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A catalog of common, intermediate and well-documented (CIWD) HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQB1 and -DPB1 alleles has been compiled from over 8 million individuals using data from 20 unrelated hematopoietic stem cell volunteer donor registries. Individuals are divided into seven geographic/ancestral/ethnic groups and data are summarized for each group and for the total population. P (two-field) and G group assignments are divided into one of four frequency categories: common (>= 1 in 10 000), intermediate (>= 1 in 100 000), well-documented (>= 5 occurrences) or not-CIWD. Overall 26% of alleles in IPD-IMGT/HLA version 3.31.0 at P group resolution fall into the three CIWD categories. The two-field catalog includes 18% (n = 545) common, 17% (n = 513) intermediate, and 65% (n = 1997) well-documented alleles. Full-field allele frequency data are provided but are limited in value by the variations in resolution used by the registries. A recommended CIWD list is based on the most frequent category in the total or any of the seven geographic/ancestral/ethnic groups. Data are also provided so users can compile a catalog specific to the population groups that they serve. Comparisons are made to three previous CWD reports representing more limited population groups. This catalog, CIWD version 3.0.0, is a step closer to the collection of global HLA frequencies and to a clearer view of HLA diversity in the human population as a whole.

Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, and -DQB1 assignments of 1472 unrelated volunteers for the unrelated donor registry in Argentina. The analysis characterized all HLA exons and introns for class I alleles; at least exons 2, 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 330 class I and 98 class II. The majority (~98%) of the cumulative allele frequency at each locus is contributed by alleles that appear at a frequency of at least 1 in 1000. Fourteen (18.2%) of the 77 novel class I and II alleles carry nonsynonymous variation within their exons; 52 (75.4%) class I novel alleles carry only single, apparently random, nucleotide variation within their introns/untranslated regions. Alleles encoding protein variation not usually detected by typing focused only on the exons encoding the antigen recognition domain are 1.0% of the class I assignments and 7.3% of the class II assignments (predominantly DQB1*02:02:01, DQB1*03:19:01, and DRB1*14:54:01). Updates to the common and well documented list of alleles include 10 alleles previously thought to be uncommon but that are found at least 30 times. Five locus haplotypes estimated using the expectation-maximization algorithm as present 3 or more times total 187. While the known HLA diversity continues to increase, the conservation of known allele sequences is remarkable. Overall, the HLA diversity observed in the Argentinian population reflects its European and Native American ancestry.

Aim Argentina represents an amalgamation of European, indigenous Amerindians, Mestizos, and non-European descendants but the contributions from each ethnic group remain relatively unexplored from a genetic perspective. We applied NGS to determine the distribution of HLA allele and haplotype frequencies in a large collection from the INCUCAI-Argentinian unrelated donor BMT registry. Methods The cohort consists of individuals (>36,000) with ancestry from 70 ethnic groups. To facilitate analyses individuals were classified into 10 broad ethnic groups; Iberian, Mixed-Hispanic, Mediterranean, Central/Western Europe, East European, English, French, Middle Eastern, API, and ‘other’ pertaining to individuals with unreported or African ancestry. Various parameters of intra-population diversity were assessed including heterozygosity, LD, and selective neutrality. Haplotype frequencies were estimated using the EM algorithm. Inter-population variance was determined using Fst distances which were used to construct phylogenetic dendrograms. Results The most frequent alleles at the class I loci were: A*02:01:01G; C*04:01:01G; B*51:01:01G in 9 groups, B*07:01:01G and B*18:01:01G were most common in East Europeans and English individuals. DQB1*02:01:01G and DQB1*03:01:01G, DRB1*07:01:01G were frequent across all groups. At the HLA-B locus, Mixed-Hispanics exhibited the greater diversity (178 alleles), and the lowest diversity was observed in the French (51 alleles). Native-American ancestry was evident as indicated by the presence of B*15, B*35, B*39, B*40, B*48, B*51, B*52 rare alleles which had the highest phenotype frequency of 20% in Iberians suggesting that patients with indigenous ancestry may find it difficult to find a suitable donor outside of Argentina. Haplotypes with distinct HLA associations common and specific to particular ethnic groups were observed. Genetic distances indicated that the Argentinian populations shared a strong HLA affinity with European populations. Conclusions The divergent HLA signatures in the Argentinian populace allude to the different migratory events of Europeans to the new world. Our data provides well-defined HLA profiles of the Argentinian registry and this information may be useful to increase the accuracy of donor selection for BMT patients.

Aim The goal of this study was to update the 2012 common and well documented (CWD) allele list using data from 3940 class I and 4124 class II assignments obtained by next generation sequencing (NGS) of two registry (USA and Argentina) populations and 360 donor-recipient pairs from the CIBMTR repository. Methods DNA was prepared from blood, buccal swabs, or cell lines. HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs. The sequences of exons and introns were determined by next generation sequencing (NGS) with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Class II NGS sequencing focused on exons 2 and 3; however, some alleles had additional exons and introns characterized. Alleles that were on the well-documented list and alleles not considered CWD based on Mack et al. Tissue Antigens 2013 were identified. Results The majority of alleles detected had previously been designated as common. For example, from the Argentine registry samples, 92% of 2944 HLA-A alleles identified were common. In the total study population, 77 class I alleles previously designated as WD and 97 alleles not defined as C or WD were observed from 1 to >5 times. Thirty-seven of the 174 class I alleles occurred greater than 5 times, e.g., previously undefined A*02:17:02, B*44:02:01:03, and C*17:01:01:05. The majority (90%) of the 19 not CWD alleles were intron variants e.g., A*03:01:01:05, B*39:01:01:03, C*06:02:01:03. Forty-five class II alleles fell into the WD or not defined categories; 29% occurred >5 times. Class II alleles occurring >5 times included DRB1*04:06:02, DQB1*03:01:01:03 and DPB1*04:02:01:02. Conclusions The NGS data expand our knowledge of the frequency of specific full length HLA alleles. The majority of the uncommon alleles characterized in this study are synonymous variations of common alleles.

Aim The aim of this study was to use next generation DNA sequencing (NGS) to characterize full length HLA class I gene diversity in 1472 volunteers for the unrelated donor registry in Argentina. Methods DNA was prepared from blood. HLA-A, -B, -C alleles were amplified with primers annealing in the 5′ and 3′ UTRs. The sequences of exons and introns were determined by NGS with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Analysis used the IPD-IMGT/HLA database from October 2016. Results Volunteers were recruited from 20 of the 23 provinces of Argentina and from its federal capital. The majority (80%) came from the central region of the country. Of the 2944 alleles identified at each locus, the majority have been designated as common (e.g., 92% HLA-A alleles) (Mack et al. Tissue Antigens 2013) and approximately 1% were not found in the IPD-IMGT/HLA database. A total of 79 different allele sequences were identified at the HLA-A locus with similar numbers at HLA-B and -C. The majority of the HLA proteins can be distinguished by sequencing only the antigen recognition domain (ARD)-encoding exons. For example, 94% of the HLA-A alleles could have been detected solely based on the sequence of the ARD-encoding exons. The remaining alleles differed only outside of these exons by either silent variation occurring mainly in introns (6%) or by nonsynonymous variation ( Conclusions NGS has allowed us to evaluate the frequency and characteristics of class I DNA sequence variation outside of the ARD-encoding exons. The additional variation detected is predominantly in noncoding regions. While NGS offers a strategy to achieve single genotype resolution, the amount of additional information gained related to HLA matching for transplantation is minimal.

Aim The aim of this study was to characterize the complete class I gene sequences of new class I alleles found in a population study of 1472 volunteers from the Argentina unrelated donor registry. Methods HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs and the sequences of exons and introns determined by next generation sequencing (NGS) with an Illumina Miseq using an in–house protocol and Conexio Assign software analysis. Initially class I and II amplicons were combined for sequencing; single locus sequencing of a second PCR amplicon was used to confirm variation. Allele sequences were submitted to the IPD-IMGT/HLA database. Results 1% of the 2944 alleles identified at each class I locus were novel. Three novel alleles were found multiple times. The majority (92%) differed by only a single nucleotide substitution from the known allele. Variants in noncoding regions predominated (79%) (T1). None of the intron variants appeared to impact RNA splicing motifs. The distribution of novel substitutions among introns is listed in T2. Unique substitutions, not shared by any other known allele at the locus, were found in 71% HLA-A, 58% HLA-B and 67% HLA-C novel alleles. Conclusions Sixty-six novel class I alleles were identified. The majority of substitutions were found in introns and appear to be random rather than resulting from gene conversion events. Download : Download high-res image (295KB) Download : Download full-size image Download : Download high-res image (73KB) Download : Download full-size image

Aim This study used next generation sequencing (NGS) in 1472 healthy volunteers from an Argentinian registry to characterize class II gene diversity. Complications of using NGS for these regions arise from large introns, different length alleles, regions with few polymorphisms, and incomplete sequences in the IPD-IMGT/HLA database. Methods Amplicons were generated to include exons 2–6 of HLA-DQB1, and exons 2–3 of -DRB1 and -DPB1 using DNA extracted from blood. Samples were combined and sequenced on an Illumina MiSeq with an in-house library construction protocol. Conexio Assign was used to identify alleles based on the IPD-IMGT/HLA database from October 2016. Full length gene sequencing of select samples was performed with two overlapping amplicons for HLA-DPB1 and -DRB1. Results The majority of the alleles identified at each locus were common with ⩽1% of alleles per gene designated as well-documented or with frequency not defined. With only exons 2–3 sequenced, 6% of HLA-DRB1 assignments and 11% of -DPB1 were ambiguous. When additional exons were sequenced, most showed the common allele; however, DPB1∗104:01 was seen in 16% of samples typed as DPB1∗03:FNVX. For HLA-DRB1, preferential amplification due to differences in allele size and presence of pseudogenes led to certain alleles with low read depth and poor sequence quality, in particular DRB1∗04. Analysis of HLA-DQB1 introns proved difficult due to insertions and deletions that poorly aligned to the reference. Intron 2 of HLA-DPB1 is large and has few variations between certain combinations of alleles that were not phased in 14% of samples. These were not resolved with full length sequencing. Conclusions Although conditions still need to be optimized, NGS of HLA class II is a powerful tool in determining genetic diversity. HLA-DRB1 ambiguities would be reduced by 82% with the initial sequencing of exons 2–4; however, longer amplicons reduce read depth. Addition of primers to maximize amplification of low amplifying alleles may be necessary to increase read depth. Alternate methodology such as sequencing of single DNA molecules is necessary to solve the ambiguities for alleles that are not phased. These proposed changes will lead to more successful sequencing of HLA class II. Even so, these complications make NGS challenging when applied to high volume typing.