Allele typing of human TNFA 5 '-flanking region using polymerase chain reaction-preferential homoduplex formation assay (PCR-PHFA): linkage disequilibrium with HLA class I and class II genes in Japanese

Tumor necrosis factor alpha plays a substantial role in a number of conditi ons such as inflammation, autoimmunity, insulin resistance and sleep. Three new single nucleotide polymorphisms, -1,031 T/C, -863 C/A and -857 T/C, we re recently identified in the upstream 5'-flanking region of TNFA in the Ja panese population. In the present study, we developed polymerase chain reac tion (PCR)-preferential homoduplex formation assay for the single-step alle le typing of TNFA, and determined the genotypes of nl healthy unrelated Jap anese individuals. Four haplotypes, -1,031/-863/-857 TCC, TCT, CAC and CCC, were found to constitute the majority, if not all, of the TNFA alleles of healthy Japanese population These alleles were designated as TNFA-U01, -U02 , -U03 and -U04, respectively, in the order of frequency. Based on HLA-A, - B and -DRB1 genotypes together with TNFA genotypes, multi-locus haplotypes were analyzed. Significant positive associations were observed between TNFA -U01 and A*3303, B*5201, B*4403, B*4601, B*0702, DRB1*1502, DRB1*0101, DRB1 *1302, between TNFA-U02 and B*5401, B*3501, DRB1*0405, DRB1*0407, between T NFA-U03 and B*4006, B*4002, DRB1*0803, DRB1*0802, DRB1*0403, DRB1*0901, and between TNFA-U04 and B*4801. Four-locus haplotype estimation revealed that A*3303-B*4403-TNFA-U01-DRB1*1302, A*2402-B*5201-TNFA-U01-DRB1*1502 and A*2 402-B*5401-TNFA-U02-DRB1*0405 constitute major extended haplotypes in Japan ese. Interestingly, TNFA alleles previously shown to have a higher promoter activity (U02, U03) were found to form haplotypes with certain DRB1 allele s associated with T helper 1 (Th1)-dominant diseases such as rheumatoid art hritis, insulin dependent diabetes mellitus and Crohn's disease in Japanese . In contrast, TNFA allele with a low promoter activity (U01) is in linkage disequilibrium with the DRB1 alleles associated with T helper 2 (Th2)-domi nant diseases such as atopic dermatitis and ulcerative colitis. These obser vations raise the possibility that TNFA upstream promoter region polymorphi sms contribute to some of the HLA-disease associations.