Affymetrix CytoScan HD array

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Affymetrix CytoScan HD array
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CytoScan HD vs current array

• Current array (CGH based)
• patient reference DNA required (two color)
• utilizes Cy dyes ozone sensitive
• copy number probes only (135 K)
• CytoScan HD array (not CGH based)
• patient DNA only (single color)
• in silico reference based on gt300 normal
  individuals and cell lines
• utilizes phycoerythrin not ozone sensitive
• copy number probes (1.9 million) SNP (750 K)

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Coverage

• Average marker spacing
• ISCA genes 384 bp
• OMIM genes 659 bp
• X chromosome OMIM genes 486 bp
• RefSeq genes 880 bp
• Intergenic backbone 1737 bp

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Single Nucleotide Polymorphisms (SNPs)
..ATGC
Allele A
..ATAC
Allele B
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Copy number SNP arrays

• SNPs limited to specific locations in genome
  SNP
• only arrays biased due to positional
  restrictions
• Non-polymorphic (copy number) probes fill gaps to
  
• allow broad coverage

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Improvements of CytoScan HD over Affy SNP 6.0

• Improved software
• Much less noise
• Probes empirically chosen based on performance
• 20 million probes screened
• All reagents centrally manufactured and provided
  as kits
• Streamlined procedure only one restriction
  digest, half the steps, less hands-on time

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Other potential benefits of CytoScan

• Affy filing for FDA clearance
• CytoScan currently has best coverage on single
  array for both constitutional and neoplastic
  cases
• Other large clinical labs switching to CytoScan
  (LabCorp, ARUP)

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• Copy number SNP arrays - detect copy number
  changes and allele frequencies
• SNPs can detect uniparental isodisomy,
  consanguinity
• more sensitive for detection of mosaicism
• independent confirmation of copy number findings
  and better breakpoint determination

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Copy number SNP array
Copy
Allele peaks
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(No Transcript)
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SNP arrays more sensitive for detection of
mosaicism
Non-mosaic deletion
Mosaic deletion
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CNC detection vs. reporting

• Cytoscan software allows differential flagging in
  known clinically signficant critical regions vs.
  backbone regions
• Can potentially detect smaller CNCs but doesnt
  mean everything should be reported
• Ex LabCorp size cut-offs for reporting in
  backbone regions
• Postnatal gt500 Kb gain, gt200 Kb loss
• Prenatal gt2 Mb gain, gt1 Mb loss

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Uniparental disomy

• Inheritance of two homologous chromosomes from
  one parent
• isodisomy two copies of the same homolog
• heterodisomy two different homologs
• UPD mechanisms
• meiotic non-disjunction with trisomy or monosomy
  rescue
• post-zygotic mitotic recombination
• Whole chromosome isodisomy vs. hetero/isodisomy

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SNPs and consanguinity or UPD
Chromosome 2
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Normal allele homozygosity
Whole chromosome isodisomy
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UPD or normal ?
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LabCorp studyPapenhausen et al. Am J Med Genet.
155A757-68, 2011

• Homozygosity profiling by SNP array is screen for
  UPD
• What LCSH size should be used as cut-off for
  recommending parental f/u for UPD?
• Determined distribution of LCSH in patient
  population
• Retrospectively analyzed eight confirmed UPD
  cases for LCSH

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Distribution of LCSH in 120 consecutive patients
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Eight known UPD cases

• Two whole chromosome homozygosity
• Six mixture of hetero/isodisomy
• Single LCSH range 13.5 48.4 Mb
• One case with two LCSH of 11 and 11.2 Mb
• Set LCSH UPD cut-off at gt13.5 Mb (two LCSH with
  total of gt 15 Mb)
• LCSH in more than one chromosome identity by
  descent

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Prospectively analyzed 13,000 patients by SNP
array

• 92 patients with UPD qualifying LCSH based on
  cut-offs
• Parental f/u on 46 cases (mostly imprinted
  chromosomes)
• Confirmed UPD in 29 cases
• 14/30 whole chromosome isoUPD
• 13/30 mixture of hetero/isoUPD
• False-positive UPD 17 cases
• Chromosome 3 and 11 pericentromeric region, 13q21

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LabCorp Study other observations

• False-positive cases had shorter average LCSH,
  greater freq near cen, no telomeric LCSH
• No false-positive cases with qualifying telomeric
  LCSH
• Sometimes see evidence of copy mosaicism in
  trisomy/monosomy rescue allele freq mosaicism in
  segmental UPD
• Low likehood of false-negatives

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LabCorp current cut-offs for UPD (combined
hetero/isodisomy or segmental UPD)

• Single LCSH in one chromosome
• gt20 Mb interstitial or gt10 Mb telomeric for
  non-imprinted chromosomes
• gt15 Mb interstitial or gt8 Mb telomeric for known
  imprinted chromosomes

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SNP detection of consanguinity
LCSH involving multiple chromosomes (regions of
identity by descent)
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LabCorp cut-offs for consanguinityLCSH gt 10 Mb
Degree of Relationship Relationship Coefficient of Inbreeding Theoretical level of LCSH (based on total of 2850 Mb minus X and Y) Empiric Level of LCSH Theoretical Percent LCSH
1ST Degree Siblings/Parent-Child 1/4 712.5 Mb 550-950 Mb 25.0
2nd Degree Half Siblings/Uncle-Niece/Aunt-Nephew/Double First Cousins 1/8 356.25 Mb 250-635 Mb 12.5
3rd Degree First Cousins/Half Uncle-Niece/Half Aunt-Nephew 1/16 178.5 Mb 100-300 Mb 6.25
4th Degree First Cousins Once Removed/Double Second Cousins 1/32 89.0 Mb 30-75 Mb 3.12
5th Degree Second Cousins 1/64 44.5 Mb gt30 Mb 1.56