Transcript Affymetrix CytoScan HD array CytoScan HD vs current array • Current array (CGH based) – patient + reference DNA required (two color) –

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
SNP
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
A
A
B
B
A
Duplication
A
A
B
B
A
A
B
A
B
AA
AB
BB
Deletion Normal
A
B
B
A
B
AAA
AAB
ABB
BBB
A
B
B
A
B
Duplication
Normal
A
B
B
A
B
A
A
B
B
A
AA
AB
BB
Normal
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 ?
13.5 Mb
Copy # = 2
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/ParentChild
1/4
712.5 Mb
550-950 Mb
25.0%
2nd Degree
Half
Siblings/UncleNiece/AuntNephew/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%