High-throughput genetic association study on candidate genes for nonsyndromic oral clefts.

摘要

With recent advances in high-throughput genotyping technology, new strategies for large scale screens of many candidate genes become feasible. However, large-scale genotyping studies may be hampered by limited amounts of genomic DNA, so an efficient and accurate method of genome amplification is desirable. Here, two topics are presented with the ultimate goal of identifying genes for nonsyndromic oral clefts. (1) The genotyping success rate and reliability of 2 MDA variants and OmniPlexRTM technology with and without 7-deaza-dGTP was determined using whole blood, buccal swabs, and dried blood spots on two SNP BeadArray panels (1,260 and 1,228 SNPs in each panel), (Illumina Inc). Both success rate and reliability were highest from DNA amplified by GenomiPhi(TM) (Amersham) and DNA extracted from whole blood. Amplification of DNA from dried blood spots was less efficient and less reliable than DNA from buccal swabs, perhaps due to its low molecular weight. (2) A set of 274 single nucleotide polymorphism (SNP) markers in 64 candidate genes was genotyped using the BeadArray approach in 58 case-parent trios from Maryland, and data were analyzed using single marker and haplotype transmission disequilibrium test (TDT). To assess whether these genes were expressed in human craniofacial structures, data from the COGENE consortium and other public databases were analyzed. Thirteen candidate genes showed significant evidence of linkage in the presence of linkage disequilibrium (P0.05), and ten of these were expressed in relevant embryonic tissues: SP100, MLPH, HDAC4, LEF1, C6orf105, CD44, ALX4, ZNF202, CRHR1, and MAPT. Three candidate genes showed statistical evidence only: ADH1C, SCN3B, and IMP5.; In conclusion, application of MDA to maximize DNA resources from whole blood and buccal swabs will be useful in future genetic studies. However, DNA isolated from dried blood spots or degraded DNA seems inappropriate for MDA. Multiple genes may be involved in the etiology of nonsyndromic oral clefts and the novel genes identified here warrant future study. This approach demonstrates how statistical evidence on large numbers of SNP markers typed in case-parent trios can be combined with comprehensive expression data in a new strategy for identifying candidate genes for complex disorders.