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The goals of the KIR component of the 17th International HLA and Immunogenetics Workshop (IHIW) were to encourage and educate researchers to begin analyzing KIR at allelic resolution, and to survey the nature and extent of KIR allelic diversity across human populations. To represent worldwide diversity, we analyzed 1269 individuals from ten populations, focusing on the most polymorphic KIR genes, which express receptors having three immunoglobulin (Ig)-like domains (KIR3DL1/S1, KIR3DL2 and KIR3DL3). We identified 13 novel alleles of KIR3DL1/S1, 13 of KIR3DL2 and 18 of KIR3DL3. Previously identified alleles, corresponding to 33 alleles of KIR3DL1/S1, 38 of KIR3DL2, and 43 of KIR3DL3, represented over 90% of the observed allele frequencies for these genes. In total we observed 37 KIR3DL1/S1 allotypes, 40 for KIR3DL2 and 44 for KIR3DL3. As KIR allotype diversity can affect NK cell function, this demonstrates potential for high functional diversity worldwide. Allelic variation further diversifies KIR haplotypes. We determined KIR3DL3 ∼ KIR3DL1/S1 ∼ KIR3DL2 haplotypes from five of the studied populations, and observed multiple population-specific haplotypes in each. This included 234 distinct haplotypes in European Americans, 191 in Ugandans, 35 in Papuans, 95 in Egyptians and 86 in Spanish populations. For another 35 populations, encompassing 642,105 individuals we focused on KIR3DL2 and identified another 375 novel alleles, with approximately half of them observed in more than one individual. The KIR allelic level data gathered from this project represents the most comprehensive summary of global KIR allelic diversity to date, and continued analysis will improve understanding of KIR allelic polymorphism in global populations. Further, the wealth of new data gathered in the course of this workshop component highlights the value of collaborative, community-based efforts in immunogenetics research, exemplified by the IHIW.

Aim The knowledge of complete haplotype structure and underlying genetic variation is often key in understanding gene function, phenotype, and medically relevant traits such as susceptibility to disease and individual response to drugs. However, genome wide SNP arrays and shotgun sequencing methods are limited in their ability to identify phased genetic variations at high resolution over extended localized regions. Methods To address this, we improved methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generation that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As specific applications, we targeted subregions of the human major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, two of the most polymorphic genomic regions in humans. Results For the MHC region, an 800 kb segment of the MHC class II region including the highly polymorphic DR, DQ, and DP genes was targeted. Complete phased sequences were obtained encompassing the DR4 common extended haplotype – a haplotype associated with autoimmune disorders - from four class II heterozygous individuals. For the KIR locus, which spans some 200 kb including tandem repeats of highly homologous polymorphic loci, we targeted several KIR haplotype heterozygous individuals. Conclusions Complete phased sequences for all haplotypes were obtained from all individuals with a high level of data accuracy. The results demonstrate the approach is amenable to cost-effective scale-up to generate scores to hundreds of phased chromosomal sequences of extended lengths that encompass targeted genomic regions associated with disease. [ABSTRACT FROM AUTHOR]

Aim: ARS identical alleles are considered high resolution matched for unrelated donor stem cell transplant; however, it is unknown if mismatches outside of the ARS region impact transplant outcome. We investigated ARS identical allele pairs, HLA-A∗02:01/09, HLA-B∗44:02/27, HLA-C∗07:01/06 and C∗07:01/18, to determine if these alleles exist on the same haplotype in the Be the Match Registry®. The goal was to identify cases for in vitro cellular assays of alloreactivity to ARS identical alleles. Methods: Over nine million donors on the Be the Match Registry were queried. Haplotypes were selected where both ARS alleles have been reported previously. These haplotypes were paired with common haplotypes within each race. High resolution sequence based typing was completed on 40 Caucasian donors for A∗02:01/09, 58 Caucasian donors for B∗44:02/27, 87 Asian Pacific Islander donors for C∗07:01/06, and 64 African American donors for C∗07:01/18. Results: A∗02:01/09 was evaluated in two haplotype pairs: 98% carried A∗02:01, 2% were neither A∗02:01 nor A∗02:09 and none carried A∗02:09. B∗44:02/27 was evaluated in two haplotype pairs: 69% were B∗44:02, 22% were B∗44:27 and 9% were neither B∗44:02 nor B∗44:27; however, B∗44:02 and B∗44:27 alleles were never found on the same haplotype. Within two haplotype pairs tested for C∗07:01/06, 98% of donors carried the C∗07:06 allele, 2% carried neither C allele of interest, while none carried C∗07:01. For the C∗07:01/18, eleven haplotype pairs were investigated, 53% of donors carried C∗07:18 and none carried C∗07:01. The remaining 47% of donors carried other C alleles. Conclusions: The results obtained from this study for A∗02:01/09, B∗44:02/27, C∗07:01/06 and C∗07:01/18 suggest that the chance of encountering an isolated mismatch between these ARS identical alleles on the haplotypes investigated in an unrelated donor transplant setting would be quite low. Work will continue to identify isolated ARS mismatches for evaluation of alloreactivity in in vitro cellular assays. [ABSTRACT FROM AUTHOR]

Aim: For hematopoietic stem cell transplantation (HSCT) HLA identical sibling donors or HLA matched unrelated donors are the most ideal donors. As 10/10 (HLA-A, -B, -C,-DRB1, -DQB1) matched donors, are not available for all patients, the identification of permissive/non-immunogenic mismatches is important to inmprove the outcome of HLA mismatched transplants. Alleles that share the amino acid sequence of the antigen recognition site (ARS)(exon 2 and 3 for HLA class I or exon 2 for HLA-DRB1) or differences outside the peptide binding groove or α-helices (as the HLA-C*03:03 vs C*03:04 mismatch) are good candidates for permissive mismatches as these differences are not likely to induce T cell alloreactivity. Methods: An inventory of the functional impact of such mismatches was performed by in vitro cellular assays including the Cytotoxic T Lymphocyte precursor test (CTLp) for HLA-class I allele differences and the MLC, IFN-γ elispot and Primed Lymphocyte Test (PLT) for HLA-DRB1 differences. Results: CTLp assays were performed for: HLA-A*23:17 vs 23:01 (differ at pos 283, exon 5), HLA-C*07:06 vs *07:01 (pos 307 & 324 exon 5 & 6), HLA-C*04:30 vs 04:01 (pos 327, exon 7), HLA-C*03:62 vs 03:03:01 (pos 197, exon 4), HLA-C*03:03 vs 03:04 (pos 91, exon 3) and HLA-B*35:02 vs 35:04 (pos 109, exon 3). The only positive CTLp test was for HLA-C*07:06 vs *07:01. DRB1*14:01 vs DRB1*14:54 differ at pos 112 and a 2nd mismatch of DRB3*02:01 vs DRB3*02:02, respectively, yielded complicated results. Five of 6 combinations tested did not respond in the MLC and 3 of 6 in the Elispot against the combined DRB1/DRB3 mismatch and only in one direction; DRB1*14:01/DRB3*02:01 against DRB1*14:54/DRB3*02:02. PLT results suggested that the DRB1* mismatch was responsible for the response. Conclusions: In conclusion, mismatches involving positions outside the ARS are not very immunogenic. However, some mismatches can lead to T cell reactivity in vitro. The impact of these mismatches on clinical outcome of HSCT remains to be established. [Copyright &y& Elsevier]

Aim: Recent data show that DP matching plays an important role in hematopoietic cell transplantation. Our previous work demonstrated that in European Americans haplotypic associations for the DP heterodimer are determined by near complete linkage disequilibrium between amino acid motifs on DPA1 (pos 31) and DPB1 (pos 85–87). We aimed to generate motif and allele-level haplotype frequencies (HF) for four race groups to extend these insights to minorities. Methods: We calculated DPA1∼DPB1 HF at allele and motif level from 16,802 Be the Match Registry® members typed by DNA methods, resolving phase, allelic and motif ambiguity using expectation maximization. Motif frequencies were calculated for position 31 on DPA1 and positions 85–87 on DPB1, which play a critical role in the P1 pocket of the peptide binding region. Normalized linkage disequilibrium (LD) was calculated on each haplotype (D′) and globally (Wn) for each population. Results: The top three allele-level haplotypes in all populations account for over 66% of the HF, with DPA1∗01:03 DPB1∗04:01 most common in the Caucasian (CAU), Asian Pacific Islander (API) and Hispanic (HIS) populations and DPA1∗02:02 DPB1∗01:01 in the African American (AFA) population. Wn for DPA1∼DPB1 allele associations for CAU, HIS, API, and AFA populations was .55, .45, .49, and .52 respectively. At the motif level there were only four possible haplotypes. M GPM and Q EAV were the two most common in every population. M GPM was the most common in CAU, HIS and API with frequencies 69%, 68% and 56% respectively, with D′= 0.97 for CAU (complete LD). The most common haplotype for AFA was Q EAV, at 48% and D′=.99. Conclusions: Low global LD was observed at the allele level for all populations. However, at the motif level haplotypes were in near complete LD suggesting that for the DPA1 DPB1 heterodimer the unit of selection for all populations is the combined amino acid motif rather than the allele, as previously observed in European Americans. [ABSTRACT FROM AUTHOR]