85-P: ALLELE LEVEL KIR TYPING BY NEXT GENERATION SEQUENCING

Abstract

Aim The killer cell Immunoglobulin-like receptors (KIR), expressed on the surface of natural killer cells, modulate cell functions by their interaction with class I human leukocyte antigen (HLA) molecules. KIR and their HLA ligands play central roles in innate immune responses; studies have shown a link between the presence or absence of KIR genes and their ligands in the response to viral infections, cancer and autoimmune diseases, as well as reproduction. Survival after bone marrow transplant (BMT) for AML has also been linked to specific KIR and HLA combinations. We are developing a next generation sequencing method to type KIR at an allelic level to understand the interaction of specific KIR alleles and their ligands in autoimmunity and BMT success. Methods We designed a set of 34 primer pairs to amplify all exons of all KIR genes. Sequencing the amplicons on a Roche GSFLX 454 instrument provides us with long (400-450bp) reads which are analyzed using Conexio Genomics Assign MPS software. To develop and validate this approach, we have sequenced 48 KIR reference samples, obtained from the National Marrow Donor Program (NMDP) and a trio based study of 354 samples. Results For the 48 KIR reference samples, a high concordance between our NGS results and the previously determined alleles from the NMDP was attained. In a few cases there was an allelic difference between our results and the NMDP. Detailed analysis of the results for each KIR gene will be discussed. Our trio study allows us to show the transmission of alleles from parents to offspring, and, in many cases, to determine haplotypes from the patterns of inheritance. Conclusions Using an amplicon-based approach, long, clonal sequencing reads and Conexio software, we have developed a method to determine KIR alleles in pooled samples. The ability to incorporate KIR allelic level typing in disease studies and BMT should further our understanding of this complex gene region and its role in the innate immune system.