Intron sequences of HLA-B*73.

Abstract

Molecular typing methods of HLA-B, like sequence-specific oligonucleotide hybridization and sequence-based typing, are based on gene-specific amplifications of exons 2 and 3 followed by probe hybridization or sequence determination. The necessary gene-specific amplification primers are often located in rather conserved regions of the introns. In several of these procedures HLA-B*73 was not amplified, resulting in drop-out of the allele. To investigate the reason for the allelic drop-out, the sequences of introns 1, 2 and 3 of HLA-B*7301 were determined. Comparison of the intron sequence of B*7301 with other HLA-B and HLA-C alleles revealed several remarkable features. The overall sequence resembles the sequence of other HLA-B alleles, although 35 differences were found with a consensus intron sequence. The insertions and deletions shown in intron 2 of B*73 were strikingly similar with the sequences of the HLA-C alleles, as was the 5' end of intron 3. Furthermore, a unique deletion was observed in the middle of intron 3, not noticed in other HLA-B or C alleles. The HLA-B-specific primers, widely used for sequence-specific oligonucleotide hybridization and sequence-based typing purposes, showed mismatches with the B*73 intron sequences, causing the allelic drop-out. Correct amplification of complete exons 2 and 3 of B*7301 was enabled by the design of new primers in intron 2 and 3.