P036 Whole gene sequencing of KIR-3DL1 with PacBio RSII and the distribution of alleleic variants in different ethic groups

Abstract

Aim The killer-cell immunoglobulin-like receptor (KIR) gene family are involved in immune modulation during viral infection, autoimmune disease and in allogeneic stem cell transplantation. Most KIR gene diversity studies and their impact on the transplant outcome is performed by gene absence/presence assays. However it is well known that KIR gene allelic variations have biological significance. Allele level typing of KIR genes has been very challenging until recently due to the homologous nature of those genes and very long intronic sequences. PacBio RSII platform’s Single Molecule, Real-Time (SMRT®) sequencing, generates average long reads of 10 to 15 kb and allows us to obtain in-phase long sequence reads. We have developed a PCR assay for PacBio RSII platform in our lab for 3DL1 whole gene sequencing. This approach allows us to obtain allele level typing for 3DL1 genes and could serve as a model to type other KIR genes at allelic level. Methods DNA samples were obtained from 41 cell lines of Caucasian ethnicity and 97 volunteers with Korean and South Asian origin. Gene absence/presence assays were done on Illumina MiSeq platform using in-house protocol.We performed whole gene 3DL1 sequencing on the genomic DNA of samples using two gene-specific primers to get two amplicons (5.5 kb and 5.3 kb, respectively) that will span all the exons and most of the Intron 5. These amplicons were sequenced on the PacBio RSII platform. We analyzed the data based on current KIR DB v 2.6.0 using our in house program. Results A total of 123 out of 138 samples had 3DL1 gene by gene absence/presence assay. Based on the exon sequences alone, 123 samples had 47 of the 110 known alleles. However when we included intron sequences to our analysis only 23 sequences out of 47 unique alleles had a match to the database for intronic sequences. The remainder 24 sequences had novel variations in the introns. The majority were in intron 6. 3DL1 ∗ 002 that has stronger inhibitory receptor was found in 13 samples and mostly in CAU/EURO samples in this study. Some ethnic clustering of certain alleles (ie: 3DL1 ∗ 01501/2 group) were observed in Korean samples. Conclusion Whole gene sequencing of KIR genes is now feasible. Allelic typing of KIR genes will allow us to better dissect their modulatory role during immune response and their distribution among diverse populations. S. Yang: Stock Shareholder; Company/Organization; PacBio.