Association of four HLA class III region genomic markers with HLA haplotypes

We have studied restriction fragment length polymorphism (RFLP) in the region 300 kb centromeric to the HLA-B locus. Four probes were used: one was genomic DNA derived from the tumor-necrosis factor (TNF)-beta gene, one was a cDNA for the BAT3 gene, and two single-copy genomic probes, R5A and M20A. The order of these markers from HLA-B towards the centromere is M20A, R5A, TNF and BAT3. The BAT3 and TNF-beta probes each detected two allelic bands with Taq I and Nco I digestion, respectively; the R5A and M20A probes each detected three polymorphic allelic bands with BstEII digestion. To determine if these restriction polymorphisms are preferentially associated with certain HLA-B and -DR haplotypes, a total of 153 HLA haplotypes was analyzed. The haplotypes A1, B8, DR3 and A3, B7, DR2 were each associated with a distinct combination of polymorphisms identified at these four sites, thereby demonstrating that the strong linkage disequilibrium characteristic of these haplotypes extends also to this segment of the class III region. In contrast, haplotypes that are not in positive linkage disequilibrium, such as A1,B8,DR4 and A2,B7,DR3, showed no preferential association with any of these polymorphisms. The antigens HLA-B27 and B35 were also found to be in positive linkage disequilibrium with RFLP patterns at three of these sites, and HLA-B14,B35,B44,Bw57 and Bw62 were found preferentially associated with polymorphisms at one or two of these sites, independent of the DR antigen present. These data further demonstrate that genetic linkage disequilibrium in the HLA class III region is complex and variable among different HLA haplotypes.