Developmental Pathology

1
Developmental Pathology

• Thursday, 10-1-07
• Michelle Dolan, M.D.
• dolan009_at_umn.edu

2
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)

3
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

4
48-hour culture overnight exposure to colcemid
5
72-hour culture several hours
colcemid ethidium bromide added
6
(No Transcript)
7
(No Transcript)
8
Basic Terminology

• Constitutional Chromosomal Abnormalities
• Acquired Chromosomal Abnormalities
• Numerical abnormalities
• Structural abnormalities
• Balanced
• Unbalanced

9
(No Transcript)
10
Karyotype -- Blood
11
Karyotype -- Marrow
12
FISH diagram (scanned)
13
Fluorescence in situ 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(1221))
• 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

14
Clinical History

• Approximately 32 weeks gestation
• Abnormalities detected on ultrasound
• Abnormal head shape
• Frontal bossing
• Clenched fists

15
(No Transcript)
16
(No Transcript)
17
(No Transcript)
18
(No Transcript)
19
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

20
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

21
(No Transcript)
22
(No Transcript)
23
Specimen to draw for a cytogenetic study?

• SODIUM heparin tube
• Recommend at least 1 ml (3 if poss)

24
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

25
(No Transcript)
26
(No Transcript)
27
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

28
Trisomy 21 (1 in 800 live births incidence
greater if mat. age gt35)

• 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 risk for leukemia

29
Thompson Thompson, Genetics in Medicine, 7th
ed, p. 91
30
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

31
Thompson Thompson, Genetics in Medicine, 7th
ed, p. 95
32
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

33
Thompson Thompson, Genetics in Medicine, 7th
ed, p. 94
34
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

35
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

36
Thompson Thompson, Genetics in Medicine, 7th
ed, p. 106
37
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)

38
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

39
Thompson Thompson, Genetics in Medicine, 7th
ed, p. 108
40
Microdeletion/microduplication syndromes

• Very small (sometimes visible by G-banding,
  sometimes not) deletions
• Result from unequal crossing over between
  homologous regions on chromosomes during meiosis
• Typically confirmed by FISH

41
Thompson Thompson, Genetics in Medicine, 7th
ed, p. 97
42
(No Transcript)
43
Microdeletion syndromes
Thompson Thompson, Genetics in Medicine, 7th
ed, p. 96
44
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

45
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 patients
  mother or the 15 that came from the patients
  father
• IMPRINTING
• Need specialized molecular studies to determine
  which homolog is deleted

46
(No Transcript)
47
(No Transcript)
48
Cassidy SB et al, 2000, Am J Med Genet 97136
49
Prader-Willi

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

50
Angelman

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

51
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

52
Marfan

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

53
(No Transcript)
54
(No Transcript)
55
(No Transcript)
56
(No Transcript)
57
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

58
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)

59
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

60
(No Transcript)
61
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
• gt200 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

62
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)

63
From geneticsmodules.duhs.duke.edu
64
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)

65
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

66
From medgen.genetics.utah.edu
67
References

• Nussbaum RL, McInnes RR, Willard HF. Thompson
  Thompson Genetics in Medicine (7th ed). Elsevier
  Saunders, 2007.
• Excellent in-depth introduction to clinical
  genetics.
• Jones KL. Smiths Recognizable Patterns of Human
  Malformation. 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).
• www.genetests.org
• Very well-written and updated reviews under the
  section, GeneReviews.