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

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.

High-Throughput MICA/B Genotyping of Over Two Million Samples: Workflow and Allele Frequencies

“…To facilitate further studies on MICA and/or MICB matching in unrelated hematopoietic stem cell transplantation, we included both genes into our high-throughput genotyping workflow for newly registered potential stem cell donors in 2017. This workflow was initially developed for the six classical HLA genes HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1 and was then gradually extended to also include CCR5, the blood groups ABO and Rh as well as the several KIR genes and HLA-E (33)(34)(35)(36)(37). Today, this workflow has been applied to genotype over seven million donors, among them more than two million including MICA and MICB.…”

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

“…With the increasing availability of next generation genotyping (NGS)‐based genotyping methods for KIR, there is a growing number of laboratories that sequence substantial numbers of KIR alleles in the context of basic research as well as first efforts to establish KIR genotyping in the context of high‐throughput stem‐cell donor registry typing . We expect that these efforts will lead to the discovery of a large number of novel KIR alleles, as was the case with the HLA genes in the previous years . However, in contrast to HLA, there is currently no software available to support the submission of full‐length KIR alleles.…”

Immunogenetics in stem cell donor registry work: The DKMS example (Part 2)

“…We published allele frequencies of several non‐HLA parameters from large samples: We estimated ABO allele and allele group frequencies from a sample of n > 113,000 German individuals (Lang et al, ). The most frequent allele groups were ABO*O.01.01 (31.8%), ABO*A1.01.01 (20.3%) and ABO*O.02.01 (18.5%). We determined KIR allele frequencies from a sample of >337,000 predominantly European individuals by applying an amplicon‐based sequencing approach on Illumina devices (Wagner et al, ). Primers were targeted at KIR exons 3, 4, 5, 7, 8 and 9 (with a combined amplicon for exons 8 and 9).…”

“…• We determined KIR allele frequencies from a sample of >337,000 predominantly European individuals by applying an amplicon-based sequencing approach on Illumina devices (Wagner et al, 2018). Primers were targeted at KIR exons 3, 4, 5, 7, 8 and 9…”