High-resolution HLA-A*0201 subtyping using directed heteroduplex analysis

HLA-A02* has become an important target for cytotoxic T lymphocyte-based im munotherapy reflecting the high prevalence of this allele in patient popula tions. There are at least 26 different A*02 alleles, and their subtype spec ificity has significant functional implications for T-cell-mediated recogni tion of immunologic targets. We have developed a novel method for HLA-A*02 allelic screening using directed heteroduplex analysis (DHDA). DNA samples from Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (EBV- B) representing 10 different HLA-A*02 alleles (0201, 0202, 0204, 0205, 0206 , 0208, 0210, 0211, 0216, 0217) were prepared. In addition, DNA was prepare d from 81 individuals representing a wide variety of A*02 subtypes previous ly determined by sequence specific primer (SSP) polymerase chain reaction ( PCR) including individuals heterozygous for two A*02 specificities. Probes and samples were generated by PCR amplification using HLA-A*02 specific pri mers encompassing exons 2 and 3, where most of the functionally significant allelic polymorphism is clustered. DHDA was performed by generating hetero duplex molecules composed of a fluorescein-labeled allelic probe sequence a nd an unlabeled allelic PCR product. Gel retardation was consistent for all ele-probe combinations. We were able to identify several A*02 alleles prepa red from EBV-B cell lines that, when used as probes, had very impressive sp ecificity and sensitivity. Combinations of two probes were identified (0205 + 0211 and 0208 + 0211) that allowed differentiation of A*0201 alleles fro m all other A*02 alleles tested. All samples typed by probe combinations ha d DHDA typing and SSP typing confirmed by DNA sequencing. This study expand s the molecular typing repertoire available to the modern HLA laboratory, a nd shows that DHDA has significant promise as a reliable screening method f or HLA A*02 subtyping.