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The killer cell immunoglobulin-like receptors (KIR) mediate human natural killer (NK) cell cytotoxicity via activating or inhibiting signals. Although informative and functional haplotype patterns have been reported, most genotyping has been performed at resolutions that are structurally ambiguous. In order to leverage structural information given low-resolution genotypes, we performed experiments to quantify the effects of population variations, reference haplotypes, and genotyping resolutions on population-level haplotype frequency estimations as well as predictions of individual haplotypes. We genotyped 10,157 unrelated individuals in 5 populations (518 African American[AFA], 258 Asian or Pacific Islander[API], 8,245 European[EUR], 1,073 Hispanic[HIS], and 63 Native American[NAM]) for KIR gene presence/absence (PA), and additionally half of the AFA samples for KIR gene copy number variation (CNV). A custom EM algorithm was used to estimate haplotype frequencies for each population by interpretation in the context of three sets of reference haplotypes. The algorithm also assigns each individual the haplotype pairs of maximum likelihood. Generally, our haplotype frequency estimates agree with similar previous publications to within <5% difference for all haplotypes. The exception is that estimates for NAM from the U.S. showed higher frequency association of cB02 with tA01 (+14%) instead of tB01 (-8.5%) compared to a previous study of NAM from south of the U.S. The higher-resolution CNV genotyping on the AFA samples allowed unambiguous haplotype-pair assignments for the majority of individuals, resulting in a 22% higher median typing resolution score (TRS), which measures likelihood of self-match in the context of population-specific haplo- and geno-types. The use of TRS to quantify reduced ambiguity with CNV data clearly revealed the few individuals with ambiguous genotypes as outliers. It is observed that typing resolution and reference haplotype set influence haplotype frequency estimates. For example, PA resolution may be used with reference haplotype sets up to the point where certain haplotypes are gene-content subsets of others. At that point, CNV must be used for all genes. [ABSTRACT FROM AUTHOR]

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5~KIR2DS3S5 and KIR2DS3S5~KIR2DL1, and telomeric KIR3DL1~KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis. [ABSTRACT FROM AUTHOR]

Highlights • Favorable killer cell immunoglobulin-type receptor unrelated donors for transplantation may improve outcome. • Prospective testing did not enrich the donor options with favorable donors: too few were genotyped. • Donor search relying on additional factors beyond HLA may require extra effort and more donors considered. Abstract We previously reported that acute myelogenous leukemia (AML) transplants using killer cell immunoglobulin-type receptor (KIR) B haplotype better or best (≥2 B activating gene loci ± Cen B/B) unrelated donors (URDs) yield less relapse and better survival. In this prospective trial we evaluated 535 AML searches from 14 participating centers with centralized donor KIR genotyping for donor selection. This represented 3% to 48% of all AML searches (median 20%) per center, totaling 3 to 172 patients (median 22) per center. Donor KIR genotype was reported at a median of 14 days after request (≤26 days for 76% of searches). In 535 searches, 2080 donors were requested for KIR genotyping (mean 4.3 per search); and a median of 1.8 (range, 0 to 4.5) per search were KIR typed. Choosing more donors for confirmatory HLA and KIR haplotype identification enriched the likelihood of finding KIR better or best donors. The search process identified a mean of 30% KIR better or best donors; the success ranged from 24% to 38% in the 11 centers enrolling ≥8 patients. More donors requested for KIR genotyping increased the likelihood of identifying KIR better or best haplotype donors. Of the 247 transplants, 9.3% used KIR best, 19% used KIR better, and 48% used KIR neutral donors while 24% used a non–KIR-tested donor. KIR genotyping did not delay transplantation. The time from search to transplant was identical for transplants using a KIR-genotyped versus a non–KIR-genotyped donor. Prospective evaluation can rapidly identify KIR favorable genotype donors, but choosing more donors per search would substantially increase the likelihood of having a KIR best or better donor available for transplantation. Transplant centers and donor registries must both commit extra effort to incorporate new characteristics (beyond HLA, age, and parity) into improved donor selection. Deliberate efforts to present additional genetic factors for donor selection will require novel procedures. [ABSTRACT FROM AUTHOR]

Abstract 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. [ABSTRACT FROM AUTHOR]

Aim The killer-cell immunoglobulin-like receptor (KIR) gene family are involved in immune modulation during viral infection, autoimmune disease and in allogeneic stem cell transplantation. Most KIR gene diversity studies and their impact on the transplant outcome is performed by gene absence/presence assays. However it is well known that KIR gene allelic variations have biological significance. Allele level typing of KIR genes has been very challenging until recently due to the homologous nature of those genes and very long intronic sequences. PacBio RSII platform’s Single Molecule, Real-Time (SMRT®) sequencing, generates average long reads of 10 to 15 kb and allows us to obtain in-phase long sequence reads. We have developed a PCR assay for PacBio RSII platform in our lab for 3DL1 whole gene sequencing. This approach allows us to obtain allele level typing for 3DL1 genes and could serve as a model to type other KIR genes at allelic level. Methods DNA samples were obtained from 41 cell lines of Caucasian ethnicity and 97 volunteers with Korean and South Asian origin. Gene absence/presence assays were done on Illumina MiSeq platform using in-house protocol.We performed whole gene 3DL1 sequencing on the genomic DNA of samples using two gene-specific primers to get two amplicons (5.5 kb and 5.3 kb, respectively) that will span all the exons and most of the Intron 5. These amplicons were sequenced on the PacBio RSII platform. We analyzed the data based on current KIR DB v 2.6.0 using our in house program. Results A total of 123 out of 138 samples had 3DL1 gene by gene absence/presence assay. Based on the exon sequences alone, 123 samples had 47 of the 110 known alleles. However when we included intron sequences to our analysis only 23 sequences out of 47 unique alleles had a match to the database for intronic sequences. The remainder 24 sequences had novel variations in the introns. The majority were in intron 6. 3DL1 ∗ 002 that has stronger inhibitory receptor was found in 13 samples and mostly in CAU/EURO samples in this study. Some ethnic clustering of certain alleles (ie: 3DL1 ∗ 01501/2 group) were observed in Korean samples. Conclusion Whole gene sequencing of KIR genes is now feasible. Allelic typing of KIR genes will allow us to better dissect their modulatory role during immune response and their distribution among diverse populations. S. Yang: Stock Shareholder; Company/Organization; PacBio. [ABSTRACT FROM AUTHOR]