Sequence-based typing of HLA-B: the B7 cross-reacting group.
The large number of polymorphic sites in the HLA-B locus makes sequencing an efficient way of detecting and analysing them. Most polymorphic sites are located in the alpha1 and alpha2 domains of the molecule, encoded by exons 2 and 3 of the gene. An HLA-B-specific sequence-based typing (SBT) strategy was designed for routine application identifying the polymorphic sites in these domains. Exons 2 and 3 were amplified separately using amplification primers located in intron 1, intron 2 and intron 3. Separate amplification of exons 2 and 3 resulted in short polymerase chain reacting (PCR) products and enabled a solid-phase sequencing approach, which made correct assignment of heterozygous positions possible due to low background. A one-step sequencing reaction was performed using fluorescent dye-labelled sequencing primers. One forward sequencing reaction was performed for exon 2, whereas for exon 3, two forward sequencing reactions were needed using two different sequencing primers located in intron 2 and exon 3. The combined sequences of exon 2 and 3 were used for automatic alignment to an HLA-B sequence database and automatic allele assignment. A total of 355 individuals with at least one allele belonging to the B7 cross-reacting group (B7, 13, 22, 27, 40, 41, 42, 47, 48, 81 and 82) were typed for HLA-B by SBT. In the B7 group 48 different alleles were identified, in the non-B7 group a further 59 alleles were sequenced, 9 new alleles were identified. The sequencing strategy described has proven to be reliable and efficient for high-resolution HLA-B typing.
Voorter
C E
CE
Tissue Typing Laboratory, University Hospital, Maastricht, The Netherlands.
van der Vlies
S A
SA
van den Berg-Loonen
E M
EM
D015235:HLA-B Antigens / Q000235:genetics*; D015793:HLA-B7 Antigen / Q000235:genetics; D006650:Histocompatibility Testing; D006801:Humans; D017422:Sequence Analysis, DNA
D015235:HLA-B Antigens; D015793:HLA-B7 Antigen