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Chromosomal Microarrays
Shashikant Kulkarni, M.S (Medicine)., Ph.D., FACMGDirector of Cytogenomics and Molecular PathologyAssociate Professor of Pathology and Immunology
Associate Professor of Pediatrics and Genetics
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Classical Cytogenetics
~5 MbGlobal survey of
genomeDetects genomic
imbalances
FISH
Needs a priori information
Cytogenomics~ 50 Kb
Global survey of genome
Detects genomic imbalances
Cytogenomic Diagnostic Tools
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Comparative Genomic Hybridization (CGH) Chromosomal
Normal Metaphases
Patient DNA
Control DNA
Gain
Loss
Resolution< 10Mb
Kallioniemi et al., Science 1992, 258:818-821
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• Detects gains or losses of small chromosomal regions by DNA differences
• The patient’s DNA is compared to a normal control in human metaphase chromosome spreads or arrays
Comparative genomic hybridization (CGH)
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Comparative Genomic Hybridization Arrays (aCGH)
Patient DNA
Control DNA
Gain
Loss
Resolution=clone size
Genomic Clones
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Single Nucleotide Polymorphism Arrays (SNP-A)
Patient DNA
In silicoReference data(hapmap)
Gain
Loss
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Various PlatformsBAC arrays Oligo arrays SNP+whole genome
BAC libraries make it easily customizable
Oligos are shorter 25-mer to 80-mer
Similar to Oligo array, but incorporate Bi-allelic probes
Tiling can make resolution better, but only to ~46Kb
Able to determine breakpoints more accurately
Able to determine breakpoints more accuratelyCan detect copy number changes and copy neutral changes (UPD, LOH)
BAC mis-localization Cross hybridization
“normal control”Interpretation can be difficult
With “correct reference data” noise can be reducedInterpretation can be difficult
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Evolution of Array Designs0 +1-1
WMS
tel
tel
cencen
0 +1-1
>3 Mb gaps
0 +1-1 0 +1-1
Targeted Targeted + 850 BAC or oligo array
Targeted + 1 Mb or 500 kb
Targeted + Whole Genome
Chr 7
0.5 -1 Mb gaps
Slide courtesy of Madhuri Hegde
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• Copy Number Variations (CNV)– DNA segment, longer than 1kb with a
variable copy number compared to reference genome
– Described both in disease and “normal” states
• Copy Number Alterations (CNA) preferred
Interpretation challenges
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• ~5-25% of human reference genome is copy number variable
• 1447 CNVs catalogued in 270 individuals (360 million bases, 12% of the genome) Redon et al Nature 2006
• Each person has ~1500 common CNVs (inherited), average size of ~20 kb- 30 million bases of CNVs/person
• CNVs- interpretation challenges
Copy Number Variants (CNV)
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CNA Interpretation-four major criteria
Pathogenic (~ 10-15%)
Variants of Uncertain Significance (VOUS)
Variant of Likely Pathologic Significance (VLPS)
Benign (~ 80%)
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Clinical Utility: Better Diagnostic YieldPo
sitiv
e C
ases
0%
5%
10%
15%
20%
ID/DD ASD MCA
KaryotypeCMA
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• CNA that overlap critical regions of known pathogenic regions
• Parental and family studies• Databases are very useful• Nature of CNA important
– Content, size, nature
Which genomic imbalances are clinically significant?
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Copy number change detected
Known pathogenic aberration?
Pathogenic if confirmed with an independent methodReported in healthy individuals?
Likely to be a benign variant
Inherited from parents
Likely to be a benign variant
Probably pathogenic gene content
Basic Algorithm for Clinical Chromosomal Microarray testing
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Gaps in standards and Opportunities in CMA testing
• Incomplete and Inaccurate databases (benign and pathogenic)
• Lack of Uniformity (various platforms with different degrees of resolution, thresholds)
• Urgent need for education• Lack of reference material