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Affymetrix CytoScan HD array. 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)

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cytoscan hd vs current array
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 >300 normal individuals and cell lines
    • utilizes phycoerythrin – not ozone sensitive
    • copy number probes (1.9 million) + SNP (750 K)
coverage
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
single nucleotide polymorphisms snps
Single Nucleotide Polymorphisms (SNPs)

……..ATGC………

Allele A

……..ATAC………

Allele B

copy number snp arrays

SNP

Non-polymorphic probes

SNP

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
improvements of cytoscan hd over affy snp 6 0
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 onerestriction digest, ~half the steps, less hands-on time
other potential benefits of cytoscan
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)
slide8
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
copy number snp array
Copy number + SNP array

Copy #

Allele

peaks

slide10

Normal

Deletion

Duplication

A

A

A

A

A

A

B

B

B

A

A

A

B

B

B

B

B

B

A

A

A

B

B

B

A

B

B

B

A

A

A

B

AAA

AA

AA

A

AAB

AB

AB

ABB

B

BB

BB

BBB

Deletion

Normal

Duplication

Normal

cnc detection vs reporting
CNC detection vs. reporting
  • Cytoscan software allows differential flagging in known clinically signficant critical regions vs. “backbone” regions
  • Can potentially detect smaller CNCs but doesn’t mean everything should be reported
  • Ex – LabCorp size cut-offs for reporting in backbone regions
    • Postnatal: >500 Kb gain, >200 Kb loss
    • Prenatal: >2 Mb gain, >1 Mb loss
uniparental disomy
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
slide14

Long continuous stretches of homozygosity

(LCSH) with normal copy number

Small

deletion

SNPs and consanguinity or UPD

Chromosome 2

slide15

Homozygous blocks of 1-3 Mb

AA

BB

Copy number = 2

Normal allele homozygosity

Whole chromosome isodisomy

slide16

13.5 Mb

Copy # = 2

UPD or normal ?

labcorp study papenhausen et al am j med genet 155a 757 68 2011
LabCorp studyPapenhausen et al. Am J Med Genet. 155A:757-68, 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
eight known upd cases
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 >13.5 Mb (two LCSH with total of > 15 Mb)

*LCSH in more than one chromosome = identity by descent

prospectively analyzed 13 000 patients by snp array
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
labcorp study other observations
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
labcorp current cut offs for upd combined hetero isodisomy or segmental upd
LabCorp current cut-offs for UPD (combined hetero/isodisomy or segmental UPD)
  • Single LCSH in one chromosome
    • >20 Mb interstitial or >10 Mb telomeric for non-imprinted chromosomes
    • >15 Mb interstitial or >8 Mb telomeric for known imprinted chromosomes
snp detection of consanguinity
SNP detection of consanguinity

LCSH involving multiple chromosomes (regions of identity by descent)