Next generation sequencing characterizes HLA diversity in a registry population from the Netherlands

Abstract

Next generation DNA sequencing is used to determine the HLA‐A, ‐B, ‐C, ‐DRB1, ‐DRB3/4/5, and ‐DQB1 assignments of 1009 unrelated volunteers for the unrelated donor registry in The Netherlands. The analysis characterizes all HLA exons and introns for class I alleles; at least exons 2 to 3 for HLA‐DRB1; and exons 2 to 6 for HLA‐DQB1. Of the distinct alleles present, there are 229 class I and 71 class II; 36 of these alleles are novel. The majority (approximately 98%) of the cumulative allele frequency at each locus is contributed by alleles that appear three or more times. Alleles encoding protein variation outside of the antigen recognition domains are 0.6% of the class I assignments and 5.3% of the class II assignments.

Common, intermediate and well‐documented HLA alleles in world populations: CIWD version 3.0.0

“…In this case, when the non‐ARD exons are being evaluated, the frequency of G group alleles A*02:179 and A*02:324 compared with that of A*02:05:01:01 can be predicted. As in the case for most G groups, the primary allele is observed most frequently ( A*02:05:01:01 appears 53 times in AFA) and secondary alleles at lower frequency ( A*02:179 appears 16 times in this group and A*02:324 is not observed). A*02:16:01G ( A*02:16 , A*02:131 ) is another example of where the comparison can be made because there are more reports of a secondary allele A*02:131 (n = 125) than the primary allele giving the G group its name ( A*02:16 , n = 2) in API.…”

“…The typing assignments submitted (Supporting Information Tables S8‐S16) illustrate the diversity of HLA assignments that arise from typing volunteer donors over time (2012‐2018) and reflect the DNA sequence‐based typing strategy, including resolution requirements, used by each registry. A good example of this is A*02:01:01:01 which is very common when individuals are typed at a consistent four‐field resolution . In this multiple registry dataset, A*02:01:01:01 was reported only 1294 times while A*02:01:01G was reported over 3.7 million times.…”

A new HLA‐C allele with an alternative splice site in exon 3: HLA‐C*03:23N

“…During the last decade, 800 552 samples were examined for HLA types, and 0.49% undetermined samples for blood donors were subjected to further examination. Among these, we found 263 (0.033%) new alleles, 258 of which were already registered in the IPD‐IMGT/HLA database (Anthony Nolan Research Institute, London, UK) . Through this large‐scale systematic HLA typing, we found a new HLA‐C allele, C*03:23N .…”

Comparison of sequence‐specific oligonucleotide probe vs next generation sequencing for HLA‐A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single‐pass high‐resolution HLA typing in support of solid organ and hematopoietic cell transplant programs

“…In this context, a continuation of new alleles is discovered, in particular with respect to the ability of NGS technologies to examine most or all exon sequences over the subset of exons examined using older methods . While currently in limited use in clinical typing laboratories, these methods have been and continue to be very effectively used in establishing more comprehensive database entries for registries . Interestingly, while the long‐range shotgun methods do examine intronic sequences, a relatively low frequency of novel intronic variation has been found, and no functional consequences have been established for the occasional new variant that has been described.…”

“…18 While currently in limited use in clinical typing laboratories, these methods have been and continue to be very effectively used in establishing more comprehensive database entries for registries. [19][20][21] Interestingly, while the long-range shotgun methods do examine intronic sequences, a relatively low frequency of novel intronic variation has been found, 21,22 and no functional consequences have been established for the occasional new variant that has been described. The current study was undertaken to examine the effectiveness of a novel amplicon-based NGS approach to improve on typing methodology for solid organ transplants, a clinical arena where SSOP HLA methods predominate.…”