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The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01, B∗45:07, B∗82:02:01 and C∗08:04:01 were notably more frequent in Cape Town compared to other populations reported in the literature. Class II loci had 21 different alleles of DPA1, 46 of DPB1, 27 of DQA1, 26 of DQB1, 41 of DRB1, 5 of DRB3, 4 of DRB4 and 6 of DRB5. The Cape Town cohort exhibited high degrees of HLA diversity and relatively high heterozygosity at most loci. Genetic distances between Cape Town and five other sub-Saharan African populations were also calculated and compared to European Americans.

Aim There is very limited information on the importance of DRB1 exon 1 variations in transplantation. Routinely, clinical laboratories do not test for exon 1 assuming it codes for the leader peptide which is cleaved off of the mature protein, and therefore not clinically important. Currently there are two CWD DRB1 alleles that differ in exon 1, DRB1∗12:01 and DRB1∗12:10, which can only be resolved by sequencing exon 1. We sought to interrogate exon 1 of DRB1 for the presence of the CWD allele, DRB1∗12:10, and to determine the HLA class II haplotypes associated with it in our local population. Methods The Immucor Mia Fora Flex 11 HLA Typing kit was used to genotype eight HSCT recipients and their unrelated donors, as well as seven additional patients. All 23 of these individuals were previously genotyped as DRB1∗12:01 using SSOP. Results All 23 individuals typed as DRB1∗12:01:01. No DRB1∗12:10 was identified in this limited cohort. Two haplotypes were identified for DRB1∗12:01:01: sixteen individuals (76%) were DRB1∗12:01:01, DRB3∗02:02:01, DQA1∗05:05:01, DQB1∗03:01:01, and five individuals (24%) were DRB1∗12:01:01, DRB3∗01:01:02, DQA1∗05:05:01, DQB1∗03:01:01. Two individuals could not have their DRB3∗01:01:01 and DRB3∗02:02:01 haplotypes identified. Conclusions The Immucor Mia Fora Flex 11 HLA Typing kit is unique among several commercial NGS kits in that it contains primers capable of sequencing exon 1 of DRB1. Traditionally, this region has been avoided because of the difficulty in primer design due to the very large 8000–10,000 bp intron 1. The CWD allele, DRB1∗12:10 originally identified in Korean population, differs from DRB1∗12:01 by one bp in exon 1, but since exon 1 is not routinely typed, the individual frequencies of these alleles are not known. Retrospective studies in Asian populations indicated that 12% of DRB1 alleles previously typed as 12:01 were in Fact 12:10. In our study, 100% of our cohort of 23 patients typed as DRB1∗12:01, which was found to be predominantly associated with DRB3∗02:02:01 (76%) and less frequently with DRB3∗01:01:02 (23%). This is in contrast to the reported preferential association of DRB1∗12:01 with DRB3∗01:01:02 (97%), while association with DRB3∗02:02:01 was very rare (2.1%). Retrospective studies may be helpful to determine the importance of exon 1 mismatches in HSCT.

Aim Africans represent the most genetically diverse population in the world, but have not been as well studied with respect to their HLA types compared to the inhabitants of western countries. Thus it was of interest to apply our high-throughput HLA sequencing technology to a cohort of 403 healthy adolescents from Cape Town, South Africa, as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with Stanford University. Methods Cryopreserved PBMC samples from 403 adolescents were collected and shipped to Stanford University from Cape Town. High resolution HLA typing was performed using MIA FORA NGS kit and analysis software. The whole gene coverage included HLA-A, -B, -C, -DPA1, -DQA1, and -DQB1; all exons and introns for -DRB1 and -DRB3/4/5 except partial coverage for exon 6 and intron 1; and all exons and introns between exons 2 and 4 for HLA-DPB1. NGS sequencing libraries were prepared using a semi-automated protocol and sequenced in high-throughput format on the Illumina NextSeq platform. HLA allele candidates were computed and final HLA typing was confirmed using MIA FORA NGS analysis software. Results HLA typing revealed a unique population, including unusual haplotypes, and novel alleles not previously reported in the IMGT/HLA database. The frequency of HLA-A, -B, -DRB1 haplotypes in Cape Town were compared to those represented in the US National Marrow Donor Program. As might be expected, 14 of the top 16 haplotypes in the Cape Town cohort were observed in the African American cohort. However there were important exceptions, including a frequent haplotype found in African American but not observed in Cape Town and two frequent Cape Town haplotypes not observed in African American samples. Conclusion High resolution HLA typing is a powerful method to characterize HLA allele and haplotype diversity in population studies. C. Wang: Consultant; Company/Organization; Immucor. M. Li: Employee; Company/Organization; Immucor. M. Fukushima: Employee; Company/Organization; Immucor. R. Kuehn: Employee; Company/Organization; Immucor. S. Krishnakumar: Employee; Company/Organization; Immucor. M. Mindrinos: Employee; Company/Organization; Immucor.