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Judith Lechner, Durga P. Dash, Kathryn P. Burdon, Jamie E. Craig, Aine Rice, Chris F. Inglehearn, Anne E. Hughes, Colin E. Willoughby; SNP Microarrays And DNA Pooling (SNP-MaP) In Sporadic Keratoconus. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1078.
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The evidence that keratoconus (KTCN) is linked to multiple chromosomal regions is consistent with an oligogenic model. Genome-wide association studies (GWAS) are a better strategy to determine the genetic basis of KTCN. One approach to address the cost, time and labour that are involved in large-scale genotyping is to carry out analyses not on individual DNA samples, but pools made up of DNA from many individuals: a method called SNP Microarrays and DNA Pooling (SNP-MaP).
DNA from Caucasian keratoconus patients and controls was accurately quantified and pools were constructed using equimolar amounts of DNA from each patient or control. A diagnosis of keratoconus was made on the basis of clinical examination, a history of penetrating keratoplasty for keratoconus and corneal topography. Pooled DNA was allelotyped on the Affymetrix SNP 6.0 Array. GPFrontend and GPGraphics software tools as well as the GenePool algorithm were used to rank all 906,600 array SNPs according to differences of relative allele signal (RAS) between cases and controls (ΔRasMean) and assess SNP clustering. Initially, genomic areas of known linkage to KTCN and genes associated with KTCN were analysed. Secondary analysis considered the biological significance of identified SNPs associated with KTCN.
Pooled DNA from 90 Caucasian patients with severe keratoconus and 90 disease-free population controls was allelotyped on the Affymetrix SNP 6.0 Array. Data analysis with the GenePool algorithm visualised with GPGraphics detected a high ranking cluster of significant SNPs on chromosome 21q22. To validate high ranking SNPs from the pooled array data, SNPs were then genotyped in each sample individually as well as in an independent cohort of Caucasian cases and controls. Several SNPs are currently undergoing further testing to validate and replicate the findings. The identified gene on chr21q22 is a plausible biologically relevant candidate for KTCN.
The pooling strategy has been validated in a number of previous studies and is effective in detecting loci of moderate to strong effect in complex diseases. Preliminary data suggests that SNP-MaP is a cost effective strategy to identify genes associated with KTCN. Understanding the genetic causes of keratoconus may lead to both predictive testing and novel therapies.
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