Widespread non‐coding polymorphism in HLA class II genes of International HLA and Immunogenetics Workshop cell lines

Abstract

As the primary genetic determinant of immune recognition of self and non‐self, the hyperpolymorphic HLA genes play key roles in disease association and transplantation. The large, variably sized HLA class II genes have historically been less well characterized than the shorter HLA class I genes. Here, we have used Pacific Biosciences Single Molecule Real‐Time (SMRT®) DNA sequencing to perform four‐field resolution HLA typing of HLA‐DRB1/3/4/5, ‐DQA1, ‐DQB1, ‐DPA1 and ‐DPB1 from a panel of 181 B‐lymphoblastoid cell lines from the International HLA and Immunogenetics Workshops. By interrogating all exons, introns, and the untranslated regions of these important reference cells, we have improved their HLA typing resolution on the IPD‐IMGT/HLA database. We observed widespread non‐coding polymorphism, with over twice as many unique genomic sequences identified compared with coding sequences (CDS). We submitted 263 unique sequences to the IPD‐IMGT/HLA Database, often from multiple cell lines, including 114 confirmations of existing alleles, of which 30 were also extensions to full‐length genomic sequences where only CDS was available previously. A total of 149 novel alleles were identified, largely differing from their closest reference allele sequences by a single nucleotide polymorphism (SNP). However, some highly divergent alleles were deemed to be recombinants, only detectable by full‐length sequencing with long, phased reads. The fourth‐field variation we observed allowed fine mapping of linkage disequilibrium patterns and haplotypes to particular ancestries. This study has highlighted the under‐appreciated non‐coding diversity in HLA class II genes, with potential implications for population genetic and clinical studies.

Fifty novel HLA‐DPB1 alleles identified in a UK cohort of unrelated hematopoietic cell donors and recipients

“…The samples used in this study are part of a larger cohort of patient/donor pairs transplanted between 1996 and 2011, with written consent obtained from all participants. Briefly, using PacBio Single Molecule Real‐Time (SMRT) sequencing and adapted full‐gene typing methodology previously described, 17,18 1470 samples, comprising 744 patients and 726 donors, were genotyped for HLA‐DPB1. Overall, 2940 full‐length HLA‐DPB1 sequences were generated, of which 107 sequences (4%) were found to contain novel variation, comprising a total of 50 unique alleles.…”

“…The samples used in this study are part of a larger cohort of patient/donor pairs transplanted between 1996 and 2011, with written consent obtained from all participants. Briefly, using Pac-Bio Single Molecule Real-Time (SMRT) sequencing and adapted full-gene typing methodology previously described, 17,18…”

Using cloud infrastructure to facilitate data collection and conversion of HLA diagnostic data for the 18th International HLA and Immunogenetics Workshop

“…8,9 HLA patterns are also collected across population groups, which sheds light on allele 10 and haplotype 11 frequencies, and variations in individuals from world populations. 12 Studies have also defined and cataloged HLA based on expression levels, 13,14 non-coding or intronic polymorphism, 15,16 as well as observed immunological compatibility. 17 In addition to characterization of HLA antigens and sequence, studies on immunization events such as transplantations, transfusions or pregnancies, produce longitudinal data on the development of anti-HLA antibodies.…”

“…HLA patterns are also collected across population groups, which sheds light on allele 10 and haplotype 11 frequencies, and variations in individuals from world populations 12 . Studies have also defined and cataloged HLA based on expression levels, 13,14 non‐coding or intronic polymorphism, 15,16 as well as observed immunological compatibility 17 …”

A pan-MHC reference graph with 246 fully contiguous phased sequences

“…A comprehensive workflow for MHC genomic analysis would entail calling all alleles of all genes, phasing all alleles, discovering novel alleles if present, and identifying CNV in C4A and C4B , all at high enough throughput to handle large numbers of samples 24 . It should also identify non-coding variants, which play a major role in HLA gene regulation 14,25 . HLA genes, for instance, are generally genotyped by commercially available kits that do not always include all genes or achieve maximum resolution 26 .…”