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………

……..ATAC………

Allele A

Allele B

Copy number + SNP arrays

Non-polymorphic probesSNP 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 #

Allelepeaks

AAABBB

Deletion Normal Duplication Normal

AAABBB

AAAA

BBBB

AABABB

BB

AA

A

ABBAB

NormalDeletion

BB

AA

AAB

BB

AA

A

DuplicationABBAB

ABBAB

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

Chromosome 2

Small deletion

Long continuous stretches of homozygosity (LCSH) with normal copy number

SNPs and consanguinity or UPD

Normal allele homozygosity

Whole chromosome isodisomy

Homozygous blocks of 1-3 Mb

AA

BB

Copy number = 2

Copy # = 2

13.5 Mb

UPD or normal ?

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

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 consanguinityLCSH > 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%