The document summarizes key differences between the Affymetrix CytoScan HD array and previous arrays for detecting copy number variations and single nucleotide polymorphisms. The CytoScan HD array has higher resolution with 1.9 million copy number probes and 750,000 SNP probes compared to older arrays. It requires only patient DNA, has less noise, and provides improved coverage of genes and the genome. The combination of copy number and SNP data enables detection of mosaicism, uniparental disomy, and consanguinity. Studies show the CytoScan HD array can reliably detect copy number changes and parental relatedness at specific size cut-offs.

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………
……..ATAC………
Allele A
Allele B

5. Copy number + SNP arrays
Non-polymorphic probes
SNP 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. AA
AB
BB
Deletion Normal Duplication Normal
AA
AB
BB
A
AAA
BBB
B
AAB
ABB
B
B
A
A
A
A
B
B
A
B
Normal
Deletion
B
B
A
A
A
A
B
B
B
A
A
A
Duplication
A
B
B
A
B
A
B
B
A
B

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

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

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

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 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%