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

Hum Immunol. 2005 May;66(5):535-42. doi: 10.1016/j.humimm.2005.01.005. Epub 2005 Feb 12.

Abstract

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.

MeSH terms

  • Alleles
  • Exons / genetics
  • Genetic Variation
  • HLA-A Antigens / genetics*
  • Heterozygote
  • Histocompatibility Testing / methods
  • Homozygote
  • Humans
  • Introns / genetics
  • Polymerase Chain Reaction
  • Pseudogenes / genetics*
  • Sequence Analysis, DNA / methods

Substances

  • HLA-A Antigens
  • HLA-A*34:01 antigen
  • HLA-A10 antigen
  • HLA-A19 antigen