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.
Swelsen
Wendy T N
WT
Tissue Typing Laboratory, University Hospital Maastricht, Maastricht, The Netherlands.
Voorter
Christina E M
CE
van den Berg-Loonen
Ella M
EM
D000483:Alleles; D005091:Exons / Q000235:genetics; D014644:Genetic Variation; D015234:HLA-A Antigens / Q000235:genetics*; D006579:Heterozygote; D006650:Histocompatibility Testing / Q000379:methods; D006720:Homozygote; D006801:Humans; D007438:Introns / Q000235:genetics; D016133:Polymerase Chain Reaction; D011544:Pseudogenes / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods
D015234:HLA-A Antigens; C505994:HLA-A*34:01 antigen; C039682:HLA-A10 antigen; C043205:HLA-A19 antigen