Next generation sequencing characterizes the extent of HLA diversity in an Argentinian registry population.

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.

Limited HLA sequence variation outside of antigen recognition domain exons of 360 10 of 10 matched unrelated hematopoietic stem cell transplant donor-recipient pairs.

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.

An update to the HLA Nomenclature Guidelines of the World Marrow Donor Association, 2012.

For more than two decades, international cooperation and information technology have been playing key roles in the identification of suitable unrelated donors and cord blood units for hematopoietic SCT. To ensure consistent coding and interpretation of HLA data among the linked computer systems, the World Marrow Donor Association has standardized the extensions of the World Health Organization (WHO) Nomenclature for factors of the HLA system applied in practice. The first version of this report published in 2007 has become the reference for the technical validation of HLA information on donors and patients in the context of search and matching and is used by registries of volunteer unrelated hematopoietic stem cell donors and umbilical cord blood banks throughout the world. The present update became necessary after the major revision of the WHO HLA nomenclature in April 2010. It now covers issues arising when alleles are withdrawn or renamed because of the continuous updating of the WHO HLA nomenclature. In addition, formal validation and interpretation rules for the so-called 'multiple allele codes' have been added.

Common and well-documented HLA alleles: 2012 update to the CWD catalogue.

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 ~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.

A one-step DNA sequencing strategy to HLA type hematopoietic stem cell donors at recruitment - rethinking typing strategies.

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.

16(th) IHIW: immunogenomic data-management methods. report from the immunogenomic data analysis working group (IDAWG).

SUMMARY: The goal of the immunogenomic data analysis working group (IDAWG) is to facilitate the consistent analysis of HLA and KIR data, and the sharing of those data among the immunogenomic and larger genomic communities. However, the data management approaches currently applied by immunogenomic researchers are not widely discussed or reported in the literature, and the effect of different approaches on data analyses is not known. With ASHI's support, the IDAWG developed a 45 question survey on HLA and KIR data generation, data management and data analysis practices. Survey questions detailed the loci genotyped, typing systems used, nomenclature versions reported, computer operating systems and software used to manage and transmit data, the approaches applied to resolve HLA ambiguity and the methods used for basic population-level analyses. Respondents were invited to demonstrate their HLA ambiguity resolution approaches in simulated data sets. By May 2012, 156 respondents from 35 nations had completed the survey. These survey respondents represent a broad sampling of the Immunogenomic community; 52% were European, 30% North American, 10% Asian, 4% South American and 4% from the Pacific. The project will continue in conjunction with the 17th Workshop, with the aim of developing community data sharing standards, ambiguity resolution documentation formats, single-task data Management tools and novel data analysis methods and applications. While additional project details and plans for the 17th IHIW will be forthcoming, we welcome the input and participation in these projects from the histocompatibility and immunogenetics community.

Extensive haplotype diversity in African American mothers and their cord blood units.

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. Seventy percent 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 show 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.

Increased HLA class I and II diversity as 72 novel alleles are identified in volunteers for the National Marrow Donor Program Registry in 2010.

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.

Conserved KIR allele-level haplotypes are altered by microvariation in individuals with European ancestry.

NK cell immunoglobulin-like receptor (KIR) haplotype-specific DNA fragments were sequenced to identify centromeric and telomeric allele-level haplotype structures and their frequencies from 76 unrelated individuals with European ancestry. Analysis was simplified by redefining the 5' boundary of the centromeric KIR gene cluster to include only exons 7-9 of KIR3DL3. Three consensus allele-level haplotypes were identified for a centromeric gene presence/absence structure designated as Cen-A1. KIR3DL3*00201 (exons 7-9)∼KIR2DL3*001∼KIR2DL1*00302 was the most frequent (37.5%) centromeric structure. Single consensus haplotypes were observed for haplotype structures Cen-B1 and Cen-B2. Six Tel-A1 and two Tel-B1 consensus haplotypes were observed; the most prevalent (23.0%) was KIR2DL4*00102∼KIR3DL1*002∼KIR2DS4*00101∼KIR3DL2*002. A small number of nucleotide substitutions (≤3) in the coding regions of the functional KIR genes created microvariants of the consensus haplotypes. Eight less common haplotype structures were also detected. Four carried hybrid genes formed during gene deletion events, two carried an insertion with a 2DL5/3DP1 fusion gene and two included a very large insertion. These data show that the KIR gene complex is composed of a limited number of conserved allele-level centromeric and telomeric haplotypes that have diversified by mutation, recombination within a locus and unequal crossing over.

Novel HLA class I and II alleles identified during routine registry typing in 2010.

Twenty-one novel human leukocyte antigen (HLA) class I and class II alleles are described: seven HLA-A alleles, five HLA-C alleles, five HLA-B alleles and four HLA-DRB1 alleles. Seventeen (∼81%) of the 21 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Nine of these single nucleotide variants cause an amino acid substitution, while eight are silent substitutions. The remaining novel alleles differ from their most similar allele by two to three nucleotide substitutions. One novel HLA-C locus allele encodes an amino acid change at codon 10 that previously has not been reported to be polymorphic for this locus. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 24 and 156) and HLA-B (codons 40 and 115) loci.

Ninety-six novel HLA class I and II alleles identified in volunteers for the National Marrow Donor Program Registry in 2009.

Ninety-six novel human leukocyte antigen (HLA) class I and class II alleles are described from volunteers for the 'Be The Match Registry®': 15 HLA-A alleles, 11 HLA-C alleles, 36 HLA-B alleles and 34 HLA-DRB1 alleles. Sixty-eight (∼71%) of the 96 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Twenty-three of these single nucleotide variants are silent substitutions and one creates a non-expressed allele (B(*) 27:59N). The remaining novel alleles differ from their most similar allele by two to five nucleotide substitutions. One of the novel HLA-A alleles (A(*) 11:52Q) is of questionable expression because of a deletion of codon 11. Three of the novel alleles encode amino acid changes at codons 63 (HLA-C), 88 (HLA-B) and 79 (HLA-DRB1) which have not previously been reported to be polymorphic in these loci. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 115, 149 and 168), HLA-C (codon 90), HLA-B (codons 23, 44, 70, 120 and 153) and HLA-DRB1 (codon 88) loci.

Seventy-eight novel HLA class I and II alleles identified during routine registry typing in 2008 and 2009.

Seventy-eight novel human leukocyte antigen (HLA) class I and class II alleles are described: 19 HLA-A alleles, 30 HLA-C alleles, 21 HLA-B alleles, 7 HLA-DRB1 alleles and 1 HLA-DPB1 allele. Eight (∼10%) of the novel alleles were found in more than one individual and may be more common in the population. Sixty-two (∼80%) of the 78 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Twelve of these single nucleotide variants are silent substitutions and one creates a null allele (C*08:26N). One of the novel HLA-C alleles, C*03:58, is a hybrid that likely resulted from an intra-locus recombination. The remaining novel alleles differ from their most similar allele by two to seven nucleotide substitutions. Four of the novel HLA-C alleles encode amino acid changes at codons 41, 42, 55 or 200 which have not previously been reported to be polymorphic. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-A (codons 55 and 120), HLA-C (codons 151, 153 and 176), HLA-B (codons 31 and 84) and HLA-DRB1 (codon 47) loci.

World Marrow Donor Association framework for the implementation of HLA matching programs in hematopoietic stem cell donor registries and cord blood banks.

A major goal of the World Marrow Donor Association (WMDA) is to foster international transplants of hematopoietic stem cells through the establishment of guidelines and recommendations in this field. In this tradition, this study defines a comprehensive framework for HLA matching programs, which use intricate algorithms to rapidly select potential donors for a patient from a database and to present these donors in a prioritized list. Starting with the comparison of single HLA markers of the donor and the patient possibly obtained using different testing methodologies at different resolutions, the more complex matching of loci and phenotypes is inductively built up. The consensus of this international collaborative group describes the state of the art in the field and points out many important design options compatible with the best practice. This should help existing registries to review and validate the most critical part of their IT systems and newly created donor registries around the world to tackle one of their real challenges.

Sixty-five novel alleles at the HLA-A, -B, and -DRB1 loci identified from National Marrow Donor Program volunteer donors.

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.

An update to HLA nomenclature, 2010.

The WHO Nomenclature Committee for Factors of the HLA System met during the 15th International Histocompatibility and Immunogenetics Workshop in Buzios, Brazil in September 2008. This update is an extract of the main report that documents the additions and revisions to the nomenclature of human leukocyte antigen (HLA) specificities following the principles established in previous reports.

African Americans exhibit a predominant allele in the midst of extensive KIR2DL1 allelic diversity.

KIR2DL1 alleles were identified by DNA sequencing of the coding region from amplified genomic DNA from 100 random African Americans. The majority of individuals (97%) carried a KIR2DL1 locus. Allele KIR2DL1*00302 was found in 68% of individuals but KIR2DL1*00401, *002, *00303, *006, and *007 were also frequent. Eleven new alleles were described: KIR2DL1*00403, *01101, *01102, *012, *013N, *014, *015, *016, *017, *018, and *019. Nine of the novel alleles encoded amino acid substitutions located throughout the receptor; one allele carried a stop codon in the exon encoding the first extracellular domain.

The profile of KIR3DL1 and KIR3DS1 alleles in an African American population resembles that found in African populations.

KIR3DL1 and KIR3DS1 allele frequencies were determined by DNA sequencing of the complete coding regions from 100 random unrelated African Americans. Alleles 3DL1*01501 (29 individuals) and 3DL1*01502 (23 individuals) were most frequently observed in addition to 21 other known alleles and 7 new alleles: KIR3DL1*01503, *03102, *064, *065, *066, *067, and *068. Two of the new alleles (KIR3DL1*064, *065) created additional variants of the chimeric KIR3DL1/KIR3DL2 gene. KIR3DS1*01301 (94% of the gene positive individuals) and KIR3DS1*049N (11%) were identified in the 18 individuals carrying this gene. Three individuals appeared to carry a killer cell immunoglobulin-like receptor haplotype with KIR3DL1/S1 duplication. The profile of alleles resembles that found in African populations but also shows signs of admixture.

Standards, regulations and accreditation for registries involved in the worldwide exchange of hematopoietic stem cell donors and products.

The World Marrow Donor Association (WMDA) is working closely with other international organizations working in cellular therapy such as the Worldwide Network for Blood and Marrow Transplantation (WBMT) to develop and maintain global recommendations and requirements for a standardized practice. WMDA launched its registry accreditation program in September 2003. Since then, 17 of the 71 hematopoietic stem cell donor registries worldwide have been accredited. These accredited registries list over 80% of the hematopoietic stem cell donors and umbilical cord blood units listed in the database of BM Donors Worldwide. The goal of the WMDA Standards and Accreditation process is to protect donors while serving the needs of patients who are urgently seeking histocompatible donors in worldwide searches. These activities address the increasing governmental regulations regarding the quality and safety of stem cells collected internationally for national patients.

Monitoring the international use of unrelated donors for transplantation: the WMDA annual reports.

The World Marrow Donor Association Annual Reports describe the current status of the use of unrelated hematopoietic cell products worldwide. In 2008, almost 1.7 million individuals were recruited into unrelated stem cell donor registries and almost 78 000 cord blood units were added to the inventory increasing the total number of available stem cell donors worldwide to over 14 million. In 2008, there were 10 481 adult stem cell donations (3221 BM and 7260 PBSC donations) provided from stem cell donor registries in 38 countries. In 2008, 3529 cord blood products were provided from 21 countries. Although the number of BM donations has been stable over the past 10 years, donations of PBSCs and umbilical cord blood are increasing.