34-OR

Abstract

Expressed by NK cells and T cells, KIR are critical for immune responses to infection, cancer and tissue transplants. KIR also contribute to autoimmunity and pregnancy syndromes. The human KIR locus is polygenic and polymorphic, with over 300 protein variants described, yet remains poorly characterized worldwide. We have developed a method to sequence complete KIR haplotypes, in parallel with HLA haplotypes, from genomic DNA. After capture of fragments with oligonucleotide probes the entire KIR locus is sequenced to high depth (>500x) using Illumina paired-end technology. In this way we analyzed 104 HLA homozygous cell lines from the 12th IHWC, for which there was limited prior KIR knowledge. To acquire these data we created a pipeline that automatically analyses the KIR -specific reads; it attributes sequencing-reads to specific KIR genes, determines presence/absence genotype by read depth, and then calls high-resolution genotypes for each of the 13 KIR and two pseudogenes that are present. The genotype calls were independently verified on eight of the IHWC cell lines using our high-resolution pyrosequencing method, also targeted to all KIR genes and alleles. The pipeline was further validated by comparison with 25 KIR-specific SNPs present in a whole-genome array (Omni1Quad). Only four IHWC cell lines are completely homozygous for all KIR genes and alleles, but ∼25% cells are homozygous for KIR gene content, due to high KIR A haplotype frequency. Accordingly, we are using population genetics to determine the composition of the 204 distinct KIR haplotypes represented in the 104 IHWC cells. All these methods were validated using the COX cell line, for which both KIR haplotypes were sequenced previously using standard techniques. Both methods gave 100% coverage and complete concordance. This versatile technique and the data so far obtained provide valuable contributions to future immunogenetic studies in transplantation and of other clinical diagnostics.