Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing

Abstract

The physiological functions of natural killer (NK) cells in human immunity and reproduction depend upon diverse interactions between killer cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands: HLA-A, HLA-B, and HLA-C. The genomic regions containing the KIR and HLA class I genes are unlinked, structurally complex, and highly polymorphic. They are also strongly associated with a wide spectrum of diseases, including infections, autoimmune disorders, cancers, and pregnancy disorders, as well as the efficacy of transplantation and other immunotherapies. To facilitate study of these extraordinary genes, we developed a method that captures, sequences, and analyzes the 13 KIR genes and HLA-A, HLA-B, and HLA-C from genomic DNA. We also devised a bioinformatics pipeline that attributes sequencing reads to specific KIR genes, determines copy number by read depth, and calls high-resolution genotypes for each KIR gene. We validated this method by using DNA from well-characterized cell lines, comparing it to established methods of HLA and KIR genotyping, and determining KIR genotypes from 1000 Genomes sequence data. This identified 116 previously uncharacterized KIR alleles, which were all demonstrated to be authentic by sequencing from source DNA via standard methods. Analysis of just two KIR genes showed that 22% of the 1000 Genomes individuals have a previously uncharacterized allele or a structural variant. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations and defining the precise HLA and KIR factors associated with disease. The methods are applicable to other highly polymorphic genes.

Deciphering the killer‐cell immunoglobulin‐like receptor system at super‐resolution for natural killer and T‐cell biology

“…(a) Number of alleles reported in the December 2015 release of the IPD KIR database 60. The advent of killer‐cell immunoglobulin‐like receptor ( KIR ) analysis by next‐generation sequencing is rapidly increasing the number of recognized KIR alleles 68. Blue are genes encoding inhibitory KIR , orange activating KIR and grey are pseudogenes.…”

“…Over 200 associations of KIR genes with disease resistance or susceptibility are published,56, 73 and the handful studied at high resolution are proving informative 58, 74, 75, 76. The field is therefore ripe for harvesting new information through in‐depth analyses 68, 77…”

“…Allele typing is achieved by filtering reads specific for genes based on alignment to all known reference alleles from KIR coding sequences (e.g. Son of Samtools ( SOS )68). In parallel, sequence data can be probed with specific sequence search strings (‘ in silico SSO ’) to determine which alleles are present (e.g.…”

“…In parallel, sequence data can be probed with specific sequence search strings (‘ in silico SSO ’) to determine which alleles are present (e.g. KIR Filter Fish ( KFF )68). …”

“…These allotypes are recognized by antibodies with specificity for receptors encoded by other KIR genes (Table 2). New next‐generation sequencing techniques will allow fast and cost‐effective KIR typing at allelic resolution and will facilitate KIR repertoire interpretation 68. In addition, high‐dimensional mass cytometry (mass cytometry by time‐of‐flight) is a powerful tool for investigating NK cell repertoire diversity through the analysis of many cellular markers simultaneously188 (Fig.…”