Molecular analysis of HLA-DRB1*08/12 alleles: identification of two additional alleles

Abstract

A nonradioactive oligotyping method that takes advantage of selective amplification using the polymerase chain reaction (PCR) and oligonucleotide probe hybridization was developed to distinguish all reported HLA-DRB1*08/12 alleles. Selective amplification was achieved using a primer complementary to the sequence encoding YSTGECY at positions 10-16 in the first hyperpolymorphic region (HPMR). This selective amplification of the HLA-DRB1*08/12 subset of alleles provides a refinement in HLA oligotyping that permits unambiguous oligotyping of many heterozygotes that cannot be resolved using less selective amplification alternatives. The amplified DNA was hybridized with a panel of then digoxigenin-labeled probes to resolve oligotypes that correspond to all reported HLA-DRB1*08/12 alleles. Oligotyping of HLA-DRB1*08/12 samples revealed two previously unknown HLA-DRB alleles. One allele, DRB1*0805, differs from DRB1*0801 by a leucine to alanine substitution at position 74. This allele is of particular interest because it is very similar to HLA-DRB1*08 alleles (YSTGECY and lack of an associated HLA-DRB3 gene), but it lacks leucine at position 74, which is characteristic of all previously reported DRB1*08 alleles. The second HLA-DRB1*08 allele, DRB1*0804, differs from DRB1*0802 by a glycine to valine substitution at position 86.