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

Abstract

The class III region of the human major histocompatibility complex (MHC) contains approximately 59 genes, many of which encode polypeptides with a variety of different functions. Eight of these genes are of particular interest because they encode novel surface molecules that could be involved in immune and/or inflammatory responses and are excellent candidates as disease susceptibility loci. These molecules are members of two different superfamilies, the immunoglobulin superfamily (1C7, G6B, and G6F genes) and the leucocyte antigen-6 superfamily (G6C, G6D, G6E, G5C, and G5B genes). Some level of variation was found when overlapping genomic DNAs from different haplotypes were compared. The present work describes a systematic search for single-nucleotide polymorphisms (SNPs) in these genes using direct sequencing and 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 θ=4.61×10^–4. The diversity in noncoding regions is 1 SNP/560 bp, but a higher frequency was detected in coding regions with 1 SNP/423 bp corresponding to θ=5.33×10^–4. Of the coding SNPs, 63.6% caused amino acid substitutions. The power of this study is emphasized by the fact that of the 37 SNPs/indels detected, only 6 can be found in the SNP database at the NCBI.