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  • Dup 15

Cyto2.ppt Cyto2.ppt Presentation Transcript

  • Cytogenetics/Behavioral Genetics II September 19,2008 Betsy Hirsch,Ph.D . [email_address]
  • 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
  • Female Carriers of Premutation
    • Overexpression of mRNA
    • Approximately 20% of female premutation carriers have premature ovarian failure (< age 40 yrs)
  • Supplemental References:
    • Garber et al, 2008, Fragile X syndrome, European Journal of Human Genetics 16:666-672
    • Koukoui SD and Chaudhuri A, 2007, Neuroanatomical, molecular genetic, and behavioral correlates of Fragile X syndrome, Brain Research Reviews 53:27-38
  • 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
  • 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
  • +18 FISH
  • Microdeletion Deletion Syndromes
    • Williams syndrome (7q11.2)
    • Prader-Willi / Angelman syndrome (15q11.2)
    • Smith Magenis syndrome (17p11.2)
    • DiGeorge/Velocardiofacial syndrome (22q11.2)
  • P-W comp 15
  • P-W FISH
  • 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
  • Prader-Willi Syndrome
    • Deletions are always of paternal origin
    • Putative PWS genes are expressed exclusively on the paternally derived homolog
    • Example of genomic imprinting
  • 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
  • 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
  • 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)
  • Paternal Maternal
  • Paternal Maternal
  • Imprinting must be a reversable process Must be able to be reset when passed from father->daughter->grandchild or mother ->son-> grandchild
  • 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
  • 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 ”
  • 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
  • a-CGH
    • Methodology for scanning the genome for regions of gain or loss:
    • (duplications, deletions, trisomy, monosomy)
  • Array-CGH Patient Genomic DNA Control Genomic DNA Restrict & Quantify, Label with different fluorochromes Hybridize to “chip” onto which DNA probes have been “spotted” Combine in 1:1 Ratio
  • Our 44K chip
  • Raw Ratio equals 0 : no copy number gains or losses < 0.3 : Loss (Deletion) > 0.3 : Gain (Duplication)
  • 10 oligos within a 1 Mb region
  • 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
  • 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)
  • 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)
  • Not all copy number gains or losses are clinically significant
  • 10 oligos within a 1 Mb region
  • Well documented CNV in healthy control populations (17/95 controls had loss)
  • 22q13.33 loss: Start point: 49427512 Stop point: 49525130 Size: 98 Kb Genes: SHANK3 (14 kb in size) ACR ADM-2 SHANK3
  • New York Times, December 28,2007