Identification of two major HLA-B44 subtypes and a novel B44 sequence (B∗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 digddUTP-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 α2 domain.