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. 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

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 one restriction 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. Deletion Duplication Normal Deletion Normal Duplication AAA AA A AABBB
Deletion
Normal
Duplication A
AAA
BBB B
AAB
ABB

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

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

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

17. 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

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 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 Mb
25.0%
2nd Degree
Half Siblings/Uncle-Niece/Aunt-Nephew/Double First Cousins
1/8 Mb Mb
12.5%
3rd Degree
First Cousins/Half Uncle-Niece/Half Aunt-Nephew
1/16
178.5 Mb 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%