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Affymetrix CytoScan HD array

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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Affymetrix CytoScan HD array

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  1. Affymetrix CytoScan HD array

  2. 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)

  3. 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

  4. Single Nucleotide Polymorphisms (SNPs) ……..ATGC……… Allele A ……..ATAC……… Allele B

  5. 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

  6. 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

  7. 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)

  8. 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

  9. Copy number + SNP array Copy # Allele peaks

  10. 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

  11. SNP arrays more sensitive for detection of mosaicism Non-mosaic deletion Mosaic deletion

  12. 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

  13. 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

  14. Long continuous stretches of homozygosity (LCSH) with normal copy number Small deletion SNPs and consanguinity or UPD Chromosome 2

  15. Homozygous blocks of 1-3 Mb AA BB Copy number = 2 Normal allele homozygosity Whole chromosome isodisomy

  16. 13.5 Mb Copy # = 2 UPD or normal ?

  17. 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

  18. Distribution of LCSH in 120 consecutive patients

  19. 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

  20. 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

  21. 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

  22. 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

  23. SNP detection of consanguinity LCSH involving multiple chromosomes (regions of identity by descent)

  24. LabCorp cut-offs for consanguinityLCSH > 10 Mb

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