 Non-carrier 1/3 1 Ȉ 2/30 1/6 10/30 5/6 The probability that their child will be affected is 1/6 * 1 * ¼ = 1/24 791 F39. The correct answer is A It is important to assess what the parents’ reaction is, before providing further information. Your plan for the session will also include further discussion of the syndrome including natural history and inheritance pattern and other testing that may be indicated for the patient or family members, and you should also have a plan in place for providing support for the family. However, in order to achieve those goals, it will be important to first assess how the parents have heard the diagnostic information that you have provided, and what their reaction is. F40. The correct answer is B Solution: Probability (Mrs. B is a carrier) = 2/3, because both of her parents are carriers, and she is unaffected. Probability (Mr. B is a carrier) = 1/4, because his aunt is an obligate carrier, and his mother has a 1/2 chance to be a carrier. Probability (affected child) = 2/3 x 1/4 x 1/4 = 1/24. F41. CORRECT ANSWER: B Keywords: Bayesian calculation Explanation: The woman is herself an obligate carrier, since she has an affected son and an affected maternal half-brother. So the daughter’s prior (Mendelian) risk to be a carrier is 1/2. Use Bayesian calculation to calculate her chance to be a carrier given that she has a healthy son. Prior Condition (healthy son) Joint Posterior Carrier 1/2 1/2 1/4 1/3 Non-carrier 1/2 1 1/2 2/3 The probability that the daughter is a carrier is 1/3. 792 Ȉ G. Cancer Genetics Questions G1 – G47 G1. Which of the following cancers is the most common malignancy after colorectal cancer seen in families with hereditary non-polyposis colon cancer (Lynch syndrome)? A. B. C. D. E. G2. A four-year-old boy is diagnosed with pulmonary pleuroblastoma and the father has multicystic goiter? In taking a family history of cancer which of the malignancies listed below might be expected to be found given this history? A. B. C. D. E. G3. Adrenal corticocarcinoma Breast cancer Lung cancer Rhabdomyosarcoma Testicular cancer A newborn boy is found to be small for gestational for both weight and length and has radial aplasia. Fanconi anemia is confirmed by laboratory studies. While counseling the parents you are most likely to mention which of the following time periods as the average age of clinical evidence for bone marrow failure in Fanconi anemia? A. B. C. D. E. G4. Ovarian cancer Bile duct cancer Endometrial cancer Breast cancer Pancreatic cancer 1 to 2 months 6 to 7 months 2 to 3 years 6 to 8 years 12 to 14 years A 2-year-old boy presents to his pediatrician with mild dysmorphic features (frontal bossing) and a family history reveals a mother with basal cell nevus syndrome (Gorlin syndrome). In addition to skin cancer which of the following malignancies is the most common pediatric malignancy typically identified in this disorder? A. B. C. D. E. Glioblastoma Medulloblastoma Neuroblastoma Wilms’ tumor Leukemia 793 G5. A twelve-year-old child presents with leukoplakia, abnormal nails and evidence of bone marrow failure. Your hematology colleague is concerned about a possible underlying genetic diagnosis. Which of the following tests is most likely to yield a diagnosis? A. B. C. D. E. G6. Based on the pedigree documented below, which of the following genes is most likely to be mutated causing the hereditary cancer syndrome in this family? A. B. C. D. E. G7. Comparative genomic hybridization Diepoxybutane breakage analysis Quantitative radiation sensitivity Sister chromatid exchange Telomere length measurement ATM BRCA1 BRCA2 P53 PTEN Approximately 10% of children with hepatoblastoma have which of the following disorders? A. B. C. D. E. Ataxia telangiectasia Familial Adenomatous Polyposis Li Fraumeni Neurofibromatosis I Von Hippel Lindau 794 G8. A five-year-old child presents with rash on the cheeks, very short stature and she was recently diagnosed with leukemia. Your hematology colleague is concerned about a possible underlying genetic diagnosis. Which of the following tests is most likely to yield a diagnosis? A. B. C. D. E. G9. Missense mutations are the most common type of constitutional mutation found in which syndrome? A. B. C. D. E. G10. Familial Adenomatous Polyposis Li Fraumeni Syndrome Multiple Endocrine Neoplasia Type I Multiple Endocrine Neoplasia Type IIA Multiple Endocrine Neoplasia Type IIB As a geneticist following patients with NF1, which of the following clinical features is the most likely to be detected only after a patient has become a teenager? A. B. C. D. E. G12. Cowden Syndrome Familial Adenomatous Polyposis Hereditary breast ovarian cancer Neurofibromatosis I von Hippel Lindau Syndrome Prophylactic thyroidectomy by age 5 is recommended for children who are diagnosed with which of the following heritable cancer syndromes? A. B. C. D. E. G11. Comparative genomic hybridization Diepoxybutane breakage analysis Quantitative radiation sensitivity Sister chromatid exchange Telomere length measurement Acoustic schwanoma Axillary freckling Learning disabilities Malignant peripheral nerve sheath tumor Optic pathway tumor In women who carry a BRCA1 mutation, the lifetime risk for ovarian cancer is closest to which of the following percentage ranges? A. B. C. D. E. 1-2% 5-15% 15-20% 25-60% 75-90% 795 G13. In men who carry a BRCA2 mutation, the lifetime risk of male breast cancer is closest to which of the following percentage ranges? A. B. C. D. E. 1-2% 5-10% 15-25% 40-50% 70-80% Questions G14-15 G14. You are asked to see the parents of a child newly diagnosed with unilateral retinoblastoma. The mother is pregnant and is concerned about the risk of having a second child with retinoblastoma. The parents have normal eye exams and have no family history of retinoblastoma. What is the approximate likelihood that the future sibling will develop retinoblastoma? A. B. C. D. E. G15. <0.1% 0.1% 1.0% 6.0% 45% The parents in question 14 return to see you again after their child with unilateral retinoblastoma underwent enucleation and genetic testing. The results of the tumor and blood analysis at the RB1 locus are shown below. Now what is the likelihood that the future sibling will develop retinoblastoma? Allele 1 Allele 2 Tumor Promoter Methylation 683delC Blood A. B. C. D. E. G16. Normal Normal <0.1% 0.1% 1.0% 6% 45% Which of the following lists gives the correct order for the risk of developing colorectal cancer in these syndromes? (HNPCC – hereditary non-polyposis colon cancer; FAP – familial adenomatous polyposis; JPC – juvenile polyposis coli; PJS – Peutz-Jeghers syndrome)? A. B. C. D. E. FAP>HNPCC>JPC>PJS FAP>HNPCC>PJS>JPC FAP>JPC>HNPCC>PJS HNPCC>FAP>JPC>PJS HNPCC>JPC>FAP>PJS 796 G17. Molecular testing identifies a nonsense mutation in the RB1 gene of a two-year-old boy with bilateral retinoblastoma. Parental molecular analyses are normal. Based on the pedigree below which of the following percentages most closely approximates the parents’ risk of recurrence in their next child? A. B. C. D. E. G18. A number of genome wide association studies have been performed to identify susceptibility genes for breast cancer. These involve using large case and control cohorts to identify which polymorphisms spread throughout the genome are differentially found in cases or controls by genotyping on SNP arrays. Which of the following breast cancer susceptibility loci was discovered using this method? A. B. C. D. E. G19. ATM CHEK2 FGFR2 PALB2 PTEN Families demonstrating low penetrance retinoblastoma are most likely to have which of the following types of mutation identified in the RB1 gene? A. B. C. D. E. G20. 1% 6% 15% 20% 25% Deletions Promoter mutations Nonsense mutations Splice site mutations Translocations Mutation or disruption of an imprinted locus has been associated with which of the following syndromes? A. B. C. D. E. Familial Adenomatous Polyposis Beckwith Wiedemann Syndrome Gorlin Syndrome Neurofibromatosis II Von Hippel Lindau 797 G21. Overall, children with Beckwith-Weidemann syndrome have an increased risk of developing Wilms tumor. Molecular testing can help to clarify the cancer risk. Which of the following results on analysis of a blood sample from a child with BWS has the highest risk for Wilms tumor? A. B. C. D. E. G22. Which of the following autosomal dominant cancer predisposition syndromes results from a mutation which activates an oncogene? A. B. C. D. E. G23. Costello Syndrome Gorlin Syndrome Juvenile Polyposis Coli Neurofibromatosis 2 von Hippel Lindau Syndrome An otherwise healthy woman with early onset breast cancer has genetic testing of the BRCA1 and BRCA2 genes. The result includes a missense mutation in BRCA2 that is a variant of uncertain significance (VUS). The testing laboratory provides you additional information about this variant. Which of the following data provides the strongest evidence against the VUS being associated with hereditary breast/ovarian cancer syndrome? A. B. C. D. E. G24. Abnormal methylation of H19/IGF2 Abnormal methylation of LIT1/KCNQ10T1 Paternal duplication of 11p15 Point mutation in CDKN1C Uniparental disomy of 11p15 The finding of a frameshift mutation in BRCA1 in the same patient. The finding of a frameshift mutation in BRCA2 in the same patient. The VUS has not been previously reported in control populations. The VUS occurs in a consensus splice site. The VUS is a nonsense mutation. In evaluating a family for hereditary non-polyposis colon cancer which test of the tumor should be performed prior to deciding about testing for constitutional mutations? A. B. C. D. E. Comparative genomic hybridization FISH analysis at MSH2 Loss of heterozygosity at the MSH2 locus Microsatellite instability Tumor cell karyotype 798 G25. A 25-year-old woman has invasive colon cancer and hundreds of polyps. She has a total colectomy. In addition to routine care for the general population, which of the following medical surveillance evaluations will she continue to require? A. B. C. D. E. G26. Which of the following explanations accounts for the increased risk of skin cancer for patients with Xeroderma Pigmentosum? A. B. C. D. E. G27. Amplification of chromosome 2 including the ALK locus Inversion of chromosome 2 involving the ALK gene Deletion of the ALK gene Missense mutation of the ALK gene Translocation between ALK and EML4 Genetic analysis of the tumor specimen of a fifty-five year old man with colon cancer reveals microsatellite instability. The likelihood that analysis of a blood sample from this patient will reveal a germline mutation in one of the mismatch repair genes is closest to which of the following percentages? A. B. C. D. E. G29. Their cells are deficient in mismatch repair. Their cells are deficient in nucleotide excision repair of UV-induced DNA lesions. Their cells are deficient in transcription-coupled repair of UV-induced DNA lesions. Their cells are sensitive to crosslinking agents. Their cells have a DNA repair gene mutation and lose the second copy frequently. The ALK gene undergoes a variety of different mutational events in cancers. Molecular analysis of a blood sample from a child with neuroblastoma is most likely to reveal the following? A. B. C. D. E. G28. Annual ophthalmic exam Breast MRI Endometrial biopsy every 2 years Screening skin exam for melanoma yearly Upper GI endoscopy every 2-3 years <1% 1% 5% 25% 50% A child is diagnosed with pulmonary pleuroblastoma. You order genetic testing. Which specific gene test is most likely to be positive? A. B. C. D. E. CTNNB1 DICER1 PTCH1 SMARCB1 SUFU 799 G30. Heterozyous mutations in CDH1 predispose to diffuse gastric cancer. About which additional cancer risk do carriers of CDH1 mutations need to be informed? A. B. C. D. E. G31. A 45-year-old man is diagnosed with chromophobe histology of kidney cancer. Considering genetic condition(s) which predispose to this type of cancer, about what other medical problem would you ask? A. B. C. D. E. G32. Embryonal rhabdomyosarcoma Chromophobe renal cell cancer Lobular breast cancer Papillary thyroid cancer Small cell lung cancer Pulmonary arteriovenous malformation Pulmonary fibrosis Pulmonary hemangiomas Recurrent pneumonias Spontaneous pneumothorax 16-year-old presents to the emergency room with intussusception. After the patient is stabilized you are asked to see the patient and recommend genetic testing? Which gene test do you order? A. B. C. D. E. APC BMPR1A PTEN SMAD4 STK11 800 G33. A 37-year-old woman was recently diagnosed with breast cancer and comes for a consultation. Which option describes the most appropriate use of the current models and guidelines? A. The BRCAPro algorithm can be used to predict the likelihood that she carries a BRCA1/2 mutation. B. The Chompret criterion requires additional family history data to determine the likelihood that she carries a BRCA1/2 mutation. C. The Claus tables can be used to predict her likelihood of developing ovarian cancer. D. The Gail model can be used to predict her likelihood of developing a second breast cancer. E. The NCCN guidelines suggest reflex TP53 testing if BRCA1/2 testing is negative. G34. Alterations in genes encoding transcription factors play an important role in the development of hematopoietic disorders. Consider an infant with Trisomy 21 who develops transient myeloproliferative disorder. A somatic mutation in which gene is most likely to be found when examining the bone marrow? A. B. C. D. E. G35. AKT ETV6 GATA1 IKZF1 RUNX1 A 6-month-old with unilateral retinoblastoma has genetic testing performed. The results of the tumor and blood analysis at the RB1 locus are shown below. What is the likelihood that a future sibling will develop retinoblastoma? Retinoblastoma tumor Blood A. B. C. D. E. Allele 1 c.7510C>T (p.R261X) Allele 2 c.7510C>T (p.R261X) normal normal Homozygous C>T substitution that causes an immediate termination codon in exon 8 of tumor DNA <1% 1% 6% 25% 50% 801 G36. A four-year-old girl presents to the emergency room with rectal bleeding and rectal prolapse. The gastroenterologist removes a bleeding polyp and stabilizes the patient. A geneticist evaluates the patient and recommends genetic testing for a next generation panel test for polyposis/GI cancer genes. Which of the following gene tests is most likely to be positive given the child’s clinical presentation? A. B. C. D. E. G37. APC MUTYH MSH2 PTEN SMAD4 You are asked by your hematology colleague to consult on a 16-year-old young man with recent diagnosis of bone marrow failure. Examination of this patient demonstrates nail dystrophy and oral leukoplakia (also present by history in an uncle who died from a different cancer). What diagnostic test is most likely to yield a positive result? A. B. C. D. E. Antibody diversity Diexpoxybutane breakage Ionizing radiation sensitivity Sister chromatid exchange Telomere flow FISH 802 G38. A 34-year-old woman was recently diagnosed with breast cancer and comes for a consultation. Which of the following options describes the most appropriate use of the current models and guidelines? A. The Amsterdam criteria can be used to predict the likelihood that she carries a PALB2 mutation. B. The Chompret criterion including additional family history data can determine the likelihood that she carries a BRCA1/2 mutation. C. The Claus tables can be used to predict her likelihood of developing ovarian cancer. D. The Gail model can be used to predict her likelihood of developing a second primary breast cancer. E. The NCCN guidelines suggest reflex TP53 testing if BRCA1/2 testing is negative. G39. A 20-year-old woman presents with a pheochromcytoma of the adrenal gland. Further work-up reveals hearing loss and an endolymphatic sac tumor. Genetic testing using an available pheochromcytoma/paraganglioma panel is ordered. A positive mutation in which of the following genes is most likely to be identified? A. B. C. D. E. G40. NF1 RET SDHB SDHD VHL The 35-year-old woman shown in the pedigree (noted by a star) is concerned about her risk of kidney cancer based on her family history of a brother who died of kidney cancer (clear cell RCC) at age 45. Given the pedigree you order genetic testing using a kidney cancer panel. Which of the following genes is most likely to have a pathogenic mutation? A. B. C. D. E. BHD FH MET PTEN VHL 803 G41. The FDA approved use of Crizotinib requires which of the following molecular tests of the tumor specimen? A. B. C. D. E. G42. Amplification of p53 in neuroblastoma Deletion of RB1 in osteosarcoma Inversion of ALK in lung cancer Missense mutation of SRC in breast cancer Translocation of ABL in chronic myelogenous leukemia You are asked by your hematology colleague to evaluate an 8-year-old boy with recent diagnosis of bone marrow failure shown in the pedigree. Examination of this patient demonstrates absence of the radius and short stature and his sister died of head and neck cancer at age 23. Which of the following diagnostic tests is most likely to yield a positive result? A. B. C. D. E. Antibody diversity Diexpoxybutane breakage Ionizing radiation sensitivity Sister chromatid exchange Telomere flow FISH 804 G43. A child is diagnosed with malignant rhabdoid tumor of the kidney. You order genetic testing. Which of the following specific gene test is most likely to be positive? A. B. C. D. E. G44. A 45-year-old woman is diagnosed with secondary glioblastoma after a prior astrocytoma and the tumor is found to carry an IDH2 missense mutation. Analysis of a germline sample from this patient is most likely to reveal? A. B. C. D. E. G45. A second germline IDH2 mutation in addition to the tumor mutation Amplification of IDH2 Deletion of one copy of IDH2 Normal sequence of the IDH2 gene The same IDH2 missense mutation in the heterozygous state Analysis of the total number of somatic mutations in a colon cancer specimen undergoing whole exome sequencing demonstrates thousands of somatic mutations. Germline genetic testing revealed a pathogenic variant in a hereditary colon cancer gene. Which of the following genes is most likely to result in predisposition to developing a hypermutated tumor? A. B. C. D. E. G46. CTNNB1 DICER1 PTCH1 SMARCB1 SUFU APC MSH2 MUTYH POLE STK11 A 25-year-old woman develops a paraganglioma. You order a 10 gene hereditary pheochromocytome/paraganglioma panel which reveals a nonsense pathogenic variant in SDHB. Which of the following clinical concerns is most compatible with this molecular result? A. The patient will require MRI screening for renal cell carcinoma B. The patient will require careful monitoring for malignant transformation of the paraganglioma C. The patient will require prophylactic thyroidectomy to reduce risk of medullary thyroid cancer D. The patient is at decreased risk of having an affected child because SDHB is maternally imprinted E. The patient will require careful monitoring of Calcium given risk of parathyroid tumors 805 G47. There is increasing use of genetic testing for cancer patients to determine treatment decisions. Which of the following pairs of genes and medications best describes this new use of genetic testing? A. B. C. D. E. APC and immune checkpoint inhibitors BRCA2 and PARP inhibitors NF1 and angiogenesis inhibitors TP53 and HER2 inhibitors VHL and mTOR inhibitors 806 Answers to Cancer Genetics Questions G1-G47 G1. The cumulative incidence of endometrial cancer in women who carry HNPCC mutations is estimated to be approximately 70%. Ovarian, bile duct and pancreatic are all increased in HNPCC. Breast cancer is not. The correct answer is C. G2. Pulmonary pleuroblastoma (PPB) is part of a cancer susceptibility syndrome due to DICER1 germline mutations which includes a variety of other benign (multinodular goiter) and malignant cancers. Rhabdomyosarcoma is found in families with PPB and DICER1 mutations. The correct answer is D. G3. For this reason the patient may present with congenital anomalies or cancer prior to bone marrow failure being clinically evident. The correct answer is D. G4. There is an estimated 4-5% risk of developing medulloblastoma in patients with Gorlin syndrome. This can represent the first clinical manifestation of the disorder and when present precedes the development of basal cell carcinomas. The correct answer is B. G5. The clinical features given are typical of DysKeratosis Congentia (DKC). Nail abnormalities are not seen in Fanconi anemia. DKC results from a telomere defect and quantitative measure of telomere length is the laboratory test which is now used to aid in making the diagnosis. The correct answer is E G6. Cowden Syndrome is due to mutations in the PTEN tumor suppressor gene and is associated with breast and thyroid cancer. Malignancies less frequently seen in Cowden Syndrome include endometrial cancer and renal cell carcinoma. The correct answer is E G7. Hepatoblastoma develops in very young children and recent publications recommend screening for hepatoblastoma in children identified to carry mutations in the APC gene. Approximately 10% of children with hepatoblastoma carry a mutation in the APC gene. Beckwith Weidemann Syndrome also carries an increased risk of hepatoblastoma. The correct answer is B. G8. The clinical features are typical of Bloom syndrome, including the butterfly rash on the face. Children with Bloom syndrome are very short. The abnormal recombination that results from mutations in the BLM gene yields an increased number of sister chromatid exchanges which can be used as a laboratory test to aid in making diagnosis. The correct answer is D. G9. A significant percentage of patients (over 35%) with VHL carry missense mutations. In particular, missense mutations are associated with Type II VHL. The Type II families demonstrate an increased risk of developing pheochromocytomas. The other syndromes listed all have truncating mutations (nonsense, frameshift and splicing) as the predominant type. The correct answer is E. 807 G10. The risk of medullary thyroid cancer in childhood is highest in MENIIA and MENIIB. However, in MENIIB the age of onset is considerably lower so that prophylactic thyroidectomy is recommended by age 1. In MENIIA it is recommended to be performed by age 5. FAP is associated with an increased risk of thyroid cancer compared to the general population but not to an extent to warrant prophylactic thyroidectomy. The correct answer is D. G11. Options B, C, E are features of NF1 but are normally diagnosed in very young or early school age children. Malignant peripheral nerve sheath tumors typically first appear during the teenage or young adult years. Acoustic lesions can be diagnosed in teenagers but occur in NF2 not NF1. The correct answer is D. G12. There significant variation among different published studies, however, most studies have resulted in estimates consistent with 25-60%. The correct answer is D. G13. Results from the Breast Cancer Linkage Consortia give a lifetime estimate of 6% for men who carry BRCA2 mutations. Although the lifetime risk is lower, several recent papers have demonstrated that some men with breast cancer carry mutations in the BRCA1 gene. The correct answer is B. G14. Overall about 15% of unilateral retinoblastoma patients carry a germline mutation. There is the risk that this mutation may be the result of germline mosaicism in the parents (which for offspring with bilateral retinoblastoma results in a 5-7% recurrence risk for another child). In the unilateral setting described here this risk is diminished but is still approximately 1% for each pregnancy that another child may develop retinoblastoma. The correct answer is C. G15. The results shown above are typical for a sporadic case of retinoblastoma. The frameshift mutation is not found in the blood sample and thus must have occurred after fertilization. Promoter methylation is an event which occurs during tumor development and is not usually inherited. With this result the likelihood that the parents carry a germline RB1 mutation is extremely low and the recurrence risk is similar to the population risk (1 in 15,000-30,000). The correct answer is A. G16. FAP and HNPCC have significantly higher risks, 95% and 70%, respectively, then JPC and PJS. Although initially controversial is it now clear that JPC families do have an increased risk of colorectal cancer (~50%) and that appears to be particularly true of families that carry mutations in the SMAD4 gene. The correct answer is A. G17. There is a significant risk of germline mosaicism in the fathers of children with constitutional RB1 mutations resulting in a 6-7% recurrence risk. Thus, even if parents test negative it is important to have all subsequent siblings tested at birth for the mutation identified in the affected child in order to determine if the newborn needs surveillance for retinoblastoma. The correct answer is B. 808 G18. Inherited mutations in all five genes result in an increased risk of developing cancer. However, in GWAS studies you examine polymorphisms that appear in the general population with a minor allele frequency of about 5% or greater. For ATM, PALB2 and PTEN there are a large number of individually rare variants that cause an increased risk of cancer. For CHEK2 the recurrent mutations in the population have a frequency of about 1% and are frameshift and deletions which are not easily detected by GWAS genotyping assays. A polymorphism in FGFR2 found in 0.38 of the case population is one of the most consistent GWAS hits for breast cancer susceptibility. The correct answer is C. G19. The splice site mutations appear to result in a small amount of correctly spliced product that is functional. There are also low penetrance families that carry missense mutations in conserved domain that result in some residual activity of the RB1 protein. The correct answer is D. G20. BWS often results from disruption of imprinting. Evidence for this includes the presence of UPD in children with BWS as well as families with affected cousins that inherit the mutation through their mothers. The correct answer is B. G21. The genetic etiology of Beckwith Weidemann syndrome is complex and there are overlapping clinical features seen in children with different underlying molecular abnormalities. The IGF2 growth factor is thought to be important for growth control and tumor development. Children with abnormal methylation of the H19/IGF2 locus have a high Wilms tumor risk compared with the other options given. Clinically, children with hemihypertrophy and organomegaly also have a high Wilms tumor risk. The correct answer is A. G22. Costello syndrome is due to classic mutations which constitutively activate the H-Ras oncogene. The second wild-type H-Ras gene is retained in the tumor and transfection assays of the mutant gene into NIH3T3 cells demonstrate transforming activity. The correct answer is A. G23. The interpretation of VUS results is quite complex. In the case of BRCA2, patients who carry two deleterious mutations demonstrate a Fanconi phenotype. Thus, the finding of a frameshift mutation in the same patient argues strongly against pathogenicity. There are a number of individuals who carry mutations in both BRCA1 and BRCA2 so (A) is not that informative. There are many rare benign variants in BRCA2 so the lack of data in a control population is not helpful. Options D and E are both findings for mutations that are associated with hereditary breast/ovarian syndrome. The correct answer is B. G24. Microsatellite instability is seen in tumors from HNPCC families. This is evidenced by increases and decreases in the size of a repeat in the tumor specimen when compared to normal DNA from the same patient. Some laboratories opt to use immunohistochemistry for the expression of the MSH2 and MLH1 proteins as an alternative to microsatellite instability testing. Methylation studies of the MLH1 gene are normally performed next as that is a common somatic change which results in microsatellite instability. The correct answer is D. 809 G25. The clinical description is classic for familial adenomatous polyposis (FAP). Adults with FAP develop gastric polyps and are at increased risk for small bowel carcinomas. Thus, they require lifelong surveillance of the upper GI tract after removal of the colon. The correct answer is E. G26. Xeroderma pigmentosum (XP) is the classic autosomal recessive cancer predisposition syndrome. Their cells do not correct UV-induced DNA lesions correctly. In Cockayne syndrome, cells are deficient for transcription-coupled repair and patients do not have a significant cancer risk. In the majority of XP subtypes, cells are deficient for nucleotide excision repair with resulting mutagenesis and cancer predisposition. The correct answer is B. G27. All of the options are rearrangements seen in ALK except C. Deletions are found in tumor suppressor genes, not oncogenes. The question asks about analysis of a blood sample, so we are looking for the cause of familial neuroblastoma which is the result of inheriting missense mutations in ALK. The inversions, translocations and amplifications are somatic rearrangements in ALK that are found in tumors. The correct answer is D. G28. The correct answer is C. Source of topic: slides Keywords: colon cancer, Lynch syndrome, MLH1 Explanation: Approximately 15-18% of colon cancers reveal microsatellite instability. However, the vast majority of these tumors have loss of mismatch repair function through epigenetic silencing of MLH1. It is only about 5% of these MIS+ tumors (or 2% of the overall population) that demonstrate germline mutation in one of the MMR genes. G29. The correct answer is B. Source of topic: slides Keywords: pulmonary pleuroblastoma, microRNA, tumor suppressor gene Explanation: The majority of patients (>80%) with pulmonary pleuroblastoma carry a germline mutation in DICER1, an enzyme involved in microRNA processing. CTNNB1, beta catenin, undergoes somatic mutation in a number of tumors; PTCH1 mutations result in Gorlin sydrome; SMARCB1 results in rhabdoid tumors and SUFU mutations result in medulloblastoma. G30. The correct answer is C. Source of topic: slides Keywords: breast cancer, gastric cancer Explanation: CDH1 encodes E-cadherin and mutations in this gene were first identified in familial diffuse gastric cancer. Analysis of these families demonstrated that there was an unexpected prevalence of lobular breast cancers in these families. Subsequently, analysis of familial lobular breast cancer kindreds also revealed CDH1 mutations. G31. The correct answer is E. Source of topic: slides Keywords: renal cell cancer Explanation: Chromophobe renal cancer is associated with the Birt-Hogg-Dube syndrome. Individuals with BHD often have a significant history of spontaneous pneumothorax as teen-agers or young adults. Pulmonary fibrosis is seen in dyskeratosis congenita and pulmonary AVMs can be seen in hereditary hemorrhagic telangiectasia. Pulmonary hemangiomas are not a typical 810 finding of VHL although hemangioms of the retina or hemangioblastoma of spine and cerebellum are seen in VHL. Patients with VHL develop clear cell RCC not chromophobe. G32. The correct answer is E. Source of topic: slides Keywords: polyposis, hamartoma Explanation: All of the genes listed can result in polyposis syndromes of different types. However, intussusception is a life-threatening complication of Peutz-Jeghers syndrome and can occur at much older ages than seen in the general population. Once the diagnosis of PJS is made it is important to warn patients and parents of this risk and the need for immediate medical care of symptoms if an acute abdomen develops. G33. The correct answer is A. Source of topic: slides. Keywords: BRCA1/2, risk prediction Explanation: BRCAPro was developed to predict the likelihood of BRCA1/2 mutation and can be used in women with or without a personal history of breast cancer. The Chompret criterion is helpful in determining the likelihood of TP53 mutations. The Claus tables are designed to determine the risk of breast cancer in healthy women based on their family history of breast cancer. The Gail model is also for healthy women (over age 35) to predict risk of breast cancer. The NCCN guidelines suggest TP53 testing for any woman diagnosed with breast cancer under age 35 if BRCA1/2 is negative. G34. The correct answer is C. Source of topic: slides Keywords: trisomy 21, leukemia Explanation: Somatic GATA1 mutations are seem in almost all cases of TMP which is a frequent complication of children with Down syndrome. AKT undergoes somatic mutation in Proteus syndrome and is not a transcription factor, ETV6 is a translocation partner in AML, IKZF1 mutations and deletions are common in ALL and RUNX1 deletions and mutations cause familial platelet disorder with AML. G35. The correct answer is A. Source of topic: Slides Keywords: retinoblastoma, genetic testing Explanation: The test results provided are consistent with sporadic retinoblastoma in the child given that both hits (a nonsense mutation in the RB1 gene followed by loss of heterozygosity) were seen in the tumor and no evidence of the nonsense mutation in the blood sample. Thus, with this information there is much less than 1% risk to subsequent siblings. 811 G36. The correct answer is E. Keywords: polyposis, hamartoma Explanation: All of the genes listed can result in polyposis syndromes of different types and are often on gene panels. However, juvenile polyposis is most likely to present first in young children with rectal bleeding due to a large polyp and thus SMAD4 is the most likely gene to have a pathogenic mutation in this scenario. The other gene underlying juvenile polyposis is BMPR1A. G37. The correct answer is E. Source of topic: Slides Keywords: dyskeratosis congenita, telomeres Explanation: The pedigree suggests an X-linked pattern of inheritance. Although bone marrow failure and head & neck cancer is also common in Fanconi anemia, it is very rarely X-linked and abnormal nails and leukoplakia are key features of dyskeratosis congenita. The most common form of DKC is X-linked and telomere flow FISH demonstrating very short telomere is the diagnostic test. G38. The correct answer is E. Keywords: TP53 guidelines, risk prediction Explanation: The NCCN guidelines suggest TP53 germline testing for any woman diagnosed with breast cancer under age 35 if BRCA1/2 is negative. A is wrong because the Amsterdam criteria are designed to determine the likelihood of Lynch syndrome. The Chompret criterion is helpful in determining the likelihood of TP53 mutations. The Claus tables are designed to determine the risk of breast cancer in healthy women (without breast cancer) based on their family history of breast cancer. The Gail model is also for healthy women (over age 35) to predict risk of breast cancer and can’t be used once a woman has a diagnosis of breast cancer. G39. The correct answer is E. Explanation: VHL is associated with pheochromocytomas of both the adrenal and extraadrenal locations. Individuals with VHL have a significantly increased risk of endolymphatic sac tumors that are not seen in any of the other genes listed here. All of the other genes are associated with pheochromcytoma or paraganglioma. G40. The correct answer is B. Explanation: The clinical description is typical of hereditary leiomyomatosis with renal cell cancer (HLRCC) which is caused by heterozygous mutations in the fumarate hydratase (FH). Birt Hogg Dube is associated with oncocytic RCC and not with uterine lesions; MET is associated with hereditary papillary renal cell cancer and not the other lesions; PTEN can be associated with RCC and endometrial cancer but the skin lesions are different; VHL is associated with RCC but no skin or uterine findings. 812 G41. The correct answer is C. Keywords: oncogenes, targeted therapeutics Explanation: C is correct. Crizotinib is an inhibitor of the ALK kinase. FDA approval was granted for treatment of non-small cell lung cancer patients if the tumor specimen is demonstrated to contain the EML4-ALK inversion. FDA approval is specific to the gene and the type of rearrangement tested. G42. The correct answer is B. Keywords: Fanconi anemia, chromosome breakage syndromes Explanation: The pedigree suggests an autosomal recessive pattern of inheritance. Most forms of Fanconi anemia are autosomal recessive except one X-linked form (FANCB). Bone marrow failure and head & neck cancer are common in both Fanconi anemia and Dyskeratosis congenita. However, radial ray anomalies are a classic feature of Fanconi anemia (as is short-stature). Thus, the diagnostic test is diepoxybutane testing of a peripheral blood sample to look for increased breakage and chromosome abnormalities. G43. The correct answer is D. Keywords: rhabdoid predisposition syndrome. Explanation: Each of genes provided is associated with a specific class of childhood cancers. Rhabdoid tumors of the solid organs and the CNS (referred to as atypical rhabdoid/teratoid tumors) are associated with both somatic and germline mutations in SMARCB1. For the other choices: CTNNB1, beta catenin, undergoes somatic mutation in a number of tumors, particularly, hepatoblastoma; the majority of patients (>80%) with pulmonary pleuroblastoma carry a germline mutation in DICER1, an enzyme involved in microRNA processing; PTCH1 mutations result in Gorlin syndrome and risk of medulloblastoma; SUFU mutations result in medulloblastoma. G44. The correct answer is D. Keywords: Pattern of mutations in oncogenes Explanation: The majority of mutations in proto-oncogenes are somatic mutations. The cancer patient does not inherit a mutation and there is a single mutant allele. Thus, analysis of the germline sample is most likely to reveal normal sequence and there is a single heterozygous missense mutation in IHD2 in the tumor sample. G45. CORRECT ANSWER: D SOURCE: Lecture/Slides KEYWORDS: Hypermutated tumors, hereditary colon cancer EXPLANATION: All of the genes provided result in an increased risk of colon cancer. Specific missense mutations in the POLE gene augment the error prone polymerase encoded by POLE and result in hypermutated tumors, with the highest number of somatic mutations currently identified. A - Tumors from patients with FAP caused by mutations in APC gene have multiple rearrangements but are not hypermutated B – Tumors from patients with Lynch syndrome were previously thought to be those with the highest rate of somatic mutation but POLE tumors are higher C – Tumors from patients with MUTYH also demonstrate diminished base excision repair but again are not hypermutated 813 E – Patients with Peutz-Jeghers syndrome from STK11 mutations do have an increased risk of colon cancer but it is not thought to be for hypermutated tumors. G46. CORRECT ANSWER: B SOURCE: Lecture/Slides KEYWORDS: SDH, hereditary paraganglioma, imprinting EXPLANATION: One of the most significant clinical concerns for carriers of SDHB pathogenic variants is the increased risk of malignant tumors which are otherwise rare in the other syndromes and may result in metastatic spread. A.– VHL is associated with PHEO and renal cell carcinoma, C – MEN2 is associated with PHEO and medullary thyroid cancer D – SDHD is paternally imprinted (not SDHB) E – MEN1 and MEN2 are associated with an increased risk of parathyroid tumors. G47. CORRECT ANSWER: B SOURCE OF ITEM TOPIC: Lecture/Slides KEYWORDS: pharmacogenetics, BRCA EXPLANATION: Tumors that are homologous recombination deficient results in sensitivity to PARP inhibitors such as Olaparib (FDA approved) which destroys the backup DNA repair pathway. A – it is Lynch syndrome genes (not APC) that results in increased sensitivity to immune checkpoint inhibitors C – it is NF2 (not NF10 and angiogenesis inhibitos like Bevacizumab D – it is ERBB2 amplified breast cancers which are sensitive to HER2 inhibitors such as trastuzumab E – it is TSC1 or TSC2 mutations that lead to sensitivity to mTOR inhibitors. 814 H. Cytogenetics Questions H1-H43 H1. Which of the following syndromes results from a chromosome deletion in a major proportion of cases? A. CHARGE syndrome B. Fragile X syndrome C. Mowat-Wilson syndrome D. Prader-Willi syndrome E. Charcot-Marie-Tooth syndrome H2. Microdeletions and duplications mediated by segmental duplications are recurrent due to their underlying mechanism. Which of the following disorders is the most common recurrent microdeletion syndrome? A. B. C. D. E. H3. Aniridia-Wilms tumor Retinoblastoma Prader-Willi syndrome VCF/DiGeorge syndrome Cri-du-chat syndrome A 2-year-old boy presents with a history of retinoblastoma, dysmorphic features and developmental delay. Which of the following molecular abnormalities is the most likely cause of his clinical findings? A. Nonsense mutation in the RB1 gene. B. Missense mutation in the RB1 gene. C. RB2 gene mutation D. Contiguous gene deletion of 13q14 E. Contiguous gene deletion of Xq27.3. H4. A young woman with severe mental retardation, short stature, and other malformations has a karyotype of 45,X/46,X,r(X). Which of the following explanations is most likely to account for the constellation of clinical features noted above? A. B. C. D. E. Both X chromosomes are likely to be active in the cells with 46 chromosomes. The normal X is likely to be inactive in most or all of the cells with 46 chromosomes. The ring X is likely to be acentric explaining its absence in the 45,X cell line. The ring X is likely to be inactive in most or all of the cells with 46 chromosomes. The XIST gene will almost certainly be present on the ring X chromosome. 815 H5. A middle-aged couple visits prenatal clinic for preconception counseling. Based on the wife’s age their risk of having a child with Down syndrome is increased. The genetic counselor explains that even if their pregnancy were to result from a trisomy 21 conception there is a reduced likelihood that the pregnancy would lead to a liveborn infant. Which of the following probabilities best represents the likelihood that a trisomy 21 conception will result in a liveborn infant? A. B. C. D. E. H6. 1% 20% 70% 90% 100% Which of the following chromosome abnormalities is mostly likely constitutional? A. t(8;21)(q22;q22) B. t(11;22)(q23;q11.2) C. del(5)(q22q33) D. inv(16)(p13q22) E. t(15;17)(q22;q21) H7. Triploidy is the result of three copies of each human chromosome and is one of the most common causes of spontaneous miscarriage. Which of the following occurrences is the most common mechanism by which triploidy arises? A. B. C. D. E. H8. chimerism duplication of an entire maternal haploid chromosome set one egg fertilized by two sperm tetraploid conception with loss of one copy of each chromosome two eggs fertilized by one sperm A newborn presents with microcephaly, hypotonia, and a high-pitched cat-like cry? Which of the following cytogenetic abnormalities is most likely to be found on chromosomal analysis? A. 4p deletion B. 5p deletion C. 5q deletion D. 8p deletion E. 11p deletion H9. A child is born with a partial deletion and partial duplication involving the same chromosome. Which of the following parental chromosomal abnormalities is most likely to lead to this occurrence? A. B. C. D. E. balanced reciprocal translocation balanced Robertsonian translocation isochromosome pericentric inversion paracentric inversion 816 H10. Chorionic villus sampling is performed on a 38-year-old woman who is concerned about having a child with a chromosomal disorder. Which one of the following karyotypes carries the highest risk for this woman to deliver a chromosomally abnormal livebom? A. B. C. D. E. 45,XX,der(13;14)(q10;q10) 45,XY,der (14;21)(q10;q10) 46,XY,inv(2)(p11.2q13) 46,XX,inv(2)(q31q35) 46,XX,t(11;22)(q23;q11.2) H11. Which of the following prenatal testing methods can detect confined placental mosaicism? A. B. C. D. E. H12. amniocentesis chorionic villus sampling fetal cells in maternal blood maternal blood percutaneous umbilical blood sampling A young woman is found to have a balanced translocation after a family history and pedigree analysis, during a preconception counseling visit, raised a red flag for a chromosomal abnormality. Based on this clinical finding, which of the following statements about chromosomal segregation and possible offspring is the most likely clinical outcome? A. Offspring resulting from alternate segregation will be chromosomally normal. B. Offspring resulting from alternate segregation are likely to be phenotypically normal. C. Offspring resulting from adjacent-1 segregation will be chromosomally normal. D. Offspring resulting from adjacent-1 segregation are likely to be phenotypically normal. E. Offspring resulting from adjacent-2 segregation are likely to be phenotypically normal. H13. During meiosis, homologs must pair in order for recombination to occur. Which of the following events allows optimal pairing and normal segregation: A. B. C. D. E. H14. One or more chiasma per chromosome Two or more chiasma per chromosome arm Distal position of chiasma on chromosome arm Medial position of chiasma on chromosome arm Centromere pairing of chromosomes Which of the following microarray confirmation methods can allow the cytogeneticist to determine the mechanism of an imbalance detected on comparative genome hybridization studies? A. Fluorescence In Situ Hybridization (FISH) B. Junction fragment PCR C. Multiplex ligation-dependent probe amplification (MLPA) D. Quantitative PCR E. Targeted microarray for the abnormal region 817 H15 If you were a T-lymphocyte destined for cytogenetic analysis, some of the treatments you would experience along the way (in correct order) are: A. B. C. D. E. H16. Hypotonic solution, acetic acid, Giemsa, trypsin Hypotonic solution, lysis buffer, trypsin, Giemsa Phytohemagglutinin, hypotonic solution, fixative, Colcemid, Giemsa Phytohemagglutinin, Colcemid, hypotonic solution, fixative, trypsin Trypsin, acetic acid, hypotonic solution, Giemsa The historical milestones in cytogenetics mark exciting intervals in the development of the field of clinical genetics. Which of the following series of events follows the chronological order in which these milestones were attained? A. Hypotonic treatment of cells > Correct chromosome number is 46 > Y chromosome identified in humans > Chromosome banding techniques>Fragile sites and fragile X syndrome B. Correct chromosome number is 46 >Hypotonic treatment of cells > Y chromosome identified in humans > Chromosome banding techniques> Fragile sites and fragile X syndrome C. Y chromosome identified in humans >Hypotonic treatment of cells > Correct chromosome number is 46 > Chromosome banding techniques> Fragile sites and fragile X syndrome D. Hypotonic treatment of cells > Y chromosome identified in humans> Correct chromosome number is 46 > Chromosome banding techniques> Fragile sites and fragile X syndrome E. Y chromosome identified in humans> Hypotonic treatment of cells > Correct chromosome number is 46 > Fragile sites and fragile X syndrome >Chromosome banding techniques> H17. A newborn presents with a cardiac defect, cleft palate and a hypoplastic thymus. Which of the following chromosome abnormalities is the most likely cytogenetic imbalance in this patient? A. A deletion of 1q.21.1 B. A deletion of 2q11.1 C. A deletion of 16p11.2 D. A deletion of 22q11.2 E. A deletion of 22q13.3 H18. The mammalian somatic cell cycle is approximately 24 hours in length. Which of the following phases of the cell cycle requires the most time? A. G1 B. S C. G2 D. Interphase E. Mitosis 818 H19. Meiotic recombination is necessary for genetic diversity and rates of genetic recombination are different in males than females and for different chromosomal regions. The highest rate of recombination is observed in which of the following situations? A. in females compared to males near centromeres B. in males compared to females near centromeres C. in males compared to females in the middle of each chromosome arm D. near centromeres compared to near telomeres E. near telomeres compared to near centromeres H20. An MRI shows lissencephaly in your patient, however the patient does not have any dysmorphic facial features. Which of the following is the most likely mutation? A. B. C. D. E. H21. G-banding has been the preferred method for cytogenetic analysis since the late 1960s. The underlying DNA content corresponds to the staining pattern observed by G-banding. Which of the following descriptions details the difference(s) between G-negative bands and G-positive bands? A. B. C. D. E. H22. Point mutation within the LIS1 gene coding region. Terminal deletion of 17p which includes the LIS1 gene Terminal deletion of 17q which includes the LIS1 gene Terminal duplication of 17p which includes the LIS1 gene Triplet repeat expansion involving the LIS1 gene G-negative bands are more gene rich than G-positive bands G-positive bands are GC rich regions and are early replicating G-negative bands are AT rich and are late replicating G-negative bands are GC rich and are late replicating G-positive bands are more gene rich than G-negative bands Comparative genomic hybridization (CGH) is one method that can be used for copy number variation (CNV) detection. Which of the following issues is a limitation of this methodology compared to a G-banded karyotype? A. B. C. D. E. Cannot detect polyploidies Cannot detect single copy differences Cannot detect unbalanced translocations Lower resolution than G-banded karyotype Requires cells to be cultured 819 CELL BIOLOGY: H23. After a normal meiosis I cell division, the resulting cells contain which of the following number of chromosomes and chromatids? A. B. C. D. E. H24. Contains 23 chromosomes and 23 chromatids Contains 23 chromosomes and 46 chromatids Contains 46 chromosomes and 23 chromatids Contains 46 chromosomes and 46 chromatids Contains 46 chromosomes and 92 chromatids Aneuploidies typically result from meiotic errors but the specific missing or additional chromosome is not necessarily derived from the maternal or paternal gametes at an equal rate. Which of the following aneuploidies virtually always arises as a result of maternal meiotic error? A. B. C. D. E. 45,X 47,XY,+16 47,XX,+21 47,XXX 47,XXY H25. A child is born with a partial deletion and partial duplication for the same chromosome. Which of the following chromosomal abnormalities represents the likely parental chromosome abnormality predisposing to this occurrence? A. B. C. D. E. H26. Balanced insertion Balanced reciprocal translocation Balanced Robertsonian translocation Paracentric inversion Pericentric inversion Chromosomal rearrangements can result from a variety of mechanisms during meiosis or mitosis. Which of the chromosome rearrangements listed below always involves at least two chromosomes? A. B. C. D. E. Balanced Translocation Duplication Interstitial deletion Isochromosome Pericentric inversion 820 H27. Carriers of balanced structural chromosomal rearrangements are at risk of having offspring with an unbalanced chromosome complement. Which of the following statements is true regarding the risk of unbalanced offspring? A. Carriers of small pericentric inversions have higher risk than large pericentric inversions B. Carriers of paracentric inversions have higher risk than pericentric inversions C. Carriers of balanced autosomal translocations have a 75% risk of having unbalanced offspring D. Female carriers of the 21;21 Robertsonian translocation have a 50% risk of having trisomy 21 offspring E. Female carriers of the 14;21 Robertsonian translocation are at higher risk than male carriers H28. The figure below depicts the various phases of the mammalian somatic cell cycle. The numbers in parenthesis represent each of the phases. What is the correct order of phases? A. B. C. D. E. 1=G1, 2=G2, 3=S, 1=G1, 2=S, 3=G2, 1=G1, 2=S, 3=G2, 1=G1, 2=S, 3=G2, 1=S, 2=G1, 3=G2, 4=prophase, 4=prophase, 4=prophase, 4=prophase, 4=prophase, 5=metaphase, 6=anaphase, 5=anaphase, 6=metaphase, 5=metaphase, 6=anaphase, 5=metaphase, 6=telophase, 5=metaphase, 6=anaphase, 821 7=telophase 7=telophase 7=telophase 7=anaphase 7=telophase H29. 41. A phenotypically normal person carries a reciprocal balanced translocation between two autosomes. When segregation occurs in this individual during meiosis I, which of the following statements regarding the eventual offspring appropriately associates the genotype with the expected phenotype? A. Alternate segregation always leads to a normal karyotype with a normal phenotype B. Alternate segregation can lead to an abnormal karyotype with a likely normal phenotype C. Adjacent-1 segregation leads to a normal karyotype with an abnormal phenotype D. Adjacent-1 segregation leads to an abnormal karyotype with a likely normal phenotype E. Adjacent-2 segregation leads to a normal karyotype with a likely normal phenotype H30. Non-allelic homologous recombination (NAHR) mediated by segmental duplications is a mechanism known to cause recurrent deletions and duplications across the genome. Deletions of 22q11.2, which have been associated with DiGeorge syndrome and Velocardiofacial syndrome, are the most common recurrent imbalance. Which of the following syndromes is also caused by a NAHR-mediated mechanism? A. 1p36 deletion syndrome B. Cri du Chat syndrome C. Miller-Dieker syndrome D. Potocki-Lupski syndrome E. Wolf-Hirschhorn syndrome H31. During meiosis, homologs must pair in order for recombination to occur. Which of the following features of homologous chromosome pairing allows optimal pairing and normal chromosome segregation? A. B. C. D. E. H32. Centromere pairing Distal position of chiasma on chromosome arm Medial position of chiasma on chromosome arm One or more chiasma per chromosome Two or more chiasma per chromosome arm In normal female somatic cells, one chromosome is active and the second remains condensed and appears in interphase as a Barr body due to X-inactivation. The number of Barr bodies in an individual depends on the chromosomal complement of the sex chromosomes. Which of the following karyotypes would result in detection of three Barr bodies? 822 A. 46,XXY B. 47,XXX C. 48,XXXY D. 48,XXXX E. 49,XXXYY H33. Two unrelated patients are evaluated in genetics clinic. One of the patients has sickle cell anemia phenotype and the other has a thalassemia phenotype. Molecular analysis detected homozygous mutations in beta globin for each patient. In the first patient, the homozygous mutation was consistent with sickle cell anemia (coding for the Glu6Val substitution), while the second patient was homozygous for a stop codon in the betaglobin gene. Which of the following concepts best describes these findings? A. Allelic heterogeneity B. Incomplete penetrance C. Locus heterogeneity D. Pleiotropy. E. Variable expressivity CLINICAL CYTOGENETICS: H34. Imprinting plays a role in determining the phenotype of many different conditions. In which of the following groups of disorders can all of the diagnoses be associated with imprinting as a major determinant of phenotype? A. B. C. D. E. Angelman syndrome, Prader-Willi syndrome, DiGeorge syndrome Ovarian teratoma, Trisomy 13 mosaicism, Beckwith-Wiedemann syndrome Triploidy, Prader-Willi syndrome, ovarian teratoma Triploidy, Williams syndrome, Beckwith-Wiedemann syndrome Prader-Willi syndrome, Trisomy 8 mosaicism, Russell-Silver syndrome H35. Which of the following recurrent translocations is diagnostic of chronic myeloid leukemia (CML)? A. B. C. D. E. t(8;21)(q22;q22) t(15;17)(q22;q21) t(12;21)(p13;q22) t(4;11)(q21;q23) t(9;22)(q34;q11.2) 823 H36. 13. A patient carries an interstitial deletion in the proximal region of the long (q) arm of the paternal homologue of chromosome 15 [del(15)(q11.2q13.1)]. This patient is expected to present with which of the following disorders? A. B. C. D. E. H37. A 10-year-old boy presents with distal muscle weakness and atrophy associated with mild glove and stocking sensory loss, depressed reflexes, and pes cavus. Array CGH analysis demonstrates an abnormality in chromosome 17 at band p12 shown in the plot below. What is the diagnosis of this patient? A. B. C. D. E. H38. Angelman syndrome DiGeorge syndrome Miller-Dieker syndrome Prader-Willi syndrome Williams syndrome Charcot-Marie-Tooth syndrome type 1A (CMT1A) Hereditary neuropathy with liability to pressure palsies (HNPP) Miller-Dieker syndrome Neurofibromatosis type 1 (NF1) Williams syndrome Recurrent microdeletions and their reciprocal microduplications are the result of which molecular mechanism? A. B. C. D. E. Allelic homologous recombination (AHR) Fork stalling and template switching (FoSTeS) Non-allelic homologous recombination (NAHR) Non-homologous end joining (NHEJ) Replication slippage 824 H39. Genome-wide chromosomal microarray analysis (CMA) detects losses and gains across the genome. Various methods have been developed for CMA, including the use of arrays that contain single nucleotide polymorphism (SNP) probes. Which of the following test characteristics represents an advantage for detecting genetic abnormalities by using an array that contains SNP probes, compared to an array that contains only copy number detection probes? A. Balanced rearrangements can be detected B. Exon-level imbalances can be detected C. Imbalances of the sex chromosomes can be detected D. Smaller imbalances can be detected E. Uniparental disomy can be detected H40. Your laboratory offers chromosomal microarray testing and you were asked to test an individual who has a known cytogenetic abnormality, which was detected by G-banding, to further define the abnormality. Which of the following karyotypes represents an individual who would not benefit from this additional characterization by microarray analysis? A. 47,XY,+mar B. 47,XY,+21 C. 46,XX,t(3;7)(p24;q22)dn D. 46,XY,add(11)(q25) E. 46,XX,der(6)t(1;3)(q32;q27) H41. Chromosomal microarray analysis (CMA) has replaced conventional G-banding as the firsttier test for cytogenetic analysis. Comparative genomic hybridization (CGH) is one type of CMA that can be used to detect copy number variants, however there are still limitations to this method. Which of the following test characteristics is a limitation of this methodology compared to a G-banded karyotype? A. All polyploidies cannot be detected B. Cells need to be cultured for DNA extraction C. Resolution is lower than a G-banded karyotype D. Single copy number differences (i.e., loss or gain) cannot be detected E. Unbalanced translocations cannot be detected 825 H42. Your laboratory offers chromosomal microarray testing and you were asked to test an individual who has a known cytogenetic abnormality, which was detected by G-banding, to further define the abnormality. Which of the following karyotypes represents an individual who would not benefit from this additional characterization by microarray analysis? A. 47,XY,+mar B. 47,XY,+13 C. 46,XX,t(3;7)(p24;q22)dn D. 46,XY,add(2)(q37) E. 46,XX,der(6)t(1;3)(q32;q27) H43. Non-allelic homologous recombination (NAHR) mediated by segmental duplications is a mechanism known to cause recurrent deletions and duplications across the genome. Deletions of 22q11.2, which have been associated with DiGeorge syndrome and Velocardiofacial syndrome, are the most common recurrent imbalance. Which of the following syndromes is also caused by a NAHR-mediated mechanism? A. 1p36 deletion syndrome B. Miller-Dieker syndrome C. Pallister-Killian syndrome D. Sotos syndrome E. Wolf-Hirschhorn syndrome 826 Answers to Cytogenetics Questions H1-H43 H1. Prader-Willi is associated with deletions in 15q. The other disorders are commonly the result of other mechanisms. The correct answer is D. H2. 22q11 deletion syndrome--including DiGeorge syndrome and VCF (velocardiofacial syndrome) is the most common deletion syndrome in humans, occurring in ~1/4000 births. This is a common, recurrent microdeletion mediated by NAHR of flanking low-copy repeats (LCRs). Other LCR genomic disorders (PWS, AS, Williams, Smith-Magenis, Sotos) occur at ~1/10,0001/20,000. Random deletion syndromes such as cri du chat, Wolf-Hirschhorn, Miller-Dieker syndrome, retinoblastoma, aniridia-Wilms tumor are less common (1/50,000-1/100,000 or less). The correct answer is D. H3. The patient has additional clinical features (dysmorphic features and developmental delay) to retinoblastoma, suggesting a larger deletion that includes more genes than just the RB1 gene The correct answer is D. H4. The abnormal phenotypes seen in association with mosaic karyotypes of the type indicated are associated with failure of X inactivation. This is usually also associated with lack of expression of the XIST gene. This means that both the normal X and the ring X are likely to be active. The XIST gene is considerably more likely to be deleted than to be present on the ring X, although deletion of the X inactivation center may be more pertinent than deletion of the XIST gene. As mentioned above, both X chromosomes are likely to be active. The ring is not likely to be acentric. The correct answer is A. H5. The probability that a trisomy 21 conception will result in live birth is approximately 20%. The correct answer is B. H6. the 11;22 translocation is the most common non-Robertsonian translocation observed in constitutional cytogenetic studies The correct answer is B. H7. Polyspermy is the most common cause of triploidy. Chimerism may caused by the fusion of two embryos and differs from mosaicism. The other options are not likely to be observed. The correct answer is C. H8. The features described fit Cri du Chat syndrome which typically results from terminal deletion 5p from band 5p15.2 to 5pter. 4p- syndrome results in Wolf-Hirschorn syndrome. 5q- and 8psyndromes do not have alternative names. 11p- deletion is associated with WAGR syndrome. The correct answer is B. H9. The occurrence of a deletion and duplication involving the terminal portions of the same chromosome is typically the result if one parent carries a pericentric inversion. This occurs if there is a cross-over within the inversion loop at meiosis. The correct answer is D. 827 H10. Both maternal and paternal carriers of rob(13q14q) have about a 1% chance of having a child with translocation trisomy 13. Fathers who carry rob(14q21q) have less than a 1% chance of having a child with translocation Down syndrome. Both types of inversions, regardless of the parental origin, have almost no chance of abnormal offspring. The pericentric inversion (C) is a common inversion found in the population and the paracentric inversion (D) would result in nonviable dicentric and acentric products. Mothers who carry the t(11;22) are at a 6% risk for an abnormal offspring typically have 47 chromosomes with a small extra der(22) and have mental retardation and other anomalies. The correct answer is E. H11. CVS samples chorionic villi from the placenta and can detect the mosaicism. The other tissues sampled do not include placental cells and will not allow for detection of mosaicism confined to the placenta. The correct answer is B. H12. Alternate segregation results in normal gametes and in gametes with balanced chromosomal translocation. Thus the offspring are phenotypically normal but not chromosomally normal in all cases. Offspring from adjacent-1 or adjacent-2 segregation will all be unbalanced and all will be chromosomally abnormal. They would also be phenotypically abnormal, except in very unusual circumstances. Adjacent segregation-1 is when homologous centromeres segregate, in contrast to adjacent-2 where the homologous centrmorees go to the same cell. Adjacent-2 is rare compared to adjacent-1. The correct answer is B. H13. Optimal pairing includes one chiasma per chromosome arm, or two per chromosome. The correct answer is A. H14. Only FISH can show the localization of the signal on chromosomes and allow for a determination of the mechanism for the imbalance. The correct answer is A H15. T-lymphocytes are stimulated to a blast phase with the red kidney bean extract phytohemagglutinin (PHA). After 48-72 hours, a mitotic poison such as Colcemid or colchicine is used to block spindle formation and attachment to “arrest” cells in mitosis, a hypotonic treatment is used to swell the cells, they are fixed in 3:1 methanol:acetic acid, put onto slides, and then most often treated with trypsin prior to Giemsa staining to produce G-banding patterns. The correct answer is D. H16. The discovery of the Y Chromosome and the XX/XY mechanism of sex determination in humans was by T. S. Painter in 1923, hypotonic treatment of cells by T. C. Hsu in 1953, the correct chromosome number by Tjio and Levan in 1956, chromosome banding techniques in 1969/70, and fragile sites and fragile X in the late 70s. The correct answer is C. H17. Deletion 22q11.2 causes VCF/DiGeorge syndrome. The three phenotypic characteristics listed are most consistent with a clinical diagnosis of 22q11.2 deletion. The correct answer is D. H18. The correct answer is D. All of the phases of mitosis take approximately one hour. 828 H19. Overall, recombination rates in human females are significantly higher (~50%) than males, except near telomeres, where male recombination rates are higher than female. Human recombination is about twice as high as that in mouse, perhaps due to our biarmed chromosomes compared to all telocentric chromosomes in mice (remember the obligatory 1 crossover per chromosome arm for stable pairing and segregation). Recombination is suppressed near centromeres, and increases greatly near telomeres in both males and females (but more so in males). The correct answer is E.. H20. Point mutations within the LIS1 gene cause Isolated Lissencephaly syndrome. These individuals can only exhibit lissencephaly without the more dysmorphic features that are associated with Miller-Dieker syndrome, which is caused by larger deletions of 17p. The correct answer is A H21. R-bands are the reverse of G-bands, so there are the same number of each. G-positive bands are AT-rich and relatively later replicating. The correct answer is A H22. The correct answer is A. H23. The correct answer is D. Keywords: meiosis Explanation: Meiosis is a reduction division. In meiosis I, homologous chromosomes replicate so each chromosome consists of two sister chromatids glued together by cohesins. In the final phases of meiosis I, homologous chromosomes segregate into separate daughter cells with each of the four resulting cells containing 23 chromosomes each of which consists of two sister chromatids. H24. The correct answer is B. Keywords: aneuploidy, nondisjunction Explanation: Trisomy 16 is a prenatal lethal aneuploidy due to a maternal meiosis I nondisjunction in 100% of cases. Turner syndrome (45,X) is 70% paternal and 30% maternal in origin. Trisomy 21 is 92% maternal and 8% paternal in origin. 47,XXX is 90% maternal and 10% paternal in origin. Klinefelter syndrome (47,XXY) is 54% maternal and 46% paternal in origin. H25. The correct answer is E. Keywords: structural chromosomal abnormalities Explanation: A pericentric inversion is the only structural chromosome abnormality that can predispose to a terminal duplication on one chromosome arm and a terminal deletion on the other chromosome arm in the offspring. H26. The correct answer is A. Keywords: structural chromosomal abnormalities Explanation: A balanced translocation in the only structural chromosomal abnormality in the list provided to involve at least two chromosomes. All other listed abnormalities involve only one chromosome. 829 H27. The correct answer is E. Keywords: risk in carriers of balanced chromosomal abnormalities Explanation: The risk of having a trisomy 21 offspring in female carriers of the 14;21 Robertsonian translocation is ~10-15%, whereas the risk in male carriers is ~0-2%. Carriers of large pericentric inversions have higher risk than small pericentric inversions. Carriers of pericentric inversions have higher risk than paracentric inversions. Carriers of balanced autosomal translocations have a 10-15% risk of having unbalanced offspring. Carriers of the 21;21 Robertsonian translocation have a 100% risk of having trisomy 21 offspring. H28. The correct answer is C. Keywords: cell cycle, interphase, M-phase, mitosis Explanation: The cell cycle is divided into interphase and M-phase. In interphase, dividing cells pass through G1, S, and G2 phases before starting the M-phase. The Mphase consists of mitosis and cytokinesis. Mitosis (or nuclear division) is divided into prophase, metaphase, anaphase, and telophase. Cytokinesis (or cytoplasmic division) finally splits the cytoplasm into two. H29. The correct answer is B. Keywords: segregation of a balanced translocation Explanation: Alternate segregation can produce either normal or balanced translocation in the offspring. If the carrier is phenotypically normal, then the offspring carrying the same balanced translocation will most likely be phenotypically normal. Adjacent-1 segregation can only produce the unbalanced versions of the translocation in the offspring with an abnormal phenotype. Adjacent-2 segregation is very rare and will also produce unbalanced products that are phenotypically abnormal. H30. The correct answer is D. Potocki-Lupski syndrome is the only syndrome listed that is mediated by NAHR. All of the distractors do not have an underlying mechanism that makes the CNVs recurrent and involving the same unique region across individuals. H31. The correct answer is D. For homologous chromosomes to pair properly, at least one or more chiasma per chromosome is needed. The distractors offer other mechanisms (centromere), positions (distal, medial) or number (two or more). H32. The correct answer is D. The number of Barr bodies is one less than the number of X chromosomes. Therefore, the correct answer is the karyotype with four X chromosomes. All of the other distractors would result in a different number of Barr bodies (A – 1, B – 2, C – 2, E – 2). H33. The correct answer is A. 830 Allelic heterogeneity refers to the situation when different alleles of a single gene produce different phenotypes, and is the correct answer to this question. Incomplete penetrance refers to the situation when an individual has a genotype known to cause a disease but does not display a phenotype. Locus heterogeneity refers to the production of identical phenotypes by mutations at two or more different loci. Pleiotropy refers to disorders in which a single gene or gene pair causes multiple phenotypes, especially when the effects are not obviously related. Expressivity is the extent to which a genetic defect is expressed, from mild to severe. H34. The correct answer is C. Keywords: imprinting Explanation: Imprinting plays a major role in the phenotype of Angelman syndrome, Prader-Willi syndrome, Beckwith-Wiedmann syndrome, Russell-Silver syndrome, ovarian teratoma, and triploidy. Imprinting does not play any role in the phenotype of Trisomy 13 and Trisomy 8 mosaicisms, DiGeorge syndrome, and Williams syndrome. H35. The correct answer is E. Keywords: cancer cytogenetics, leukemia Explanation: The t(9;22)(q34;q11.2) is diagnostic of CML and a subset of B-precursor ALL. The t(8;21)(q22;q22) is diagnostic of AML-M2. The t(15;17)(q22;q21) is diagnostic of acute promyelocytic leukemia (AML-M3). The t(12;21)(p13;q22) is diagnostic of a subset of B-precursor ALL. The t(4;11)(q21;q23) is diagnostic of a subset of B-precursor ALL observed mostly in infants. H36. The correct answer is D. Keywords: microdeletion, genomic disorders Explanation: 15q11.2q13.1 deletion on the paternal chromosome 15 causes Prader-Willi syndrome, whereas the same deletion on the maternal chromosome 15 causes Angelman syndrome. DiGeorge syndrome is due to a 22q11.2 deletion. Miller-Dieker syndrome is due to a 17p13.3 deletion. Williams syndrome is due to a 7q11.23 deletion. H37. The correct answer is A. Keywords: microdeletion, microduplication, genomic disorders Explanation: This array CGH plot shows a 17p12 duplication that encompasses the PMP22 gene. The phenotype of the patient and the 17p12 duplication is consistent with the clinical diagnosis of Charcot-Marie-Tooth syndrome type 1A (CMT1A). Williams syndrome is due to a 7q11.23 deletion. HNPP is due to a 17p12 deletion. Miller-Dieker syndrome is due to a 17p13.3 deletion. NF1 is due to a 17q11.2 deletion. H38. The correct answer is C. 831 Keywords: molecular mechanisms, recurrent CNVs Explanation: Recurrent microdeletions/microduplications are caused by NAHR, which is mediated by the homologous flanking segmental duplications. FoSTeS, NHEJ, and replication slippage, all result in non-recurrent copy number changes. AHR is a normal recombination mechanism that does not result in copy number changes. H39. The correct answer is E. The use of SNP probes allows uniparental disomy (UPD) to be detected. UPD cannot be detected on arrays that only have DNA probes that do not contain polymorphic markers. All of the other distractors listed can be detected on array platforms that contain or do not contain SNP probes. H40. The correct answer is B. Microarray analysis cannot detect the mechanism for a cytogenetic imbalance. Therefore, since a diagnosis of trisomy 21 by G-banding is already established, microarray analysis will not provide any additional information about this imbalance. Microarray analysis could help to: identify the origin of a marker chromosome (A) or additional material on a chromosome (D), look for an imbalance at the breakpoint of an apparently balanced de novo translocation (C), or define the size and gene content of an unbalanced translocation (E). H41. The correct answer is A. CMA using CGH cannot detect all polyploidies since the data is normalized to a copy number normal reference DNA sample. Therefore, tetraploidies, and many triploidies cannot be detected using this method. Cells do not need to be cultured, DNA can be extracted from blood samples. The resolution is higher than a G-banded karyotype and single copy number difference can be detected. Unbalanced translocations can also be detected by CMA/CGH. H42. The correct answer is B. Microarray analysis cannot detect the mechanism for a cytogenetic imbalance. The diagnosis of trisomy 13 by G-banding is established, so microarray analysis will not provide any additional information about this imbalance. Microarray analysis could help to: identify the origin of a marker chromosome (A) or additional material on a chromosome (D), look for an imbalance at the breakpoint of an apparently balanced de novo translocation (C), or define the size and gene content of an unbalanced translocation (E). H43. The correct answer is D. Sotos syndrome is the only syndrome listed that is mediated by NAHR. All of the distractors do not have an underlying mechanism that makes the CNVs recurrent and involving the same unique region across individuals. 832 I. Prenatal/Reproductive Genetics Questions I1-I46 I1. Chorionic villus sampling (CVS) occasionally produces cytogenetic results that are somewhat ambiguous such as mosaicism. The finding of a mosaic trisomy 15 in a CVS leads to a secondary amniocentesis, which demonstrated a normal diploid karyotype. In such a case which of the following clinical options would be the best response? A. B. C. D. E. I2. Which of the following chromosome studies would most likely be detected in a phenotypically normal woman with infertility? A. B. C. D. E. I3. 46,XY 45,X 46,XX/45,X 47,XXY 47,XYY Fragile X testing should be considered during infertility evaluations when you encounter which of the following clinical findings? A. B. C. D. E. I4. Consider uniparental disomy molecular cytogenetic studies for chromosome 15 Monitor the pregnancy with targeted ultrasound for cardiac malformations. Perform cordocentesis (funicentesis) to confirm normal karyotype in fetal blood. Reassure the parents that the baby will be normal during a discussion of the results. Suggest consideration of termination of pregnancy for fear of hidden ‘tissue mosaicism” azoospermia oligospermia partial androgen insensitivity polycystic ovarian syndrome premature ovarian failure The risk of pregnancy loss is considered to be the highest with which of the following prenatal procedures? A. B. C. D. E. amniocentesis - standard (> 15 weeks) amniocentesis - early (< 12 weeks) chorionic villus sampling fetal magnetic resonance imaging study first trimester nuchal lucency determination 833 I5. The laboratory at your center is seeking your advice regarding introduction of array Comparative Genome Hybridization (aCGH) as an option for prenatal diagnostic testing from chorionic villus sampling and amniocentesis. You can counsel the laboratory administration that, when compared to conventional karyotype analysis, aCGH is associated with which of the following features? A. B. C. D. E. I6. When noted as an isolated finding on second trimester ultrasound, findings such as choroid plexus cyst, echogenic bowel, echogenic cardiac focus, or short femur are most likely occur in a fetus with which of the following outcomes? A. B. C. D. E. I7. cystic fibrosis Down syndrome trisomy 18 normal infant tuberous sclerosis Among causes of premature ovarian failure, which of the following would be most commonly encountered by the infertility specialist? A. B. C. D. E. I8. Increases detection for submicroscopic deletions Produces more uninterruptible results from stillbirth specimens Requires a greater volume of amniotic fluid Requires greater technician skill Requires longer cell culture time 46, XY female 45, X fragile X premutation carrier galactosemia myotonic dystrophy Mr and Mrs Smith each are heterozygotes for delta F508 mutation of the CFTR gene and are undergoing Preimplantation Genetic Diagnosis(PGD) to avoid transfer of embryos homozygous for delta F508. Mrs Smith who is 39, inquires about her increased of Down syndrome and asks if screening also can be undertaken for aneuploidy. Which of the following statements can you report to her about using fluorescence in situ hybridization (FISH) technologies for aneuploidy detection for Preimplantation Genetic Screening (PGS) ? A. B. C. D. E. decreases Down syndrome miscarriages decreases Down syndrome livebirths increases implantation rates removes the need for CVS or amniocentesis has not yet shown any proven benefit 834 I9. Mrs. Smith has an 11-week ultrasound with a fetus with a nuchal translucency measurement of 4.5 mm. Her diagnostic testing by chorionic villlus sampling (CVS) reveals 46, XY. At second trimester ultrasound, the risk is greatest for identification of anomalies in which of the following systems? A. B. C. D. E. I10. Among male causes of infertility, which of the following would be most likely to be encountered in the man with oligospermia? A. B. C. D. E. I11. Balanced translocation Fragile X mutation Delta F508 mutation Sex chromosome aneuploidy Sertoli cell only syndrome Among male causes of infertility, which of the following would be most likely to be encountered in the man with non-obstructive azoospermia? A. B. C. D. E. I12. Cardiac Central nervous system Gastrointestinal Genitourinary Skeletal Balanced translocation Fragile X premutation Delta F508 mutation Sertoli cell only syndrome Sex chromosome aneuploidy Mrs. Smith is scheduled for a chorionic villus sampling (CVS) at 11-weeks gestation as she and her husband are carriers of cystic fibrosis. They are aware of their 25% risk and are seeking early diagnosis. However, while she is aware of the risk of miscarriage associated with CVS, she also inquires as to which of the following complications have been associated with CVS? A. B. C. D. E. Abruption Hemangioma Placenta previa Preterm delivery Preterm premature rupture of membranes 835 I13. Among male causes of infertility, which of the following would be most likely to be encountered in the man with obstructive azoospermia? A. B. C. D. E. I14. If a fetus has a nuchal translucency increased to 2 standard deviations at 11-14 weeks, which of the karyotype results is the most likely to be found on amniocentesis? A. B. C. D. E. I15.. 45, X 45, X/46,XX 46,XX 47,XX, +18 47, XX, +21 Mrs Jones is 35-years-old and has an amniocentesis for an increased risk of Down syndrome based on her maternal serum screening. The ultrasound at the time of the amniocentesis was unremarkable without structural abnormalities noted in the fetus, nor ultrasound markers for Down syndrome. The amniotic fluid was normal and the placenta was posterior without hematomas noted. Two days following the amniocentesis she is diagnosed with premature rupture of membranes. Which of the following outcomes are you going to counsel her is the most likely? A. B. C. D. E. I16. Balanced translocation DAZ deletion Delta F508 mutation Fragile X premutation Sex chromosome aneuploidy Chorioamnionitis Chorionamnion separation Fetal demise Miscarriage Reaccumulation of amniotic fluid In order to reach 80% detection of trisomy 21 in women under the age of 35 with nuchal translucency and first trimester serum markers, approximately how many women will have positive screening results? A. B. C. D. E. <10 % 20-30% 30-40% 40-50% > 55 % 836 I17. John and Mary present for an infertility evaluation. John is diagnosed with oligospermia (a low sperm count). Which of the following genetic conditions is associated with oligospermia? A. B. C. D. E. I18. As part of an infertility evaluation, Mrs. Smith at age 35 years is diagnosed with premature ovarian failure based on an elevated follicle stimulating hormone. She is otherwise healthy with an unremarkable clinical history. Which of the following clinical disorders is a likely diagnosis to consider in your differential diagnosis? A. B. C. D. E. I19. Cystic Fibrosis Huntington Disease Kennedy Disease Myotonic Dystrophy Premutation Fragile X Susan is being cared for by her gynecologist. She carries a diagnosis of late onset congenital adrenal hyperplasia based on clinical exam and endocrinologic studies. Treatment with steroids has regulated her menstrual cycle and she is now contemplating pregnancy. Obtaining her specific molecular diagnosis is important in order to determine which of the following management issues? A. B. C. D. E. I20. Balanced translocation carrier Cystic fibrosis Fragile X Syndrome 47,XXY 47,XYY Correctly adjust her steroid medication during pregnancy Determine whether she is at risk for severe salt wasting during pregnancy Establish whether she can become pregnant without assisted reproduction Identify risks for adverse pregnancy outcomes Identify the potential risk for a child with classic salt wasting CAH Congenital bilateral absence of the vas deferens (CBAVD) is present in almost all males with classic cystic fibrosis. Among men with CBAVD alone, what is the likelihood that they will have at least one mutation of the CFTR region (including mutations in 5T)? A. B. C. D. E. 25% 50% 70% 85% 100% 837 I21. Mr. Jones and his wife are a Northern European couple who have experienced two years of infertility. Mr. Jones visits his urologist and is diagnosed with bilateral congenital absence of the vas deferens (CBAVD). He is otherwise well with an unremarkable clinical history. Analysis of his cystic fibrosis transmembrane receptor gene (CFTR) most likely reveals which of the following molecular findings? A. B. C. D. E. I22. Among children born following assisted reproduction, the risk of a major congenital malformation is approximately how much greater than the general population risk? A. B. C. D. E. I23. 10% 30% 50% 60% 70% Early amniocentesis (9.0-12.9 weeks) is associated with the highest risk of fetal loss when compared to any other diagnostic modalities (CVS, standard amniocentesis and PUBS). The increased fetal loss rate is most likely the consequence of which of the following events associated with the procedure? A. B. C. D. E. I24. Absence of mutations on a common 23 mutation panel Absence of mutations on an expanded mutation panel Double heterozygosity for classic CFTR mutations Double heterozygosity for poly T variants Heterozygosity for a classic CFTR mutation Difficulty piercing the membranes leading to tears and fluid leaks Increased susceptibility for infection at early gestational age Poor ultrasound resolution at early gestation leading to fetal injury Proportionately a greater volume of amniotic fluid removed Incomplete conversion of corpus luteal to placental progesterone support Mrs Smith is a 35 year old G1 at 11 weeks gestation with a fetal ultrasound which demonstrates a nuchal lucency measurement of 3.0 mm. Which of the following karyotypes are you most likely to find on analysis of this fetus’ chromosomes? A. B. C. D. E. 45, X 46, XX 47,XX,+21 47,XX, +18 47, XX, +13 838 I25. Assisted reproductive technologies are associated with an increased risk of congenital malformations. Based on animal and human studies, which of the following categories of genetic conditions has been reported most frequently? A. B. C. D. E. I26. Mrs Jones is interested in an early pregnancy, noninvasive method to determine if her current fetus has Down syndrome. Free fetal nucleic acids are most uniquely differentiated from fetal cells in the maternal circulation by which of the following features? A. B. C. D. E. I27. Chromosomal breakage disorders DNA expansion disorders Imprinting disorders Point mutations Segmental duplication/deficiency disorders ability to cross from the fetus to the mother ability to identify fetal sex clearance from the mother within hours of delivery capacity for identifying fetal aneuploidy identification in the maternal circulation in the first trimester A 40-year-old woman carries a fetus with increased nuchal translucency of 3.0 mm at her 12-week ultrasound. This clinical finding poses the highest risk for which of the following fetal abnormalities? A. B. C. D. E. Aneuploidy Developmental delay Fetal demise Growth restriction Neural tube defect I28. You are a molecular/cytogenetic laboratory director and would like to convince your hospital to provide chromosome microarray analysis. Potential benefits include an increase in the numbers of clinically-significant diagnoses above that obtained from routine karyotype in several settings in which patients are seen by the obstetrics department. Which of the following indications for testing will have the lowest increase in new diagnoses? A. B. C. D. E. Amniocentesis for anomalies Amniocentesis for maternal age > 35 Chorionic villus sampling for increased nuchal translucency Miscarriages Stillbirths 839 I29. A couple presents to your office and informs you the wife carries a balanced translocation between chromosomes 5 and 12. Which of the following options would not be an appropriate reproductive genetic technology to address their risks? A. B. C. D. E. I30. A 36-year-old patient is now pregnant following 4 years of infertility and three cycles of IVF. She is concerned about her risks of delivering a child with Down syndrome but is anxious to avoid a miscarriage. Which of the following screening tests is associated with the highest positive predictive value in women over 35 years? A. B. C. D. E. I31. Amniocentesis Chorionic villus sampling Noninvasive free fetal DNA Percutaneous umbilical blood sampling Preimplantation genetic diagnosis Combined first trimester screen Free fetal dna screen Integrated serum screen Quad serum screen Triple serum screen A couple presents to your office with a pregnancy at 10 weeks gestation. Using assisted reproductive technology (ART), the pregnancy was conceived by in vitro fertilization as the husband has oligospermia. They are also each carriers of cystic fibrosis and preimplantation genetic diagnosis (PGD) was performed for their specific CFTR mutations. The couple asks “Is this pregnancy at greater risk for birth defects?” Which of the following responses is most appropriate? A. No, there is no evidence that either PGD or ART is associated with increased risks of birth defects. B. No, your PGD was performed to allow only healthy embryos to develop. C. Yes, ART is associated with an approximate 30% increase in congenital malformations D. Yes, for imprinted gene abnormalities but these can be tested for at amniocentesis. E. Yes, for imprinted gene abnormalities but these were tested for at PGD. 840 I32. A 40-year-old woman presents for prenatal care with questions concerning her risk of delivering a child with Down syndrome. She would like to proceed with the screening test that has the lowest false positive rate. You should offer which of the following prenatal screening tests? A. B. C. D. E. I33. A 30-year-old patient returns from her 18-week ultrasound visit with a verbal report stating she was told there were “birth defects” noted in her developing fetus. As you are obtaining the report, your concern for the presence of possible chromosome abnormality would be highest if the report indicates which of the following ultrasound findings? A. B. C. D. E. I 34. Atrial septal defect Gastroschisis Hydronephrosis Omphalocele Upper limit normal ventriculomegaly (10 mm) A 28-year-old woman with a BMI of 40 presents for her 18-week ultrasound. Her chances of having an incomplete fetal survey are increased due to her higher BMI. Her fetal survey should be repeated as she faces increased risks for congenital anomalies. She is at the highest relative risk is for which of the following anomalies? A. B. C. D. E. I35. Combined first trimester screen Free fetal DNA screen Intergrated serum screen Quad serum screen Triple serum screen Cardiac anomaly Cleft lip/palate Gastroschisis Limb anomalies Neural tube defect A couple transfers to your practice and reports a history of two miscarriages during their ten years of marriage, as well as difficulty conceiving. The initial evaluation reveals oligospermia. On further study of the husband, which of the following karyotypic abnormalities are you most likely to find? A. B. C. D. E. A balanced translocation 45, X 45, X/46, XX 47, XXY 47, XYY 841 I36. You offer your patient noninvasive prenatal testing (NIPT) as a screening test for aneuploidy at 10 weeks into her pregnancy. Which of the following conditions would have excluded her from this testing? A. B. C. D. E. I37. Mrs Smith is a 40-year-old G2 P1 at 18-week gestation, with an ultrasound report indicating a neural tube defect was found in the fetus. She had a 12-week cell free fetal DNA (cffDNA) study which was "normal' for chromosomes 13, 18, 21 and X, Y. She remains concerned about the fetus so she decides to proceed with an amniocentesis. Which of the following findings is most likely from her amniocentesis? A. B. C. D. E. I38. Demise of twin gestation at 8 weeks Diabetes type 1 Diabetes type 2 Miscarriage within past year Obesity (BMI > 30) Autosomal aneuploidy (other than chromosome 13, 18, 21) Autosomal aneuploidy (of chromosome 13, 18, 21) Microdeletion syndrome Normal karyotype Sex chromosome aneuploidy In your obstetric practice, you obtain a detailed second trimester fetal ultrasound survey to assess for the risk of Down syndrome. Which of the following ultrasound findings would have the highest relative risk for trisomy 21? A. Choroid plexus cyst B. Echogenic bowel C. Echogenic cardiac focus D. Renal pyelectesis (4.0 mm) E. Shortened femur I39. Mr and Mrs Jones are both in their early thirties and seeking prenatal genetic counseling at 18 weeks gestation. This is their first pregnancy, conceived by IVF following 4 years of infertility, etiology undetermined. They are worried about the possibility of abnormal findings on their upcoming ultrasound. If an ultrasound abnormality were identified, which of the following findings is most likely to be seen on their pending ultrasound? A. B. C. D. E. Dysplastic kidney and absent stomach bubble Omphalocele and nephromegaly suggesting Beckwith Weideman Syndrome Shortened long bones consistent with a skeletal dysplasia Tetralogy of Fallot Ventriculomegaly and adducted thumbs suggesting X-linked aqueductal stenosis 842 I40. Susan Smith is a G1 at 22 weeks gestation. Her medical history is significant for obesity (BMI 40), Type 2 diabetes and anxiety. She drinks a glass of wine with most dinner meals. She started metformin preconception and has continued on it through the pregnancy. First trimester hemoglobin A1C was slightly elevated (7.0, normal < 6.1). She took Ampicillin for 7 days at 10 weeks gestation for a urinary tract infection. She presents to your office with an ultrasound finding of a fetus with a neural tube defect. From her history, which of the following factors conveys the highest relative risk for a fetus with a birth defect, especially a neural tube defect? A. B. C. D. E. I41. Ms. Jones is 25-years old with her first pregnancy. She is 10 weeks pregnant and elects cfDNA for non-invasive screening for aneuploidy which returns as negative for chromosomes 13, 18, 21. At 18 weeks, her ultrasound for a complete fetal survey indicates the fetus is growing appropriately, no structural abnormalities are identified but an echogenic focus is noticed in the heart. Which of the following next steps is most consistent with current guidelines? A. B. C. D. E. I42. Alcohol consumption during pregnancy Ampicilin use Maternal obesity (BMI > 40) Metformin use Type 2 diabetes A1C = 7.0 Amniocentesis Echocardiograph (ECHO) No further action Quad serum screen Third trimester fetal growth ultrasound 17. Mrs. Brown is 40-years old with her first pregnancy. She received counseling on the various options for screening as well as diagnosis in order to detect cytogenetic abnormalities. After counseling and reading, she feels that only a diagnostic procedure will provide her the information she is seeking. She elects to proceed with an amniocentesis. This is performed at 17 weeks, 20 ml clear fluid is sent for chromosome microarray analysis. She calls the next morning reporting that on awakening, she noted a gush of fluid from her vagina. She returns to the office for an ultrasound which reveals a 17-week fetus, positive heart beat and mild oligohydramnios. Clinical exam is positive for rupture of membranes. Which of the following outcomes is the most likely with rupture of membranes after an amniocentesis? A. B. C. D. E. Contractures in the neonate Intrauterine growth restriction Late preterm delivery Miscarriage Pulmonary hypoplasia in the neonate 843 I43. Mr. and Mrs. Smith present to your office with questions concerning possible exposures that could result in a child with a birth defect. They are particularly concerned as they want to conceive soon and their rental apartment was recently painted. Their awareness of congenital abnormalities has heightened as their neighbor just delivered an infant with a neural tube defect. Mrs Smith is 25 years old, has a BMI of 28, is healthy, had a RouxN-Y gastric bypass 4 years ago, takes no medication, and reports no remarkable family history. Mr Smith is also 28 years old, has a BMI of 30, is healthy with no surgeries, takes a daily SSRI for anxiety/depression and reports no significant family history. Both are Northern European in ancestry. Both have jobs they describe as “stressful” and they enjoy a bottle of wine many nights of the week to unwind. Which of the following exposures to Mrs. Smith has the highest relative risk of contributing to the development of a birth defect? A. B. C. D. E. I44 Mrs. Smith is a 40-year-old woman at 10 weeks gestation in her first pregnancy. Her obstetrician discusses screening options and she elects NIPT screening for Down syndrome. The result returns as uninterpretable, on repeat sampling at 12 weeks, the NIPT is also uninterpretable. Which of the following circumstances is most compatible with an uninterpretable NIPT result? A. B. C. D. E. I45. Alcohol exposure Folate deficiency Obesity Paint fume exposure Selective serotonin receptor uptake BMI > 40 Confined placental mosaicism Maternal malignancy Maternal mosacism Vanished twin Mrs Smith is 36-years old and pregnant with her first child. She has a first trimester screening evaluation for Down syndrome (nuchal translucency measurement and serum biomarkers) and the report indicates an increased risk for Down syndrome. Her neighbor, also 36-years old and expecting her first child, has a noninvasive screening test (NIPT) from a sample of her blood and the result is also reported as high risk. Which of the following when comparing screening tests for Down syndrome, is most consistent with NIPT compared to serum screening? A. B. C. D. E. Better approach for twins Higher false positive rate Higher sensitivity rate Lower chance of non-informative results Lower positive predictive value 844 I46. Mrs Jones with her first pregnancy has her 18-week ultrasound planned. She mentions to you that her sister had a child with congenital cytomegalovirus (CMV) who had deafness, developmental and growth delay and passed away at age 3. Which of the following ultrasound findings increases is the chance there is fetal infection with CMV? A. B. C. D. E. Gastroschesis Large for gestational age fetus Polydactyly – post axial Polyhydramnios Ventriculomegaly 845 Answers to Prenatal/Reproductive Genetics Questions I1-I46 I1. The correct answer is A. – one etiology of mosaicism is considered to be an initially trisomic conception which undergoes trisomic rescue, this would put the pregnancy at risk for retention of two chromosomes from the same parent, uniparental disomy testing would be indicated. Ultrasound surveillance is indicated for growth restriction but not for congenital anomalies such as cardiac malforamation. Low level tissue mosaicism can never be disproven in the fetus. I2. The correct answer is C. – 46,XX/45,X can present as a phenotypically normal female with infertility, Answer A could be a phenotypically normal female but genetically be a male with complete androgen insensitivity and would have amenorrhea.but is less common. Answer A would present with Turner syndrome phenotype. Answers D and E present as males. I3. The correct answer is E. – POF is the only impact on reproductive capacity recognized among fragile X carriers I4. The correct answer is B. – at a loss rate of 2-3%, early amniocentesis out paces the risk of losses from CVS (1.0%) and standard amniocentesis (0.5%), MRI and nuchal lucency measurement are noninvasive and not associated with pregnancy loss I5. The correct answer is A. -- Implementation of aCGH in the prenatal setting may be advantageous for several reasons including increased detection of genomic disorders when ultrasound malformations are present and the conventional karyotype is normal and when poor cell growth is anticipated (stillbirth or miscarriage). The ability of aCGH to utilize smaller volumes of fluid and the applicability for automation may also prove valuable components. Current trials are underway to address aCGH versus conventional karyotype in routine amniocentesis with the issue of CNV to be addressed. I6. The correct answer is D. – each of these findings in isolation can occur in as many as 1-5% of pregnancies; in the majority of instances the karyotype is normal. As aneuploidy is a relatively rare event, although the relative risk of aneuploidy is indeed increased (1.2 to 5.0 fold), the majority of pregnancies are karyotypically normal. Echogeneic bowel is associated with cystic fibrosis with an increased relative risk but again the majority of infants with echogenic bowel are normal. None of the findings are seen in fetuses with tuberous sclerosis. I7 The correct answer is C. – Amenorrhea would be expected with A (Androgen insensitvity) and B (Turner syndrome) Galactosemia is rare in itself , so an infertility specialist will only rarely encounter a patient. Myotonic dystrophy may impact fertility but not specifically though PFO. I8. The correct answer is E. -- Aneuploidy is known to increase as women age and theoretically screening the embryos of women of women of advanced maternal age prior to transfer should decrease their rate of aneuploid conceptions. This should indirectly increase their implantation rate, lower their miscarriage rate and increase their delivery rate. However, while initial studies with FISH were promising, randomized controlled trials found no benefit to screening embryos prior to transfer and even detriment to implantation rates. Advances in technology to allow application of aCGH to PGS may overcome these difficulties. 846 I9. The correct answer is A. -- With increasing nuchal lucency in the first trimester the risk of a structural malformation being detected on the second trimester ultrasound increases. The association between cardiac anomaly and nuchal edema may be physiologic although there does not appear to be good correlation between type of cardiac defect and presence of increased edema. Among fetuses with trisomy 21, increased nuchal edema in the first trimester is not predictive of a second trimester cardiac anomaly despite the high prevalence of cardiac malformations in Down syndrome. I10. The correct answer is A. – based on frequencies of infertility identified in each category, A (balanced translocation) would be most commonly encountered with oligospermia. Sex chromosome aneuploidy and Sertoli cell only syndrome are commonly associated with azoospermia; Delta F508 mutuation with obstructive azoospermia and CBAVD. I11. The correct answer is E. – balanced translocation is associated with oligospermia; cystic fibrosis mutation associated with obstructive azoospermia (CBAVD) and sex chromosome aneuploidy associated with non-obstructive azoospermia. Sertolic cell only syndrome is a rare cause of azoospermia. Fragile X is not associated with male infertility. I12. The correct answer is B. -- Adverse pregnancy outcomes with regard to aberrant placentation, preterm labor or rupture of membranes have not been associated with CVS. However, concern has been raised as to the possible effects on the fetus and in particular most recently an increase in hemangiomas. This increased risk in hemangiomas appears to be concentrated in transcervical CVS and is not gestational age dependent (in contrast to the limb reduction abnormalities which were concentrated in the < 10 week CVS cases). I13. The correct answer is C. - balanced translocation is associated with oligospermia; cystic fibrosis mutation associated with obstructive azoospermia (CBAVD) and sex chromosome aneuploidy associated with non-obstructive azoospermia. DAZ deletion is a cause of azoospermia, fragile X premutation is not a associated with infertility in the male, I14. The correct answer is C. – as a screening test, an enlarged NL at the second standard deviation increases the relative risk for aneuplopidy but will be encountered most commonly in chromosomally normal pregnancies. I15. The correct answer is E. -- Premature rupture of membranes following amniocentesis occurs in approximately 1% of cases. Reaccumulation of the amniotic fluid occurs in > 90% of patients though on average takes 3 weeks. The outcomes are generally favorable which is in contrast to spontaneous rupture of membranes in the second trimester which is associated with fetal demise, miscarriage and chorioamnionitis. I16. The correct answer is A. – highlights the need to have a fairly significant portion of the population screen positive in order to detect 80% of trisomy 21 fetuses I17. The correct answer is A. 3-5% of men with oligospermia carry a balanced translocation. Cystic fibrosis is associated with male infertility with obstructive azoospermia, Fragile X is associated with decreased ovarian reserve in female premutation carriers and 47, XXY would classically present with azoospermia. 47,XYY syndrome individuals are typically fertile. 847 I18. The correct answer is E. -- Premutation fragile X carriers are noted in 2-5 % of women with premature ovarian failure varying with the strength of their family history of POF. Myotonic dystrophy is associated with male infertily, Kennedy Disease is a rare condition not associated with POF and Cystic Fibrosis is assopciated with male infertility and CBAVD. Huntington Disease is not associated with infertility. I19. The correct answer is E. -- Some women with late onset CAH will carry a classic salt wasting CAH mutation as well as an atypical allele. If the partner is also a carrier for salt wasting CAH, the risk of an affected child would be 25%. Her specific molecular diagnosis would not impact her steroid medication during pregnancy, alter whether she can become spontaneously pregnant or her pregnancy complications. I20. The correct answer is D. -- If 5T is included, approximately 32% of men with CBAVD are heterozygotic for a mutation in CFTR and an additional 53% are double heterzygotes. I21. The correct answer is E. -- The majority of men with a clinical diagnosis of CBAVD who are otherwise asymptomatic have either a double heterozygosity or heterozygosity for CF mutations. In individuals who are asymptomatic from a respiratory standpoint, if they are double heterozygotes then typically non-classic mutations are involved. Double heterozygosity for classic mutations would be associated with an earlier onset of typical cystic fibrosis symptoms. I22. The correct answer is B. -- Recent meta-analysis suggests that once large, studies could be assessed; an increased congenital malformation rate of approximately 30% over background is noted following assisted reproduction techonologies. I23. The correct answer is D. For an amniocentesis at 10 weeks, the removal of the needed 20 cc amniotic fluid represents almost half of the total volume. Removal of 20 ccs at 16 weeks represents only about 12% of the total volume. I24. The correct answer is B. -- While enlargement of the nuchal space in the first trimester fetus is a sensitive marker for aneuploidy, in most fetuses it represent a variation of normal. Thus the need to add serum markers to first trimester nuchal translucency measurements in order for these findings to be used as a screening tool without a high screen positive rate I25. The correct answer is C. -- Evidence for increased congenital malformations is based on several population based studies with the highest rates seen for infants with multiple malformations. Based on animal and human data, work is accumulating that alterations of imprinting may contribute to this increase as seen in specific recognized syndromes ( Beckwith Weidemann for example). However, whether the imprinting errors occur related to the ART and ovulation induction, or are related to the underlying infertility which brings the couple to the ART program remains an area of investigation. I26. The correct answer is C. --. Cell free nucleic acids are ubiquitous in humans and reflect cell apotosis due either to inflammation, necrosis or programmed cell death. Fetal cell free nucleic acids are most often of placental origin and are of shorter length then the cell free nucleic acids derived form the mother’s own cells undergoing cell turnover and death. Fetal cf nucleic acids rapidly clear from the maternal circulation within hours of delivery which is a differentiating property form intact fetal cells which may be ahrobroed for decades in the mother. The persistence of fetal cells in the maternal circulation was one factor which hindered progression of noninvasive prenatal testing which has been overcome by the rapid clearance of the cell free nucleic acids 848 I27. The correct answer is A. Source : Reproductive Genetics – 1 Preconception Explanation: A. Aneuploidy – with an NT of 3.0 approximately 17% of fetuses have an aneuploidy, this is likely higher given her maternal age of 40. B. Developmental delay – among fetuses with increased NT who are found to be euploid and without structural malformations, approximately 6% were found to have developmental delay at later ages though these children were clustered among those with markedly enlarged NT (> 6.0 mm) C. Fetal demise – this can be associated with increased NT but typically at increased dimensions of > 6.0 and those which persist into the second trrimester D. Growth restriction – not classically associated with increased NT E. Neural tube defect - while the risk of congenital malformations are increased in euploid fetuses with an increased NT, the predominant malformation is cardiac anomaly I28. The correct answer is B. Source : Reproductive Genetics – 2 Prenatal Explanation: A. Amniocentesis for anomalies – increases in clinically significant diagnoses beyond that afforded by the conventional karyotype occur in almost 10% of samples sent for ultrasound anomalies B. Amniocentesis for maternal age > 35 – increased clinically significant diagnoses are reported in approximately 1% of women C. Chorionic villus sampling for increased nuchal translucency – a portion of increased diagnoses would be anticipated given the increased association of large NT with later structural anomalies, especially cardiac and 22q-. D. Miscarriages - – increases in diagnostics are expected from the application of chromosome microarray to stillbirths due to both the ability to utilize nonviable tissues and the greater degree of diagnostic resolution E. Stillbirths – increases in diagnostics are expected from the application of chromosome microarray to stillbirths due to both the ability to utilize nonviable tissues and the greater degree of diagnostic resolution 849 I29. The correct answer is C. Source : Reproductive Genetics – 2 Prenatal Explanation: A. Amniocentesis - amniocentesis is a earliest diagnostic test later in pregnancy (> 15 weeks) which would also provide definitive information to address the increased risk of an unbalanced conception to the parent with a balanced translocation B. Chorionic villus sampling – CVS represents the earliest diagnostic test in pregnancy which would provide definitive information to address the increased risk of an unbalanced conception to the parent with a balanced translocation C. Noninvasive free fetal DNA – currently the available technologies for noninvasive screening with fetal DNA have focused on the common aneuploides (trisomy 21, 13, 18) and sex chromosome abnormalities and would not detect abnormalities in other chromosomes D. Percutaneous umbilical blood sampling - a earliest diagnostic test late in pregnancy (> 18 weeks) which would also provide definitive information to address the increased risk of an unbalanced conception to the parent with a balanced translocation. May be chosen over amniocentesis as while there is a higher risk of miscarriage with PUBs, the rapid return of a full karyotype (48 hours) may be of greater importance. E. Preimplantation genetic diagnosis – chromosome rearrangements are ideal candidates for assessment by PGD either through individually designed FISH probers or the use of chromosome microarray technologies I30. The correct answer is B. Source : Reproductive Genetics – 2 Prenatal Explanation: A. Combined first trimester screen - the chances of a true positive from among the screen positive population is low, approximately 3% B. Free fetal DNA screen – among women older than 35 years of age, the chances of a true positive from among the screen positive population is high, greater than 97% C. Intergrated serum screen – the chances of a true positive from among the screen positive population is low, approximately 4% D. Quad serum screen - the chances of a true positive from among the screen positive population is low, approximately 2% E. Triple serum screen – the chances of a true positive from among the screen positive population is low, approximately 2% 850 I31. The correct answer is C. Source : Reproductive Genetics – 1 Preconception Explanation: A. No, there is no evidence that either PGD or assisted reproduction is associated with increased risks of birth defects – less research has been compiled on the population utilizing PGD but meta-analyses support a 30% increased rate of congenital malformations with ART. B. No, your preimplantation genetic diagnosis was performed to allow only healthy embryos to develop – PGD is a testing modality which tests for specific conditions and the intention is not to screen for numerous conditions to assure a healthy fetus. C. Yes, ART is associated with an approximate 30% increase in congenital malformations – recent meta-analysis supports this estimate of risk although the contribution of underlying sub/infertility among the couples remains an unanswered question. D. Yes, for imprinted gene abnormalities but these can be tested for at amniocentesis imprinted abnormalities are not the only form of genetic disease or congenital anomaly for which the ART exposed fetus faces an increased risk. As the number of potential changes to imprinted genes may be numerous and the connection to clinical disease still evolving, they are not screened for at amniocentesis. E. Yes, for imprinted gene abnormalities but these were tested for at PGD – imprinted abnormalities are not the only form of genetic disease or congenital anomalies for which the ART exposed fetus faces an increased risk. The number of potential changes to imprinted genes are numerous and the connection to clinical disease still evolving. PGD is applicable for this testing. I32. The correct answer is B. Source : Reproductive Genetics – 2 Prenatal Explanation: A. Combined first trimester screen – ultrasound and serum analytes used to screen for Down syndrome which moves screening into the first trimester but can have as high as a 15% screen positive rate in > 35 year old women. B. Free fetal DNA screen – less than 1% of women (0.02% reported in studies of women > 35 years of age). C. Integrated serum screen – a combined first and second trimester ultrasound and serum screening for Down syndrome with the lowest screen positive rate of the serum analyte programs. D. Quad serum screen - a second trimester serum analyte screening for Down syndrome with a screen positive rate in women < 35 of approximately 5%, increased detection of trisomy 21 compared to triple screen. E. Triple serum screen - a second trimester serum analyte screening for Down syndrome with a screen positive rate in women < 35 of approximately 5%. 851 I33. The correct answer is D. Source : Reproductive Genetics – 2 Prenatal Keywords: Birth defects, chromosomal abnormality, ultrasound detection Explanation: A. Atrial septal defect – an isolated cardiac defect with < 10 % association with aneuploidy B. Gastroschisis – considered a vascular defect typically unassociated with aneuploidy C. Hydronephrosis – one of the soft markers associated with an increase in aneuploidy in conjunction with other markers D. Omphalocele – a single anomaly with a strong association with aneuploidy trisomy 21,18 and 13 (30%) E. Ventriculomegaly (10 mm) – a finding usually detected in normal fetuses but with an association with trisomy 21 I34. The correct answer is E. Source : Reproductive Genetics – 1 Preconception Keywords: Neural tube defect, BMI, fetal survey Explanation: A. Cardiac anomaly – increased risk among those with obesity but not with relative risks as high as for NTD B. Cleft lip/palate – increased risk with higher levels of obesity C. Gastroschisis – not seen in recent studies D. Limb anomalies - variably associated at low levels in some studies E. Neural tube defect – highest relative risks which increase as BMI increases I35. The correct answer is A. Source : Reproductive Genetics – 1 Preconception Keywords: Infertility, oligospermia Explanation: A. A balanced translocation – as sperm levels decrease, men are at increased risk to be identified as carriers of balanced translocations. B. 45, X – would present with Turner syndrome phenotype typically C. 45, X/46, XX - typically presents with Turner syndrome phenotype D. 47, XXY – azoospermia is typically identified when men carry sex chromosome abnormalities E. 47, XYY – not typically associated with alterations in sperm levels 852 I36. The correct answer is A. Keywords: Noninvasive prenatal testing (NIPT) Explanation: Source : Reproductive Genetics – 2 Prenatal Explanation: A. Demise of twin gestation at 8 weeks – a proposed contributor to false positives or discordant results when the NIPT is positive and the fetal testing does not confirm the aneuploidy B. Diabetes type 1 – not shown to affect free fetal DNA levels C. Diabetes type 2 - not shown to affect free fetal DNA levels D. Miscarriage within past year – fetal fragments of DNA clear the maternal circulation within hours of delivery so prior pregnancies do not interfere with the test E. Obesity (BMI > 30) – shown to decrease free fetal DNA levels as BMI increases, a reason to note BMI but not withhold testing I37. The correct answer is D. Keywords: cell free DNA, aneuploidy screening, ultrasound anomalies Explanation: The overall sensitivity and specificity for cell free DNA panels are > 98% although variation by the specific chromosomes do exist. Thus while it is possible that the "normal" cffDNA result may represent a false negative, the likelihood of this occurrence in the setting of a fetus with a neural tube defect is low. Most fetuses with a NTD are chromosomally normal. Similarly, while microdeletions have been identified in 3-7% of fetuses with ultrasound anomalies, this would not represent the most likely finding. I38. The correct answer is B. Keywords: aneuploidy screening, ultrasound anomalies Explanation: Echogenic bowel is represented by gastrointestinal images which appear as bright as bone. Such a finding has one of the highest likelihood ratios for trisomy 21, on the order of 5 - 7 fold. This is higher than the likelihood ratios for most other "soft markers" such as shortened femur, (LR approximately 2.0) and renal pyelectesis and echogenic intracardiac focus, both of the latter with likelihood ratios < 2.0. Choroid plexus cyst is a soft marker for trisomy 18 and not a marker for trisomy 21. 853 I39. The correct answer is D. Keywords: assisted reproduction, ultrasound anomalies, imprinting disorders Explanation: Pregnancies conceived by assisted reproduction (ART) are at increased risk for isolated congenital anomalies, multiple congenital anomalies and disorders of imprinting. The association of imprinting disorders such as Beckwith Weidman syndrome with IVF has received attention as a means to understanding the biology during assisted reproduction. However, the chance of a child with BWS is actually low (estimated at 1 in 4,000 ART pregnancies). Conversely, the relative risk of isolated congenital anomalies has been in the 6-8% range when background risks for isolated congenital anomalies have been in the 5-6% range. For multiple defects, while the relative risk is larger, their overall background rate is lower (0.5% increasing to 2.0% in the ART population). I40. The correct answer is C. Keywords: ultrasound anomalies, teratogenicity Explanation: Teratogenicity from fetal exposure to fairly common agents and conditions is underappreciated. These include the effects from maternal obesity, hyperglycemia in poorly controlled diabetes and alcohol exposure. Of the three above - maternal obesity has the highest odds of producing a congenital malformation in the fetus - as much as four fold higher in women with a BMI of > 40. Poorly controlled diabetes also increases risk with hyperglycemia reflected by hemoglobin A1C levels. However even with elevated levels of 9.0 - 12.0, the associated risk congenital malformation is a doubling of the background rate. Alcohol consumption during pregnancy is associated most often with the development with delayed growth, neurocognitive delays and a characteristic facial appearance. Medication exposures remain relatively poorly studied during pregnancy. However for more commonly encountered medications, such as a common antibiotic (ampicillin) and an oral antigylcemic (metformin) , the risks of a birth defect have not been found to be significantly elevated. I41. The correct answer is C. SOURCE: Lecture/Slides/Syllabus KEYWORDS: aneuploidy, cell free DNA screening, ultrasound “soft markers” Ultrasound “soft markers” were introduced at a time when other options for screening for trisomy 21 were limited. “Soft markers” are subtle imaging findings (such as echogenenic intracardiac focus (EIF), renal pyelectasis, choroid plexus cyst) with an increased association with aneuploidy. However, these “soft markers” also occur in 1-4% of fetuses with normal chromosomal content. Given a very low relative risk of trisomy 21 based on the presence of an isolated EIF, with a negative cell free DNA study, the recommendation is to classify EIF as a normal variant. EIF has no association with cardiac anomaly, cardiac function or other altered fetal development such as growth restriction. A. Amniocentesis – with a negative cfDNA for trisomy 21, an EIF does not contribute further to the baseline risk and diagnostic testing for trisomy 21 is not recommended. Even prior to the introduction of cfDNA, EIF in isolation carried a very small increased relative risk and in women under the age of 35, diagnostic testing was not recommended. B. ECHO – EIF does not have any association with cardiac anomalies which would be the focus for an echocardiography study of the fetus C. Correct answer 854 D. Quad screening is a second trimester maternal serum test (MSAFP, HcG, estriol and inhibin) with relatively less sensitivity for trisomy 21 (70-80%) than NIPT (> 98%). With the higher sensitivity (>98%) of cfDNA, screening with a second, lower sensitivity test would not provide helpful information. E.Third trimester fetal growth ultrasound – EIF is not associated with other alterations in fetal growth or development. One “soft marker” however, echogenic bowel, can herald the development of fetal growth restriction in settings where its presence is likely associated with placental insufficiency. I42. CORRECT ANSWER: C SOURCE OF ITEM TOPIC: Lecture/Slides/Syllabus KEYWORDS: amniocentesis, rupture of membranes, oligohydramnios EXPLANATION: Premature preterm rupture of membranes (PPROM) following a procedure has a different prognosis than when membranes rupture spontaneously in the second trimester. The majority of pregnancies (90%) with PPROM after an amniocentesis deliver at a late preterm (average 34 weeks) gestation. This is in comparison to the higher risk of loss (in some series over 80%) and very early preterm delivery (< 26 weeks) noted when PPROM occurs spotnaneously. The difference may reflect the underlying causes with inflammation / infection occurring more commonly in the spontaneous rupture group. A. Contractures occur more commonly following spontaneous PPROM as the chance of reaccumulation of amniotic fluid is lower. With continued oligohydramnios from the time of spontaneous PPROM to delivery, this places the infant at higher risk of contractures. With post amniocentesis PPROM, the likelihood of reaccumulation of the amniotic fluid within 1 -2 weeks is high. B. Fetal growth restriction may be slightly increased in this population but does not occur in the majority. D. Miscarriage is more commonly seen following spontaneous PPROM likely related to the differences in underlying causation of the membrane rupture. Spontaneous PPROM has a more inflammatory/infectious nature which is the driver then for the pregnancy loss. With PPROM after amniocentesis, the majority of women reaccumulate the amniotic fluid and the overall likelihood of miscarriage is low. E. Pulmonary hypoplasia occurs after persistent oligohydramnios during the second trimester. As the amniotic fluid often reacccumulates within a week or two following an amniocentesis related PPROM, pulmonary hypoplasia is uncommon. The ongoing oligohydramnios with spontaneous PPROM contributes to a high rate of pulmonary hypoplasia, similar to that seen in other conditions without amniotic fluid such as those with renal agenesis. I43. CORRECT ANSWER: A SOURCE: Lecture/Slides/Syllabus KEYWORDS: teratogenicity, environmental exposures EXPLANATION: There is not a recognized level of alcohol exposure during pregnancy which is considered safe and without effect on the developing fetus. With higher levels of alcohol consumption, fetal alcohol syndrome can be seen which is often characterized by facial dysmorhpia, growth restriction, congenital anomalies and cognitive/behavioral effects. However, beyond FAS, there is now animal and human data supporting a fetal alcohol disease spectrum. 855 The risks of fetal effects from alcohol are highest with increased volumes, binge drinking and early first triemster exposures. There is no safe level of alcohol consumption during pregnancy with increasing efforts focused on preconception / gynecologic / primary care screening and education. B. Folate deficiency – although her bypass surgery can place her at risk for iron and other vitamin deficiencies, lower levels of folate have not been reported. Additionally, many flours and grain products in the United States are now supplemented with folate. Women are encouraged to take a prenatal vitamin with folate when attempting conception to further lower their background risk of about 1 in 1,500. C. Obesity – a high BMI is associated with over a 2-fold increase in risk of neural tube defect, and shows an increasing risk profile with increasing BMI. Her BMI currently is not elevated and her prior obesity prior to her gastric bypass has not been associated with an increased risk of NTD. D. Paint fume exposure – the toluene components of aerosolized paint at high levels can produce features similar to fetal alcohol syndrome. However, an isolated exposure to paint in a well ventilated room does not reach the levels of concern which are obtained only from ongoing, recreational inhalation of toluene fumes (“huffing spray paint”). E. Selective serotonin uptake inhibitors - paternal use of SSRIs would not be expected to have a teratogenic effect on the fetus. I44. CORRECT ANSWER: A SOURCE OF ITEM TOPIC: Lecture/Slides/Syllabus KEYWORDS: NIPT, biologic discordancy, low fetal fraction EXPLANATION: Women with an elevated BMI have a higher rate of uninterruptable results due to low fetal fraction. Some of the lowering of the fetal fraction may be related to increased volume distribution in women with increased BMI, similar to the process known to impact the analytes for serum screening. Additionally, however, as the fetal fraction is a portion of the maternal fraction of free DNA, in women with an increased BMI, their inflammatory state is higher resulting in a higher baseline level of cell free DNA from cell apoptosis. This then lowers the ability to detect the relatively lower level of fetal free DNA. B. Confined placental mosaicism (CPM) can result in false positive NIPT results when the aneuploid and not the normal cell line are detected in the maternal circulation (and the fetus is euploid). With CPM, false negative results can also occur when the normal but not aneuploidy line is detected in the maternal circulation and the fetus possesses the aneuploid line (seen with trisomy 18 and 13) C. NIPT results which are positive for more than one chromosome abnormality have been associated with maternal malignancy. The malignancies themselves have been shown to be responsible for the chromosomally abnormal cell free fragments which have a maternal and not fetal origin. D. The cell free DNA methodology used by most NIPT programs reflects changes in the expected number of DNA fragments for each chromosome but does not specifically identify which are maternal as opposed to fetal. NIPT results with both high and low counts the sex chromosomes have been reported to also occur with a normal fetal karyotype and a mother with an unrecognized sex chromosome aneuploidy (47, XXX or 45, X /46, XX). 856 E. Vanished twins occur more commonly than realized. When SNP methodology is used for the NIPT assay, cell free fragments from two distinct fetal genotypes have been reported. Notably, the presence of free fetal DNA fragments from a co-twin can persist for more than 8 weeks after early fetal demise. I45. CORRECT ANSWER: C SOURCE OF ITEM TOPIC: Lecture/Slides/Syllabus KEYWORDS: noninvasive prenatal testing, aneuploidy screening, first trimester screening EXPLANATION: The overall sensitivity for cell free DNA panels for trisomy 21 is > 98%; lower but still high sensitvity is also present for the other common aneuploidies. This exceeds the sensitivity for serum screening for trisomy 21 (about 80%). A. NIPT is not currently supported for twins with concerns for unequal levels of cell free fetal DNA from each placenta. Serum screening in twins has a lower sensitivity than in singletons but has been validated in clinical studies. B. Serum screening results in a 5-10% false positive rate (rate at which women are called positive), this rate is < 1.0% with NIPT. D. NIPT has rates of noninformative results which can vary between 3-7% and are associated with a higher rate of aneuploidy; noninformative results from serum screening are rare. E. The positive predictive value for trisomy 21 in a woman over 35 years of age is over 80% for NIPT and 4-6% for serum screening. I46. CORRECT ANSWER: E SOURCE OF ITEM TOPIC: Lecture/Slides/Syllabus KEYWORDS: viral infection, birth defects EXPLANATION: Following primary cytomegalovirus (CMV) infection during pregnancy, there is a relatively high rate of transmission of the virus to the fetus and an increased risk of fetal infection especially during the first and second trimesters. As with other viral infections, the consequences of CMV fetal infection are often marked by CNS involvement including ventriculomegaly, abnormal gyration patterns and abnormal development of CNS structures. Isolated ventriculomegaly can occur as a normal variant but also be associated with aneuploidy, single gene disorders and genetic syndromes. The prognosis for isolated ventriculomegaly depends on whether additional CNS involvement is evident on MRI and the underlying etiology. A. Gastroschisis - this abdominal wall abnormality occurs to the right of the umbilicus, with extrusion of GI contents without a peritoneal covering. Conditions which increase the risk of vascular events such as maternal cocaine have been associated with gastroschisis. B. Large for gestation age – growth parameters > 90% in the second trimester are unusual and may raise the concern for a genetic overgrowth syndrome. However much of fetal growth and overgrowth occurs during the third trimester whether due to an overgrowth syndrome, exposure to maternal diabetes or as part of the normal distribution of the population. CMV fetal infection typically leads to growth restriction which can present in the second trimester as CMV infection of the placenta impedes fetal support. C. Polydactyly – while associated with some chromosome and genetic syndromes, in isolation postaxial polydactyly is often a familial trait. D. Polyhydramnios – altered amniotic fluid levels (both high and low) in the second trimester are more highly associated with underlying fetal concerns then when noted after 28 weeks. High levels of amniotic fluid may be due to obstruction of the GI tract at several levels 857 (tracheoesophageal fistula, small bowel obstruction). High amniotic fluid levels due to glucose imbalance from maternal diabetes usually presents in the third trimester. CMV infection can lead to low amniotic fluid (oligohydramnios) thought to be due to two mechanisms – early inflammation of renal cells with infection and ongoing placental infection leading to decreased placental perfusion. 858 J. Mendelian Genetics Questions J1-J29 J1. In successive pregnancies, two healthy parents have two children with osteogenesis imperfecta, type 1 There is no family history of this disorder. Which of the following concepts is most likely to explain this situation in this family? A. B. C. D. E. J2. Ben and his wife Jennifer come in for preconception counseling. They met at a local benefit for cystic fibrosis (CF). Ben is healthy, but has a brother with CF and Jennifer has CF. Which of the following percentages best represents their risk for having a child with CF? A. B. C. D. E. J3. 25% 33% 50% 67% 100% Mary was recently diagnosed with Duchenne Muscular Dystrophy (DMD), an X-linked disorder. Her brother died of the condition. Which of the following explanations is the most likely cause of Mary’s clinical findings? A. B. C. D. E. J4. Genomic imprinting Germline mosaicism Multifactorial inheritance Parental consanguinity Uniparental disomy Her father has germline mosaicism for Duchenne Muscular Dystrophy. Her father is not her biological father (non-paternity). Her mother has germline mosaicism for Duchenne Muscular Dystrophy. She has a comorbid disorder, androgen insensivity syndrome. She has an X;21 translocation, with the normal X carrying the mutant DMD gene. A 14-year-old African-American girl has the early signs of a rare neurodegenerative disease that was first described in members of a small village in Africa. Nothing is known about the cause of the disease, although it is suspected that it is genetically determined. Her parents are healthy, but her paternal grandfather, who came to the United States from that same region in Africa, died at 56 from the same disease. Which of the following assumptions about this disease can be definitively concluded based on this history? A. B. C. D. E. A multifactorial etiology for this disease is likely. An autosomal dominant inheritance pattern can be excluded. An autosomal recessive inheritance pattern is likely. An X-linked recessive inheritance pattern can be excluded. The underlying cause is certainly genetic. 859 J5. Billy has Branchio-Oto-Renal syndrome (BOR). He has bilateral renal hypoplasia and requires dialysis. He is also deaf. His mother has a mild hearing loss and is missing one kidney. Her 80-year-old father is well except for a history of having had a neck cyst resected when he was a boy. Which of the following genetic concepts best explains this situation? A. B. C. D. E. J6. Cleidocranial dysplasia (CCD) is caused by mutations in the gene RUNX2. A small number of CCD patients with a more severe phenotype (including mental retardation), typically have a microdeletion of the 6p21 region that includes RUNX2. The nature of RUNX2 mutations is best described by which of the following explanations below? A. B. C. D. E. J7. RUNX2 mutations result in a loss of function of the protein. RUNX2 mutations result in a triplet repeat expansion. RUNX2 mutations result in excessive activity the protein product. RUNX2 mutations result in the disruption of an imprinted gene. RUNX2 mutations result in the protein having a novel function. Triplet repeat disorders can be characterized by several features that become evident when evaluating an affected family’s pedigree. Which of the following characteristics is a geneticist most likely to find only within a family harboring a disorder caused by expansion of triplet nucleotide repeats? A. B. C. D. E. J8. Allelic heterogeneity Heteroplasmy Locus heterogeneity Pleiotropy Variable expressivity The disorder appears to be more likely with increased paternal age. The disorder is transmitted only through the females in the pedigree. The disorder may appear to have skipped generations in the family. The severity of the phenotype worsens in subsequent generations. There is a reduced number of male offspring compared to female offspring. Assume that hypophosphatemic rickets is an X-linked dominant trait with normal fitness and complete penetrance in both sexes. Which of the following ratios represents the expected male:female sex ratio in the affected population? A. B. C. D. E. 1:3 1:2 1:1 2:1 3:1 860 J9. Robert is a three-month-old infant with ectrodactyly (a rare malformation of the hands and feet) who is brought to you by his parents, Marsha and Todd. Robert has one older sibling who has normal hands and feet. Although neither parent has hand or foot abnormalities, other members of Todd’s family are also affected with ectrodactyly. A family pedigree is below, where symbols representing individuals affected with ectrodactyly are shaded and symbols representing individuals without clinical symptoms are unshaded. Which of the following modes of inheritance is the best described by this pedigree? Todd Marsha Robert A. B. C. D. E. J10. Autosomal dominant with reduced penetrance Autosomal recessive with variable expressivity X-linked dominant with reduced penetrance X-linked recessive with a high degree of pleiotropy Y-linked with evidence of allelic heterogeneity Mark’s father was affected with type 1 albinism, an autosomal recessive disorder that results from a mutation in tyrosinase. Mark and his wife Annette show no signs of the disease. Annette's maternal grandmother was also affected with type I albinism. Annette is pregnant. Which of the following probabilities represents the risk that this fetus will be affected with type 1 albinism? A. B. C. D. E. 1/4 1/8 1/16 1/24 1/64 861 J11. Based on the pedigree below, which of the following inheritance patterns best describes the disease represented by the shaded members of the family? A. B. C. D. E. J12. Autosomal dominant Autosomal recessive Mitochondrial X-linked dominant X-linked recessive Bobby and Donna are the parents of Stephen, a child affected with a fully penetrant, autosomal recessive disorder that occurs in the population with an incidence of 1/6400 and is easily diagnosed at birth. Neither parent has this disorder and their next child, Brad, is born without any apparent signs of the disease. Brad grows up and marries Anita, a woman with no known family history of the disorder. Which of the following ratios best represents the chance that a child of Brad and Anita will be affected with the same disorder that affects Stephen? A. B. C. D. E. 1/60 1/120 1/180 1/240 1/320 862 J13. A significant proportion of nonsyndromic holoprosencephaly is caused by mutations in the sonic hedgehog gene (SHH), which codes for a secreted signaling protein required for developmental patterning. Some individuals with SHH mutation have only mild clinical symptoms, such as midface hypoplasia. Other individuals with SHH mutations may have severe CNS and facial malformations that are incompatible with life. Which of the following genetic concepts best describes this spectrum of clinical features? A. B. C. D. E. J14. Digenic inheritance Locus heterogeneity Reduced penetrance Sex-influenced expression Variable expressivity The ABO blood group is coordinated by three alleles (A, B, O) at the blood group locus. The A and B alleles function in a codominant manner, and both are dominant to the O allele. The pedigree below shows the blood groups from a three-generation family. I Type A Type A Type A Type ? Type B Type B II III Type B Type A Type O Based on this information, which of the following allele pairs represents individual II-1’s most likely genotype at the ABO blood group locus? A. B. C. D. E. AA AB AO BO OO 863 J15. Janet and Jim are married and both have achondroplasia. Each always knew they would only marry someone who also had achondroplasia. This type of choice, which at the population level can disturb Hardy-Weinberg equilibrium, is an example of which of the following genetic concepts? A. B. C. D. E. J16. Bethany is affected with a rare form of retinitis pigmentosa (a progressive retinal degeneration) that only occurs when individuals are heterozygous for mutations in two different unlinked genes, ROM1 and peripherin. Individuals who carry a mutation in only one of these genes are not affected; only patients heterozygous for mutations in both genes develop the disease. Which of the following genetic concepts explains this complex inheritance pattern? A. B. C. D. E. J17. Co-dominant inheritance Digenic inheritance Genetic heterogeneity Pleiotropy Triallelic inheritance An autosomal recessive disorder has a frequency of 1 per 2500 in the Caucasian population. All known cases of this disorder result from an identical mutation in the causative gene. Which of the following percentages represents the approximate proportion of this population with two copies of the normal allele for this gene? A. B. C. D. E. J18. Assortative mating Codominance Consanguinity Linkage Disequilibrium Positive selection 2% 4% 90% 96% 98% A three allele locus on the X chromosome controls the green-sensitive pigment for color vision. The normal allele “G” is dominant to the other two alleles, both of which are mutations. The g1 mutation leads to a condition called green shift – the presence of a weakened green-sensitive pigment where only certain shades of green are indistinguishable from browns. The g1 mutation is dominant to the g2 mutation, which results in green blindness, where reds, greens and yellows cannot be distinguished. A man with green shift vision marries a woman with green blindness. Which of the following types of green vision will the sons born to this couple have? A. B. C. D. E. All will have green blindness All will have green shift All will have normal green vision Half will have green shift; half will have green blindness Half with have normal vision; half will have green shift 864 J19. Which of the following modes of inheritance is most likely to explain the findings for this rare disorder shown in the pedigree below? A. B. C. D. E. J20. Autosomal dominant with imprinting Autosomal recessive Digenic Mitochondrial X-linked dominant Which of the following modes of inheritance is most likely to explain the findings for this rare disorder shown in the pedigree below? A. B. C. D. E. Autosomal dominant with imprinting Autosomal dominant with male limitation X-linked dominant X-linked recessive Y-linked 865 J21. Which of the following modes of inheritance is most likely to explain the findings for this rare disorder shown in the pedigree below? A. B. C. D. E. J22. Autosomal dominant with incomplete penetrance Autosomal dominant with sex limitation Autosomal recessive X-linked dominant X-linked recessive Which of the following modes of inheritance is most likely to explain the findings for this rare disorder shown in the pedigree below? A. B. C. D. E. Autosomal dominant with imprinting Autosomal dominant with incomplete penetrance Pseudodominant X-linked recessive X-linked dominant 866 J23. Which of the following modes of inheritance is most likely to explain the findings for this rare disorder shown in the pedigree below? A. B. C. D. E. Autosomal dominant Autosomal dominant with imprinting Mitochondrial X-linked dominant X-linked recessive 867 J24. Which of the following modes of inheritance is most likely to explain the findings for this rare disorder shown in the pedigree below? A. B. C. D. E. J25. Autosomal dominant with imprinting Autosomal recessive Mitochondrial X-linked dominant X-linked recessive Differences in the degree to which different tissues are impaired in an individual with a mitochondrial disorder is best explained by which of the following genetic concepts? A. B. C. D. E. Germline mosaicism Heteroplasmy Heterozygosity Incomplete penetrance New mutation 868 J26. Which of the following modes of inheritance is most likely to explain the findings for this rare disorder shown in the pedigree below? A. B. C. D. E. J27. Autosomal dominant with imprinting Autosomal dominant with sex limitation Digenic X-linked dominant X-linked recessive Which of the following genetic concepts is most likely to explain the findings for this rare disorder shown in the pedigree below? A. B. C. D. E. Digenic inheritance Germline mosaicism Imprinting Recurrent new mutation Triplet repeat expansion 869 J28. Which of the following disorders is most is most consistent with the findings shown in the pedigree below? A. B. C. D. E. J29. Hemochromatosis Incontinentia pigmenti MELAS Neurofibromatosis 1 Tuberous sclerosis Which of the following disorders is most is most consistent with the findings shown in the pedigree below? A. B. C. D. E. Duchenne muscular dystrophy Hemochromatosis MERFF Rett syndrome X-linked hypophasphatemic rickets 870 Answers to Mendielian Genetics Questions J1-J29 J1. Answer: B. Type I OI is an autosomal dominant condition. Germline mosaicism is a common explanation for the apparent recessive transmission of a known dominant disorder, and OI is one of the more common syndromes to manifest germline mosaicism. 1st cousins with Angelman syndrome are most likely due to an imprinting or UBE3A mutation; father to son transmission of sickle cell disease is most likely due to pseudodominant inheritance, with the mother being a SS carrier; deafness in siblings who are heterozygous for a GJB2 mutation can occur due to several reasons – another genetic cause, a shared environmental agent (e.g., maternal CMV infection), or compound heterozygosity of the GJB6 deletion (“Digenic” inheritance). J2. Answer: B. Jennifer will pass on a CF allele. There is a 2/3rds chance that Ben is a carrier, and 1 in 2 (50%) chance that he will pass on the abnormal allele. J3. Answer: D. In androgen insensitivity syndrome an affected individual will have the phenotypic appearance of a female but the chromosomal compliment (46,XY) of a male. Non-paternity and paternal germline mosaicism have no bearing, as the altered X is transmitted from the mother; maternal germline mosaicism cannot cause this to occur either. An X;21 translocation would cause skewed X-inactivation, but as described the normal X chromosome would be preferentially inactivated (would have the DMD gene), while the translocated X has the normal dystrophin gene and would not cause DMD. J4. Answer: D. It is unlikely that this disorder is X-linked because of the male to male transmission (the girl’s grandfather to father) it would require. While it may be genetic, it is still possible that the disorder is caused by an environmental agent that is shared by members of this village, such as a particular food. Autosomal dominant and autosomal recessive inheritance are both possible. J5. Answer: E. This is a classic example of variable expressivity. While there are several genes that can cause BOR, it is safe to assume that the same gene and the same mutation are present in all members in this family. Therefore, neither locus nor allelic heterogeneity would be correct. Heteroplasmy refers to mitochondrial genomic mutations, which present differently than a developmental disorder such as BOR. Pleiotropy refers to the wide range of effects caused by a genetic mutation. J6. Answer: A. Mutations that result in premature termination upstream or within the runt domain produce classic CCD by abolishing the transactivation activity of the mutant protein with consequent haploinsufficiency. Hypomorphic mutations (Arg391X, Thr200Ala, and 90insC) result in a clinical spectrum ranging from isolated dental anomalies without the skeletal features of CCD to mild CCD to classic CCD. Intrafamilial variability is significant (genereviews.org). J7. Answer: D. Worsening in the severity of the phenotype in subsequent generations is also called genetic anticipation, and this is a distinguishing feature of triplet repeat expansion disorders. Maternal transmission only is seen with mitochondrial inheritance. A reduced number of males compared to females is seen with X-linked lethal disorders. Increased paternal age is seen with some autosomal dominant disorders. “Skipped generation” is seen with autosomal dominant disorders that feature non-penetrance. J8. Answer: B. Frequency of males = q. Frequency for females = 2pq + q2 which will generally be close to 2q for rare traits. Ratio for male to female about 1:2. 871 J9. Answer: A. Males and females are affected in roughly equal proportions, there are affected individuals in each generation and there is transmission by individuals of both sexes – all characteristics of autosomal dominant inheritance. It appears that Todd inherited the mutation causing ectrodactyly, but did not express clinical symptoms, a finding consistent with reduced penetranceOther choices: A – E-linked inheritance can be excluded because Todd's sister inherits ectrodactyly from her father (who passed an X and not a Y chromosome to her). B – Affected individuals in multiple generations makes this option unlikely, especially given that the disorder is rare (the chance that four carriers married into this family would be very low). C & D – The male-to-male transmission (from Todd's father to Todd's brother) excludes X-linked inheritance patterns. J10 Answer: B. -Individuals with autosomal recessive disorders have mutations in each copy of the causative gene. For a child of Mark and Annette to have type 1 albinism, each parent must inherit one copy of the mutation and then pass it to the child. Mark's father had type 1 albinism so Mark inherited one copy of the tyrosinase mutation from him. There is a 1/2 chance he will pass this along to a child. We know Annette’s mother is an obligate carrier for type 1 albinism (she had to inherit one of the two mutant copies from Annette’s grandmother, who was affected.) Subsequently, there is a 1/4 chance Annette will pass along a tyrosinase mutation to a child – a 1/2 chance she received the mutation her mother inherited from Annette's grandmother multiplied by a 1/2 chance that she would pass along that mutation to a child. So there is a 1/8 total chance the fetus will inherit two copies of the tyrosinase mutation. J11. Answer: D. Notice that individuals are affected in each generation – a hallmark of dominant disorders. In addition, there is no male-to-male transmission, there are approximately twice as many affected females as males, and affected fathers transmit the disease to all of their daughters – all of which suggest a sex-linked dominant disorder. Other choices: A – The lack of male-tomale transmission and the presence of affected individuals in each generation make this an unlikely option. B – Although the pedigree certainly suggests a dominant disorder, the lack of male-to-male transmission and the excess of affected females suggests a sex-linked dominant disorder is a better choice. C – In a sex-linked recessive disorder, there is usually an excess of affected males – females who inherit one copy of the mutation are generally not affected (although it is possible that under certain conditions some carrier females exhibit clinical symptoms). J12. Answer: D. Three things must occur for Brad and Anita to have an affected child. 1. Brad must be a carrier for the disorder. As Brad doesn't show any symptoms of the disorder (which is fully penetrant), he does not carry both copies of the mutation. Subsequently, there are three equally likely remaining genotype options for Brad – homozygous normal, heterozygous with the mutation received from his mother, or heterozygous with the mutation received from his father. The overall probability that Brad is a carrier is therefore 2/3. 2. Anita must also be a carrier. Because she has no family history of disorder, we assume her risk of being a carrier is equivalent to the carrier frequency in the population. This can be deduced using Hardy-Weinberg equilibrium. The incidence of this disorder in the population is 1/6400. A rough estimate of the carrier frequency can be obtained by doubling the square root of the population frequency – § u 1/80 = 1/40. This is the chance that Anita is a carrier. 3. Both Brad and Anita must pass their mutation on to a child. If they are both carriers, the chance they would have an affected child is 1/4. To obtain the final probability for this problem, multiply the probabilities from all three events – 2/3 u 1/40 u 1/4 = 2/480 = 1/240. 872 J13. Answer: E. Variable expressivity refers to the variation observed in clinical symptoms among individuals with an identical genetic disorder. Other choices: A – Digenic inheritance is a situation where a disease is the result of double heterozygosity at two different genes acting in an additive fashion. . B – Locus heterogeneity occurs when a disorder can be caused by mutations in more than one gene. C – Reduced penetrance describes an individual who inherits a diseasecausing mutation, but exhibits no clinical symptoms whatsoever. This is believed to be the result of modifying effects of other genes or environmental factors D – Sex-influenced expression refers to disorders that are more frequently expressed in one sex than another, probably due to the influence of sex-specific hormones. J14. Answer: C. Individual II-1's parents have type A blood. Their genotypes are either AA or AO. Depending on their specific genotypes, Individual II-1 could either be AA, AO or OO. Individual II-1's wife has type B blood and is either BB or BO at the blood group locus. Based on her father's type A blood, we can infer that Individual II-1's wife is BO at the ABO locus – she inherits a B from her mother and an O from her father. Individual II-1 and his wife have three children. The second child has type A blood. Individual II-1's wife can pass on an O or B allele to her child. In order to have type A blood, the child must have inherited an O from his mother and an A allele from Individual II-1. The third child has type O blood, which can only occur when an OO genotype is present. So Individual II-1 must also have an O allele. That means Individual II-1 is AO at the blood group locus and has type A blood. J15. Answer: A. One of the assumptions underlying Hardy-Weinberg equilibrium is

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