SELECTION OF UNRELATED BONE-MARROW DONORS BY PCR-SSP TYPING AND SUBSEQUENT NONRADIOACTIVE SEQUENCE-BASED TYPING FOR HLA DRB1/3/4/5, DQB1, AND DPB1 ALLELES

HLA incompatibility between bone marrow recipients and unrelated donors is one of the main obstacles in bone marrow transplantation. HLA class I and generic class II DR and DQ typing is generally performed by serology. Precise subtyping of HLA class II genes, however, can only be achieved by molecular genetic methods. Hen, the final selection of serologically pretyped unrelated bone marrow donors by confirmatory PCR-SSP (PCR-sequence-specific primers) typing and subsequent nucleic acid sequence analysis of the second exon of DRB1, DRB3, DRB4, DRB5, DQB1, and DPB1 alleles is presented. Serologically identical potential marrow donors and their corresponding recipients were analyzed for HLA-DRB identity by PCR-SSP analysis. After solid-phase single-strand separation, direct sequencing of the allele- or group-specific DRB amplified products was performed by applying fluorophor-labelled sequencing primers. Electrophoretically separated sequencing products were detected by means of an automated DNA sequencer. Group-specific amplification and sequencing of DQB1 alleles was carried out for all potential bone marrow donors and recipients, while only the final donor-recipient pair was analyzed for DPB1 alleles. Thus, the presented amplification strategy in combination with direct sequencing of PCR products allows matching of bone marrow transplant pairs with the highest degree of reliability for the assessment of HLA class II identity.