HLA‐B locus DNA typing: Detection of B*7801 and seven additional alleles by BW6‐specific exon 2 amplification

Abstract

A molecular approach to type a new HLA-B5 antigen, HLA-BSNA, characterized by its unusual association with the public determinant BW6, referred to as B*7801, has been designed. Antigens disclosing serological identity with SNA, BX1 and Te76 were also investigated. Based upon HLA-B exon 2 group-specific PCR, the following procedure was established: 5' and 3' primers were designed by targetting the codons 11-12 (AM) and 81-83 (LRG), respectively, in exon 2 (alpha 1 domain). The 5' primer discriminates with HLA-A, -C genes and pseudogenes, while the 3' primer detects the sequence encoding the BW6 epitope (NLRG) and discriminates it from the BW4 epitope. The combination of this pair of primers specifically amplifies 26 HLA-B alleles. Oligotyping for B*7801 was performed using a combination of two non-radioactively labeled SSO probes identifying positions T45 and D74 in exon 2. To resolve ambiguous hybridization patterns, an additional set of probes was used. The specificity of this BW6-group-specific amplification procedure was investigated on 150 genomic DNA samples. Among them, we obtained 94 amplified DNA products which were tested with eight SSOs. Beside B*7801, the following B alleles could be defined: B*0801, B*35, B*5401, B*5501-2, B*5601-2, B*1501-7 (including B62, B75 and B72) and B*4601. SNA, BX1 and Te76 DNA samples gave similar hybridization pattern providing a clue to the identity of these antigens. This "one PCR and two probes" procedure represents a simple oligotyping strategy which can also be applied to type many other HLA-B specificities.