CytogeneticsBehavioral Genetics II

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Cytogenetics/Behavioral Genetics II

• September 19,2008
• Betsy Hirsch,Ph.D.
• hirsc003_at_umn.edu

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Fragile X-Associated Tremor/Ataxia Syndrome
(FXTAS)

• Affects approximately 30 of premutation male
  carriers
• Adult onset progressive tremor and ataxia
• Attributed to over-expression of FMR1 mRNA (5
  fold to 8 fold) rather than to the decreased
  levels of FMRP

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Female Carriers of Premutation

• Overexpression of mRNA
• Approximately 20 of female premutation carriers
  have premature ovarian failure (lt age 40 yrs)

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Supplemental References

• Garber et al, 2008, Fragile X syndrome, European
  Journal of Human Genetics 16666-672
• Koukoui SD and Chaudhuri A, 2007,
  Neuroanatomical, molecular genetic, and
  behavioral correlates of Fragile X syndrome,
  Brain Research Reviews 5327-38

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Microdeletion/Microduplication Syndromes

• Typically involve loss or gain of a portion of
  one G-band, generally involving approximately 2-3
  Mb of DNA
• Can be very difficult to detect even by high
  resolution G-banding
• Most frequently diagnosed and characterized by
  FISH

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Fluorescence-in-situ-hybridization(FISH)

• Technique in which a DNA probe containing one or
  more genes (or a small chromosomal region) is
  labeled with a fluorescent dye, and then
  hybridized to interphase or metaphase cells
• Use to detect small regions of deletions or
  duplications
• Used to examine specific regions of the genome in
  non-dividing tissues
• Used to map genes

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18 FISH
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Microdeletion Deletion Syndromes

• Williams syndrome (7q11.2)
• Prader-Willi / Angelman syndrome (15q11.2)
• Smith Magenis syndrome (17p11.2)
• DiGeorge/Velocardiofacial syndrome (22q11.2)

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P-W comp 15
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P-W FISH
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Prader-Willi / Angelman Syndromes

• In 70 of cases of PWS and AS, apparently
  identical deletions of 15q11.2 are detected
• Deletion encompasses 3.5 megabases of DNA
• 10 genes have been identified within this
  deleted region

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Prader-Willi Syndrome

• Deletions are always of paternal origin
• Putative PWS genes are expressed exclusively on
  the paternally derived homolog
• Example of genomic imprinting

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Prader Willi Syndrome

• As more than one gene within the deleted region
  shows such a paternal expression pattern, PWS may
  be a contiguous gene disorder

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Deletions may result in clinical syndromes
because

• Single putative gene is contained within the
  deleted region
• or
• Several contiguous genes, each contributing some
  aspect of the phenotype, are contained within the
  deleted region

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Angelman Syndrome

• Deletions are always of maternal origin
• A single putative AS gene, UBE3A, has been
  identified, which is expressed in brain tissue
  exclusively from the maternally derived homolog
  (biparental expression of UBE3A has been shown in
  other tissues)

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Paternal
Maternal
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Paternal
Maternal
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Imprinting must be a reversable processMust be
able to be reset when passed from
father-gtdaughter-gtgrandchild ormother -gtson-gt
grandchild
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Uniparental Disomy in PWS

• In 28 of PWS cases, the syndrome is caused not
  by a deletion, but by maternal uniparental disomy
  for chromosome 15

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Uniparental Disomy

• Both chromosomes of a given chromosome pair are
  inherited from ONE parent
• Most likely generated by an initial
  non-disjunction event leading to trisomy,
  followed by loss of one of the trisomic
  chromosomes and restoration of a disomic state
  trisomic rescue

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Genotype/Phenotype Correlations

• Microdeletions typically associated with
  specific constellation of clinical findings,
• e.g. Williams syndrome, DiGeorge Syndrome,
  Prader Willi syndrome
• Microduplications less likely to be
  associated with specific clinical syndrome
  however, have been associated with developmental
  disabilities, autism spectrum disorders

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a-CGH

• Methodology for scanning the genome for regions
  of gain or loss
• (duplications, deletions, trisomy, monosomy)

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Array-CGH
Control Genomic DNA
Patient Genomic DNA
Restrict Quantify, Label with different
fluorochromes
Combine in 11 Ratio
Hybridize to chip onto which DNA probes have
been spotted
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SCAN and ANALYZE
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Our 44K chip
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Raw
Ratio equals 0 no copy number gains or losses lt
0.3 Loss (Deletion) gt 0.3 Gain (Duplication)
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10 oligos within a 1 Mb region
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Start point 15,443,578 Stop point
17,021,468 Size 1,577,890 bp
CESK1 XKR3 GAB4 IL17RA CECR6 CECR5 CECR1 SLC25A18
ATP6V1E1 BCL2L13 BID PEX26 TUBA8 USP18
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Copy Number Variants

• In 2004, another level of variation in the human
  genome was demonstrated
• Differences in the number of copies of a given
  sequence
• (versus base pair composition of the sequence)
• Iafrate et al (Nature Genetics) and Sebat
  et al (Science)

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CNV

• DNA segment 1Kb or larger
• Has a variable copy number compared with a
  reference genome
• Current Estimates
• 5000 CNV loci encompassing approximately 6 Mb
  of the genome (range 5 24 Mb)
• http//projects.tcag.ca/variation
• http//paralogy.gs.washington.edu/structuralvariat
  ion

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Not all copy number gains or losses are
clinically significant
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10 oligos within a 1 Mb region
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Well documented CNV in healthy control populations
(17/95 controls had loss)
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ADM-2
22q13.33 loss Start point 49427512 Stop point
49525130 Size 98 Kb Genes SHANK3 (14 kb in
size) ACR
SHANK3
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New York Times, December 28,2007