P062Coding and non-coding variation in 60 novel full-length sequences of KIR2DL1

Abstract

Aim KIR2DL1 is a member of the killer-cell immunoglobulin-like receptor (KIR) family, which is an important factor of the human immune system. KIR genes are key regulators of natural killer cell activity and partly bind to proteins of the human leukocyte antigen (HLA) family, e.g. HLA-B and -C in the case of KIR2DL1. Likely due to the complexity of the KIR locus with its extensive genetic variation, only little is known about the impact of allelic variation. Here, we identified novel KIR2DL1 alleles by routine high-throughput exon-based KIR genotyping and subsequently created full-length reference sequences. We analysed coding and non-coding variation to identify possible systematic differences between alleles. Methods We sequenced whole 16 kb amplicons of KIR2DL1 using shotgun sequencing (Illumina MiSeq) and single molecule real time (SMRT) sequencing (PacBio Sequel). Using the R package DR2S, we combined phase information from SMRT sequencing with the accuracy of shotgun sequencing to generate phased full-length sequences. Results We successfully generated 60 distinct KIR2DL1 allele sequences from 45 specifically selected samples. This includes 41 novel alleles and 17 distinct alleles that were previously only partially characterised. The sequence analysis revealed three deep allelic groups separated by 164 variable positions. Allelic variants of two groups harbour mutually exclusive nucleotides, while a third group of alleles may harbour nucleotides of both the other groups. The three groups are mostly separated by non-coding SNPs, indels, and a T homopolymer of fixed length in one and variable length in two groups. Some separating variants exist in most exons and alter the amino acid sequence of the leader peptide, the extracellular D1- and D2-domains containing the HLA binding sites as well as the cytoplasmatic domain. Conclusions Our sequencing efforts resulted in a 4-fold increase in known full-length sequences of KIR2DL1, enabling further research on this KIR gene. We gained insights into systematic differences at the sequence level which might be responsible for or indicative of medically relevant allelic differences. Especially variation in the D2-domain has been shown to be involved in binding to HLA proteins and may as such be clinically relevant.