HLA-B leader genotypes in a clinical population.

The -21 dimorphism in the leader sequences of HLA-B exon 1 is associated with risk of graft-versus-host disease (GVHD), relapse and overall survival after unrelated donor hematopoietic cell transplantation (HCT), haploidentical HCT and cord blood transplantation. Consideration of the leader dimorphism in the prospective selection of allogeneic donors for HCT may help to lower risks for patients, but requires understanding of the frequencies of the leader in patients and candidate transplant donors. We defined the frequencies of the HLA-B leader, and its association to HLA-B Bw4/Bw6 and C1/C2 KIR epitopes. Sequence variants of rs1050458 of exon 1 position -21 for 11,126 haplotypes were analyzed from high resolution HLA typing of over 5500 study subjects. HLA typing was performed by TruSight/AlloSeq NGS and analyzed using TruSight/AlloSeq Assign software. HLA-B Bw4/Bw6 and C1/C2 KIR epitopes were defined based on established sequence alignments and nomenclature. Alleles at rs1050458 of HLA-B exon 1 were validated as dimorphic: rs1050458-C or -T variants encoding threonine (T) or methionine (M) at anchor position 2 (P2) of nonameric HLA-B leader peptides, respectfully. No additional variants were observed. Among study subjects, 70% of HLA-B haplotypes encoded T-leader and 30% encoded M-leader sequences. The genotype frequencies of TT, MT, and MM were consistent among patient, related, and unrelated donor groups. The associations of M/T leader, Bw4/Bw6, and C1/C2 enhanced understanding of the Class I features involved in the innate immune response. A population of patients and transplant donors confirms the rs1050458 leader dimorphism and its association with HLA-B Bw4/Bw6 and C1/C2 KIR features.

Identification of the rs9277534 HLA-DP expression marker by next generation sequencing for the selection of unrelated donors for hematopoietic cell transplantation.

Mismatching of an unrelated donor against a high-expression HLA-DPB1 recipient allele is associated with a high risk of graft-versus-host disease and mortality. The Seattle Cancer Care Alliance (SCCA) and Fred Hutchinson Cancer Research Center transplant program employs an algorithm to match for HLA-A, B, C, DRB1, DQB1 and DPB1 alleles (12/12) and to avoid, whenever possible, donor mismatching against a recipient high-expression HLA-DPB1 allele. HLA-DPB1 expression is associated with the rs9277534 A/G polymorphism located in the 3'UTR of the HLA-DPB1 gene. Next generation sequencing of HLA-DPB1 using the Illumina TruSight HLA V2 Sequencing Panel and Conexio Assign software analyses provides information on rs9277534 variants without the need for any additional SNP testing. Here we present the molecular location of rs9277534 in NGS data and discuss the challenges to resolve HLA-DPB1 ambiguities.

Next generation sequencing to determine HLA class II genotypes in a cohort of hematopoietic cell transplant patients and donors.

Current high-resolution HLA typing technologies frequently produce ambiguous results that mandate extended testing prior to reporting. Through multiplex sequencing of individual amplicons from many individuals at multiple loci, next generation sequencing (NGS) promises to eliminate heterozygote ambiguities and extend the breadth of genetic data acquired with little additional effort. We report here on assessment of a novel NGS HLA genotyping system for resequencing exons 2 and 3 of DRB1/B3/B4/B5, DQA1 and DQB1 and exon 2 of DPA1 and DPB1 on the MiSeq platform. In a cohort of 2605 hematopoietic cell transplant recipients and donors, NGS achieved 99.6% accuracy for DRB1 allele assignments and 99.5% for DQB1, compared to legacy genotypes generated pretransplant. NGS provided at least single 4-digit allele resolution for 97% of genotypes at DRB1 and 100% at DQB1. Overall, NGS typing identified 166 class II alleles, including 9 novel sequences with greater than 99% accuracy for DRB1 and DQB1 genotypes and elimination of diploid ambiguities through in-phase sequencing demonstrated the robust reliability of the NGS HLA genotyping reagents and analysis software employed in this study.