Development of a totalplex amplification and bead array-based genotyping method for killer cell immunoglobulin-like receptor

Abstract

Natural killer cells have Killer cell Immunoglobulin-like Receptors (KIR) that interact with human class I leukocyte antigens. The KIR genes belong to a multigene family that contains 16 members which display a high level of sequence similarity. The composition of KIR genes in each individual is highly polymorphic so that there can be 186 different genotypes. Recently, it has been reported that the interactions between KIR and human leukocyte antigens affect infection resistance, autoimmune disease, cancer, reproduction and the outcome of hematopoietic stem cell transplantation, increasing the importance of KIR genotyping. Among the various KIR genotyping methods currently used, sequence-specific primer directed polymerase chain reaction (SSP-PCR) technology has been widely used because of its simplicity. But it is a time- and labor-consuming method and needs genomic DNA at the μg level. In addition, SSP-PCR method has the disadvantage of being partially or totally based on the amplification of long DNA fragments which requires high quality DNA. Therefore, it is necessary to develop more sensitive, accurate and less sample consuming methods for KIR genotyping. In this study, I developed a novel technique for KIR genotyping using totalplex amplification and a Luminex bead array. In stage of KIR gene amplification, totalplex amplification was performed to amplify 16 KIR genes using specific bulge specific primers in two separate reactions. Then amplified products were mixed and labeling was performed using gene specific primer extension primers. Amplified KIR gene signals were detected using Luminex bead array analysis and then the results were translated to individual KIR genotypes by median fluorescence intensity. For validation of totalplex/bead array-based KIR genotyping, a NK/KIR reference panel Ι consisting of 48 cell types provided by the 13th the International Histocompatibility Working Group was used. The KIR genotyping results of the totalplex/bead array-based method coincided with the information provided with the NK/KIR reference panel Ι. When KIR genotyping was performed using the DNA samples from 8 human cell lines, 8 buccal samples and 17 peripheral blood samples, the results from the newly developed method had a 100% correlation with those obtained by SSP-PCR. The reproducibility of the newly developed method was verified by showing that 6 repetitive genotypings of the same DNA samples produced the same KIR genotype results. The sensitivity of the method was confirmed by demonstrating that accurate KIR genotyping results were obtained when DNA samples of 5 ng or more were used. These results indicate that the newly developed totalplex/bead array-based KIR genotyping method is a sensitive and reproducible method that has the advantage of saving on reagent cost, time, labor and DNA amount necessary for KIR genotyping.