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This is an open access article under the CC BY license. The etiology of myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is unknown, but involvement of the immune system is one of the proposed underlying mechanisms. Human leukocyte antigen (HLA) associations are hallmarks of immune-mediated and autoimmune diseases. We have previously performed high resolution HLA genotyping and detected associations between ME/CFS and certain HLA class I and class II alleles. However, the HLA complex harbors numerous genes of immunological importance, and there is extensive and complex linkage disequilibrium across the region. In the current study, we aimed to fine map the association signals in the HLA complex by genotyping five additional classical HLA loci and 5,342 SNPs in 427 Norwegian ME/CFS patients, diagnosed according to the Canadian Consensus Criteria, and 480 healthy Norwegian controls. SNP association analysis revealed two distinct and independent association signals (p ≤ 0.001) tagged by rs4711249 in the HLA class I region and rs9275582 in the HLA class II region. Furthermore, the primary association signal in the HLA class II region was located within the HLA-DQ gene region, most likely due to HLA-DQB1, particularly the amino acid position 57 (aspartic acid/alanine) in the peptide binding groove, or an intergenic SNP upstream of HLA-DQB1. In the HLA class I region, the putative causal locus might map outside the classical HLA genes as the association signal spans several genes (DDR1, GTF2H4, VARS2, SFTA2 and DPCR1) with expression levels influenced by the ME/CFS associated SNP genotype. Taken together, our results implicate the involvement of the MHC, and in particular the HLA-DQB1 gene, in ME/CFS. These findings should be replicated in larger cohorts, particularly to verify the putative involvement of HLA-DQB1, a gene important for antigen-presentation to T cells and known to harbor alleles providing the largest risk for well–established autoimmune diseases.

Immune dysfunction plays a role in the development of Parkinson disease (PD). NK cells regulate immune functions and are modulated by killer cell immunoglobulin-like receptors (KIR). KIR are expressed on the surface of NK cells and interact with HLA class I ligands on the surface of all nucleated cells. We investigated KIR-allelic polymorphism to interrogate the role of NK cells in PD. We sequenced KIR genes from 1314 PD patients and 1978 controls using next-generation methods and identified KIR genotypes using custom bioinformatics. We examined associations of KIR with PD susceptibility and disease features, including age at disease onset and clinical symptoms. We identified two KIR3DL1 alleles encoding highly expressed inhibitory receptors associated with protection from PD clinical features in the presence of their cognate ligand: KIR3DL1*015/HLA-Bw4 from rigidity (pc = 0.02, odds ratio [OR] = 0.39, 95% confidence interval [CI] 0.23–0.69) and KIR3DL1*002/HLA-Bw4i from gait difficulties (pc = 0.05, OR = 0.62, 95% CI 0.44–0.88), as well as composite symptoms associated with more severe disease. We also developed a KIR3DL1/HLA interaction strength metric and found that weak KIR3DL1/HLA interactions were associated with rigidity (pc = 0.05, OR = 9.73, 95% CI 2.13–172.5). Highly expressed KIR3DL1 variants protect against more debilitating symptoms of PD, strongly implying a role of NK cells in PD progression and manifestation.

The human leukocyte antigen (HLA) genes are extremely polymorphic and are useful molecular markers to make inferences about human population history. However, the accuracy of the estimation of genetic diversity at HLA loci very much depends on the technology used to characterize HLA alleles; high-resolution genotyping of long-range HLA gene products improves the assessment of HLA population diversity as well as other population parameters compared to lower resolution typing methods. In this study we examined allelic and haplotype HLA diversity in a large healthy European American population sourced from the UCSF-DNA bank. A high-resolution next-generation sequencing method was applied to define non-ambiguous 3- and 4-field alleles at the HLA-A, HLA-C, HLA-B, HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 loci in samples provided by 2248 unrelated individuals. A number of population parameters were examined including balancing selection and various measurements of linkage disequilibrium were calculated. There were no detectable deviations from Hardy-Weinberg proportions at HLA-A, HLA-DRB1, HLA-DQA1 and HLA-DQB1. For the remaining loci moderate and significant deviations were detected at HLA-C, HLA-B, HLA-DRB3/4/5, HLA-DPA1 and HLA-DPB1 loci mostly from population substructures. Unique 4-field associations were observed among alleles at 2 loci and haplotypes extending large intervals that were not apparent in results obtained using testing methodologies with limited sequence coverage and phasing. The high diversity at HLA-DPA1 results from detection of intron variants of otherwise well conserved protein sequences. It may be speculated that divergence in exon sequences may be negatively selected. Our data provides a valuable reference source for future population studies that may allow for precise fine mapping of coding and non-coding sequences determining disease susceptibility and allo-immunogenicity.

The ‘Immunogenetics of Aging’ project is a component introduced in the 14th International HLA and Immunogenetics Workshop (IHIW) and developed further within subsequent workshops. The aim was to determine the relevance of immunogenetic markers, focusing on HLA, cytokine genes, and some innate immunity genes, for successful aging and an increased capacity to reach the extreme limits of life-span. Within the 17th IHIW we applied Next Generation Sequencing methods to refine further HLA associations at allele level in longevity, and to extend our knowledge to additional loci such as HLA-DQA1, HLA-DPB1 and HLA-DPA1. Analysis of relatively small number of healthy elderly and young controls from four populations showed that some HLA class I and class II alleles were significantly positively associated with healthy aging. Additionally we observed statistically significant differences in HLA allele distribution when the analysis was performed separately in elderly females and males compared to sex-matched young controls. Haplotypes, probably associated with better control of viral and malignant diseases were increased in the elderly sample. These preliminary NGS data could confirm our hypotheses that survival and longevity might be associated with selection of HLA alleles and haplotypes conferring disease resistance or susceptibility. Therefore HLA alleles and haplotypes could be informative immunogenetic markers for successful ageing.

Next-generation sequencing (NGS) at the HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1 and -DRB3/4/5 loci was performed on 282 healthy unrelated individuals from different major regions of Spain. High-resolution HLA genotypes defined by full sequencing of class I loci and extended coverage of class II loci were obtained to determine allele frequencies and also to estimate extended haplotype frequencies. HLA alleles were typed at the highest resolution level (4-field level, 4FL); with exception of a minor deviation in HLA-DPA1, no statistically significant deviations from expected Hardy Weinberg Equilibrium (HWE) proportions were observed for all other HLA loci. This study provides new 4FL-allele and -haplotype frequencies estimated for the first time in the Spanish population. Furthermore, our results describe extended haplotypes (including the less frequently typed HLA-DPA1 and HLA-DQA1 loci) and show distinctive haplotype associations found at 4FL-allele definition in this Spanish population study. The distinctive allelic and haplotypic diversity found at the 4FL reveals the high level of heterozygosity and specific haplotypic associations displayed that were not apparent at 2-field level (2FL). Overall, these results may contribute as a useful reference source for future population studies, for HLA-disease association studies as a healthy control group dataset and for improving donor recruitment strategies of bone marrow registries. HLA genotyping data of this Spanish population cohort was also included in the 17th International Histocompatibility and Immunogenetics Workshop (IHIW) as part of the study of HLA diversity in unrelated worldwide populations using NGS., This study was supported by Stanford Blood Center (as the official organizer and sponsor of the 17th International Histocompatibility and Immunogenetics Workshop-2017 (17th IHIW)) and by United States National Institutes of Health (NIH) grant U19NS095774.

Significance Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease with both familial and sporadic forms and a clear genetic component. In addition, underlying immunoregulatory dysfunction and inflammatory processes have been implicated in PD pathogenesis. In this study, deep sequencing of HLA genes, which encode highly variable cell surface immune receptors, reveals specific variants conferring either risk or protection in PD. Because a history of cigarette smoking is known to be protective in PD, we analyzed the interaction of these genetic variants with smoking history in PD patients and healthy controls and found that the genetic effects are modified by history of cigarette smoking. These results provide a molecular model that explains the unique epidemiology of smoking in PD., Parkinson’s disease (PD) is a neurodegenerative disease in which genetic risk has been mapped to HLA, but precise allelic associations have been difficult to infer due to limitations in genotyping methodology. Mapping PD risk at highest possible resolution, we performed sequencing of 11 HLA genes in 1,597 PD cases and 1,606 controls. We found that susceptibility to PD can be explained by a specific combination of amino acids at positions 70–74 on the HLA-DRB1 molecule. Previously identified as the primary risk factor in rheumatoid arthritis and referred to as the “shared epitope” (SE), the residues Q/R-K/R-R-A-A at positions 70–74 in combination with valine at position 11 (11-V) is highly protective in PD, while risk is attributable to the identical epitope in the absence of 11-V. Notably, these effects are modified by history of cigarette smoking, with a strong protective effect mediated by a positive history of smoking in combination with the SE and 11-V (P = 10−4; odds ratio, 0.51; 95% confidence interval, 0.36–0.72) and risk attributable to never smoking in combination with the SE without 11-V (P = 0.01; odds ratio, 1.51; 95% confidence interval, 1.08–2.12). The association of specific combinations of amino acids that participate in critical peptide-binding pockets of the HLA class II molecule implicates antigen presentation in PD pathogenesis and provides further support for genetic control of neuroinflammation in disease. The interaction of HLA-DRB1 with smoking history in disease predisposition, along with predicted patterns of peptide binding to HLA, provide a molecular model that explains the unique epidemiology of smoking in PD.

The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01, B∗45:07, B∗82:02:01 and C∗08:04:01 were notably more frequent in Cape Town compared to other populations reported in the literature. Class II loci had 21 different alleles of DPA1, 46 of DPB1, 27 of DQA1, 26 of DQB1, 41 of DRB1, 5 of DRB3, 4 of DRB4 and 6 of DRB5. The Cape Town cohort exhibited high degrees of HLA diversity and relatively high heterozygosity at most loci. Genetic distances between Cape Town and five other sub-Saharan African populations were also calculated and compared to European Americans.

The primary goal of the unrelated population HLA diversity (UPHD) component of the 17th International HLA and Immunogenetics Workshop was to characterize HLA alleles at maximum allelic-resolution in worldwide populations and re-evaluate patterns of HLA diversity across populations. The UPHD project included HLA genotype and sequence data, generated by various next-generation sequencing methods, from 4,240 individuals collated from 12 different countries. Population data included well-defined large datasets from the USA and smaller samples from Europe, Australia, and Western Asia. Allele and haplotype frequencies varied across populations from distant geographical regions. HLA genetic diversity estimated at 2- and 4-field allelic resolution revealed that diversity at the majority of loci, particularly for European-descent populations, was lower at the 2-field resolution. Several common alleles with identical protein sequences differing only by intronic substitutions were found in distinct haplotypes, revealing a more detailed characterization of linkage between variants within the HLA region. The examination of coding and non-coding nucleotide variation revealed many examples in which almost complete biunivocal relations between common alleles at different loci were observed resulting in higher linkage disequilibrium. Our reference data of HLA profiles characterized at maximum resolution from many populations is useful for anthropological studies, unrelated donor searches, transplantation, and disease association studies. (c) 2021 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

International audience; With the aim to understand how next-generation sequencing (NGS) improves both our assessment of genetic variation within populations and our knowledge on HLA molecular evolution, we sequenced and analysed 8 HLA loci in a well-documented population from sub-Saharan Africa (Mandenka). The results of full-gene NGS-MiSeq sequencing compared with those obtained by traditional typing techniques or limited sequencing strategies showed that segregating sites located outside exon 2 are crucial to describe not only class I but also class II population diversity. A comprehensive analysis of exons 2, 3, 4 and 5 nucleotide diversity at the 8 HLA loci revealed remarkable differences among these gene regions, notably a greater variation concentrated in the antigen recognition sites of class I exons 3 and some class II exons 2, likely associated with their peptide-presentation function, a lower diversity of HLA-C exon 3, possibly related to its role as a KIR ligand, and a peculiar molecular diversity of HLA-A exon 2, revealing demographic signals. Based on full-length HLA sequences, we also propose that the most frequent DRB1 allele in the studied population, DRB1*13:04, emerged from an allelic conversion involving 3 potential alleles as donors and DRB1*11:02:01 as recipient. Finally, our analysis revealed a high occurrence of the DRB1*13:04-DQA1* 05:05:01-DQB1*03:19 haplotype, possibly resulting from a selective sweep due to protection to Onchorcerca volvulus, a prevalent pathogen in West Africa. This study unveils highly relevant information on the molecular evolution of HLA genes in relation to their immune function, calling for similar analyses in other populations living in contrasting environments.

The 17(th) International HLA and Immunogenetics Workshop (IHIW) conducted a project entitled “The Study of Haplotypes in Families by NGS HLA”. We investigated the HLA haplotypes of 1,017 subjects in 263 nuclear families sourced from five US clinical immunogenetics laboratories, primarily as part of the evaluation of related donor candidates for hematopoietic stem cell and solid organ transplantation. The parents in these families belonged to five broad groups – African (72 parents), Asian (115), European (210), Hispanic (118) and “Other” (11). High-resolution HLA genotypes were generated for each subject using next-generation sequencing (NGS) HLA typing systems. We identified the HLA haplotypes in each family using HaplObserve software that builds haplotypes in families by reviewing HLA allele segregation from parents to children. We calculated haplotype frequencies within each broad group, by treating the parents in each family as unrelated individuals. We also calculated standard measures of global linkage disequilibrium (LD) and conditional asymmetric LD for each ethnic group, and used untruncated and two-field allele names to investigate LD patterns. Finally we demonstrated the utility of consensus DNA sequences in identifying novel variants, and confirming them using HLA allele segregation at the DNA sequence level.

The Mestizos of Oaxaca resulted from the admixture of Zapotecan Natives with Spaniards and Africans. We selected 112 donors from Oaxaca and applied next-generation sequencing to characterize exon and intron variants in complete or extended HLA genes. Some alleles found, are unique to Mexican Natives and most likely will be absent in most major ethnicities, namely: Caucasians, Africans or Asians. Among these are HLA-A*68:03:01, HLA-A*68:05:01, HLA-C*03:04:01:02, HLA-C*15:09, HLA-C*3:05, HLA-C*03:06:01, HLA-B*39:05:01, HLA-B*35:14:01, HLA-B*35:12:01, HLA-B*35:43:01, HLA-B*40:05, HLA-B:40:08, HLA-B*51:02:01, HLA-B*35:24:01 and HLA-B*39:08. HLA-DQA1*05:05:01:05 and some HLA-DRB1 alleles were only present in Amerindians/Mestizos. Three haplotypes are unique to Mexican Natives, five to Middle-Eastern and Sephardi-Jews. We detected a novel HLA-DQA1*04:01:01 exon 4 variant. Any novel allele may have been positively selected to enlarge the peptide-binding repertoire, and some, like HLA-B*39:02:02 and HLA-B*39:05:01 were found with unique haplotype associations, suggesting convergent evolution events and/or allele lineage diversification. The allele frequencies were fairly evenly distributed in most HLA loci with the exception of HLA-DPB1. The application of NGS in Oaxaca is novel and will lead to better use in the clinical setting. It offers deep knowledge on the population structure, origins, migration, and discovery of new alleles and haplotypes that other techniques did not achieve.

Background: The association between HLA-DRB1*15:01 with multiple sclerosis (MS) susceptibility is well established, but the contribution of the tightly associated HLA-DRB5*01:01 allele has not yet been completely ascertained. Similarly, the effects of HLA-DRB1*04:01 alleles and haplotypes, defined at the full-gene resolution level with MS risk remains to be elucidated. Objectives: To characterize the molecular architecture of class II HLA-DR15 and HLA-DR4 haplotypes associated with MS. Methods: Next-generation sequencing was used to determine HLA-DQB1, HLA-DQA1, and HLA-DRB1/4/5 alleles in 1403 unrelated European-American patients and 1425 healthy unrelated controls. Effect sizes of HLA alleles and haplotypes on MS risk were measured by odds ratio (OR) with 95% confidence intervals. Results: HLA-DRB1*15:01:01:01SG (OR = 3.20, p Conclusion: HLA-DR15 haplotypes, including genomic variants of HLA-DRB5, and HLA-DR4 haplotypes affect MS risk.

Many patients lack a fully HLA-matched donor for hematopoietic cell transplantation (HCT), and HLA mismatch is typically associated with inferior outcomes. Total lymphoid irradiation and antithymocyte globulin (TLI-ATG) is a nonmyeloablative conditioning regimen that is protective against graft-versus-host disease (GVHD), and we hypothesized that the protective effect would extend beyond HLA-matched donors. We report outcomes for all consecutively transplanted patients at Stanford University from December 2001 through May 2015 who received TLI-ATG conditioning and HCTs from 8 to 9 out of 10 HLA-mismatched unrelated donors (MMUDs, N = 72) compared with 10 out of 10 HLA-matched unrelated donors (MUDs, N = 193). The median age of the patients was 60 years with a median follow-up of 2 years, and there was a similar distribution of lymphoid and myeloid malignancies in both cohorts. There were no significant differences between MMUD and MUD cohorts in overall survival (46% vs 46% at 5 years, P = .86), disease-free survival (38% vs 28% at 5 years, P = .25), nonrelapse mortality (17% vs 12% at 2 years, P = .34), acute GVHD grades III-IV (6% vs 3% at day +100, P = .61), or chronic GVHD (39% vs 35% at 5 years, P = .49). There was a trend toward less relapse in the MMUD cohort (45% vs 60% at 5 years, hazard ratio: 0.71, P = .094), which was significant for patients with lymphoid malignancies (29% vs 57% at 5 years, hazard ratio: 0.55, P = .044). Achieving full donor chimerism was strongly associated with lower relapse rates. TLI-ATG conditioning may overcome the traditionally poorer outcome associated with HLA-mismatched donors and may be particularly well suited for patients with lymphoid malignancies who lack HLA-matched donors.

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17(th) International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17(th) IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.

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 Genetic recombination occurs at specific hotspots within the MHC, leading to widely dispersed block-like structures; the alpha block containing HLA-A, beta block (HLA-C and HLA-B) and delta block (HLA-DR and HLA-DQ). Linkage disequilibrium (LD) is much stronger within than across these blocks. The HLA-A locus (most telomeric) has weaker LD with extended HLA haplotypes. Additionally, a recombination hotspot exists between HLA-DP genes and other class II loci, which leads to an HLA-DPB1 match rate of only 10–15% in 10/10 HLA-matched donor/recipient pairs. This report describes an extended 9 locus HLA haplotype found in 2 HCT and 1 kidney transplant recipients and their potential donors (8, 2 and 1 donors, respectively). Methods 1. Case review of 2 unrelated HCT recipients, both European Caucasian (CAU) and their related and unrelated potential donors. 2. Family study of HCT recipient and a sibling to identify the haplotype by segregation. 3. Determine frequency of observed haplotype in different populations through Haplostats.org (6 locus at 2 field resolution could yield higher estimate than the 9 locus 3 field resolution haplotype described here). 4. Searching lab database for incidence of this haplotype. Results All identified cases shared the following haplotype HLA-A*31:01:02∼C*07:02:01∼B*07:02:01∼DRB5*01:01:01∼DRB1*15:01:01∼DQA1*01:02:01∼DQB1*06:02:01∼DPA1*01:03:01∼DPB1*04:01:01. Although most alleles in this haplotype are fairly common, with HLA-A*31:01 least common (∼5%), Haplostats indicated that the haplotype HLA-A*31:01∼C*07:02∼B*07:02∼DRB5*01:01∼DRB1*15:01∼DQB1*06:02 ranks 61898th & 170477th in CAU & African Americans (AFA), respectively and is not seen in other populations. Searching our laboratory database for subjects A31 positive, 382 subjects out of 8340 typed between 2015 & 2017 were identified. Of those, 40 were positive for the haplotype HLA-A*31:01∼C*07:02∼B*07:02∼DRB5*01:01∼DRB1*15:01∼DQB1*06:02 and of these 17 were typed for HLA-DPB1. All were positive for the entire extended 9 locus haplotype and were either CAU (n = 6) or of unknown ethnicity (n = 11). Conclusions Although the 6 locus haplotype portion of this extended haplotype seems fairly uncommon, the extended 9 locus subset of that haplotype seems to be conserved especially in Caucasians.

Aim The primary goal of the 17th IHIWS was to advance the field of Histocompatibility and Immunogenetics research as well as clinical practices through the application of NGS technologies. Analyses of very-high resolution NGS HLA profiles sampled from different populations will be useful for reconstructing human history and may improve match predictions for unrelated donor selection algorithms. Methods In the unrelated population HLA diversity project, 15 laboratories from 10 different countries contributed HLA genotype data generated by various NGS methods. HLA population data included well-defined large datasets from the USA and East Asia and smaller samples from Europe, Australia, and Western Asia. Participants uploaded data in HML/XML format into the 17th IHIW database, data was systematically reviewed, and statistical analysis was performed using the genetic software PyPop v.0.7.0 to estimate; (i) allele frequencies, (ii) heterozygosity, (iii) conformity to HWE, (iv) selective neutrality, (v) 2-locus haplotypes and (vi) LD. The software BIGDAWG was used to estimate extended haplotype frequencies by an EM algorithm. Inter-population variance was determined using Nei’s genetic distances which were then used to construct phylogenetic dendrograms. Results Amongst each population, test of selective neutrality often revealed an excess of heterozygous compared with neutral expectations. A comparison of the HLA genetic diversity estimated at the 4 and 2 fields revealed that diversity at the majority of loci, particularly for populations of European descent, was lower at the 2-field resolution. African descent populations had a lower number of distinct HLA alleles at class I and II loci compared to other groups but exhibited higher levels of heterozygosity. Two-locus haplotype analyses revealed distinct HLA associations at the 4 − field. Analyses of extended haplotype frequencies, estimated both in the presence and absence of DP loci, showed unique haplotypes that were common to each ethnic group although some haplotypes were shared amongst different ethnic groups. Conclusions This workshop project provided a vast amount of new information about HLA genetic diversity at the intronic level and will be a useful resource for anthropological studies, disease association studies and transplantation.

Aim HLA alleles are observed in specific haplotypes because of linkage disequilibrium between particular alleles. Multiple sclerosis (MS) has been associated with HLA genes and haplotypes. Our goal was to identify candidate HLA gene alleles and haplotypes that are susceptible or protective to MS. Methods We applied a high-throughput, high-resolution NGS method to type 11 HLA loci in 481 trio families comprising of 962 unaffected parental controls and 481 children diagnosed with MS. We developed an in-house computer program named HaplObserve that builds haplotypes by comparing offspring and parents HLA genotypes from nuclear families. HLA haplotypes were built from the trio families using HaplObserve that also allows tracing HLA allele/haplotype transmissions from the parents to the offspring. We dissected the extended haplotypes generated into smaller haplotypes and alleles, then explored associations with MS using standard transmission disequilibrium test (TDT) and multiallelic TDT analyses. Results We built a pipeline to systematically analyze HLA alleles and various segments of haplotypes using standard TDT and multiallelic TDT tests. Standard TDT and multiple allelic TDT showed that the DRB5∗01:01:01 ∼ DRB1∗15:01:01:01 haplotype was significantly predisposing (standard TDT: p 2.2e−16). We identified that DRB1∗01:01:01 ∼ DQB1∗05:01:01:03 (standard TDT: p = 8.63e−06), C∗04:01:01:01 ∼ B∗35:01:01:02 (p = 0.001645) and A∗02:01:01:01 ∼ C∗03:04:01:01 ∼ B∗40:01:02 (p = 0.008318) haplotypes were protective. Our analysis indicated that DRB1∗11:01:01:01 (p = 0.001738), B∗27:05:02 (p = 0.0004531), B∗38:01:01 (p = 0.0031953) and C∗07:04:01:01 (p = 0.004126) were protective. Conclusions We have shown the suitability of the HaplObserve to efficiently build haplotypes from NGS data in a large number of families, as well as the efficacy and power of TDT analysis for MS association studies. By comparing statistical significance of various haplotype segments, we were able to fine-map MS candidate alleles/haplotypes while eliminating false signals resulting from ‘hitchhiking’ alleles.

BACKGROUND: A serious constraint in the investigation of the human platelet antigen (HPA) status of potential neonatal alloimmune thrombocytopenia (NAIT) cases is the limited amount of DNA available from the neonates. Whole genome amplification (WGA) of these DNA samples could overcome this problem, but requires validation to ensure that it is sufficiently sensitive and accurate before its application in a clinical diagnostic setting. STUDY DESIGN AND METHODS: This study has validated the use of WGA DNA for HPA-1, -2, -3, -4, -5, and -15 genotyping with a panel of six controls and 13 previously HPA-typed samples from neonates together with parental DNA, using a 5′-nuclease (TaqMan) assay. WGA was performed using titrated amounts of genomic and WGA DNA template. HPA typing was performed on genomic and amplified DNA using a 5′-nuclease assay or polymerase chain reaction with sequence-specific primers (PCR-SSP). RESULTS: WGA DNA yields were in the suggested range of 400× to 800×, as assessed by spectrophotometry and gel analysis, and did not require further purification. HPA genotyping showed 100 percent concordance when using down to 5 ng of genomic or WGA template. CONCLUSION: This study demonstrates that WGA can be used for HPA typing using PCR-SSP or plate-based 5′-nuclease assays. The use of WGA for HPA typing in clinical samples from NAIT patients was validated with 100 percent concordance, and it is suggested that this technology can be used for other analyses where DNA amounts are limited.

High levels of fetal haemoglobin (HbF) are protective in beta-haemoglobinopathies. The proportion of erythrocytes containing HbF (F-cells, FC) was measured in healthy adults of African and Caucasian ancestry to assess the feasibility of localizing genes for the FC trait using admixture mapping. Participants were Afro-Caribbean (AC) blood donors and residents of a rural enclave with a history of recent German admixture (Afro-German, AG) recruited in Jamaica, and Caucasian Europeans recruited in Jamaica and the UK. FC levels were significantly different between groups (P < 0.001); the geometric mean FC level in the AC sample (n = 176) was 3.75% [95% confidence interval (CI) 3.36-4.18], AG sample (n = 631) was 2.77% (95% CI 2.63-2.92), and among Caucasians (n = 1099) was 3.26% (95% CI 3.13-3.39). After adjustment for age, sex, haemoglobin electrophoresis pattern, and HBG2 genotype, FC levels in the AC group remained significantly different (P < 0.001) from those in the Caucasian and the AG group but the difference between the Caucasian and AG groups became non-significant (P = 0.46) despite substantial differences in average ancestry. The data confirm ethnic differences in FC levels and indicate the potential usefulness of these populations for admixture mapping of genes for FC levels.

BACKGROUND: The outcome of clinical transplantation and a number of disease susceptibilities show very strong associations with genetic variants within the major histocompatibility complex, particularly in the human leukocyte antigen (HLA) genes. A problem with many association studies is the lack of sufficient DNA to perform multiple genetic analyses, particularly with transplantation outcomes where donor and recipient DNA are often in short supply. This study assesses whether a multiple-strand displacement whole genome amplification (WGA) method could generate sufficient template of high quality to perform unbiased amplification for analysis of the HLA-A, -B, -C, -DRB1, and -DQB1 genes. STUDY DESIGN AND METHODS: A panel of DNA samples from various biological sources was subjected to WGA reaction using Φ29 DNA polymerase. The HLA genotypes were subsequently determined using standard polymerase chain reaction (PCR)-based methods including sequence-specific oligonucleotide probes (PCR-SSOP, Luminex, Luminex Corp.) and sequence-based typing (PCR-SBT). WGA products and original DNA samples were used to determine the sensitivity of the Luminex assay; in addition, reamplified WGA products were also genotyped. RESULTS: The WGA templates, as well as serially amplified DNA for two successive rounds, yielded HLA genotypes fully concordant with those determined for the original DNA samples. WGA products and original DNA gave reproducible HLA-DQB1 genotypes with 100 to 10 ng of template. Purification of the WGA products was required for successful PCR-SBT, but not for the PCR-SSOP method. CONCLUSION: Our study suggests that WGA can be a reliable method for generating unlimited DNA for medium- or high-resolution HLA typing using the techniques described above.

Aim HLA alleles are observed in specific haplotypes because of linkage disequilibrium between particular alleles. HLA typing using Next Generation Sequencing (NGS) platforms allows generating nearly full-length gene sequences and detecting novel alleles. NGS technologies also permit high-throughput HLA typing for many samples in a cost effective manner. To establish HLA haplotypes from families for the 17th International HLA and Immunogenetics Workshops (IHIW), we developed the Family HLA Haplotype (FamHLAHap) software that compares offspring and parents’ HLA genotypes and builds haplotypes in an automated fashion. To optimize and validate the software, we generated haplotypes from 200 quartet and 1500 trio nuclear families. Methods The quartet families include both parents and two children, and occasional extended family members. A trio contains a child who is diagnosed with multiple sclerosis (MS) and two unaffected parental controls. DNA sequencing library was prepared using MIA FORA NGS kits, and sequenced on either MiSeq or NextSeq platforms. HLA types were obtained using MIA FORA software. The FamHLAHap software reports HLA haplotypes according to the order of genes along the chromosome to facilitate identifying potential meiotic recombination, and also compares to the previously established haplotypes. Results We built haplotypes from a total of 1700 families using the FamHLAHap software. Typing of DPB1 locus by NGS-shotgun sequencing and Sanger often results in ambiguous genotypes because of lack of phasing between exons 2 and 3. In the present study virtually all ambiguities were resolved through the analyses of segregation of sequences. Conclusions We have shown the suitability of the FamHLAHap software to efficiently build haplotypes from NGS data in a large number of families included in the 17th IHIW. Understanding haplotype structures allows delineating evolutionary pathways of the HLA region that include both divergent and consequent evolution. The haplotypes generated from family study provide valuable resources for hematopoietic stem cell donor match prediction, and identification of haplotypes associated with autoimmune diseases.

HLA alleles are observed in specific haplotypes, due to linkage disequilibrium (LD) between particular alleles. Haplotype frequencies for alleles have been established for specific ethnic groups and racial categories from population studies. HLA typing using Next Generation Sequencing (NGS) platforms allows generating full-length gene sequences, obtaining four-field HLA types and detecting novel alleles. NGS technologies also permit high-throughput HLA typing for large numbers of samples in a cost effective manner. Aim To establish HLA haplotypes with four-field resolution from quartet (two children and two parents) families. Methods We selected quartet families from our clinical database. Extended family members, aunt, uncle, cousin and grandparents, were also included if available. HLA types generated using SBT, SSP and SSO were reviewed for selecting subjects. Two children were selected if they did not share two HLA haplotypes. DNA was processed using MIA FORA NGS kits, and sequenced on either MiSeq or NextSeq sequencer. HLA types were obtained using MIA FORA 2.0 software. HLA haplotypes were built by comparing children’s and parents’ HLA types. The HLA haplotypes were reported according to the order of genes along the chromosome to identify potential meiotic recombination, and were also compared to the previously established haplotypes. Results Of 100 selected families, we sequenced 50 families. We built 200 haplotypes consisting of four-field HLA types from these. We developed automated haplotype building software. It was feasible to resolve almost all ambiguities including phase ambiguities and to generate completely phased full-length gene sequences from the consensus sequences generated from these studies. Conclusions The haplotypes generated from quartet families will be important resources for hematopoietic stem cell donor match prediction and play a role as reference haplotypes for expanding a collection of HLA haplotypes.

Introduction NGS allows the generation of a large number of clonal sequences in a single run. We can handle the tight MHC Δ between genes, polymorphism and heterozygosity. We showed a non-homogenous combination of Amerindian, Caucasian and African genes in Mestizos, depending upon the region. Oaxaca, is additionally, admixed with 18 Native local groups. Methods 197 donors from Oaxaca, registered at DONORMO were chosen. HLA typing was achieved by amplification by long range PCR with full gene coverage for class I loci and large coverage of class II loci including all exons with exception of exon-2. All amplicons for each sample were pooled and tagged for sequencing and sample identification; 24 samples at a time were pooled and sequenced with an Illumina Miseq instrument. Analyses of sequences was determined by the application of ad hoc develop programs that utilized three logic components including mapping of sequences to references and by performing full assembly. With exception of DPB1, genotypes at all loci, were assigned unambiguously. Allele (AF) and (HF) frequencies were assessed with the Arlequin 3.5.1.2. Results We show here some prevalent alleles (AF > 6%): A ∗ 02:01:01, ∗ 24:02:01:01; B ∗ 39:05:01e,39:02:02e1(AF > 8%); C ∗ 07:02:01:01, ∗ 04:01:01:01(AF > 18%); DRB1 ∗ 04:07:01, ∗ 08:02:01, ∗ 16:02:01e2(AF > 7%); DRB3 ∗ 02:02:01:02, ∗ 01:01:02e3(AF > 20%); DRB4 ∗ 01:03:01:01, ∗ 01:03:01:01/03(AF = 25%); DRB5 ∗ 02:02e, ∗ 01:01:01e1(AF > 20%); DQA1 ∗ 03:01:01, ∗ 04:01:01e- IV(AF > 9%); DQB1 ∗ 03:02:01, ∗ 03:01:01 (AF > 13%); DPA1 ∗ 01:03:01:05, ∗ 01:03:01:02(AF > 24%); DPB1 ∗ 04:02:01:02, ∗ 04:01:01:01; ∗ 04:02:01:02-IV, (AF > 10%). Of 3516 A ∗ -B ∗ -C ∗ -DRB1 ∗ -DQB1 ∗ -DQA1 ∗ haplotypes ( > 1.5%): 68:03-39:05-07:02-04:07-03:02-03:01; 02:01-39:02-07:02-04:07-03:02-03:01 and of 1184 DRB1 ∗ -DQB1 ∗ -DQA1-DPB1 ∗ -DPA1 ∗ : 04:07-03:02-03:01-04:02;01-03; 08:02-04:02-04:01-04:02-01;03, prevailed. Conclusions Analyzing the whole gene, will lead to a better understanding of the role of HLA in transplantation and disease and NGS brings about less ambiguities. Some issues will be solved establishing a complete database of HLA alleles and will overcome the present errors in assembling single reads to allelic sequences. Many allele frequency estimation errors exist in population frequencies that NGS will clarify, as shown in this group. M. Mindrino : Employee; Company/Organization; President . M. Fernandez-Vina : Consultant; Company/Organization; NGS .

Aim Africans represent the most genetically diverse population in the world, but have not been as well studied with respect to their HLA types compared to the inhabitants of western countries. Thus it was of interest to apply our high-throughput HLA sequencing technology to a cohort of 403 healthy adolescents from Cape Town, South Africa, as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with Stanford University. Methods Cryopreserved PBMC samples from 403 adolescents were collected and shipped to Stanford University from Cape Town. High resolution HLA typing was performed using MIA FORA NGS kit and analysis software. The whole gene coverage included HLA-A, -B, -C, -DPA1, -DQA1, and -DQB1; all exons and introns for -DRB1 and -DRB3/4/5 except partial coverage for exon 6 and intron 1; and all exons and introns between exons 2 and 4 for HLA-DPB1. NGS sequencing libraries were prepared using a semi-automated protocol and sequenced in high-throughput format on the Illumina NextSeq platform. HLA allele candidates were computed and final HLA typing was confirmed using MIA FORA NGS analysis software. Results HLA typing revealed a unique population, including unusual haplotypes, and novel alleles not previously reported in the IMGT/HLA database. The frequency of HLA-A, -B, -DRB1 haplotypes in Cape Town were compared to those represented in the US National Marrow Donor Program. As might be expected, 14 of the top 16 haplotypes in the Cape Town cohort were observed in the African American cohort. However there were important exceptions, including a frequent haplotype found in African American but not observed in Cape Town and two frequent Cape Town haplotypes not observed in African American samples. Conclusion High resolution HLA typing is a powerful method to characterize HLA allele and haplotype diversity in population studies. C. Wang: Consultant; Company/Organization; Immucor. M. Li: Employee; Company/Organization; Immucor. M. Fukushima: Employee; Company/Organization; Immucor. R. Kuehn: Employee; Company/Organization; Immucor. S. Krishnakumar: Employee; Company/Organization; Immucor. M. Mindrinos: Employee; Company/Organization; Immucor.

Aim Next generation sequencing of extended long-range HLA gene products provides better molecular characterization of HLA genes and improves the assessment of HLA population diversity than traditional HLA genotyping methods. In addition, accurate comprehensive analyses of HLA diversity would be expected to increase the efficiency of unrelated donor selection. In this study we assessed the genomic and haplotype diversity in European Americans (EA) and African Americans (AFA). Methods We used a high-throughput NGS HLA typing method developed by Sirona Genomics to genotype class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA, -DQB, -DRB1, -DRB3, -DRB4, -DRB5) loci in >2400 EAs and >840 AFAs unrelated healthy individuals. Sample libraries were sequenced using Illumina HiSeq and NextSeq instruments. Sequence data was analyzed and genotypes were assigned using the MIA FORA NGS HLA software. Statistical analyses was performed using the GENE[RATE] computer pipeline (hla-net.eu). Results The distribution of allele frequencies in both EA and AFA populations is similar across all loci with an extreme right skew in each case. Rare alleles and novel DPA and DQB alleles were detected in both populations. The AFA population contained higher levels of mean heterozygosity at class I loci and most class II loci compared to the EA population. Tests of selective neutrality revealed a significant excess of homozygosity in B, C, and DPB1 in EAs and significant excess of heterozygosity in A and DQB1 in the AFA group. Haplotype analyses of neighbouring loci pairs in positive LD, such as B ∼ C and B ∼ DRB1, revealed novel associations at the 4th field level of resolution. Conclusions Our findings show unique 4-field associations that were not apparent in results obtained using testing methodologies with limited sequence coverage and phasing. In addition, our results provide new 4-field allele and haplotype frequencies. Our data provides a useful reference source for future population studies that in turn may allow for precise fine mapping of coding and non-coding sequences determining disease susceptibility and allo-immunogenicity. S. Krishnakumar: Employee; Company/Organization; Immucor. C. Wang: Employee; Company/Organization; Immucor. M. Li: Employee; Company/Organization; Immucor. M.N. Mindrinos: Employee; Company/Organization; Immucor.

Fetal haemoglobin (HbF) is a major ameliorating factor in sickle cell disease. We investigated if a quantitative trait locus on chromosome 6q23 was significantly associated with HbF and F cell levels in individuals of African descent. Single nucleotide polymorphisms (SNPs) in a 24-kb intergenic region, 33-kb upstream of the HBS1L gene and 80-kb upstream of the MYB gene, were typed in 177 healthy Afro-Caribbean subjects (AC) of approximately 7% European admixture, 631 healthy Afro-Germans (AG, a group of African and German descendents located in rural Jamaica with about 20% European admixture), 87 West African and Afro-Caribbean individuals with sickle cell anaemia (HbSS), as well as 75 Northern Europeans, which served as a contrasting population. Association with a tag SNP for the locus was detected in all four groups (AC, P = 0.005, AG, P = 0.002, HbSS patients, P = 0.019, Europeans, P = 1.5 x 10(-7)). The association signal varied across the interval in the African-descended groups, while it is more uniform in Europeans. The 6q QTL for HbF traits is present in populations of African origin and is also acting in sickle cell anaemia patients. We have started to distinguish effects originating from European and African ancestral populations in our admixed study populations.

During human development, the switch from fetal to adult hemoglobin (Hb) is not complete with the residual gamma-globin expression being restricted to a subset of erythrocytes termed "F cells" (FC). Statistical analyses have shown the FC trait to be influenced by a common sequence variant (C--T) at position -158 upstream of the Ggamma-globin gene, termed the "XmnI-Ggamma polymorphism." The XmnI-Ggamma site is believed to be involved in the expression of the Ggamma-globin gene through interaction with transcription factors, and polymorphisms in the transcription factors could be influencing fetal Hb expression, conditional on the XmnI-Ggamma site. Using a two-locus model, in which the second locus was the known quantitative-trait locus (QTL) at the XmnI-Ggamma site, we showed suggestive linkage to chromosome 8q. A maximum single-point LOD score of 4.33 and a multipoint LOD score of 4.75 were found in a 15-20 cM region of chromosome 8q. A single-locus analysis failed to show linkage of FC to the region when the XmnI-Ggamma site was accounted for by removing its effects from the data or including it as a covariate. Results of the single-locus analysis were significant when the effects of the XmnI-Ggamma site were not accounted for in any way. The results of analysis in a large Indian kindred indicate that there is an interaction between the XmnI-Ggamma site and a QTL on chromosome 8q that is influencing the production of fetal Hb.