Abstract
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.
Highlights
The human killer cell immunoglobulin-like receptor (KIR) gene family resides on the long arm of human chromosome 19 in a region that varies between 100 and 200 kb in size
Given the complexity of killer-cell immunoglobulin-like receptor (KIR) genotyping at the allelic level, thorough verification of the workflow and algorithm is essential
During setup, testing, validation, and optimization of the workflow and algorithm we extensively took advantage of 93 International Histocompatibility Working Group (IHWG) reference samples that had been previously genotyped for all KIR genes at allelic resolution [31]
Summary
The human killer cell immunoglobulin-like receptor (KIR) gene family resides on the long arm of human chromosome 19 in a region that varies between 100 and 200 kb in size. This region is characterized by highly diverse haplotypes, that differ both in gene content and copy number of the KIR genes [1,2,3,4,5]. KIR binding specificity and affinity to HLA class I molecules, and patterns of resistance to specific diseases are influenced by complex interactions of allelic polymorphisms of both KIR and HLA class I genes [13,14,15,16,17,18]
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