Single-nucleotide polymorphism detection by denaturing high-performance liquid chromatography and direct sequencing in genes in the MHC class III region encoding novel cell surface molecules

The class III region of the human major histocompatibility complex (MHC) co ntains approximately 59 genes, many of which encode polypeptides with a var iety of different functions. Eight of these genes are of particular interes t because they encode novel surface molecules that could be involved in imm une and/or inflammatory responses and are excellent candidates as disease s usceptibility loci. These molecules are members of two different superfamil ies, the immunoglobulin superfamily (1C7, G6B, and G6F genes) and the leuco cyte antigen-6 superfamily (G6C, G6D, G6E, G5C, and G5B genes). Some level of variation was found when overlapping genomic DNAs from different haploty pes were compared. The present work describes a systematic search for singl e-nucleotide polymorphisms (SNPs) in these genes using direct sequencing an d denaturing high-performance liquid chromatography (DHPLC) in 24 unrelated healthy individuals. We validated the DHPLC methodology by first studying the 1C7 gene. This gene was directly sequenced in all 24 samples, and DHPLC was found to resolve all the polymorphic sites present in the heterozygote samples tested. We screened the rest of the genes by DHPLC only, and only those chromatograms that revealed a polymorphic profile were sequenced. We detected one SNP every 489 bp in the 18 kb of DNA studied, corresponding to theta =4.61x10 (4). The diversity in noncoding regions is 1 SNP/560 bp, bu t a higher frequency was detected in coding regions with 1 SNP/423 bp corre sponding to theta =5.33x10 (4). Of the coding SNPs, 63.6% caused amino acid substitutions. The power of this study is emphasized by the fact that of t he 37 SNPs/indels detected, only 6 can be found in the SNP database at the NCBI.