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Genetics Dr. Joseph de Nanassy Associate Professor, uOttawa Chief of Pathology, CHEO [email protected] 737-7600 x 2897 Objectives ☺ Develop a basic understanding of the genetic apparatus ☺ Comprehend definitions of major genetic abnormalities ☺ Correlate molecular abnormalities and genetic defects Outline I. Definitions Genetic code Chromosomes, Genes, Cell Division Molecular mechanisms II. Abnormal fetal development Malformations, deformations, dysplasias, disruptions III. Perinatal pathology Birth defects Metabolic disorders The Cell Nucleus ☺ DNA: arranged in chromosomes (network of granules = nuclear chromatin) ☺ RNA: spherical intranuclear structure(s) - nucleolus / nucleoli Genetic Code ☺ A series of messages contained in the chromosomes ☺ This code regulates cell functions by way of directing the synthesis of cell proteins ☺ The code corresponds to the structure of the DNA ☺ The code is transmitted to new cells during cell division DNA structure DNA replication mRNA and tRNA Chromosomes ☺ Exist in pairs – homologous: 22a + 1s ☺ Composed of double coils of DNA ☺ Basic unit: nucleotide phosphate group deoxyribose sugar base: purine (A, G) pyrimidine (T, C) Genes ☺ Basic units of inheritance ☺ Segments of the DNA chain ☺ Beads on a (chromosome) string ☺ Determine cell properties, both structure and functions unique to the cell Genome ☺ Sum total of all genes contained in a cell’s chromosomes ☺ Identical in all cells ☺ Not all genes are expressed in all cells ☺ Not all genes are active all the time ☺ May code for enzymes or other functional proteins, structural proteins, regulators of other genes Gene Product ☺ A protein specified by a gene ☺ Transcribed into mRNA ☺ Translated through tRNA and cytoplasmic ribosomes into protein Human Genome ☺ 3 billion pairs of DNA nucleotides ☺ 50,000 – 100,000 genes ☺ Genes = 10% of human genome ☺ Exons: parts of the DNA chain that code for specific proteins ☺ Introns: the parts in-between the exons ☺ Both exons and introns are transcribed but only the exons are translated (introns are removed from mRNA before leaving nucleus) Sex chromosomes ☺ Genetic sex = composition of X and Y ☺ Large X: many genes, many activities ☺ Small Y: almost entirely male sexual diff. ☺ Female: XX, male XY ☺ One X randomly inactivated and nonfunctional after first week of embryonic development ☺ Same inactivated X in descendant cells Lyon hypothesis Barr body Y chromosome ☺ Stains with some fluorescent dyes - bright fluorescent spot in the nucleus ☺ Normal female: sex chromatin body but no fluorescent spot ☺ Normal male: fluorescent spot but no sex chromatin body Cell Division ☺ Mitosis: somatic cells (PMAT) Daughter cells have the same number of chromosomes as the parent cell. ☺ Meiosis: gametogenesis (1st and 2nd div) Number of chromosomes reduced by half. Chromatids ☺ Paired chromosomes Before mitosis, the DNA chains duplicate to form new chromosome material. The duplicated chromosomes lie side by side = chromatid. Mitosis = the process by which chromatids separate into chromosomes. Mitosis ☺ Interphase: DNA duplication to form chromatids just before mitosis ☺ Prophase: centriole migration, mitotic spindle ☺ Metaphase: chromosomes line up in centre, chromatids still joined at centromere ☺ Anaphase: chromatids separate into chromosomes ☺ Telophase: new nuclear membranes form, cytoplasm divides Mitosis Meiosis ☺ First meiotic division: duplication of chromosomes to form chromatids ☺ Prophase of meiosis: homologous chromosomes lie side by side over entire length = synapse. Interchange of segments of homologous chromosomes = crossover. 2 Xs side by side just like the autosomes. X and Y end to end – no crossover. Meiosis ☺ Metaphase: paired chromosomes arrange in middle of cell ☺ Anaphase: homologous chromosomes migrate to opposite poles of the cell; each chromosome is composed of two chromatids, the chromatids are not separated ☺ Telophase: two new daughter cells form; each contains half the chromosome number = reduction of chromosomes by half; interchange of genetic material occurred during synapse Meiosis ☺ Second meiotic division = mitotic division 2 chromatids separate, 2 new daughter cells are formed with half the normal number of chromosomes Meiosis Gametogenesis ☺ Gonads: testes, ovaries; contain ☺ Precursor cells or germ cells; mature into ☺ Gametes: sperm, ova; in gametogenesis ☺ Spermatogenesis, oogenesis Gametogenesis Primary follicles Oogenesis vs. spermatogenesis ☺ One ovum (+ 3 polar bodies) vs. four spermatozoa ☺ Oocytes formed before birth vs. continuous spermatogenesis (‘fresh’ sperm) Prolonged prophase of first meiotic division until ovulation – more frequent congenital abnormalities in ova of older women (longer exposure to potentially harmful environmental influences until meiotic division resumes at ovulation) Chromosome Analysis Karyotype Genes and Inheritance ☺ Locus: specific site of a gene on the chromosome. Since the chromosomes exist in pairs, genes are also paired. ☺ Alleles: alternate forms of a gene can occupy the same locus (homo, hetero) ☺ Recessive gene: expressed only when homozygous ☺ Dominant gene: homo or hetero or co☺ Sex-linked gene: X, recessive, hemi Gene Imprinting ☺ Genes occur in pairs on homologous chromosomes, one from each parent ☺ Different effects of gene whether ♀ or ♂ ☺ Genes modified during gametogenesis ☺ Gene imprinting: additional methyl groups added to DNA molecules ☺ Basic structure identical; in some diseases different expression (behaviour) depending on parent of origin: hereditary disease as a result of imprinting Genetic Engineering ☺ Insertion of a gene encoding a desired product (e.g. insulin) into a bacterium ☺ Bacterial gene spliced enzymatically, recombinant DNA inserted into plasmid (circular DNA segment in bacterium), dividing bacterial population produces desired protein Gene Therapy ☺ Normal gene inserted into defective cell ☺ Compensates for the missing or dysfunctional gene, in somatic cells only ☺ Can be inserted into mature cell (ly) ☺ Can be inserted into stem cell (bone marrow) ☺ Used to treat e.g. ADA deficiency, CF, … Congenital / Hereditary Diseases ☺ Congenital: present at birth ☺ Hereditary (genetic): result of chromosome abnormality or defective gene Causes of malformations 1. Chromosomal abnormalities 2. Gene abnormalities 3. Intrauterine injury (e.g. drugs, radiation, infection, environmental, etc) 4. Environmental effect on genetically predisposed embryo Chromosomal abnormalities ☺ Nondisjunction: failure of homologous chromosomes in germ cells to separate from one another during 1st or 2nd meiotic division ☺ Sex chromosomes or autosomes ☺ Extra chromosome: trisomy (24 or 47) Absent chromosome: monosomy (22 or 45) Nondisjunction in meiosis ☺ Chromosome Deletion: Broken piece of chromosome is lost from cell ☺ Translocation: Not lost, just misplaced and attached to another chromosome - reciprocal: between two nonhomologous chromosomes (no loss or gain of genetic material - no loss of cell function) - in germ cells: deficient or excess chromosome material – abnormal zygote Translocation in gametes Sex chromosome abnormalities Turner syndrome Klinefelter syndrome Autosomal abnormalities ☺ Loss: aborted embryo ☺ Deletion: congenital anomalies ☺ Trisomy: syndromic, e.g. 21, 13, 18 Trisomy 21 (Down) T21 causes 1. Nondisjunction during gametogenesis (95%) 2. Translocation (few) 3. Nondisjunction in zygote (rare) Translocation T21 Zygote nondisjunction T21- Mosaic Abnormal gene diseases ☺ Individual gene abnormalities ☺ Hereditary diseases transmitted mostly on autosomes, only a few on sex chromosomes. ☺ Gene mutation: spontaneous environmental ☺ Minor structural change may result in major functional abnormality (e.g. SSD) Modes of Inheritance ☺ Autosomal dominant (a dominant gene expressed in the heterozygous state) ☺ Autosomal recessive (expressed only in homozygous individual, disease only if both alleles are abnormal) ☺ Codominant (full expression of both alleles in heterozygous state) ☺ X-linked (usually affects male offspring; the abnormal X-linked gene acts as dominant gene when paired with the Y chromosome) Intrauterine Injury 1. Drugs: thalidomide (phocomelia), DES (cervical cancer), street drugs (IUFD), smoking (IUGR), alcohol (FAS), etc 2. Radiation: x-rays 3. Maternal infections: - Rubella virus (CVS, CNS, chr. infection) - CMV (microcephaly, chronic infection) - Toxoplasma gondii (hydrocephalus, systemic infection) Thalidomide baby Prenatal CMV infection Multifactorial Inheritance ☺ Combined effect of multiple genes interacting with environmental agents, e.g. cleft palate, cardiac malformations, club foot, hip dislocation, spina bifida, etc ☺ Cause: developmental sequence fails to reach a certain point at an appropriate time (threshold) Genetically determined variation in rate of development Effect of harmful environmental agents on susceptibility to congenital malformations Interaction of genetic predisposition and environmental factors in cleft palate Prenatal Diagnosis of Congenital Abnormalities 1. Examination of fetal cells for chromosomal, genetic or biochemical abnormalities 2. Examination of amniotic fluid for products secreted by the fetus 3. Ultrasound of the fetus to detect malformations (NTD, hydrocephalus, …) Prenatal Diagnosis of Congenital Abnormalities Main indications for amniocentesis 1. Maternal age (>35) 2. Previous infant with T21 or other chromosomal abnormality 3. Known translocation T21 carrier 4. Other chromosomal abnormality in either parent, e.g. t(7;21) 5. Risk of genetic disease in the fetus that can be detected prenatally (thalassemia) 6. Previous infant born with neural tube defect (multifactorial inheritance, ~5%) Methods of fetal DNA analysis 1. Enzyme analysis of DNA: resultant DNA fragments different in health and disease, e.g. sickle cell anemia 2. DNA probes: same complementary nucleotide arrangement as in defective DNA gene – binds to mutant gene Molecular Genetics of Solid Pediatric Tumors ☺ Mechanisms for tumor development 1. Creation of novel fusion proteins 2. Loss of tumor suppressor genes 3. Activation of proto-oncogenes Translocations, Oncogenes, Tumor suppressor genes NB: MYCN amplification and 1p deletion by FISH NB: Double-minute chromosomes by FISH RB: MYCN probe to detect homogeneously staining region in metaphase spread and interphase nuclei Ewing sarcoma: t(11;22) EWS green, FLI-1 pink, t yellow E-RMS: Spectral karyotype t(1;3), t(1;15), t(1;21) Abnormal Fetal Development ☺ ☺ ☺ ☺ Malformation Deformation Dysplasia Disruption Prenatal development, pre-embryonic Prenatal development, early embryonic Prenatal development, late embryonic Fetal development Normal gametogenesis Meiosis Abnormal gametogenesis ♂ & ♀ gametes Sperm penetrating oocyte Fertilization Causes of human congenital anomalies Malformations ☺ Intrinsic abnormalities of blastogenesis and organogenesis affecting the morphogenetically reactive fields of the embryo = developmental field defects ☺ Occur alone or in combination (syndromes or associations) ☺ Severe (spina bifida aperta) or mild (spina bifida occulta) Malformations ☺ Causally heterogeneous ☺ Intrinsic causes: mendelian mutations, chromosome abnormalities, environmental interactions (multifactorial), mitochondrial mutations Disruptions ☺ Environmental (exogenous) causes producing abnormalities of morphogenetic field dynamics ☺ E.g. rubella, thalidomide, isotretinoin, alcohol, etc Rubella embryopathy Diabetic embryopathy Dysplasias ☺ Disturbances of histogenesis, occurring later and somewhat independently of morphogenesis ☺ Morphogenesis is prenatal, histogenesis continues postnatally in all tissues that have not undergone end differentiation ☺ Dysplasias may predispose to cancer Neurofibromatosis Tuberous sclerosis Deformities ☺ Secondary changes in form or shape of previously normally formed organs or body parts ☺ Caused by extrinsic forces (e.g. Potter syndrome) or intrinsic defects (e.g. fetal akinesia syndrome with congenital arthrogryposis) Oligohydramnios (Potter) sequence Arthrogryposis Sequences ☺ Secondary consequences of malformations, disruptions, dysplasias, or deformities ☺ E.g. renal adysplasia leads to Potter oligohydramnios sequence DiGeorge anomaly leads to tetany, hypoparathyroidism, heart failure, conotruncal congenital heart defect Minor Anomalies ☺ Disturbance of phenogenesis in fetal life ☺ Phenogenesis: the process of attaining final quantitative anthropometric traits of the race and family (variant familial developmental pattern) ☺ Causes: intrinsic (chromosome imbalance) extrinsic (teratogens) Syndromes ☺ Patterns of anomalies proven or presumed causally related ☺ Causes: - chromosome mutations - imprinting defects - aneuploidy - multifactorial disorders - teratogenic sequences Treacher-Collins syndrome (mandibulofacial dysostosis) AD Leprechaunism (defective insulin binding) AR Associations ☺ Idiopathic multiple congenital anomalies of blastogenesis Vertebral anomalies V Anorectal anomalies A TracheoEsophageal defects TE Radial and Renal defects R ☺ Single hit during gastrulation affecting multiple, morphogenetically closely related structural primordia Metabolic Disorders ☺ Most are inherited as AR, some are X-linked, a few are AD. ☺ Great variability in presentation ☺ Some present with dysmorphic features ☺ Storage material in RES and other tissues Storage Diseases ☺ Lysosomal Lipid Storage Diseases Nieman-Pick: sphyngomyelin Gaucher disease: glucocerebrosidase Tay-Sachs disease: Gangliosidoses Metachromatic leukodystrophy ☺ Mucopolysaccharidoses (I, II, III, VII) Hurler syndrome COH Disorders ☺ Glycogen Storage Diseases ☺ Galactosemia Glycogen storage disease type II Amino Acid Disorders Misc. ☺ Fatty Acid Beta-Oxidation Defects (LCAD, MCAD, SCAD) ☺ Organic Acidemias ☺ Defects in Purine Metabolism ☺ Carnitine Deficiency ☺ Peroxisomal Disorders ☺ Disorders in Metal Metabolism ☺ Defects in Copper Metabolism References ☺ Wigglesworth: Textbook of Fetal and Neonatal Pathology ☺ Moore, Persaud: The Developing Human ☺ Perspectives in Pediatric Pathology, Volume 21, Society for Pediatric Pathology ☺ Gilbert-Barness: Potter’s Atlas of Fetal and Infant Pathology ☺ Crowley: An Introduction to Human Disease, Pathology and Pathophysiology Thank you