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The development of a standardized HLA nomenclature has been critical in our understanding of the HLA system and in facilitating the clinical applications of HLA. The Nomenclature Committee for Factors of the HLA System, established in 1968, has overseen the development and usage of nomenclature based on serologic specificities, cellular responses, and DNA sequences. Their decisions have been guided by community consensus reached through 17 international workshops beginning in 1964 and continuing today. Two websites provide a curated database of the sequences of over 26,000 HLA alleles and a reference site for the current nomenclature. This review covers the major steps in the development of the HLA nomenclature as well as the efforts of other groups to extend its usefulness for research and clinical applications.

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.

Next generation DNA sequencing has facilitated the routine characterization of complete HLA gene sequences. These data complement structural and functional studies of HLA elements encoded outside of the exons specifying the antigen recognition domain. This commentary is focused on evaluating whether the interpretation of HLA clinical typing results should expand the region of the HLA gene considered in the assignment from the exon(s) encoding the antigen recognition domain to the full gene sequence. Our recommendation is that, at present, there is insufficient data to support considering variation in the regions outside of the antigen recognition domain in clinical decision-making.

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 Sanger-based DNA sequencing of exons 2 + 3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. In order to reduce the time and effort required to produce a single high resolution HLA genotype, samples were typed in parallel by Sanger sequencing and probe hybridization to obtain G level typing resolution. Methods The panel of probes used was designed to yield high to intermediate resolution typing and to provide the phasing necessary to resolve common alternative genotypes. Class I assignments from more than 30,000 samples from registry donors were typed using this strategy supported by an in-house data merger and management software. To monitor accuracy and resolution, 552 samples were retyped by a next generation full length gene sequencing (NGS) strategy. Results More than 95% of the class I assignments were at the level of a single G-level genotype compared to ∼ 26% for European Americans when probe data were not included. The work load was reduced by at least 70% by eliminating secondary sequencing assays and by dramatically reducing the time needed for data review since the combined method addresses the weaknesses of each single method. Typing accuracy was increased. 99% of Sanger + probe typing results were concordant with NGS; all 32 allele discrepancies were attributed to weaknesses in the NGS data. Furthermore, NGS also demonstrated that only 0.9% of the G level assignments resulted in different full length protein sequences (e.g., C∗07:01:01G yielded C∗07:18 with NGS). The combined method had a rapid turnaround time compared with NGS and previous methods applied alone. Conclusions Our combined method routinely provides biologically relevant typing resolution at the level of the antigen recognition domain. It can be applied to both single sample or to large volume typing supporting either bone marrow or solid organ transplantation using technology currently available in many HLA laboratories and is cost effective when compared with current NGS kits. B. Tu: Other (Identify); Company/Organization; Intellectual property. C. Masaberg: Other (Identify); Company/Organization; Intellectual property. J. Lee: Employee; Company/Organization; Thermo Fisher. D. Behm: Employee; Company/Organization; Data Blueprint. J. Sells: Employee; Company/Organization; Data Blueprint. P. Tausch: Employee; Company/Organization; Thermo Fisher. L. Hou: Other (Identify); Company/Organization; Intellectual property. J. Ng: Other (Identify); Company/Organization; Intellectual property. C.K. Hurley: Other (Identify); Company/Organization; Intellectual property.

HLA class I assignments were obtained at single genotype, G-level resolution from 98 855 volunteers for an unrelated donor registry in the United States. In spite of the diverse ancestry of the volunteers, over 99% of the assignments at each locus are common. Within this population, 52 novel alleles differing in exons 2 and 3 are identified and characterized. Previously reported alleles with incomplete sequences in the IPD-IMGT/HLA database (n = 519) were selected for full gene sequencing and, from this sampling, another 27 novel alleles are described.

Two novel HLA class II alleles, DRB4*03:01N and DQB1*03:276N, containing large deletions were identified during routine typing. Extraction of DNA encompassing the deletions was carried out with a panel of capture oligonucleotides followed by whole genome amplification. Next generation DNA sequencing was then used to characterize the sequences. DRB4*03:01N has a 16 kilobase pair deletion stretching upstream from intron 2 toward centromeric DRB8. DQB1*03:276N has two deletions separated by 844 nucleotides. The first deletion (3.7 kilobase pairs) is upstream of intron 1 and the second deletion removes 3.3 kilobase pairs further upstream towards centromeric DQA2.

KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules.

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of (similar to)140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.

In order to reduce the time required to identify a match for unrelated donor hematopoietic stem cell transplantation, a one-step DNA sequencing strategy was employed at the time of recruitment. The impact of this strategy on human leukocyte antigen (HLA) typing resolution and the effect of current registry requirements on resolution and coding of assignments were evaluated. Sanger-based DNA sequencing was used to obtain diploid exons 2 and 3 HLA-A, -B and -C assignments of 2747 unrelated African American and 1822 European American volunteers at recruitment. The results demonstrate the high resolution of the approach and challenge several aspects of the current registry typing strategy. Of the 46% of African American and 74% of European American individuals whose HLA typing resulted in alternative genotypes, the majority (≥93%) was predicted to have only a single 'common' genotype among the alternatives. The common practice of adding secondary assays to resolve alternative genotype assignments that include more than two antigen groups was also evaluated. While the percentage of assignments with greater than two antigen groups reached as high as 21% (HLA-A in European Americans), only 1.8% of individuals at most carried two common genotypes encompassing three antigen groups. The assignment of (National Marrow Donor Program) NMDP-designated allele codes to the one-pass results reduced the resolution substantially and introduced genotypes that were not included in the laboratory's assignments. We suggest the alternative strategy of using the exons 2-3 diploid nucleotide sequence as the assignment submitted to the registry with the added benefit of immortalizing the assignment in time regardless of the introduction of novel alleles. To keep pace with current donor selection criteria and with the increasing number of new alleles, it is time to rethink our recruitment typing strategies.

Traditional DNA-based typing focuses primarily on interrogating the exons of human leukocyte antigen (HLA) genes that form the antigen recognition domain (ARD). The relevance of mismatching donor and recipient for HLA variation outside the ARD on hematopoietic stem cell transplantation (HSCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in 10 of 10 (HLA-A, -B, -C, -DRB1, -DQB1) matched unrelated donor transplant pairs (n = 360). Next-generation DNA sequencing was used to characterize both HLA exons and introns for HLA-A, -B, -C alleles; exons 2, 3 and the intervening intron for HLA-DRB1 and exons only for HLA-DQA1 and -DQB1. Over 97% of alleles at each locus were matched for their nucleotide sequence outside of the ARD exons. Of the 4320 allele comparisons overall, only 17 allele pairs were mismatched for non-ARD exons, 41 for noncoding regions and 9 for ARD exons. The observed variation between donor and recipient usually involved a single nucleotide difference (88% of mismatches); 88% of the non-ARD exon variants impacted the amino acid sequence. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D-R pairs will be difficult to assess in HSCT outcome studies because these mismatches do not occur very frequently.

Correspondence to: Dr Steven G. E. Marsh Anthony Nolan Research Institute Royal Free Hospital Pond Street Hampstead London NW3 2QG UK Tel.: þ442072848321 Fax: þ442072848331 e-mail: marsh@ebi.ac.uk Following the decision to hold their next full meeting after the 14th International Histocompatibility Workshop in 2005, the WHO Nomenclature Committee for Factors of the HLA System has decided to publish an interim report listing updated tables of alleles including those assigned since the publication of the last full report in 2002 (1). The alleles named during the period follow the principles established in previous reports (1–17).

HLA-A, -B, -C, -DRB1, -DQB1 assignments were obtained for 374 pairs of African American mothers and their umbilical cord blood units (CBU) by DNA sequencing. An algorithm developed by the National Marrow Donor Program was used to assign 1122 haplotypes by segregation. 70% of the haplotypes carried assignments at all five loci. In the remainder, alleles at various loci, most often DQB1 in 48% of the haplotypes with a missing assignment, could not be assigned due to sharing of both alleles by mother and CBU. There were 652 haplotypes carrying a unique combination of alleles at the five loci; the majority (74%) were singletons. Novel B~C and DRB1~DQB1 associations were observed. The results demonstrate the genetic diversity in this population and provide validation for a publically available tool for pedigree analysis. Our observations underscore the need for procurement of increased numbers of units in the national cord blood inventory in order to identify matching donors for all patients requiring hematopoietic stem cell transplantation.

Seventy-two novel human leukocyte antigen (HLA) class I and class II alleles are described from volunteers for the 'Be The Match Registry®': 17 HLA-A alleles, 12 HLA-C alleles, 31 HLA-B alleles and 12 HLA-DRB1 alleles. Forty-six (≈ 64%) of the 72 novel alleles are single-nucleotide substitution variants when compared with their most homologous allele. Five of these single-nucleotide variants are silent substitutions and one creates a non-expressed allele (B*44:108N). The remaining novel alleles differ from their most similar allele by two to five nucleotide substitutions. One of the novel HLA-C alleles (C*07:150Q) is of questionable expression due to an insertion of 21 nucleotides starting at codon 143 that adds seven amino acids to exon 3. An inter-locus gene conversion may have created the novel allele HLA-A*23:31 that shares its codon differences with HLA-B*07:28. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 116 and 150), HLA-C (codon 114), HLA-B (codons 11, 21, 35, 42, 48, 73, 98 and 170) and HLA-DRB1 (codon 29) loci.

The following commentary was developed by the National Marrow Donor Program Cord Blood Advisory Group and is intended to provide an overview of umbilical cord blood (UCB) processing, summarize the current state of potency assays used to characterize UCB, and define limitations of the assays and future needs of the cord blood banking and transplant community. The UCB banking industry is eager to participate in the development of standardized assays to uniformly characterize cellular therapy products that are manufactured in a variety of ways. This paper describes the desired qualities of these assays and how the industry proposes to co-operate with developers to bring relevant assays to market. To that end, the National Marrow Donor Program (NMDP) Cord Blood Bank Network is available to serve as a resource for UCB testing material, research and development consulting, and product/assay testing in an accredited UCB manufacturing environment.

High-resolution DNA sequencing was used to identify the human leukocyte antigen (HLA) –A, -B, -C, and -DRB1 alleles found in 552 individuals from the United States indicating Southern European (Italian or Spanish) heritage. A total of 46 HLA-A, 80 HLA-B, 32 HLA-C, and 50 DRB1 alleles were identified. Frequent alleles included A*02:01:01G (allele frequency = 0.26 in Italian Americans and 0.22 in Spanish Americans); B*07:02:01G (Italian Americans allele frequency = 0.11); B*44:03 (Spanish Americans allele frequency = 0.07); C*04:01:01G and C*07:01:01G (allele frequency = 0.13 and 0.16, respectively, in Italian Americans; 0.15 and 0.12, respectively, in Spanish Americans); and DRB1*07:01:01 (allele frequency = 0.12 in each population). The action of balancing selection was inferred at the HLA-B and -C loci in both populations. The A*01:01:01G-C*07:01:01G-B*08:01:01G-DRB1*03:01:01 haplotype was the most frequent A-C-B-DRB1 haplotype in Italian Americans (haplotype frequency = 0.049), and was the second most frequent haplotype in Spanish Americans (haplotype frequency = 0.021). A*29:02:01-C*16:01:01-B*44:03-DRB1*07:01:01 was the most frequent A-C-B-DRB1 in Spanish Americans (haplotype frequency = 0.023), and was observed at a frequency of 0.015 in Italian Americans. Pairwise F′ st values measuring the degree of differentiation between these Southern European American populations as well as European and European American populations suggest that Spanish Americans constitute a distinct subset of the European American population, most similar to Mexican Americans, whereas Italian Americans cannot be distinguished from the larger European American population.

Aim This study examined the variation observed when typing exons 2 + 3 of HLA-DQB1 using reverse sequence specific oligonucleotides (rSSO) as compared to typing exon 2 alone in volunteers from an unrelated hematopoietic stem cell donor program. New alleles and those with incomplete sequences were characterized with next generation sequencing (NGS) to update the IPD-IMGT/HLA database. Methods Samples were typed for HLA-DQB1 using One Lambda’s SSO LabType with additional phasing probes for exon 2 ( ∼ 50,000 donors) or for exons 2 + 3 ( ∼ 15,000). Full-length HLA-DQB1 sequences (40) were obtained using an Illumina platform and in-house protocol. Results Overall, 38 incomplete DQB1 alleles were identified and sequenced with NGS, including 6 that could only be detected by rSSO with exon 3 probes. The majority of these alleles appear only 1–2 times in the sample set and were not listed as common or well-documented in Mack et al. 2013. The addition of exon 3 in rSSO typing complicated analysis since many alleles lack sequence data for exon 3, and the software assumes no probe binding if there is no known sequence. An example is DQB1*06:08 ( ∼ 10), which binds to one of the exon 3 probes and therefore is not assigned (i.e., false positive). Two alleles showed discrepant results between rSSO and NGS due to novel sequences. The full-length sequences obtained for all alleles sequenced demonstrate high conservation of the regions outside of exon 2. Conclusions There are advantages and disadvantages from screening exons 2 + 3 of HLA-DQB1. It elucidates greater variation not identified from screening exon 2 alone but has a more complicated analysis due to incomplete DNA sequences. Updating the IPD-IMGT/HLA database is critical for improved accuracy. C. Masaberg: 7. Other (Identify); Company/Organization; Intellectual property typing workflow, One Lambda licensing. C.K. Hurley: 7. Other (Identify); Company/Organization; Intellectual property typing workflow, One Lambda licensing.

Aim The aim of this study was to complete full gene sequences for previously reported HLA class I sequences for the IPD-IMGT/HLA database. Methods We identified 569 class I alleles in ∼ 99,000 individuals typed for a U.S. donor registry that had incomplete sequences in the IPD-IMGTHLA database. The alleles had been typed by a combined Sanger SBT-SSOP workflow focused on exons 2 + 3. Single locus alleles were amplified with primers annealing in the 5’ and 3’ UTR and the sequences of exons and introns determined with an Illumina Miseq using an in-house protocol. Results Most (423, 74%) of the 569 alleles were found in a single cell. The majority of the class I full length sequences were identical to the most similar HLA allele (e.g., C*01:70 differed by only a single exon 2 nucleotide when compared to C*01:02:01:01). Two novel HLA-A alleles with exon 2/3 variation were not detected by SBT/SSO due to inability to phase key polymorphic nucleotides [A*03:150, *26:01:01G vs. A*03:01:01:01 + A*26:149 (new); A*02:01:55 + *33:03:01G vs. A*02:01:01:01 + *33:03:33 (new)]. Overall, new alleles were found in 18 (3.2%) samples; most in the targeted incomplete allele. Most (14, 78%) showed variation in intron and UTR regions. Some of the incomplete alleles (49/569) could not be characterized because the allele combination did not allow phasing of the sequences (e.g., C*07:01:01:01 + *07:27:01 had no polymorphic nucleotides within a span of 2298 bp (introns 2–7)). Conclusions Class I gene sequences outside exons 2 + 3 are conserved. Genotypes with novel alleles can be missed by typing assays that do not provide comprehensive phasing. Increasing the knowledge of full sequences for all alleles will improve the accuracy of HLA typing. Download : Download high-res image (153KB) Download : Download full-size image J.S. Ng: 4. Scientific/Medical Advisor; Company/Organization; Georgetown University. C.K. Hurley: 4. Scientific/Medical Advisor; Company/Organization; Georgetown University.

Aim Long range next generation sequencing was used to characterize the HLA class I (HLA-A,B,C) and class II (DRB1, DRB3/4/5, DQB1) alleles of a hematopoietic stem cell registry population (N = 643) from The Netherlands (Matchis). Methods The in-house typing protocol included several new strategies to reduce errors, add automation, and balance the yield of all loci when 96 samples are sequenced in one 600 cycle paired-end Illumina MiSeq sequence run. Results Of the 22 new class I alleles, three contain a nonsynonymous substitution in an exon (1, 4 or 7). The remainder are single nucleotide intron variants. Thirty-two class I alleles are incomplete in the IMGT/HLA database. The majority of the alleles in the population are common; the three most common alleles at each locus are listed in the table. Alleles not previously listed as common or well-documented include mainly 4th field variations; exceptions included single copies of B*51:20, C*07:100, and DRB1*04:92. Alleles encoding nonsynonymous substitutions compared to the primary allele in the G group include, as examples, A*23:17 (1 vs. 20 A*23:01:01:01), B*44:27:01 (1 vs 83 B*44:02:01:01), C*07:06 (3 vs 190 C*07:01:01:01), DRB1*14:54:01 (44 vs 3 DRB1*14:01:01), DQB1*02:02:01 (84 vs 175 DQB1*02:01:01). Non-expressed alleles were not observed. DRB3/4/5 associations were identified. Conclusions Long-range sequencing of HLA alleles from world-wide populations will continue to build our understanding of HLA diversity and its impact on the immune response. Download : Download high-res image (231KB) Download : Download full-size image C.K. Hurley: 7. Other (Identify); Company/Organization; Intellectual property sequencing Thermo Fisher.

Sixty-five novel human leukocyte antigen (HLA) alleles are described from volunteer donors of the ‘Be The Match Registry®’: 29 HLA-A alleles, 24 HLA-B alleles, and 12 HLA-DRB1 alleles. Eight (∼12%) of the novel alleles were found in more than one individual and may be more common in the population. Forty (∼62%) of the 65 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Two of these single nucleotide variants are silent substitutions and one creates a null allele. The remaining novel alleles differ from their most similar allele by 2–10 nucleotide substitutions. Some of the novel alleles encode amino acid changes at codons not previously reported to be polymorphic, such as codons 23, 93, 129, and 155 in HLA-A alleles and codon 3 in HLA-B alleles.

The WHO Nomenclature Committee for Factors of the HLA System met following the 14th International HLA and Immunogenetics Workshop in Melbourne, Australia in December 2005 and Buzios, Brazil during the 15th International HLA and Immunogenetics Workshop in September 2008. This report documents the additions and revisions to the nomenclature of HLA specificities following the principles established in previous reports (1–18).

The five two-domain stimulatory KIR genes carried by 100 random African Americans were characterized by DNA sequencing of genomic DNA covering the majority of coding exons. The frequency of individual loci was similar to that found in European Americans, with the exception of a reduced frequency for KIR2DS1 in African Americans. New alleles were identified at the KIR2DS1 (*008), KIR2DS2 (*006), KIR2DS3 (*00104, *00105, *00106, *004), KIR2DS4 (*00103, *00104, *009, *011, *012, *013), and KIR2DS5 (*006, *007, *00801, *00802, *009) loci. The distribution of alleles at each locus was similar to that found in a European American population except for KIR2DS5. KIR2DS5 exhibits a single allele in European Americans; the same allele is found at a reduced frequency (41% of gene-positive individuals), accompanied by KIR2DS5*006 (18%), KIR2DS5*007 (26%), and six other alleles (25%), in African Americans.

One hundred and four novel human leukocyte antigen (HLA) alleles are described from volunteer donors of the National Marrow Donor Program: 37 HLA-A alleles, 37 HLA-B alleles, and 30 HLA-DRB1 alleles. Seventeen ( approximately 16%) of the novel alleles were found in multiple individuals and likely are relatively common in the population. Seventy-two ( approximately 69%) of the 104 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Nine of these single nucleotide variants are silent substitutions and three create null alleles. The remaining novel alleles differ from their most similar allele by two to seven nucleotide substitutions. Some of the novel alleles encode amino acid changes at positions not previously reported to be polymorphic, such as codons 6 and 11 in HLA-A alleles and codons 5, 105, and 141 in HLA-B alleles. Interestingly, one of the novel HLA-DRB1 alleles (*1471) has a change that is not the typical glycine/valine dimorphism at codon 86, which plays a key role in peptide binding to DR molecules. This is only the second DRB1 allele described that encodes an amino acid other than glycine or valine at this position.

Sequence-based typing was used to identify human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 alleles from 558 consecutively recruited US volunteers with Eastern European ancestry for an unrelated hematopoietic stem cell registry. Four of 31 HLA-A alleles, 29 HLA-C alleles, 59 HLA-B alleles, and 42 HLA-DRB1 alleles identified (A*0325, B*440204, Cw*0332, and *0732N) are novel. The HLA-A*02010101g allele was observed at a frequency of 0.28. Two-, three-, and four-locus haplotypes were estimated using the expectation-maximization algorithm. The highest frequency extended haplotypes (A*010101g-Cw*070101g-B*0801g-DRB1*0301 and A*03010101g-Cw*0702-B*0702-DRB1*1501) were observed at frequencies of 0.04 and 0.03, respectively. Linkage disequilibrium values (Dij') of the constituent two-locus haplotypes were highly significant for both extended haplotypes (P values were less than 8 x 10(-10)) but were consistently higher for the more frequent haplotype. Balancing selection was inferred to be acting on all the four loci, with the strongest evidence of balancing selection observed for the HLA-C locus. Comparisons of the A-C-B haplotypes and DRB1 frequencies in this population with those for African, European, and western Asian populations showed high degrees of identity with Czech, Polish, and Slovenian populations and significant differences from the general European American population.

Several overlapping amplicons were used to obtain the sequence of genomic DNA covering most of the coding regions of KIR3DL1 and KIR3DS1 from a family and 77 bone marrow transplant patients and their unrelated donors. Alleles 3DL1*00101 and *002 were most frequently observed in addition to 12 other known 3DL1 alleles. A single 3DS1 allele, 3DS1*01301, was identified in the 31 of 32 individuals carrying this gene. Two new alleles, 3DL1*01702 and 3DS1*058, were characterized. Three samples appeared to carry the duplicated killer cell immunoglobulin-like receptor (KIR) haplotype observed in other studies based on the presence of 3DS1 and two 3DL1 alleles. Additionally, one sample appeared to carry a novel KIR haplotype containing one 3DL1 and two 3DS1 alleles.

Twenty-eight novel human leukocyte antigen-B alleles are described: one B*07 (*0745), two B*08 (*0826 and *0831), one B*27 (*2731), four B*35 (*350106, *350402, *3566, and *3568), two B*37 (*370102 and *3711), two B*38 (*380102 and *3813), two B*39 (*3935 and *3939), one B*41 (*4108), one B*42 (*4209), two B*44 (*4444 and *4448), two B*48 (*480302 and *4815), one B*51 (*5140), one B*55 (*5523), one B*56 (*5617), two B*57 (*570103 and *5710), and three B*15 (*9505, *9510, and *9519). Sixteen of the variants are single-nucleotide substitutions from their most homologous allele, of which 10 result in amino acid changes (B*0745, *0831, *3813, *3935, *3939, *4815, *9505, *9509, *9510, and *9519) and 6 are silent substitutions. The remaining alleles (B*0826, *2731, *3566, *3568, *3711, *4108, *4209, *4444, *4448, *5140, *5523, *5617, and *5710) differ from their most similar alleles by two to seven nucleotides substitutions, altering from one to five amino acids.

Human leukocyte antigen typing of 2578 donor-recipient pairs whose transplantation was facilitated by the National Marrow Donor Program allowed for an in-depth analysis of the accuracy of high-volume allele level testing data. The methods employed provided allele level typing at DRB1/3/5, DQA1, DQB1, DPA1, and DPB1 using sequence-specific oligonucleotide probe hybridization (SSOPH), polymerase chain reaction (PCR) restriction fragment length polymorphism analysis, sequence specific PCR, and direct sequence-based typing (SBT). Each typing was independently tested by two laboratories in Phase 1, and in subsequent phases targeted samples were typed in duplicate by SBT to monitor typing quality. Comparison with prior transplant center typing was also evaluated. SSOPH detected discrepancies ranged from 0.6% at DPB1 to 5.1% at DQB1 in Phase 1. The majority of discrepancies, 62%, resulted from human error such as sample handling, result interpretation, or clerical errors. Alleles that are frequently discrepant have been identified in this predominantly white population.

Genomic sequencing was used to characterize most of the coding regions of the five two-domain stimulatory killer cell immunoglobulin-like receptor (KIR) loci from 80 unrelated, primarily Caucasian, individuals. Specific loci were present in from 26% (KIR2DS3) to 98% (KIR2DS4) of individuals. The number of known alleles present varied from one (KIR2DS1, KIR2DS5) to five (KIR2DS4). The frequencies of loci and alleles were similar to observations made in populations of European and Asian ethnicities. New alleles were found at 2DS1 (*00202, *00302, *005, *006, *007) and 2DS4 (*008) loci.

Exons 2-9 of KIR3DL3 alleles were characterized by genomic DNA sequencing in two families and in 78 bone marrow transplant samples. Several strategies were used to isolate single alleles for characterization and to resolve alternative allele combinations. We describe 17 novel 3DL3 alleles carried by 30 individuals. Compared with the most closely matched alleles, the new alleles differ by from one to three nucleotides and from zero to three amino acids. The majority of the substitutions were shared with other 3DL3 alleles although three novel polymorphic codons, 151 (exon 4), 327 (exon 7) and 352 (exon 9), are described. Of the 36 different 3DL3 alleles detected in the transplant population, the three most common alleles accounting for 47% of the total were KIR3DL3*00101 (13.5%), KIR3DL3*003 (21.2%) and KIR3DL3*00901 (12.2%).

Large registries of volunteer hematopoietic stem cell donors typed for HLA contain potentially valuable data for studying haplotype frequencies in the general population. However the usual assumptions for use of the expectation-maximization (EM) algorithm are typically violated in these registries. To avoid this problem, previous studies using registry data have reduced the HLA typings to low-resolution and/or excluded subjects who were selected for testing on behalf of a specific patient ("patient-directed" typings). These restrictions, added to avoid bias from selection of nonrepresentative volunteers for higher-resolution typing, have limited previous results to haplotypes defined at low resolution. In this article we eliminate the need for such restrictions by formally relaxing the assumptions necessary for the EM algorithm. We show mathematically and through simulation that varying levels of resolution can be incorporated even if the level of typing resolution is chosen based on the HLA type. This allows use of intermediate and high resolution data from patient-directed typings to extend haplotype frequency estimates to the allele level for HLA-DRB1. We demonstrate the feasibility of using this computationally demanding algorithm on large datasets by applying it to more than 3 million volunteers listed in the National Marrow Donor Program Registry.

Exons 2 through 9 of KIR3DL2 were amplified from genomic DNA from 79 bone marrow transplantation patients and their unrelated donors. Sequencing of heterozygotes and isolated alleles identified 9 of the 17 known alleles. The alleles provide confirmation of previously submitted sequences and are carried by transformed B-cell lines that can be used as references for assay development. Alleles 3DL2*001, *002, *007 and *009 accounted for 111 of the total 144 possible alleles and were the only ones found in a homozygous state. New alleles (3DL2*017, *018, *019, *020, and *021) were found in seven transplant samples and one workshop cell. This study describes the development of reagents and protocols for sequencing of KIR3DL2 alleles from genomic DNA.

Sequence-based typing was used to identify human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 alleles from 564 consecutively recruited African American volunteers for an unrelated hematopoietic stem cell registry. The number of known alleles identified at each locus was 42 for HLA-A, HLA-B 67, HLA-C 33, and HLA-DRB1 44. Six novel alleles (A*260104, A*7411, Cw*0813, Cw*1608, Cw*1704, and DRB1*130502) not observed in the initial sequence-specific oligonucleotide probe testing were characterized. The action of balancing selection, shaping more 'even' than expected allele frequency distributions, was inferred for all four loci and significantly so for the HLA-A and DRB1 loci. Two-, three-, and four-locus haplotypes were estimated using the expectation maximization algorithm. Comparisons with other populations from Africa and Europe suggest that the degree of European admixture in the African American population described here is lower than that in other African American populations previously reported, although HLA-A:B haplotype frequencies similar to those in previous studies of African American individuals were also noted.

Genetic polymorphisms found in the killer Ig-like receptor (KIR), two domains, long cytoplasmic tail 2/3 (KIR2DL2/3) locus are responsible for the differential binding of KIR2DL2/3 allelic products with their HLA-C ligands and have been associated with the resolution of hepatitis C infection. In our study, a KIR CD3ζ fusion-binding assay did not detect any interaction between the KIR2DL2*004 extracellular domain and several putative KIR2DL2/3 ligands. To determine the amino acid polymorphism(s) responsible for the KIR2DL2*004 phenotype, we mutated the polymorphic residues of full-length KIR and expressed them in human Jurkat cells. Flow cytometry analysis failed to detect the surface expression of receptors containing a threonine at position 41 (T41), a polymorphism specific to KIR2DL2*004. Confocal microscopy showed that receptors containing T41 were retained inside the cell and had a perinuclear localization, possibly indicating that their extracellular domain was misfolded. Most KIR2DL2/3 alleles possess an arginine at position 41 (R41), and we predicted through molecular modeling and demonstrated by mutagenesis that R41 most likely interacts with the nearby residues Y77 and D47. Interaction between these residues would maintain C strand contact with the C′ and F strands of the D1 domain β-sheet. Furthermore, R41 and Y77 are conserved in the C and F strand amino acid alignments of Ig-like superfamily members, and may therefore be necessary for the structural integrity of other immune response proteins. Our data indicate that the extracellular T41 polymorphism encoded by the KIR2DL2*004 allele most likely results in misfolding of the D1 domain and complete intracellular retention of the receptor.

KIR3DL3 alleles were characterized in two families and one unrelated individual. Based on exon 2-9 nucleotide sequences, six novel alleles, 3DL3*00402, *005, *006, *007, *00801, *00802, were identified bringing the total number of known alleles to 11. Compared with 3DL3*001, the six new alleles differ by from three to nine nucleotides and from three to four amino acids. The new alleles double the number of known polymorphic positions to 18 with variation in exons encoding the extracellular domains, transmembrane region, and a portion of the cytoplasmic tail. Many of the nucleotide substitutions are shared among alleles of 3DL3 or other KIR loci, but five were found only in single 3DL3 alleles. Comparison of intron sequences among individuals carrying the same allele showed a modest number of substitutions with the exception of 3DL3*001 which differed substantially in its intron sequences. Two alleles sharing coding region sequences, 3DL3*00201 and 3DL3*00202, were also substantially different in intron sequences.

Twenty-five novel HLA-B alleles are described in this paper: B*0729, B*1309, B*1814, B*1815, B*2724, B*2725, B*3539, B*3926, B*4037, B*4040, B*4042, B*4043, B*4044, B*4204, B*440203, B*4428, B*4429, B*4430, B*4505, B*5308, B*5309, B*5510, B*5511, B*570102, and B*5709. Most of the variants are single nucleotide substitutions. Two involve variants at the Bw4/Bw6 epitope. Two alleles carry substitutions of conserved amino acids.

The frequencies of DRB1*08 alleles within four major United States populations found within a hematopoietic stem cell volunteer donor database were determined by DNA sequencing of over 60 DRB1*08 positive individuals from each group. Seven of 30 known DRB1*08 alleles were identified within this study population (080101, 080201, 080302, 080401, 0806, 0807, and 0811). Each ethnic group was characterized by a different highly prevalent allele: DRB1*080101 in Caucasians; DRB1*080401 in African-Americans; DRB1*080302 in Asians; and DRB1*080201 in Hispanics. The alleles DRB1*080101, DRB1*080201, and DRB1*080401 were present in all four populations. This report also describes five novel DRB1*08 alleles uncovered during routine human leukocyte antigen typing.

At least 60 DRB1*02 positive individuals from each of four US population groups found within a hematopoietic stem cell volunteer donor registry – Caucasoids, African Americans, Asians/Pacific Islanders, and Hispanics – were randomly selected from a database of 82,979 individuals. DRB1*02 alleles were identified by DNA sequencing. A total of five of 23 known DRB1*02 alleles were detected. DRB1*15011 was the predominant DRB1*02 allele in Caucasoids and Hispanics. The most common DRB1*02 allele observed in African Americans was DRB1*1503, and DRB1*15021 in Asians/Pacific Islanders. Caucasoids were found to be the least diversified; only DRB1*15011 and DRB1*16011 were observed. A subset of individuals was also typed for DRB5 alleles by DNA sequencing. DRB5*01011, DRB5*0102, DRB5*0103, DRB5*0108N and DRB5*0202 were detected and nine DRB1-DRB5 haplotypes defined.

Killer-cell immunoglobulin-like receptors (KIRs) are a newly described family of polymorphic and highly homologous genes that have been difficult to classify and characterize. Before comprehensive analyzes of the genes are completed, researchers must struggle with completing the task of classifying and characterizing what is currently known. A collection and alignment of all KIR sequences found in GenBank was created to facilitate oligonucleotide reagent development and to provide an overall picture of this complex gene system. Two methods, a direct measurement of homology and phylogenetic analysis, were used to categorize sequences previously not specifically identified as belonging to a particular locus. The two methods agreed for 64.2% of sequences. A further 22.6% of uncategorized sequences were specified by only one method, although the assignments were consistent. Some sequences (11.3%) could not be assigned to a locus by either method. For one sequence, the two methods disagreed as to the locus assignment (1.9%). The failure to categorize a sequence was usually related to the short length of the sequence and the similarity among KIR loci. The tools developed in this study have been valuable in the analyses of KIR sequences and can be used for any complex gene system.

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 In order to reduce the time and effort required to produce a single high resolution HLA class I genotype, a workflow combining one-step Sanger sequencing with probe hybridization has been successfully used in HLA-A, -B, -C typing for a hematopoietic stem cell donor registry (Tu et al. HLA 89:90, 2017). This workflow has now been applied to type HLA-DRB1, -DQB1 and -DPB1. Methods DNA was prepared from buccal swabs from 184 registry donors. Sanger sequencing used Thermo Fisher Scientific SeCore® HLA-DRB1, -DQB1, and -DPB1 kits. Simultaneous hybridization used One Lambda LABType® probes. Assignments were merged with in-house software. Interpretation used IPD-IMGT/HLA database 3.19. Results Use of both DNA sequencing and SSO (without additional phasing probes) hybridization resulted in assignment of a single G-level genotype for the majority of samples compared to sequencing alone (Table). Ambiguous genotypes observed for DRB1 and DQB1 will be addressed by additional phasing probes to raise the frequency of single genotype resolution for these loci closer to 100%. Conclusions This workflow complements that described by us for HLA class I alleles and provides a comprehensive strategy for complete HLA typing. The SBT+SSO combination workflow with genotype assignments at the level of the antigen recognition domain is a powerful typing strategy, merging strengths of both methods and improving accuracy and reducing turnaround time and typing cost. While applied to registry typing, this workflow also can be applied to a small number of samples. Download : Download high-res image (155KB) 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.

This paper describes 29 novel HLA-B locus alleles identified during low-resolution typing. The majority of the novel alleles carry new patterns of previously known polymorphic motifs or codons. Three alleles carry alterations in the Bw4/Bw6 epitope. Five alleles carry novel substitutions.

Twelve new B*15 alleles are described. All of the known B*15 alleles are divided into subgroups based on serologic assignments and/or nucleotide sequence polymorphisms. These groups might be used as a reference for DNA-based testing at an intermediate (i.e. “serologic”) level of resolution.

Direct automated DNA sequencing was used to analyze exons 2 and 3 of HLA-B alleles present in forty-four unrelated individuals residing in the village of Adiopodoume, Cote d'Ivoire (Ivory Coast). Of the 23 HLA-B alleles observed, the most frequently detected allele was HLA-B*5301 (22.7%), which is believed to confer resistance to severe Plasmodium falciparum malaria. B*4501 (9.1%), B*1503 (8.0%), B*0705 (5.7%), B*1510 (5.7%) and B*3501 (5.7%) occurred frequently in the population. A second allele of B53 was identified; B*5302 contains a single amino acid variation at residue 171 (Y-->H). Two additional novel alleles, B* 1405 (a single amino acid variant of B*1402) and B*4410 (a five amino acid variant of B*4403) were characterized.

Seven new HLA-B locus alleles have been described. B*44022 and B*44032 are silent substitutions altering known alleles. B*4411 carries a unique Bw4-like epitope. B*4420, B*4421, and B*4424 carry new combinations of motifs previously observed in other alleles. B*8301 appears to be the result of the replacement of exon 2 from B*4402 with exon 2 from B*5603.