HLA‐DRB1 DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors

Abstract

The HLA-DRB1 sequencing based typing strategies reported to date require separate amplifications of each sample with a series of group-specific primers followed by sequencing of any resulting polymerase chain reaction (PCR) products. Whilst this results in high resolution typing in the majority of cases, a number of unnecessary amplifications are performed. We report here a novel approach where amplification of the second exon of HLA-DRB1 is performed in a single tube for all alleles. Retrospective analysis of 642 consecutive Western Australian unrelated bone marrow registry donors has shown that this approach results in unambiguous typings in 71.1% of cases. Ambiguities can be readily resolved if necessary with a single additional sequencing reaction on the original PCR product.

Improved outcome in HLA-identical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by KIR and HLA genotypes

“…43 When needed for unambiguous determination of HLA identity between donor and recipient, additional HLA allele-level typing was performed with direct, sequenced-based typing. 44 KIR genotyping was performed according to methods previously described. 26 …”

Contributions of vitamin D response elements and HLA promoters to multiple sclerosis risk

“…High-resolution 4-digit HLA-DRB1 genotyping was performed in the Department of Clinical Immunology, PathWest, Royal Perth Hospital by DNA sequencing for both MS cases and control samples, using a previously reported method. 16 Automated sequencing was carried out on the ABI Prism 3730 and 3730xl Genetic Analyzers and HLA-DRB1 analysis was carried out using ASSIGN V4.0.1.36 (Conexio Genomics). All HLA typing results for MS samples were resolved to the 4-digit level using heterozygous ambiguity resolving primers (HARPs) where applicable.…”

Allergic women show reduced T helper type 1 alloresponses to fetal human leucocyte antigen mismatch during pregnancy

“…Extracted DNA from mothers and infants was sent to the Department of Clinical Immunology, Royal Perth Hospital, Perth, Western Australia. HLA-DRb1 and HLA-C typing was performed by DNA sequencing using protocols established previously at the laboratory [11].…”