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9/7/2012 Topics Principle of Genetics Introduction Genetic disorders Single gene disorders Chromosomal disorders Multifactorial diseases Somatic cell genetic disorders นายแพทย ดร. ณตพล ศุภณัฐเศรษฐกุล ภาควิชาพยาธิวิทยา คณะแพทยศาสตร มหาวิทยาลัยนเรศวร Email : [email protected] 12 กันยายน พ.ศ. 2555 http://www.uga.edu/srel/kidsdoscience/images/genetics-puppets.gif http://www.uwo.ca/biology/images/undergraduate/chromosome.jpg Topics Clinical genetics and Molecular Diagnosis Genetic testing for carrier detection Prenatal diagnosis Newborn screening Molecular diagnosis Genetic counseling Gregor Mendel ค.ศ.1822-1884 http://history.nih.gov/exhibits/nirenberg/images/photos/01_mendel_pu.jpg http://www.scq.ubc.ca/wp-content/mendel.jpg 1 9/7/2012 1990 – 2003 James D. Watson 1928 - Structure of DNA 1953 Nobel prize in 1962 Francis H.C. Crick 1916 - 2004 http://tesla.desy.de/~rasmus/media/Coherence/PDFs%20and%20high%20res olution/watson%20and%20crick.jpg http://www.sanger.ac.uk/Info/Press/gfx/030414_hgp_300.jpg Structure of DNA “Central Dogma” Transcription DNA Translation RNA Protein Replication http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/16-05-doublehelix.jpg 2 9/7/2012 Human Genome (chromosomes) In Nucleus In Mitochondria 3.1 x 109 bp 16,569 bp 10% 90% Gene Non-gene (Extragenetic) <10% Coding Sequences (Exon) Cell cycle >90% Non coding Sequences (Intron) 50% 50% Repetitive DNA Non repetitive DNA http://ghs.gresham.k12.or.us/science/ps/sci/soph/cells/cycle/cycle.gif Tandem repeats (satellite, minisatellite, microsatellite) Interspersed repeats (SINES, LINES) http://www.cbp.pitt.edu/faculty/yong_wan/images/main_cell_cycle.jpg Mutation หมายถึงกระบวนการที่กอใหเกิดการเปลี่ยนแปลง ทุกรูปแบบ ที่เกิดขึ้นกับโมเลกุลของ DNA “Wild type” and “mutant allele” Normal variation, polymorphism, inherited disease Types of mutation http://homepage.mac.com/enognog/cell%20cycle.jpg http://tainano.com/Molecular%20Biology%20Glossary.files/image040.gif Base substitution => silent mutation, missense, nonsense Insertion => frameshift mutation Deletion => frameshift mutation Chromosomal abnormality http://www.layevangelism.com/bastxbk/images/mutation.jpg 3 9/7/2012 http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=112100&screenWidth=1280&screenHeight=800 Genetic disorders Terminology Single gene disorders Mendelian inheritance Non-Mendelian inheritance Chromosomal disorders Polygenic or multifactorial disorders Somatic cell genetic disorders (Neoplasm) Genotype = genetic constitution of an individual Phenotype = observed characteristic Homozygote = identical alleles at a given locus Heterozygote = two different alleles at a given locus Autosomes = any chromosomes other than sex chromosomes Autosomal inheritance = involve any chromosomes other than sex chromosomes Pedigree charts = to illustrate the inheritance 4 9/7/2012 Terminology Genotype = genetic constitution of an individual Phenotype = observed characteristic Homozygote = identical alleles at a given locus Heterozygote = two different alleles at a given locus Autosomes = any chromosomes other than sex chromosomes Autosomal inheritance = involve any chromosomes other than sex chromosomes Pedigree charts = to illustrate the inheritance http://naturalsciences.sdsu.edu/ta/classes/lab2.4/trait.jpg Terminology Locus = position of DNA sequences, or gene on a chromosome Allele = possible alternative form of a given gene DNA polymorphism = the occurrence in a population of two or more alternative genotypes Gene pool = the study of frequency of total alleles on a given locus Population genetics = the study of the genetic composition of populations Cystic Fibrosis Transmembrane Regulator, CTFR http://ghr.nlm.nih.gov/handbook/illustrations/chromosomallocation.jpg 5 9/7/2012 Gene loci http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromo7.gif http://www.geneticsandhealth.com/wp-content/allele.jpg Terminology Genotype = genetic constitution of an individual Phenotype = observed characteristic Homozygote = identical alleles at a given locus Heterozygote = two different alleles at a given locus Autosomes = any chromosomes other than sex chromosomes Autosomal inheritance = involve any chromosomes other than sex chromosomes Pedigree charts = to illustrate the inheritance http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/Images/autosome.gif 6 9/7/2012 Terminology Genotype = genetic constitution of an individual Phenotype = observed characteristic Homozygote = identical alleles at a given locus Heterozygote = two different alleles at a given locus Autosomes = any chromosomes other than sex chromosomes Autosomal inheritance = involve any chromosomes other than sex chromosomes Pedigree charts = to illustrate the inheritance http://www.geneticsandhealth.com/wp-content/pedigree%20image_1.gif http://www.geneticsandhealth.com/wp-content/pedigree%20image_2.gif http://www.migeneticsconnection.org/genomics/Family%20History/pedigreeSymbols.jpg 7 9/7/2012 Introduction Single gene disorders are caused by individual mutant genes There are approximately 11,000 single gene disorders, affecting 1% of the populations Follow Mendelian patterns of inheritance Autosomal dominant inheritance (AD) Autosomal recessive inheritance (AR) X-linked recessive inheritance (X-linked) X-linked dominant inheritance Y-linked (Holandic) inheritance A “dominant phenotype” is one that is expressed in heterozygotes, whereas a “recessive trait” is expressed only in homozygotes If the expression of each allele can be detected in the presence of the other, the two alleles are termed “co-dominant” Autosomal Dominant Phenotypically expressed in homozygotes and heterozygotes for that gene There is vertical inheritance (affected child usually has an affected parent) Unaffected family members usually have unaffected partners, and they produce normal children Affected family members, usually have unaffected partners, and they produce a 1:1 ratio of normal and affected children Usually both sexes are equally affected, and they are equally likely to pass on the disease http://www.uic.edu/classes/bms/bms655/gfx/pedigree1.gif 8 9/7/2012 Achondroplasia Marfan syndrome http://www.sciencemuseum.org.uk/on-line/genes/images/1-3-5-1-4-2-1-1-1-3-0.jpg http://www.nhlbi.nih.gov/health/dci/images/Marfan_lowres.gif http://www.moondragon.org/obgyn/graphics/achondroplasiacomparison.jpg http://www.commonwealthadoption.org/images/johnston.jpg http://www.homoeopathiker.de/homoeopathik/syphilis/bilder/marfan3.jpg Examples Skeletal: Marfan syndrome, Achondroplasia, Osteogenesis imperfecta Nervous system: Huntington disease, Neurofibromatosis Urinary: Polycystic kidney disease (Adult) Gastrointestinal: Familial polyposis coli Hematopoietic: Von Willebrand disease Metabolic: Familial hypercholesterolemia, Acute intermittent porphyria http://www.uic.edu/classes/bms/bms655/lesson5.html 9 9/7/2012 Autosomal Recessive Expressed only in homozygotes, otherwise can be trait There is horizontal inheritance (normal parents often have more than one affected child) Affected individuals have phenotypically normal parents Affected individuals usually have unaffected partners and all their children will be carriers If a carrier has an unaffected partner, there is a 50% chance of the children being carriers Only mating between heterozygotes (carrier) will produce affected individuals, with an expected frequency of 1 in 4 There is an association with consanguinity due to sharing of genes in families (rare recessive genetic disorders are more likely to arise through consanguinity) Both sexes are equally affected and equally likely to pass the mutation to the next generation http://healthnet.md.chula.ac.th/text/forum1/thalassemia2/picture/thalassemia.jpg http://upload.wikimedia.org/wikipedia/commons/thumb/3/3e/Autorecessive.svg/300px-Autorecessive.svg.png 10 9/7/2012 http://www.microscopyu.com/galleries/pathology/images/sicklecell/sicklecellanemia40x02small.jpg http://home.kku.ac.th/acamed/kanchana/p7.jpg http://www.irondisorders.org/News/SickleCell.jpg Example Metabolic: Cystic fibrosis, Phenylketonuria, Hemochromatosis, Glycogen storage diseases, Galactosemia, Homocystinuria, Lysosomal storage diseases Hematopoietic: Thalassemia, Sickle cell disease Endocrine: Congenital adrenal hyperplasia Nervous: Friedreich ataxia http://www.uic.edu/classes/bms/bms655/lesson7.html#RECESSIVE 11 9/7/2012 X-linked Recessive Many more males than females show the recessive phenotype The disease is transmitted by a carrier female, who is usually asymptomatic If a mother is a carrier, her son have a 50% chance of being affected and her daughters a 50% chance of being carriers An affected male will usually have no affected offspring, but all his daughters will be carriers and, in turn, 50% of their sons will be affected No sons of the affected male will inherit the gene (there is no male-to-male transmission) http://www.uic.edu/classes/bms/bms655/lesson7.html#RECESSIVE Gower’s sign Example Duchenne muscular dystrophy http://www.neurologyarticles.com/sitebuilder/images/Scan10106-176x315.jpg Musculoskeletal: Duchenne muscular dystrophy, Becker muscular dystrophy Blood: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, Hemophilia A and B Metabolic: Diabetes insipidus, Lesch-Nyhan syndrome Nervous: Fragile-X syndrome (Atypical) Immune: Agammaglobulinemia http://medicalimages.allrefer.com/large/x-linked-recessive-genetic-defects.jpg 12 9/7/2012 II. Chromosomal Abnormalities Terminology Terminology Introduction Numerical Chromosome abn. Structural chromosome abn. Chromosome instability synd. Karyotype = the chromosome constitution of an individual, normal human karyotype is 46,XY (male) and 46,XX (female) Polyploidy = the number of hapoloid chromosome sets is greater than two (2n), triploidy, tetraploidy Aneuploidy = chromosome number is not an exact multiple of haploid number, monosomies, trisomies Trisomy = three representatives of a given chromosome, trisomy 21 in Down dyndrome Monosomy = one member of a chromosome pair is missing, Turner syndrome (45,X) Translocation = the transfer of one segment of a chromosome to another http://images.wikio.com/images/p/51ca/the-world-s-worst-genetic-mutation-in-a-dog.jpg Terminology Karyotype = the chromosome constitution of an individual, normal human karyotype is 46,XY (male) and 46,XX (female) Polyploidy = the number of hapoloid chromosome sets is greater than two (2n), triploidy, tetraploidy Aneuploidy = chromosome number is not an exact multiple of haploid number, monosomies, trisomies Trisomy = three representatives of a given chromosome, trisomy 21 in Down dyndrome Monosomy = one member of a chromosome pair is missing, Turner syndrome (45,X) Translocation = the transfer of one segment of a chromosome to another http://www.asklenore.info/miscarriage/bick/images/fig3.jpg 13 9/7/2012 Terminology Karyotype = the chromosome constitution of an individual, normal human karyotype is 46,XY (male) and 46,XX (female) Polyploidy = the number of hapoloid chromosome sets is greater than two (2n), triploidy, tetraploidy Aneuploidy = chromosome number is not an exact multiple of haploid number, monosomies, trisomies Trisomy = three representatives of a given chromosome, trisomy 21 in Down dyndrome Monosomy = one member of a chromosome pair is missing, Turner syndrome (45,X) Translocation = the transfer of one segment of a chromosome to another http://www.ucl.ac.uk/~ucbhjow/bmsi/lec7_images/47_xx_21.gif Terminology Karyotype = the chromosome constitution of an individual, normal human karyotype is 46,XY (male) and 46,XX (female) Polyploidy = the number of hapoloid chromosome sets is greater than two (2n), triploidy, tetraploidy Aneuploidy = chromosome number is not an exact multiple of haploid number, monosomies, trisomies Trisomy = three representatives of a given chromosome, trisomy 21 in Down dyndrome Monosomy = one member of a chromosome pair is missing, Turner syndrome (45,X) Translocation = the transfer of one segment of a chromosome to another http://www.myoops.org/cocw/tufts/courses/20/content/D293242/C293223.jpg 14 9/7/2012 Spectral Karyotyping (SKY) Analysis Introduction Maybe numerical or structural Nomenclature Numerical disorders : [47, XY,+21], [45,X] Structural disorders : [46,XY,t(14;21)(q11,p10)], [46,XY,dup(5) (q20-q30)], [46,XY,del(15)(q11-q13)], (break point, margin, or region) http://www.nature.com/onc/journal/v24/n33/images/1208711f1.jpg Numerical Chromosome Abnormalities Concern : Extra single chromosome, Missing single chromosome, Extra haploid sets Polyploidy Fertilization by two sperm A diploid sperm (2n) due to failure in meiosis A diploid ovum (2n) due to failure in meiosis Mechanisms Polyploidy Trisomies Monosomies 15 9/7/2012 Trisomies Failure of separation (nondisjunction) of homologous chromosomes at meiosis I Failure of separation of chromatids in meiosis II Advancing maternal age is associated with increased incidence of trisomy http://www.anselm.edu/homepage/jpitocch/genbio/nondisjunction.JPG Examples Autosomal disorders : Sex chromosome disorders : Trisomy 21 (Down syndrome) Trisomy 18 (Edwards syndrome) Trisomy 13 (Patau syndrome) Klinefelter syndrome (47,XXY) Turner syndrome (45,X) XXX = female with an extra X chromosome XYY = male with an extra Y chromosome XX male = Y sequences are transferred to the X chromosome, look like male with Klinefelter syndrome http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=108200&screenWidth=1280&screenHeight=800 16 9/7/2012 Down’s syndrome CVS : Endocardial cushion defect (Atrioventricular septal defect) most common GI : increase risk of Hirschprung’s diseases (intestinal stenosis) Down’s syndrome facies Simian crease Gap between 1st and 2nd toes http://www.suriyothai.ac.th/en/node/1005 http://upload.wikimedia.org/wikipedia/commons/c/c3/Trisomy21_graph.jpg http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm 17 9/7/2012 http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=109400&screenWidth=1280&screenHeight=800 http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm Structural Chromosome Abnormalities Result from breakage and limitations of DNA repair systems Mechanisms Translocation Inversion Duplication Deletion and ring chromosome Isochromosome http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=107050&screenWidth=1280&screenHeight=800 18 9/7/2012 III. Multifactorial Disorders Terminology Multifactorial disorders Threshold model of multifactorial disorders Examples of multifactorial disorders Cri du chat syndrome (cat-like cry) : Deletion of the region on 5p15.2 or the whole short arm of chromosome 5 mental retardation, low birth weight, low set ears, cat-like cry http://cas.bellarmine.edu/tietjen/HumanBioogy/Finished%20Images/gen30.gif Multifactorial Inheritance Normal human characteristics : Blood pressure, height, finger ridges, and intelligence Heritability and Environmental factors Heritability : percentage denoting that the genetic contribution of a given disease Congenital malformations : neural tube defects, cleft lip and palate, and congenital heart disease Common disorders of adult life : DM, Hypertension, peptic ulcer, and schizophrenia If heritability is high, there is a high correlation in relatives Environmental factors : can be manipulated to reduce an individual’s susceptibility below than the threshold 19 9/7/2012 Threshold model for Multifactorial disorders Example (Tendency) Diabetes mellitus, type I (Insulin-dependent) Essential hypertension (62% heritability) Atherosclerosis (65% heritability) Peptic ulcer (37% heritability) Schizophrenia (85% heritability) Asthma (80% heritability) Alzheimer disease http://www.uic.edu/classes/bms/bms655/gfx/figure18.gif Clinical genetics and Molecular Diagnosis The age of expression of the major types of genetic disease Genetic testing for carrier detection Prenatal diagnosis Newborn screening Molecular diagnosis Genetic counseling 20 9/7/2012 Genetic Testing for Carrier Detection To identify asymptomatic heterozygotes for AR traits, or AD disorders that have limited penetrance or late onset Confined to small ethnic populations in which there is an anomalously high incidence of a particular disease Examples : Thalassemia, Cystic fibrosis, ADPKD, Cascade screening Prenatal diagnosis To assure of having unaffected children when the risk of having an affected child is unacceptably high NOT equivalent to the assurance of having normal children, It does not address all possible birth defects Noninvasive techniques : ultrasonography, maternal serum screening Invasive technique : amniocentesis, chorionic villus sampling, cordocentesis DNA analysis (PCR, Southern blot), Cytogenetic analysis Cordocentesis http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/1062.jpg http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19177.jpg http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19175.jpg 21 9/7/2012 Chorionic villi sampling http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/19163.jpg http://www.pennhealth.com/health_info/pregnancy/graphics/images/en/19166.jpg Newborn screening To identify treatable genetic disorders such as phenylketonuria, galactosemia, maple syrup urine disease, and congenital adrenal hyperplasia in newborn infants Dietary management is usually an early intervention to eliminate clinical symptoms that would otherwise lead to severe disability, mental impairment, or dealth Prevalence, severity of a condition, availability, effectiveness of a treatment, and cost will determine which disorder will be screened http://www.health.state.mn.us/divs/fh/mcshn/ncfu/images/pkupath.gif 22 9/7/2012 Molecular techniques DNA , RNA Extraction Polymerase Chain Reaction (PCR) Gel Electrophoresis Direct Hybridization Method Inborn error of metabolism - PKU, galactosemia Hormonal abnormality - congenital hypothyroidism - congenital adrenal hyperplasia Southern blot analysis Northern blot analysis Fluorescence In situ Hybridization (FISH) DNA Microarrays Cytogenetics Karyotyping Spectral karyotyping (SKY analysis) http://www.qctimes.com/content/articles/2005/07/22/features/health/doc42e079564f5f3070410001_thumb.jpg Polymerase Chain Reaction (PCR) DNA Extraction Taq DNA Polymerase http://www.favorgen.us/sitebuilder/images/Blood_Genomic_DNA_Mini_chart-437x463.jpg http://www.bio.miami.edu/~cmallery/150/gene/c7.20.7.pcr.jpg http://www.obstbau-kompetenzzentrum.de/arbeitsbereiche/Folder.2006-11-07.1358/Thermocycler_.jpg 23 9/7/2012 DNA Gel Electrophoresis http://users.ugent.be/~avierstr/principles/pcrcopies.gif http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg http://universe-review.ca/I11-50-gelectrophoresis.jpg Southern Blot Analysis http://images.google.co.th/imgres?imgurl=http://users.ugent.be/~avierstr/principles/pcrcopies.gif&imgrefurl=http://users.ugent.b e/~avierstr/principles/pcr.html&usg=__kCFieOdf4YXbyVmdNjY2uR9bquI=&h=306&w=598&sz=14&hl=th&start=9&um=1&tbnid =3Kupvmv7CJ2gM:&tbnh=69&tbnw=135&prev=/images%3Fq%3Dpcr%26ndsp%3D18%26hl%3Dth%26sa%3DN%26um%3D1 http://www.mmi.mcgill.ca/mmimediasampler2002/images/5no15/5no15overview.gif 24 9/7/2012 Blot Detection Method Southern blot = DNA detection (DNA probe) Edwin Southern, Professor of Biochemistry, 1975 Northern blot = RNA detection (RNA probe) Western blot = Protein detection (Antibody) Southwestern blot = DNA binding protein (DNA probe) Eastern blot = Protein post translation modification (probe detect CHO, lipid, etc.) Dot blot (Slot blot) = Nucleotide or Protein Fluorescent In situ Hybridization Localized specific DNA sequences on chromosomes using fluorescent probes Fluorescence microscopy Localized specific mRNA = gene expression http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/images/fluoromicrofigure1.jpg http://en.wikipedia.org/wiki/File:FISH_(Fluorescent_In_Situ_Hybridization).jpg http://www.nature.com/modpathol/journal/v18/n12/images/3800465f1.jpg 25 9/7/2012 DNA Microarrays http://img.medscape.com/fullsize/migrated/430/330/cc430330.fig2.gif http://en.wikipedia.org/wiki/File:NA_hybrid.svg http://www.fao.org/DOCREP/003/X6884E/x6884e00.jpg http://en.wikipedia.org/wiki/File:Affymetrix-microarray.jpg http://neurophilosophy.files.wordpress.com/2006/08/cdna-array.jpg http://farm3.static.flickr.com/2077/2034113679_ac5a5c688d_m.jpg 26 9/7/2012 Cytogenetics : Karyotyping Spectral Karyotyping (SKY) Analysis http://www.cityofhope.org/PublishingImages/Research/Shared% 20Resources%20%20Equipment/Cytogenetics%20Core%20Laboratory/HumanSK Y_Large.jpg http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t2/dwn_chromo.gif Genetic Counseling Indications for genetic counseling Information conveyed in genetic counseling Indications for Genetic Counseling Known or suspected hereditary disease in a patient or family Birth defects Mental retardation Advanced maternal age Family history of early onset cancer Recurrent pregnancy loss Teratogen exposure Consanguinity http://www.gen.vcu.edu/images/counseling.jpg 27 9/7/2012 Information Conveyed in Genetic Counseling References The magnitude of the risk of occurrence or recurrence The impact of the disease on the patient and the family The possibility of modification of either the impact or the risk Anticipated future development คัมภิรานนท, อมรา., “พันธุศาสตรมนุษย (Human Genetics), พิมพครัง้ ที่ 2.” เท็กซ แอนด เจอรนลั พับลิเคชัน่ , กรุงเทพมหานคร, 2546. บุญแสง, วิชยั ., และคณะ., “ลายพิมพดเี อ็นเอ..จากสารพันธุกรรมสูเ ทคโนโลยีพสิ จู นบคุ คล, พิมพครัง้ ที่ 2.” สํานักงานพัฒนาวิทยาศาสตร และเทคโนโลยีแหงชาติ, ปทุมธานี, 2545. Gelehrter, T.D., Collins, F.S., Ginsburg, D., “Principles of Medical Genetics, 2nd edition.” Williams & Wilkins, Maryland, 1998. Lamb, N.E., “Crash course : Cell Biology and Genetics.” Mosby-Elsevier, Inc., Philadelphia, 2007. Kumar, V., et al., “Robbins and Cotran Pathologic Basis of Disease, 7th edition.” Elsevier Inc., Philadelphia, 2005. Rimoin, D. L., editors, et al., “Emery and Rimoin’s Principles and Practice of Medical Genetics, 4th edition.” Churchill Livingstone, London, 2002, volume 1. 28