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Developmental Pathology INDIAN DENTAL ACADEMY Leader in continuing dental education

Cytogenetics • Examination of chromosomes under the microscope • Necessary to induce cells to undergo mitosis in order to see individual chromosomes • Molecular cytogenetic techniques (e.g., FISH) can be performed on interphase cells (cells that are not actively dividing)

Reasons to do a cytogenetic study • Diagnose constitutional disorders – I.e., disorders present at birth -- classic example is trisomy 21 (Down syndrome) – Typically involve more than one cell line

• Add further diagnostic or prognostic information to a diagnosis of an acquired disorder – I.e., diseases that are NOT constitutional -- these are most commonly malignancies – Typically involve only the cell line or tissue involved by the malignancy 48-hour culture; overnight exposure to colcemid

72-hour culture; several hours’ colcemid; ethidium bromide added

Basic Terminology • Constitutional Chromosomal Abnormalities • Acquired Chromosomal Abnormalities • Numerical abnormalities • Structural abnormalities – Balanced – Unbalanced

Karyotype -- Blood

Karyotype -- Marrow

FISH diagram (scanned)

Fluorescence in s itu hybridization (FISH) • Specimen is collected as previously described for each tissue type • Indications for FISH include: – Microdeletions (e.g., Prader-Willi, Angelman and DiGeorge syndromes) – Cryptic translocations (e.g., t(12;21)) – Cancer translocations (e.g., BCR-abl, PML-RARA) and rearrangements (e.g., MLL) – Enumeration of chromosomes or detection of translocations or rearrangements in interphase nuclei

Clinical History • Approximately 32 weeks’ gestation • Abnormalities detected on ultrasound: – Abnormal head shape – Frontal bossing – Clenched fists

Newest method -- array-based Comparative Genomic Hybridization • DNA is extracted from patient • DNA from patient and sex-matched control are labeled in different colors • Labeled DNA is hybridized to a chip (microarray) on which are oligonucleotides spaced across the genome (density or spacing of oligos depends on platform) • Results in ratio of patient to control at these loci

Array CGH • Used to detect abnormalities too small to be seen under the microscope (each G-band can contain hundreds of genes) • Can detect only unbalanced rearrangements (e.g., deletions, duplications) • Balanced rearrangements (e.g., inversions, insertions) will NOT be detected by array-CGH

Specimen to draw for a cytogenetic study? • SODIUM heparin tube • Recommend at least 1 ml (3 if poss)

Trisomy (one extra chromosome) • Typically arises from a nondisjunction error in either meiosis I, meiosis II or mitosis (if due to amitotic error, the trisomy is mosaic) • Most autosomal trisomies arise from maternal nondisjunction errors – Strong correlation between increasing maternal age and risk for nondisjunction – Advanced Maternal Age (AMA) is the most common reason for referral for a prenatal chromosome study – 95% of trisomy 21 is due to maternal nondisjunction errors

Major “viable” trisomies • Chromosome 13 (obsolete name, Patau synd) • Chromosome 18 (obsolete name, Edward synd) • Chromosome 21 (Down synd) • All other chromosomes have been reported in trisomic state; unless mosaic, virtually uniformly fatal in utero or shortly after birth

Trisomy 21 (1 in 800 live births; incidence greater if mat. age >35) • • • • • •

Hypotonia Short neck with loose skin at nape Flat nasal bridge Brushfield spots around edge of iris Epicanthal folds Short, broad hands with single transverse palmar crease • Congenital heart disease • Mental retardation • Increased for leukemia Thompson & Thompson, Genetics in Medicine, 7th ed, p. 91

Trisomy 13 (1 in 15-25000 live births) • Growth retardation • Severe central CNS malformations (e.g., holoprosencephaly) • Microcephaly • Cleft lip, cleft palate • Polydactyly • Congenital heart, renal and genitourinary malfomations Thompson & Thompson, Genetics in Medicine, 7th ed, p. 95

Trisomy 18 (1 in 7500 live births) • Severe cardiac malformations • Low-set, malformed ears • Characteristic clenched fist (2nd and 5th digits overlap) • Rocker-bottom feet • Mental retardation • Increased maternal age is a risk factor

Thompson & Thompson, Genetics in Medicine, 7th ed, p. 94

Sex chromosome numerical abnormalities • Male

– Klinefelter (47,XXY): 1/1000 males – 47,XYY: 1/1000 males – 46,XX males: 1/20,000 males

• Female – – – –

Turner (45,X): 1/5000 females Trisomy X (47,XXX): 1/1000 females 46,XY females: 1/20,000 females Androgen insensitivity (testicular feminization): 1/20,000 females

Klinefelter syndrome • Tall, thin body habitus; long legs • Signs of hypogonadism at puberty – Small testes, underdeveloped secondary sex characteristics

• May have gynecomastia • Almost always infertile • May be mosaic for a normal (or other abnormal) cell line

Thompson & Thompson, Genetics in Medicine, 7th ed, p. 106

Turner syndrome • Approx. 99% of 45,X conceptions die in utero; livebirth approx 1/4000 females • Approx. 50% cases 45,X; remainder are mosaic for another cell line, either 46,XX or with a structurally abnormal X (e.g., isochromosome Xq)

Turner syndrome • • • •

Short stature Broad chest with widely spaced nipples Gonadal dysgenesis (e.g., streak gonads) Webbed neck (from lymphedema during fetal life) • Lymphedema of dorsum of feet • Low posterior hairline • Renal and cardiovascular abnls, incl. coarctation of aorta Thompson & Thompson, Genetics in Medicine, 7th ed, p. 108

Microdeletion/microduplication syndromes • Very small (sometimes visible by Gbanding, sometimes not) deletions • Result from unequal crossing over between homologous regions on chromosomes during meiosis • Typically confirmed by FISH Thompson & Thompson, Genetics in Medicine, 7th ed, p. 97

Microdeletion syndromes

Thompson & Thompson, Genetics in Medicine, 7th ed, p. 96

DiGeorge/velocardiofacial syndromes • Characteristic craniofacial features • Varying degrees of mental retardation may be a feature • Conotruncal heart defects (e.g., tetralogy of Fallot, pulmonary atresia, absent pulmonary valve) • Over 30 different genes in this region, so phenotype dependent on size of deletion

Prader-Willi/Angelman syndromes • Both due to a deletion within the proximal long arm of a chromosome 15 • Manifestations depend on which chromosome 15 is deleted: the 15 that came from the patient’s mother or the 15 that came from the patient’s father: – IMPRINTING – Need specialized molecular studies to determine which homolog is deleted Cassidy SB et al, 2000, Am J Med Genet 97:136

Prader-Willi • • • •

Severe hypotonia in infancy Hypogonadism Feeding difficulties in infancy Over time, feeding difficulties resolve and hyperphagia ensues --> extreme foodseeking behavior • Obesity • Mild mental retardation, learning difficulties, behavioral issues

Angelman • Severe mental retardation and developmental delay • Jerky, ataxic gait (“puppet”-like) together with characteristic arm position • Paroxysms of inappropriate laughter • Virtually absent speech

Marfan syndrome • Autosomal dominant connective tissue disorder due to mutations in fibrillin 1 (FBN1) gene • FBN1 encodes an extracellular matrix glycoprotein • Wide-ranging systemic effects: – Skeletal, ocular, pulmonary, skin

• Clinical diagnosis; heterogeneity of gene makes identification of causative gene extremely difficult

Marfan • • • • • • • • •

Tall stature, arachnodactyly Pectus excavatum Joint laxity Narrow palate Ectopia lentis Mitral valve prolapse Aortic dilatation, dissection Pulmonary blebs, pneumothorax Striae

Cystic Fibrosis • Autosomal recessive: patients have mutations in both CFTR (CF transmembrane conductance regulator gene) alleles • Predominantly dz of northern Europeans, with carrier rate approx 1 in 29 (incidence of dz approx 1/2500) • Lungs and exocrine pancreas primarily affected • Increased sweat chloride concentrations

CF Clinical Features • Pulmonary findings

– Very thick secretions, recurrent infections, COPD and bronchiectasis

• Pancreatic findings

– Decreased secretion of pancreatic enzymes such as trypsin and lipase (pts can take supplements)

• Other features: meconium ileus in 10-20% newborns with CF • CBAVD: Congenital bilat absence of vas deferens (some pts with absent to very mild features of CF may present with infertility)

Fragile X syndrome • X-linked mental retardation syndrome due to unstable CGG repeats in promoter region of FMR1 gene on very distal long arm of X chromosome • Prevalence 16-25/100,000 in gen pop; most common cause of inherited mental retardation

Fragile X • Due to expansion of repetitive sequences (similar disorders: Huntington, myotonic dystrophy, various ataxias) • CGG repeat in 3’ untranslated region of FMR1 gene: – – – –

5-40 repeats: normal 41-58 repeats: gray zone 59-200 repeats: premutation >200 repeats: full mutation

• This expansion occurs during maternal meiosis (so mothers of Fragile X pts have premutations); risk of expansion to full mutation increases with size of premutation

Fragile X clinical features • Both males and females can manifest features (usually more pronounced in males as no other copy of normal X) • Moderate mental retardation (usu. milder in females) • Hyperactivity, hand flapping or biting, temper tantrums • Post-pubertal males: long face, prominent jaw and forehead, large ears, large testes (FMR1 is normally expressed in testes) From:

Duchenne Muscular Dystrophy • X-linked progressive myopathy due to mutations or deletions within the DMD gene • Incidence: approx 1/3500 male births • DMD encodes dystrophin, expressed in muscle (smooth, cardiac and skeletal) • Mutations lead to partially functional or nonexpressed dystrophin (severity of disease based in part on expression status of dystrophin)

DMD clinical features • Progressive muscle degeneration and weakness • Begins with hip girdle and neck flexors, begins to spread distally • Usually manifests by age 5 (Gowers maneuver) and have calf pseudo-hypertrophy • Cardiac findings present in approx 95% pts; chronic heart failure in 50% • Confined to wheelchair by age 12 or so • Median age at death is 18 years


References • Nussbaum RL, McInnes RR, Willard HF. Tho m p s o n & Tho m p s o n Ge ne tic s in Me dic ine (7th e d). Elsevier Saunders, 2007. – Excellent in-depth introduction to clinical genetics.

• Jones KL. Sm ith’s Re c o g niz ab le Patte rns o f Hum an Malfo rm atio n. Elsevier Saunders, 2006.

– Outstanding guide, with many pictures and differential diagnoses, of many genetic syndromes and abnormalities. Lists of syndromes associated with various clinical findings (e.g., dental and maxillofacial abnormalities).


– Very well-written and updated reviews under the section, GeneReviews.

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