Abstract The class III region of the human major histo- compatibility complex (MHC) contains approximately 59 genes, many of which encode polypeptides with a variety of different functions. Eight of these genes are of particu- lar interest because they encode novel surface molecules that could be involved in immune and/or inflammatory responses and are excellent candidates as disease suscep- tibility loci. These molecules are members of two differ- ent superfamilies, the immunoglobulin superfamily (1C7, G6B, and G6F genes) and the leucocyte antigen-6 super- family (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 liq- uid chromatography (DHPLC) in 24 unrelated healthy in- dividuals. 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 polymor- phic 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. Keywords MHC · Autoimmunity · Receptors · SNP Introduction The most abundant type of DNA sequence variation in the human genome consists of single-nucleotide poly- morphisms (SNPs). An SNP is estimated to be present once every 500–1000 bp when any two chromosomes are compared (Cooper et al. 1985; Kruglyak 1997; Landegren 1996). SNPs have gained popularity in re- cent years and are now being touted as the genetic mark- ers of choice for the study of complex genetic traits (Chakravarti 1998, 1999; Collins et al. 1997; Kruglyak 1997; Lander 1996; Risck and Merikangas 1996). How- ever, some concerns have been raised about how many SNPs would be needed to track down susceptibility genes. Some studies have pointed out the necessity of maps of 500,000 SNPs with an average spacing of 6 kb across the genome (Kruglyak 1999). International efforts such as the SNP Consortium (TSC) (International SNP Map Working Group 2001; Marshall 1999) have created a public library of 1.42 million SNPs, though most of these require validation. Because of their expected num- ber, some SNPs will be located in regulatory and coding regions of genes directly affecting gene expression lev- els, or the structure and function of the encoded proteins. These may, therefore, represent candidate alterations for genetic mechanisms in disease. A wide range of diseases has been linked to the MHC, which is located on human Chromosome 6. Although many of these appear to be due to allelic differences in class I and class II antigens, there is evidence to suggest that additional loci within the central class III region may contribute to disease susceptibility as well. Recent studies using microsatellite markers (Hsieh et al. 1997) G. Ribas · R.D. Campbell ( ✉ ) MRC UK HGMP Resource Centre, Genome Campus, Hinxton, Cambridge CB10 1SB, UK e-mail: rcampbel@hgmp.mrc.ac.uk Tel.: +44-1223-494511, Fax: +44-1223-494512 M.J. Neville MRC Immunochemistry Unit, Department of Biochemistry, University Oxford, Oxford, OX1 3QU, UK Immunogenetics (2001) 53:369–381 DOI 10.1007/s002510100343 ORIGINAL PAPER Gloria Ribas · Matthew J. Neville R. Duncan Campbell 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 Received: 26 April 2001 / Revised: 28 May 2001 / Published online: 10 July 2001 © Springer-Verlag 2001