DNA typing of the HLA-A gene: Population study and identification of four new alleles in Japanese

With the use of polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe (SSOP), we established a DNA typing method of the HLA-A locus. A pair of primers to amplify the highly polymorphic region of HLA-A gene including exon 2 and exon 3 was designed and the amplified DNAs were hybridized with 91 types of P-32 labeled SSOPs. This method allowed discrimination of all known HLA-A alleles except for two combinations, A*0201 or A*0209 and A*0207 or A*0215N, which have identical sequences in exon 2 and exon 3. Another pair of primers was designed for amplification of exon 4 and the PCR products were hybridized with 5 SSOPs to distinguish A*0201 and A*0207 from A*0209 and A*0215N, respectively. In this study, 81 B-lymphoblastoid cell lines (BLCL) homozygous for HLA and 553 unrelated healthy Japanese individuals were determined for their HLA-A genotypes. Based on the genotyping results, frequency of HLA-A alleles and linkage disequilibrium between HLA-A and HLA-B in the Japanese population were investigated. In addition, four new HLA-A alleles were identified and their nucleotide sequences in exon 2 and exon 3 were determined to confirm the typing results.