IDENTIFICATION OF 2 MAJOR HLA-B44 SUBTYPES AND A NOVEL B44 SEQUENCE (B-ASTERISK-4404) - OLIGOTYPING AND SOLID-PHASE SEQUENCING OF POLYMERASE CHAIN-REACTION PRODUCTS

PCR-based analyses were performed for the identification of HLA-B44 subgroups. Genomic DNA from six homozygous cell lines and 44 healthy individuals who had serologically tested positive for HLA-B44 was investigated for polymorphism in exons 2 and 3 of the HLA-B44 genes. Two primers were designed for specific amplification of the B(*)4401 allele in exon 2. None of the tested genomic DNAs, including the cell line ''BAU-J'' from which the sequence of B(*)4401 was derived, was amplified successfully using these primers, indicating that the B(*)4401 sequence may not be correct in position 242-244. For identification of the B(*)4402 and (*)4403 subtypes we specifically amplified the B44 gene in exon 3 using two sequence-specific primers. The PCR products, which were obtained from all B44-positive samples (n = 50) and from none of the B44-negative controls (n = 20), were subsequently hybridized with the dig-ddUTP-labeled oligonucleotides. The base substitution at position 146, as described previously for B(*)4401 and (*)4402 (C for G), could not be confirmed by oligonucleotide hybridization. In contrast, the oligonucleotide typing for G in position 146 gave positive signals in all B44-positive samples. Except for one, HLA-B44-positive DNAs from LCLs and healthy individuals could be divided into two subgroups according to the polymorphic region in position 195-197. Out of 44 unrelated individuals with B44, 27 (61%) were positive for B(*)4402 and 16 (36%) were positive for B(*)4403. Nonradioactive solid phase sequencing of PCR products was performed to analyze B44 genes in exons 2 and 3 in order to resolve the discrepancies with published sequences observed in allele-specific amplification and oligotyping, and to validate the new oligotyping pattern in one individual (BEB). Sequencing confirmed that base differences between B(*)4401 and B(*)4402/(*)4403 in position 242-244 of exon 2 do not exist. The base substitution C for G at position 146 in exon 3 was also not observed by sequencing. Moreover, we report a novel B44 variant (B(*)4404), which has nucleotide substitutions at positions 195-197 and 216-217. These substitutions lead to a change of the deduced amino acid sequence at codons 156 and 163 of the alpha(2) domain.