Killer Cell Immunoglobulin-like Receptors Alleles Research Articles

Genetic complexity of killer-cell immunoglobulin-like receptor genes in human pangenome assemblies.

The killer-cell immunoglobulin-like receptor (KIR) gene complex, a highly polymorphic region of the human genome that encodes proteins involved in immune responses, poses strong challenges in genotyping owing to its remarkable genetic diversity and structural intricacy. Accurate analysis of KIR alleles, including their structural variations, is crucial for understanding their roles in various immune responses. Leveraging the high-quality genome assemblies from the Human Pangenome Reference Consortium (HPRC), we present a novel bioinformatic tool, the structural KIR annoTator (SKIRT), to investigate gene diversity and facilitate precise KIR allele analysis. In 47 HPRC-phased assemblies, SKIRT identifies a recurrent novel KIR2DS4/3DL1 fusion gene in the paternal haplotype of HG02630 and maternal haplotype of NA19240. Additionally, SKIRT accurately identifies eight structural variants and 15 novel nonsynonymous alleles, all of which are independently validated using short-read data or quantitative polymerase chain reaction. Our study has discovered a total of 570 novel alleles, among which eight haplotypes harbor at least one KIR gene duplication, six haplotypes have lost at least one framework gene, and 75 out of 94 haplotypes (79.8%) carry at least five novel alleles, thus confirming KIR genetic diversity. These findings are pivotal in providing insights into KIR gene diversity and serve as a solid foundation for understanding the functional consequences of KIR structural variations. High-resolution genome assemblies offer unprecedented opportunities to explore polymorphic regions that are challenging to investigate using short-read sequencing methods. The SKIRT pipeline emerges as a highly efficient tool, enabling the comprehensive detection of the complete spectrum of KIR alleles within human genome assemblies.

High-resolution KIR and HLA genotyping in three Chinese ethnic minorities reveals distinct origins.

Polymorphism of killer-cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands impacts the effector activity of cytotoxic NK cell and T cell subsets. Therefore, understanding the extent and implications of KIR and HLA class I genetic polymorphism across various populations is important for immunological and medical research. In this study, we conducted a high-resolution investigation of KIR and HLA class I diversity in three distinct Chinese ethnic minority populations. We studied the She, Yugur, and Tajik, and compared them with the Zhejiang Han population (Zhe), which represents the majority Southern Han ethnicity. Our findings revealed that the Tajik population exhibited the most diverse KIR copy number, allele, and haplotype diversity among the four populations. This diversity aligns with their proposed ancestral origin, closely resembling that of Iranian populations, with a relatively higher presence of KIR-B genes, alleles, and haplotypes compared with the other Chinese populations. The Yugur population displayed KIR distributions similar to those of the Tibetans and Southeast Asians, whereas the She population resembled the Zhe and other East Asians, as confirmed by genetic distance analysis of KIR. Additionally, we identified 12.9% of individuals across the three minority populations as having KIR haplotypes characterized by specific gene block insertions or deletions. Genetic analysis based on HLA alleles yielded consistent results, even though there were extensive variations in HLA alleles. The observed variations in KIR interactions, such as higher numbers of 2DL1-C2 interactions in Tajik and Yugur populations and of 2DL3-C1 interactions in the She population, are likely shaped by demographic and evolutionary mechanisms specific to their local environments. Overall, our findings offer valuable insights into the distribution of KIR and HLA diversity among three distinct Chinese ethnic minority populations, which can inform future clinical and population studies.

Killer cell immunoglobulin-like receptor alleles influence susceptibility to occult hepatitis B infection in West African population.

Occult hepatitis B infection (OBI) is a public health problem in Burkina Faso. OBI represents a risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). OBI could be due to mutant viruses undetectable by HBsAg assays or a strong suppression of viral replication and gene expression under the pression of the host immune system. To investigate the role of killer cell immunoglobulin-like receptor (KIR) gene polymorphisms in patients with OBI in Burkina Faso compared to healthy and chronic hepatitis B subjects. A total of 286 participants was recruited, including 42 cases of OBI, 110 cases of chronic hepatitis B and 134 HBV negative subjects. SSP-PCR was performed to search for the presence of KIR genes. The HBV viral load was determined by qPCR. The frequencies of the activator gene KIR2DS5 (P=0.045) and the pseudogene KIR2DP1 (P<0.001) in patients with OBI were higher than those in patients with chronic hepatitis B. These genes are associated with susceptibility of occult hepatitis B infection. The frequencies of the inhibitory KIR gene KIR2DL3 (P=0.01) of patients with occult hepatitis B were lower than those in chronic hepatitis B patients. This gene KIR2DL3 is associated with protection against occult hepatitis B infection. Also, the frequencies of the inhibitory KIR genes KIR2DL2 (P<0.001), KIR2DL3 (P<0.001) and activators KIR2DS2 (P<0.001) in chronic hepatitis B patients were higher compared to the frequencies of the KIR genes in healthy subjects. These genes KIR2DL3, KIR2DL5 (A, B), KIR3DL3, KIR3DS1, KIR2DL2 and KIR2DS2 are thought to be genes associated with the susceptibility to OBI. The KIR2DS5 and KIR2DP1 genes could be associated with susceptibility to OBI. As for the KIR gene KIR2DL3 could be associated with protection against occult hepatitis B infection.

Efficient and accurate KIR and HLA genotyping with massively parallel sequencing data.

Killer cell immunoglobulin like receptor (KIR) genes and human leukocyte antigen (HLA) genes play important roles in innate and adaptive immunity. They are highly polymorphic and cannot be genotyped with standard variant calling pipelines. Compared with HLA genes, many KIR genes are similar to each other in sequences and may be absent in the chromosomes. Therefore, although many tools have been developed to genotype HLA genes using common sequencing data, none of them work for KIR genes. Even specialized KIR genotypers could not resolve all the KIR genes. Here we describe T1K, a novel computational method for the efficient and accurate inference of KIR or HLA alleles from RNA-seq, whole-genome sequencing, or whole-exome sequencing data. T1K jointly considers alleles across all genotyped genes, so it can reliably identify present genes and distinguish homologous genes, including the challenging KIR2DL5A/KIR2DL5B genes. This model also benefits HLA genotyping, where T1K achieves high accuracy in benchmarks. Moreover, T1K can call novel single-nucleotide variants and process single-cell data. Applying T1K to tumor single-cell RNA-seq data, we found that KIR2DL4 expression was enriched in tumor-specific CD8+ T cells. T1K may open the opportunity for HLA and KIR genotyping across various sequencing applications.

Association of iKIR-mismatch model and donor aKIRs with better outcome in haploidentical hematopoietic stem cell transplantation for acute myeloid leukemia.

Killer cell immunoglobulin like receptor (KIR) can trigger the alloreactivity of NK cells. However, there is no clear consensus as to their function. Here, we investigated the potential influence of KIR mismatch and KIR alleles on the outcome of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) in acute myeloid leukemia (AML) patients. Data from 79 AML patients treated with haplo-HSCT were retrospectively analyzed. HLA-C genotyping was determined by the PCR-rSSO method. KIR, HLA-A and HLA-B genotyping was performed by the PCR-SSP method. Cox proportional hazards model and Kaplan-Meier survival curves were used for analysis. Both KIR ligand mismatch (KLM) group and KIR receptor-ligand mismatch (RLM) group were associated with a decreased risk in aGVHD and relapse rate (RR), and better overall survival (OS) compared to the KIR ligand matching and receptor-ligand matching groups, respectively (aGVHD: KLM: p=0.047, HR:0.235; RLM: p<0.001, HR:0.129; RR: KLM: p=0.049, HR:0.686, RLM: p=0.017, HR:0.200;OS:KLM: p=0.012, HR: 0.298, RLM: p=0.021, HR:0.301). RLM was more accurate at predicting relapse and aGVHD compared with KLM (aGVHD: p=0.009; RR: p=0.039). Patients with greater number of donor activating KIRs (aKIR) had a lower incidence of aGVHD and relapse, and the benefits correlated with the increase in the number of donor aKIRs (aGVHD: p=0.019, HR:0.156; RR: p=0.037, HR:0.211). Patients with RLM and the highest number of donor aKIRs had the lowest RR, lowest incidence of aGVHD and best OS. Both KLM and RLM reduced the risk of aGVHD and relapse after haplo-HSCT in AML patients, and RLM showed superiority in predicting HSCT outcome. The synergistic effects of RLM and donor aKIRs can provide a better donor selection strategy to improve haplo-HSCT outcome in AML patients.

Donor with HLA-C2 is associated with acute rejection following liver transplantation in Southern Chinese.

Apart from presenting peptides to T cells, class I HLA molecules serve as ligands for killer cell immunoglobulin-like receptor (KIRs) and regulate the response of natural killer (NK) cells. The role played by HLA and KIR in the acute rejection (AR) following liver transplantation has been controversial. In this retrospective study, we assessed the influence of class I HLA alleles, HLA matching between donor-recipient pairs, recipient KIR and donor HLA ligands on AR following liver transplantation in southern Chinese. In total, 143 recipients and 78 donors obtained from a single transplant center were included in the study cohort. Thirty-three recipients with histologically confirmed AR were observed. We found that the incidence of AR did not correlate with donor or recipient class I HLA alleles and HLA matching. Neither recipient KIR gene nor the KIR genotype was associated with AR, moreover, high-resolution genotyping of 14 functional KIR genes of recipients showed that no KIR allele was independently associated with AR. However, the frequency of HLA-C2+ donor significantly increased in AR group compared with NAR group (52.9% vs. 24.6%, p=0.03). In the presence of HLA-C2 by the donor allograft, AR was more frequently observed in recipients with normal expressed KIR2DS4 (43.8% vs. 15.0%, p=0.03). Donor with HLA-C2 is therefore a major determinant of AR, which can confer risk effect in liver transplantation. Our findings can provide valuable clues for better understanding pathogenesis of AR and have important clinical implications in liver transplantation for Chinese.

Inhibitory KIR2DL2 receptor and HHV-8 in classic or endemic Kaposi sarcoma

KIR2DL2, an inhibitory Killer cell Immunoglobulin-like Receptor (KIR), has been shown to predispose to the development of several herpesvirus-associated diseases by inhibiting the efficiency of Natural Killer (NK) cells against virus-infected cells. The aim of this observational study was to assess the prevalence of KIR2DL2 and Human Herpes Virus 8 (HHV8) in patients affected with classical and endemic Kaposi sarcoma (KS), as well as in controls. Blood samples collected from 17 Caucasian, HIV-negative, immunocompetent patients affected with classical KS (c-KS), 12 African, HIV-negative patients with endemic KS (e-KS), 83 healthy subjects and 26 psoriatic patients were processed for genotypization by PCR for two KIR alleles, such as KIR2DL2 and KIR2DL3 and analyzed for HHV-8 presence. The totality of both c-KS and e-KS patients presented HHV-8 infection, whereas HHV8 was found in 26.9% of psoriatic subjects and 19.3% of healthy subjects. KIR2DL2 was found in the 76.5% of c-KS subjects, while the receptor was found in 41.7% of the e-KS group, 34.6% of psoriatic patients and 43.4% of healthy controls (p < 0.0001). A significantly higher prevalence of KIR2DL2 in c-KS patients than in all the other subjects was also confirmed comparing age-matched groups. Based on these results, the inhibitory KIR2DL2 genotype appears to be a possible cofactor which increases the risk of developing c-KS in HHV8-positive, immunocompetent subjects, while it seems less relevant in e-KS pathogenesis.

Association of Ikir-Mismatching and Donor Akirs with Better Outcomes in Haploidentical Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia

Objectives: Natural killer (NK) cells are the first donor-derived lymphocytes to be reconstituted after transplantation and play a critical role in improving transplantation outcomes. Their surface receptor, killer cell immunoglobulin like receptor (KIR), can trigger the alloreactivity of NK cells and have been shown to be protective for acute and chronic graft-versus-host disease (aGVHD, cGVHD) while retaining graft-versus-leukemia (GVL) effect. However, different results have been reported about KIR matching models and KIR alleles based on patient, donor and transplant characteristics, resulting in significant controversy about the best donor selection strategy. Here, we investigated the potential influence of KIR matching and KIR alleles on GVHD prophylaxis, overall survival (OS) and relapse rate (RR) of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) in acute myeloid leukemia (AML) patients. Methods：Data from 79 patients with AML treated with haplo-HSCT between May 2015 and May 2021 in the transplantation unit of the Fujian Medical University Union Hospital were retrospectively analyzed. The cohort included 49 male patients (62.0%) and 30 female (38.0%), with a median age of 25 years (1-68 years). KIR genotyping was performed by the PCR-SSO method. The amplicons were quantified on the Luminex 200 flow analyzer and analyzed using the Quick-Type for Lifecodes software for generating KIR data. HLA-A and -B alleles were typed by polymerase chain reaction using sequence-specific primers (PCR-SSP) (TBG, Taipei, Taiwan). HLA-C genotyping was determined by reverse hybridization with sequence specific oligonucleotides probes (rSSO) Line probe assay. Cox proportional hazards model and Kaplan-Meier survival curves were used for analysis. Results：At the time of transplantation, 49 cases (62.0%) were at CR1, while 30 (38.0%) were not. aGVHD occurred in 16 patients (20.3%) and recurrence arose in 10 patients (12.7%), 4 (5.1%) cases cGVHD was observed. After adjusting for age, disease-risk, disease-status, HLA-match, donor gender, conditioning regimen intensity and type of post-grafting GVHD prophylaxis, Cox regression analysis revealed that both KIR ligand-ligand mismatching (KLM) and KIR receptor-ligand matching (RLM) was associated with an decreased risk of aGVHD and relapse compared to KIR ligand-ligand matching and receptor-ligand matching group, respectively (aGVHD: KLM: p=0.047, RLM: p＜0.001; RR: KLM: p=0.049, RLM: p=0.017). Furthermore, RLM shows more accurate in prediction of relapse and aGVHD compared with KLM in both aGVHD and relapse. (aGHVD: p=0.009; RR: p=0.039). After taking activating KIR (aKIR) into consideration to compensate the defect of only inhibitory KIR (iKIR) dominant model, we found that patients with more donor activating KIRs can reach the lower incidence of aGVHD and relapse, and the benefit can gradually increase parallel with the increase in donor activating KIRs. (aGVHD：p=0.019；RR：p=0.037). Patients with both receptor-ligand mismatch and the most donor aKIRs can reach the lowest relapse, lowest incidence of aGVHD and best overall survival (OS). Conclusions: KIR-mismatch, both KLM and RLM significantly reduced the risk of aGVHD and relapse after halpo-HSCT in AML patients and RLM show more superiority in the prediction of HSCT outcome. The synergistic effects of receptor-ligand mismatch and more donor aKIRs can provide a better clinically applicable donor selection strategy to improve haplo-HSCT outcome in AML patients. DisclosuresHu: Astellas Pharma, Inc.: Research Funding.

The combinatorial diversity of KIR and HLA class I allotypes in Peninsular Malaysia.

Killer cell immunoglobulin-like receptors (KIRs) interact with polymorphic human leucocyte antigen (HLA) class I molecules, modulating natural killer (NK) cell functions and affecting both the susceptibility and outcome of immune-mediated diseases. The KIR locus is highly diverse in gene content, copy number and allelic polymorphism within individuals and across geographical populations. To analyse currently under-represented Asian and Pacific populations, we investigated the combinatorial diversity of KIR and HLA class I in 92 unrelated Malay and 75 Malaysian Chinese individuals from the Malay Peninsula. We identified substantial allelic and structural diversity of the KIR locus in both populations and characterized novel variations at each analysis level. The Malay population is more diverse than Malay Chinese, likely representing a unique history including admixture with immigrating populations spanning several thousand years. Characterizing the Malay population are KIR haplotypes with large structural variants present in 10% individuals, and KIR and HLA alleles previously identified in Austronesian populations. Despite the differences in ancestries, the proportion of HLA allotypes that serve as KIR ligands is similar in each population. The exception is a significantly reduced frequency of interactions of KIR2DL1 with C2+ HLA-C in the Malaysian Chinese group, caused by the low frequency of C2+ HLA. One likely implication is a greater protection from preeclampsia, a pregnancy disorder associated with KIR2DL1, which shows higher incidence in the Malay than in the Malaysian Chinese. This first complete, high-resolution, characterization of combinatorial diversity of KIR and HLA in Malaysians will form a valuable reference for future clinical and population studies.

Centromeric KIR AA Individuals Harbor Particular KIR Alleles Conferring Beneficial NK Cell Features with Implications in Haplo-Identical Hematopoietic Stem Cell Transplantation.

Simple SummaryWe have recently shown a broad disparity of Natural Killer (NK) cell responses against leukemia, highlighting good and bad responders resting on the Killer cell Immunoglobulin-like Receptors (KIR) and HLA genetics. In this study, we deeply investigated KIR2DL NK cell repertoire in combining high-resolution KIR allele typing and multicolor flow cytometry from a cohort of 108 blood donors. Our data suggest that centromeric (cen) AA individuals display more efficient KIR2DL alleles (L1*003 and L3*001) to mount a consistent frequency of KIR2DL+ NK cells and to confer an effective NK cell responsiveness. The transposition of our in vitro observations in T-replete haplo-identical Hematopoietic Stem Cell Transplantation (HSCT) context led us to observe that cenAA HSC grafts limit significantly the incidence of relapse in patients with myeloid diseases after T-replete haplo-identical HSCT. As NK cells are crucial in HSCT reconstitution, one could expect that the consideration of KIR2DL1/2/3 allelic polymorphism could help to refine scores used for HSC donor selection.We have recently shown a broad disparity of Natural Killer (NK) cell responses against leukemia highlighting good and bad responders resting on the Killer cell Immunoglobulin-like Receptors (KIR) and HLA genetics. In this study, we deeply studied KIR2D allele expression, HLA-C recognition and functional effect on NK cells in 108 blood donors in combining high-resolution KIR allele typing and multicolor flow cytometry. The KIR2DL1*003 allotype is associated with centromeric (cen) AA motif and confers the highest NK cell frequency, expression level and strength of KIR/HLA-C interactions compared to the KIR2DL1*002 and KIR2DL1*004 allotypes respectively associated with cenAB and BB motifs. KIR2DL2*001 and *003 allotypes negatively affect the frequency of KIR2DL1+ and KIR2DL3+ NK cells. Altogether, our data suggest that cenAA individuals display more efficient KIR2DL alleles (L1*003 and L3*001) to mount a consistent frequency of KIR2DL+ NK cells and to confer an effective NK cell responsiveness. The transposition of our in vitro observations in the T-replete haplo-identical HSCT context led us to observe that cenAA HSC grafts limit significantly the incidence of relapse in patients with myeloid diseases after T-replete haplo-identical HSCT. As NK cells are crucial in HSCT reconstitution, one could expect that the consideration of KIR2DL1/2/3 allelic polymorphism could help to refine scores used for HSC donor selection.

Estimation of German KIR Allele Group Haplotype Frequencies.

The impact of the highly polymorphic Killer-cell immunoglobulin-like receptor (KIR) gene cluster on the outcome of hematopoietic stem cell transplantation (HCST) is subject of current research. To further understand the involvement of this gene family into Natural Killer (NK) cell-mediated graft-versus-leukemia reactions, knowledge of haplotype structures, and allelic linkage is of importance. In this analysis, we estimate population-specific KIR haplotype frequencies at allele group resolution in a cohort of n = 458 German families. We addressed the polymorphism of the KIR gene complex and phasing ambiguities by a combined approach. Haplotype inference within first-degree family relations allowed us to limit the number of possible diplotypes. Structural restriction to a pattern set of 92 previously described KIR copy number haplotypes further reduced ambiguities. KIR haplotype frequency estimation was finally accomplished by means of an expectation-maximization algorithm. Applying a resolution threshold of ½ n, we were able to identify a set of 551 KIR allele group haplotypes, representing 21 KIR copy number haplotypes. The haplotype frequencies allow studying linkage disequilibrium in two-locus as well as in multi-locus analyses. Our study reveals associations between KIR haplotype structures and allele group frequencies, thereby broadening our understanding of the KIR gene complex.

Human leucocyte antigen but not KIR alleles and haplotypes associated with chronic HCV infection in a Chinese Han population.

The host immune system plays a key role in the elimination of infected cells which depend on killer-cell immunoglobulin-like receptors (KIR), human leucocyte antigen (HLA) class I molecules and their combinations. To evaluate the roles of HLAclass I, KIR genes and their combination in Chronic hepatitis C virus (HCV) infection (CHC), a total of 301 CHCs and 239 controls in a Chinese Han population were included for HLA and KIR genotyping using next-generation sequencing and multiplex PCR sequence-specific priming, respectively. The allele frequency of HLA-C*08:01 was significantly higher in the CHCs than that of the controls (0.088 vs. 0.040, OR=2.332, 95%CI: 1.361-3.996, p=0.022), while the frequencies of B*13:01 (0.032 vs. 0.084, OR=0.357, 95%CI: 0.204-0.625, p=0.009) and C*08:04 (0.008 vs. 0.038, OR=0.214, 95%CI: 0.079-0.581, p=0.022) were significantly lower in the CHCs. The frequencies of haplotype A*11:01-C*08:01 were higher in the CHCs (0.058 vs. 0.019, OR=3.096, 95%CI: 1.486-6.452, p=0.026), while haplotype B*13:01-C*03:04 were lower in the CHCs compared to the controls (0.028 vs. 0.071, OR=0.377, 95%CI: 0.207-0.685, p=0.012). No association of CHC with KIR genes, genotypes, or haplotypes, as well as HLA/KIR combinations was observed. Our results indicated that HLA-C*08:01 was a risk factor for CHC, while HLA-C*08:04 and HLA-B*13:01 were protective factors against CHC. Haplotypes HLA-A*11:01-C*08:01 could increase susceptibility to CHC, while HLA-B*13:01-C*03:04 could be protective against CHC in the Chinese Han population.

TypeLoader2: Automated submission of novel HLA and killer-cell immunoglobulin-like receptor alleles in full length.

The Immuno Polymorphism Database (IPD) databases provide global, curated repositories for information regarding polymorphisms of genes of the immune system, thereby generating immense value for the research and clinical communities. The advent of high‐throughput genotyping in immunogenetics has led to dramatically growing numbers of heretofore unknown HLA and lately also killer‐cell immunoglobulin‐like receptor (KIR) alleles, which are to be curated and deposited in the IPD‐IMGT/HLA and IPD‐KIR databases, respectively. It is highly desirable that these novel alleles are characterised and submitted in full length, and that known alleles are extended to cover the complete gene sequence.However, the manual annotation and submission of sequences to European Molecular Biology Laboratory's European Nucleotide Archive and the IPD‐IMGT/HLA and IPD‐KIR databases is time‐consuming and error‐prone. Here, we report the substantial extension of the HLA allele submission tool TypeLoader, which now also supports the annotation and submission of KIR alleles. To enable a more widespread use of this tool, we have made it available as a stand‐alone application that can easily be installed on standard Windows or Linux computers. Furthermore, an internal SQLite database was added to store a wide range of metadata about each allele. This allows TypeLoader2 to be used as a lab's central information platform for the annotation, curation and submission of full‐length HLA and KIR allele sequences. The software is freely available from GitHub (https://github.com/DKMS-LSL/typeloader).We hope that the increased convenience and scope of TypeLoader2 will foster the submission of more full‐length sequences to the IPD‐IMGT/HLA and IPD‐KIR databases, ultimately promoting the use of full‐length sequencing for genotyping both HLA and KIR.

Allele-Level KIR Genotyping of More Than a Million Samples: Workflow, Algorithm, and Observations.

The killer-cell immunoglobulin-like receptor (KIR) genes regulate natural killer cell activity, influencing predisposition to immune mediated disease, and affecting hematopoietic stem cell transplantation (HSCT) outcome. Owing to the complexity of the KIR locus, with extensive gene copy number variation (CNV) and allelic diversity, high-resolution characterization of KIR has so far been applied only to relatively small cohorts. Here, we present a comprehensive high-throughput KIR genotyping approach based on next generation sequencing. Through PCR amplification of specific exons, our approach delivers both copy numbers of the individual genes and allelic information for every KIR gene. Ten-fold replicate analysis of a set of 190 samples revealed a precision of 99.9%. Genotyping of an independent set of 360 samples resulted in an accuracy of more than 99% taking into account consistent copy number prediction. We applied the workflow to genotype 1.8 million stem cell donor registry samples. We report on the observed KIR allele diversity and relative abundance of alleles based on a subset of more than 300,000 samples. Furthermore, we identified more than 2,000 previously unreported KIR variants repeatedly in independent samples, underscoring the large diversity of the KIR region that awaits discovery. This cost-efficient high-resolution KIR genotyping approach is now applied to samples of volunteers registering as potential donors for HSCT. This will facilitate the utilization of KIR as additional selection criterion to improve unrelated donor stem cell transplantation outcome. In addition, the approach may serve studies requiring high-resolution KIR genotyping, like population genetics and disease association studies.

OR36 NGS Allele-level typing strategy for nine KIR genes

Aim Killer-cell Immunoglobulin-like Receptors (KIRs) are encoded by 17 genes in the Leukocyte Receptor Complex (LRC). These genes are highly polymorphic, on the level of structural variants, and copy-number variation. These characteristics make it challenging to design specific amplifications, and to perform reliable analysis of each KIR allele present in a sample. To enable allele-level resolution KIR genotyping, we developed an NGS-based strategy that involves whole gene amplification of KIR genes. Methods We developed KIR-gene specific amplification primers, with specificity for one or more KIR genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DS1, KIR3DL1, KIR3DL2, KIR3DL3 and KIR3DP1). Amplicons were processed in the NGSgo® (GenDx) library preparation procedure (GenDx) and paired-end sequenced on an Illumina MiSeq platform (2 × 151 bp). Data was genotyped with NGSengine® software (GenDx), using KIR database IPD-KIR 2.7.1. Results Multiple samples with known and unknown KIR genotypes were analyzed using this new strategy. KIR amplification, sequencing and subsequent typing was successful for the majority of samples. Many samples could be typed unambiguously, despite the fact that phasing was not fully accomplished due to the length of the genes (up to 17 kb). Remaining challenges in analysis are samples with KIR copy number variation and high degree of homology, which can hamper the data analysis. Conclusions KIR genotyping at allelic level is feasible using the short read sequencing technology of Illumina. This new typing strategy may be an attractive alternative to existing KIR genotype assays that only determine KIR gene content at a limited resolution, or complex sequencing methods aiming to sequence the entire LCR. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. K. van Ham: 5. Employee; Company/Organization; GenDx. S. Vendelbosch: 5. Employee; Company/Organization; GenDx. M.T. Penning: 5. Employee; Company/Organization; GenDx. E.H. Rozemuller: 5. Employee; Company/Organization; GenDx. 6. Stock Shareholder; Company/Organization; GenDx. 7. Other (Identify); Company/Organization; Founder of GenDx.

Next-generation sequencing technology a new tool for killer cell immunoglobulin-like receptor allele typing in hematopoietic stem cell transplantation

Killer cell Immunoglobulin-like Receptor (KIR) genes are a family of genes located together within the leukocyte receptor cluster on human chromosome 19q13.4. To date, 17 KIR genes have been identified including nine inhibitory genes (2DL1/L2/L3/L4/L5A/L5B, 3DL1/L2/L3), six activating genes (2DS1/S2/S3/S4/S5, 3DS1) and two pseudogenes (2DP1, 3DP1) classified into group A (KIR A) and group B (KIR B) haplotypes. The number and the nature of KIR genes vary between the individuals. In addition, these KIR genes are known to be polymorphic at allelic level (907 alleles described in July 2017). KIR genes encode for receptors which are predominantly expressed by Natural Killer (NK) cells. KIR receptors recognize HLA class I molecules and are able to kill residual recipient leukemia cells, and thus reduce the likelihood of relapse. KIR alleles of Hematopoietic Stem Cell (HSC) donor would require to be known (Alicata et al. Eur J Immunol 2016) because the KIR allele polymorphism may affect both the KIR+ NK cell phenotype and function (Gagne et al. Eur J Immunol 2013; Bari R, et al. Sci Rep 2016) as well as HSCT outcome (Boudreau et al. JCO 2017). The introduction of the Next Generation Sequencing (NGS) has overcome current conventional DNA sequencing method limitations, known to be time consuming. Recently, a novel NGS KIR allele typing approach of all KIR genes was developed by our team in Nantes from 30 reference DNAs (Maniangou et al. Front in Immunol 2017). This NGS KIR allele typing approach is simple, fast, reliable, specific and showed a concordance rate of 95% for centromeric and telomeric KIR genes in comparison with high-resolution KIR typing obtained to those published data using exome capture (Norman PJ et al. Am J Hum Genet 2016). This NGS KIR allele typing approach may also be used in reproduction and to better study KIR+ NK cell implication in the control of viral infections.

OR27 Detection of thousands of novel KIR alleles with high-throughput NGS typing via neXtype

Aim The killer cell immunoglobulin-like receptor (KIR) gene family is seen to play an important role in unrelated hematopoietic stem cell transplantations. Allele level KIR genotyping may improve the donor selection process. The IPD-KIR database currently comprises 753 alleles. With our newly established high-throughput workflow for KIR allele-level typing, we discovered a vast number of previously unknown variations of KIR alleles. Methods We perform cost efficient KIR allele level genotyping based on a paired-end short-amplicon approach by NGS using Illumina HiSeq 2500 instruments. The amplicons cover exons 3, 4, 5, 7, 8 and 9 of each KIR gene. Our neXtype software was adapted to report KIR genotyping results at allelic Level. Results Starting in October 2016, we added KIR allele-level typing to the DKMS standard genotyping profile for new potential donors and already applied it to more than 500,000 samples. Since then, we have detected more than 5000 distinct novel SNP sequences, see Fig. 1, i.e. sequences that differ from the reported alleles by at least one nucleotide. About half of the novel sequences exhibit new protein sequences. In addition, we identified many alleles with so far unreported combinations of described exon sequences. We analyzed the distribution of new variants according to ethnic self-assessment of recruited donors. Conclusions The ability to genotype KIR genes at allelic level in a high-throughput framework gives the opportunity to detect a huge number of unknown sequences on a rather short time scale. Consequently, the database of currently named alleles can be extended significantly and thus gives input to studies around the complex KIR gene Family.

P108 Phased whole-gene characterization of novel KIR3DL1, KIR3DL2, and KIR3DL3 alleles using a dual redundant sequencing strategy

Aim The human killer-cell immunoglobulin-like receptors (KIR) forms a complex receptor family expressed on the surface of natural killer (NK) cells. KIR genotypes have been demonstrated to affect disease predisposition and transplantation outcome. To facilitate the selection of donors based on KIR genotypes, we added KIR to the genotyping profile for newly registered potential stem cell donors in 2014. Up to now we have KIR genotyped more than 2 million samples based on our next-generation sequencing approach. This resulted in the discovery of thousands of novel alleles. For the submission of novel alleles to the IPD-KIR Database, full-length phased sequence characterization is desirable. Therefore, we initiated a project for high quality full-length sequence characterization of novel KIR3DL1 , KIR3DL2 , and KIR3DL3 alleles. Methods We established three long-range PCRs targeting the whole genes including both UTRs resulting in PCR products of 13.5 kb ( KIR3DL1 ), 17.5 kb ( KIR3DL2 ), and 16 kb ( KIR3DL3 ). Redundant sequencing strategies were applied to sequence two independent PCR products per target: Shotgun sequencing on Illumina MiSeq instruments and Single Molecule Real Time (SMRT) sequencing on Pacific Biosciences Sequel Systems. Using the Dual Redundant Reference Sequencing software (DR2S, DKMS Life Science Lab), phase-defined consensus sequences were generated from the SMRT data and confirmed with the short, high-fidelity reads. Applying this approach we analyzed a set of 364 samples targeting 112 distinct novel KIR alleles (41 KIR3DL1 , 37 KIR3DL2 , and 34 KIR3DL3 ). Results The generated sequencing data revealed high specificity of the assays for KIR3DL1 , KIR3DL2 and KIR3DL3 . The approach proved capable of generating fully phased sequences spanning the entire 13 to 17 kb genes. By applying two complementary technologies, rare sequencing errors could be corrected. This enabled high-quality phase-defined characterization of novel KIR alleles without the requirement for time consuming cloning steps. Conclusions We established a method for highest quality phased whole-gene KIR sequencing and applied it to characterize more than 100 novel KIR3DL1 , KIR3DL2 , and KIR3DL3 alleles. By submitting these sequences to IPD-KIR we will complement the database for frequent but so far undescribed KIR alleles.

OR28 High-throughput KIR sequencing by NGS: 500,000 registry samples genotyped at allelic resolution

Aim The human killer-cell immunoglobulin-like receptor (KIR) family of genes is a key regulator of natural killer cell activity. Several studies have indicated that KIR genotypes affect hematopoietic stem cell transplantation outcome and the evidence is rising that the extensive allelic diversity discovered in KIR genes may be an important component to consider. However, high-resolution characterization of KIR genotypes has so far been applied only to smaller cohorts. Therefore, we developed a KIR genotyping approach based on NGS that would be cost-effective and amendable for high-throughput application. Methods We amplified KIR exons 3, 4, 5, 7, 8 and 9, targeting one to two exons per PCR reaction but multiplexed across all KIR genes. Amplicons of 2 × 3760 samples were combined for sequencing on Illumina HiSeq 2500 instruments together with amplicons for HLA and blood groups. The reads were mapped against the described KIR alleles (IPD-KIR Release 2.6). Based on the read coverage, we estimated the genomic copy number of each sequence feature. These sequence copy calculations enabled precise allele calling and in addition empowered us to determine gene copy numbers. Results After successful validation, we applied this workflow to genotype 500,000 registry samples. Despite the focus on throughput and cost efficiency, we achieved mostly allotype (3 digit) resolution with the exception of certain allotypes differing only in the short non-targeted exon regions. In addition, certain phasing ambiguities remained. We report the observed allele diversity and the relative abundance of alleles for a mainly Caucasian cohort. Conclusions We demonstrate that high-resolution KIR genotyping is feasible using a very cost-efficient workflow. This approach enables KIR genotyping for population genetics, disease association studies and other applications requiring large cohorts. As of October 2016 we have been applying this workflow to the analysis of all volunteer samples registering with DKMS as potential donors for hematopoietic stem cell transplantation. High-resolution KIR genotyping results will thus become available to search coordinators and may soon be used as an additional selection criterion to improve transplantation outcome.

Association of killer cell immunoglobulin-like receptor (KIR) genes and their HLA ligands with susceptibility to Behçet’s‎ disease

Objectives: Behçet’s disease (BD) is a systemic inflammatory disorder with remissions and exacerbations. It is thought that defects in the natural killer (NK) cell repertoire may be involved in BD through killer cell immunoglobulin-like receptors (KIRs). This study aimed to evaluate KIR and HLA genes, their interactions in BD patients, and their associations with clinical manifestations.Method: The presence or absence of KIR and HLA alleles and genotypes was analysed by polymerase chain reaction sequence-specific primer on genomic DNA of 397 BD patients and 300 healthy controls.Results: None of the KIR genes showed significant effects on BD susceptibility. HLA-C1Asn80 showed a protective effect against BD, whereas HLA-C2Lys80, HLA-B-Bw4Ile80, HLA-B5, and HLA-B51 were associated with a susceptibility risk for BD. In the combination of KIR and HLA genes, the frequencies of HLA genotypes no. 2, 3, 5, and 8, and inhibitory KIR no. 4 were significantly higher in patients than in controls. The frequencies of KIR genotype no. 3 and HLA genotypes no. 1, 4, 6, 7, and 9 were significantly lower in patients than in controls. There were many associations between KIR and HLA genes with clinical features of BD.Conclusion: Differences in the frequency of HLA genes, KIR–HLA interactions, and genotypes between BD and healthy controls and their associations with clinical manifestations indicate that NK cells are involved in BD pathogenesis. The observed differences indicated an NK cell activity imbalance in BD patients, and suggest a role of the KIR-HLA repertoire in the development of BD.