Polymerase chain reaction analysis of the Xba I polymorphism of the human complement C4 genes provides evidence for strong haplotype conservation.

The genes coding for the two isotypes of the fourth component of human complement, C4A and C4B, are located between the HLA-B and -DR loci of the MHC. We studied the linkage relationship of the previously described XbaI RFLP to obtain further insight into the evolution of the tandemly arranged C4 genes. Using exon-specific PCR amplification followed by restriction analysis and direct DNA sequencing, the polymorphic site could be located in exon 40 of the C4 gene (cDNA position 5095). The polymorphism does not change an amino acid residue. Using nested PCR amplification with isotype-specific primers to amplify either C4A or C4B alleles the haplotype arrangement of the XbaI sites in both isotypic C4 genes was analyzed independently. It was observed that the XbaI restriction site was either present or absent in both C4 genes of a given haplotype. In a study of 106 Caucasian haplotypes, only two different haplotypes could be identified carrying a C4A gene with and a C4B gene without the XbaI restriction site. Also, the XbaI site could only be detected in long C4 genes possessing the 6.5-kb insertion in intron 9. Our findings provide evidence that the mutation creating the XbaI polymorphism occurred in an ancestral C4 gene already carrying the long intron 9. The duplicating resulting in the presence of two isotypic genes, C4A and C4B, must have taken place subsequently giving rise to haplotypes with or without the XbaI site.