Polymerase-chain-reaction-based analysis of polymorphism in the HLA-B gene

Abstract

The polymerase chain reaction (PCR) in combination with the sequence-specific oligonucleotide probe (SSOP) was applied to analyze the polymorphism in the exon 2 of the HLA-B gene. In this study, genomic DNAs from 85 B-lymphoblastoid cell lines homozygous for HLA and peripheral blood granulocytes of 156 Japanese individuals were investigated. Two HLA-B-specific 5'-sided primers (CG4 and CG5) and two 3'-sided primers (CG2 and CG3) were designed for specific amplification of the exon. HLA-B alleles were classified into two groups (groups I and II) according to specific amplification with two types of the 3'-sided primers. The amplified DNAs were hybridized with 23 nonradioactively labeled SSOPs. Based on the hybridization patterns with the SSOPs, 34 HLA-B specificities were divided into 26 epitope combination (EC) groups. Fifteen HLA-B specificities were classified into four EC groups and these HLA-B specificities could not be distinguished from one another in the same EC group. Another 16 HLA-B specificities corresponded one by one to 16 distinct EC groups, and two subtypes of HLA-Bw75, B27, and Bw48 were also identified enabling the accurate typing of 22 HLA-B alleles at the DNA level. Single-strand conformation polymorphism (SSCP) of the PCR products from group I HLA-B alleles was also investigated. The HLA-B alleles showed distinct electrophoretic patterns in nondenaturing polyacrylamide gels, depending on the nucleotide sequences of the exon 2, indicating that the SSCP analysis may be an alternative, useful and practical HLA-matching system of HLA-B specificity in tissue transplantation.