Sequence-based typing of the HLA-A10/A19 group and confirmation of a pseudogene coamplified with A*3401.

The strategy for sequencing human leukocyte antigen (HLA)-A was based on separate amplification of exons 2 and 3, followed by forward and reverse heterozygous sequencing of the alleles. Validation of the method was obtained by sequencing 11 individuals carrying alleles from all different HLA-A allele groups, except *43. All alleles could be correctly identified except A*3401. Unexpected polymorphic positions were identified in exon 3, even in individuals homozygous for A*3401. In addition, the pseudogene HLA-COQ or HLA-DEL linked to A*3401 was coamplified and sequenced. The problem was solved by using different amplification primers for exon 3 with mismatches for the two pseudogenes. A total of 252 unrelated individuals with at least one allele belonging to the A10 or A19 group were typed for HLA-A by this strategy. Ten different alleles were identified in the A10 group and 14 in the A19 group. As second allele a further 30 different subtypes from all different groups were sequenced. In 21 individuals, sequencing exon 1 was necessary to distinguish A*7401 from A*7402. The sequencing strategy, with separate amplification of the exons, has proven to be a robust method, resulting in reliable and efficient high-resolution HLA-A typing.