Identification and Typing of Killer-Cell Immunoglobulin-Like Receptors (KIRs) Using Quantitative PCR Assay Reagents for Research

Aim:Recent publications demonstrate after an allogeneic hematopoietic cell transplant (HCT), anti-leukemia effects are primarily provided by T cells and Natural Killer (NK) cells. NK cells play a role in reduced graft versus host disease and in increased graft versus leukemia effects predominantly due to their cell surface receptors namely Killer-cell immunoglobulin-like receptors (KIRs). Conventional KIR genotyping methods involve sequence-specific primer (SSP)-PCR or sequence specific oligonucleotide hybridization PCR followed by visualization of PCR products by agarose gel electrophoresis or using sequence-based typing. Lack of a universal test to type KIR combined with the low throughput and high costs of existing methodologies prompted the development of a prototype quantitative PCR (qPCR) assay for KIR genotyping. The prototype assay uses fluorescent hydrolysis probes and may provide accurate and fast KIR genotyping with minimal DNA to aid in KIR genotyping for pre and post-transplantation research.

 

Methods: Panel of 24 DNA containing common KIR genotypes was obtained from UCLA DNA exchange. 240 ng total DNA for 16 KIR reactions (each reaction run in triplicate) was used for the prototype qPCR assay and results were compared with a PCR-SSP KIR genotyping kit utilizing 3 ug total DNA. The prototype assay consists of reagents to perform multiplexed KIR genotyping which is amplification of a KIR gene and an internal qPCR reference gene for normalizations, quantification, and confirmation of true absence of KIR genes.

 

Results: The prototype qPCR assay results were 100% concordant with the commercial PCR-SSP kit. The qPCR assay accurately detected each KIR gene with a 12 fold reduction in the amount of DNA required. There was no-post PCR analysis or additional specialized equipment with all KIR genes being amplified under the same thermocycling conditions.

 

Conclusions: We have shown feasibility for a prototype qPCR assay for KIR genotyping that is robust and flexible for both small and large scale genetic analysis studies. With implications of KIR in transplantation (particularly in AML HCT), autoimmune and infectious diseases, and pregnancy-related complications, faster detection might aid earlier therapy intervention and a more sensitive assay can potentially identify previously unidentified novel associations. Genotyping KIRs using qPCR based assays could be useful in both clinical and basic research settings.