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) 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 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) ……..ATGC……… Allele A ……..ATAC……… Allele B

Copy number + SNP arrays Non-polymorphic probes SNP 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 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

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)

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 # Allele peaks

Deletion Duplication Normal Deletion Normal Duplication AAA AA A AAB BB Deletion Normal Duplication A AAA BBB B AAB ABB

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

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 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

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

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

UPD or normal ? Copy # = 2 13.5 Mb

LabCorp study Papenhausen 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

Distribution of LCSH in 120 consecutive patients

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 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 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) 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 LCSH involving multiple chromosomes (regions of identity by descent)

LabCorp cut-offs for consanguinity LCSH > 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 >30 Mb 1.56%