Co-amplification and sequencing of C4 genes to identify block specific SNPs

The MHC consists of several large segments or blocks of DNA within which recombination is rare. Within these blocks are replicated DNA fragments containing highly polymorphic genes. The C4 genes (C4A and C4B) are located within the same block within the central or Class III region of the MHC. The expressed C4A and C4B genes are highly polymorphic. Haplotypes also differ in their gene copy number with most containing either 1, 2 or 3 C4 genes. Either the C4A or the C4B genes can be replicated. The C4 region has been implicated in susceptibility to SLE and cannot be excluded from susceptibility to other MHC associated disorders but has largely been ignored for extensive study due to the difficulty in characterising the C4 genes at the molecular level. This difficulty is largely due to the high sequence similarity between the 2 genes (C4A3 from the A3, B7, S31, DR15 haplotype and the C4B1 from the A1, B8, SQ01, DR3 are 99.75% similar in the coding regions). The high sequence similarity and the presence of replicated genes virtually prohibit gene/locus specific amplification. Therefore to characterise the C4 region at the molecular level we have adopted a unique approach where all C4 genes are amplified and sequenced simultaneously. Automated DNA sequencing facilitates quantitative DNA sequencing. Thus polymorphisms are identified that are block rather than gene specific. The C4A*3 and C4B*1 sequences and other partial C4 sequences were aligned to identify variable regions. Exons 9, 12, 17, 21 and the complete region between exons 24 and 28 were selected and amplified in 5 PCRs. To date we have sequenced these regions in 8 haplotypes from DNA of the 4AOH panel. Of the 8 haplotypes the A1, B8, SQ01, DR3 haplotype was included. Fifteen polymorphic regions have been identified to date. Each of the 8 DNA contain unique sequence arrangements spanning the 15 polymorphic sites. This included three haplotypes containing the serological defined C4A3 and C4B1 specificities (the A3, B7, S31, DR15, the A30, B13, S31, DR7 and the A29, B44, F31, DR7 haplotypes). Thus approach has demonstrated that serologically defined complement specificities can be further split at the molecular level. This has implications in determining the role of the complement region in MHC associated disease.