Generating full-length Killer-cell Immunoglobulin-like Receptor (KIR) gene sequences using Third Generation long-amplicon sequencing to assess the impact of KIR polymorphism on Haematopoietic Cell Transplantation outcomes

Killer-cell Immunoglobulin-like Receptor (KIR) polymorphism is extensive in both allelic and copy number variation. Although multiple assays have been designed to assess the latter form of polymorphism, KIR allelic diversity is less well understood owing to the homologous nature of different KIR genes and, until recently, limitations in sequencing technology. To better understand KIR allelic diversity in the UK, and its impact on haematopoietic cell transplant (HCT) outcomes, I have designed and validated a whole-gene, fully-phased allele sequencing strategy that encompasses third generation sequencing technology to deliver unambiguous genotypes for several different KIR genes. Subsequently, this strategy was applied to a novel, largely T cell deplete UK cohort of patients receiving HCT to treat acute myeloid leukaemia, and their respective donors. This assay utilises a semi-generic targeted polymerase chain-reaction amplification prior to multiplexed library preparation and sequencing, providing a relatively high-throughput methodology amenable to clinical laboratories. Initially, to assess the relevance of presence/absence KIR polymorphism on HCT outcomes, I utilised existing genotyping methods to establish baseline characteristics. In contrast to previous publications, relapse was largely unaffected by KIR polymorphism. However, striking differences related to preparative conditioning regimen were observed in KIR-mediated effects in other HCT outcomes. Presence of donor-encoded centromeric (Cen)-B motifs relayed increased risk of detrimental non-relapse mortality following myeloablative conditioning, whereas the opposite appeared to be true of reduced-intensity conditioning transplants. When the impact of allelic diversity at the KIR2DL1, KIR2DL2/3 and KIR3DL1/S1 loci on HCT outcomes was assessed in my cohort, allelic differences within the Cen-A, Cen-B and telomeric A haplotype motifs provided additional insight into the possible mechanisms of KIR-mediated influence on HCT outcomes. By estimating frequencies of different KIR alleles within a UK population, I have demonstrated that donor selection algorithms could feasibly incorporate allelic KIR genotypes that may improve quality of life after HCT.