Using denaturing HPLC for SNP discovery and genotyping, and establishing the linkage disequilibrium pattern for the all-trans-retinol dehydrogenase (RDH8) gene

Abstract

All-trans-retinol dehydrogenase (RDH8) is a visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol. As part of an on-going effort to map genes involved in complex eye diseases, myopia in particular, using association studies, single nucleotide polymorphisms (SNPs) were identified and linkage disequilibrium (LD) pattern was established within and around the RDH8 gene. We used denaturing high-performance liquid chromatography (DHPLC) to screen SNPs in four DNA pools each consisting of DNA from five individuals and genotyped the identified SNPs in 150 Chinese subjects from Hong Kong. Fifteen SNPs were identified: seven were common with the minor allele frequency > 0.05 and ten were novel. Common SNPs were included in LD and haplotype analysis using the ASSOCIATE and EH programmes. Four SNPs in the 3′ region exhibited significant LD and formed a haplotype block, while three common SNPs in the 5′ region did not exhibit useful LD. The LD pattern around the RDH8 gene suggested that one SNP from the 3′region and two to three SNPs from the 5′ region were needed in association studies involving RDH8. Our results demonstrated the efficiency of DHPLC in screening SNPs when coupled with DNA pooling strategy and in genotyping SNPs.