High‐resolution HLA typing for the DRB3/4/5 genes by sequence‐based typing

Abstract

The high degree of polymorphism of the HLA genes at the nucleotide sequence level has proven sequence-based typing a major typing strategy. For DRB1 the allelic variability is predominantly present in the second exon and by DNA sequencing of exon 2 all hitherto known DRB1 alleles can be detected. For the associated genes DRB3, DRB4 and DRB5 the situation is slightly different. Allelic differences are not limited to exon 2 and the sequence of exon 3 and sometimes exon 4 is needed for complete subtyping. Oligonucleotides to amplify the exons needed for subtyping of DRB3, DRB4 and DRB5 were designed. Gene-specific products were generated to make simultaneous detection of alleles in heterozygous combinations possible. In this way 238 individuals were fully typed for their DRB3, 4 and 5 subtypes. Additional samples were typed for only one of the genes. All samples had been previously typed by PCR-SSP. Concordant typing results were obtained for all individuals tested. The DRB3 alleles typed for included *0101, *0201, *0202 and *0301, for DRB4 they were *01011, *0102 and *0103 and for DRB5 *0101, *0102, *0103, *0105, *0201, *0202 and *0203. All alleles were easily detected by the protocol described except for DRB5*0201. Sequencing of exon 3 and 4 of the DRB5*0201 allele showed this allele to be a sequencing error and the sequences obtained were identical to the exon 2, 3 and 4 sequences of DRB5*0202. Two new alleles were identified in the samples studied, DRB4*0105 and DRB3*0207. Sequence based typing has been recognized as a valuable tool for HLA typing of DRB1, DQB1 and DPB1 since several years. It is shown to be a superior typing method as well in the detection of the different DRB3, 4 and 5 subtypes.

Severe Pulmonary Sarcoidosis Is Strongly Associated With the Haplotype HLA-DQB1*0602–DRB1*150101

“…Sequencing was accomplished by the Autoread sequencing kit (Amersham Pharmacia Biotech) as previously described [22][23][24][25]. Sequence data were processed automatically and evaluated manually.…”

Helper and cytotoxic T cell precursor frequencies are not predictive for development of acute graft-versus-host disease after partially T cell-depleted HLA-identical sibling BMT

“…For sequencing a solid phase approach was used as described previously using the Autoread Sequencing kit from Pharmacia Biotech (Uppsala, Sweden). 19,20 Typing was performed using Pharmacia Typing Software.…”

“…Subsequently, to determine whether HLA-DPB mismatches were responsible for the induction of aGVHD, HLA-DPB was analysed by a sequence-based typing method. 19,20 Furthermore, HTLp-f and CTLp-f were determined to investigate whether the presence of alloreactive T cells before transplantation was predictive for development of aGVHD after BMT. Our data show that neither the functional assays (HTLp-f/CTLp-f/MLC) nor detailed HLA typing is predictive for the development of aGVHD in recipients of a partially T cell-depleted HLA-identical sibling graft.…”

Sequence-Based Typing of the HLA-A10/A19 Group and Confirmation of a Pseudogene Coamplified With A*3401

“…The presence of PCR products with the correct base length was checked by agarose gel electrophoresis. A solid phase sequencing approach was used as previously described [28, [32][33][34]. The nonbiotinylated DNA strand was removed by alkaline denaturation after attachment of 40 l of biotinylated product to streptavidin-coated beads (Dynal, ITK diagnostics, Uithoorn, The Netherlands).…”