Novel multiplex PCR-SSP method for centromeric KIR allele discrimination

Jean-Benoît Le Luduec,Jeanette E Boudreau,Anupa Kudva,Katharine C Hsu

doi:10.1038/s41598-018-33135-1

Abstract

Allelic diversity of the KIR2DL receptors drive differential expression and ligand-binding affinities that impact natural killer cell function and patient outcomes for diverse cancers. We have developed a global intermediate resolution amplification-refractory mutation system (ARMS) PCR-SSP method for distinguishing functionally relevant subgroups of the KIR2DL receptors, as defined by phylogenetic study of the protein sequences. Use of the ARMS design makes the method reliable and usable as a kit, with all reactions utilizing the same conditions. Six reactions define six subgroups of KIR2DL1; four reactions define three subgroups of KIR2DL2; and five reactions define four subgroups of KIR2DL3. Using KIR allele data from a cohort of 426 European-Americans, we identified the most common KIR2DL subtypes and developed the high-throughput PCR-based methodology, which was validated on a separate cohort of 260 healthy donors. Linkage disequilibrium analysis between the different KIR2DL alleles revealed that seven allelic combinations represent more than 95% of the observed population genotypes for KIR2DL1/L2/L3. In summary, our findings enable rapid typing of the most common KIR2DL receptor subtypes, allowing more accurate prediction of co-inheritance and providing a useful tool for the discrimination of observed differences in surface expression and effector function among NK cells exhibiting disparate KIR2DL allotypes.

Highlights

Allelic diversity of the KIR2DL receptors drive differential expression and ligand-binding affinities that impact natural killer cell function and patient outcomes for diverse cancers
The KIR2DL receptors exclusively recognize HLA-C molecules: KIR2DL1 recognizes HLA-C allotypes characterized by Lys[80]; KIR2DL3 recognizes predominantly HLA-C allotypes characterized by Asn[80] (HLA-C group 1); while KIR2DL2 recognizes members of both HLA-C groups 1 and 22
We present a similar approach for the centromeric inhibitory killer cell immunoglobulin-like receptors (KIR) genes KIR2DL1, KIR2DL2, and KIR2DL3, identifying the nucleotide sites potentially important for functional discrimination among receptor alleles and devising an amplification-refractory mutation system (ARMS) PCR-SSP typing methodology

Summary

Introduction

Allelic diversity of the KIR2DL receptors drive differential expression and ligand-binding affinities that impact natural killer cell function and patient outcomes for diverse cancers. Our findings enable rapid typing of the most common KIR2DL receptor subtypes, allowing more accurate prediction of co-inheritance and providing a useful tool for the discrimination of observed differences in surface expression and effector function among NK cells exhibiting disparate KIR2DL allotypes. As key members of the innate immune response, natural killer (NK) cells survey surrounding cells, discriminating damaged or infected cells from healthy cells, in part via receptor recognition of altered self-MHC on damaged cells[1] This process, termed “education” or “licensing” is enabled through interactions between inhibitory receptors on NK cells with “self ” MHC, that permit cytotoxic granule release for target cell killing, and inhibition of the NK cell upon binding to cognate MHC. Primers name ControlF ControlR 2DL1R1F 2DL1R1R 2DL1R2F 2DL1R2R 2DL1R3F 2DL1R3R 2DL1R4F 2DL1R4R 2DL1R5F 2DL1R5R 2DL1R6F 2DL1R6R 3DP1F 3DP1VF 3DP1R 2DL1O1F 2DL1O1R 2DL1O2F 2DL1O2R 2DL1O3F 2DL1O3R 2DL1O4F 2DL1O4R 2DL2R1F 2DL2R1R 2DL2R2F 2DL2R2R 2DL2R3F 2DL2R3R 2DL2R4F 2DL2R4R 2DL2O1F 2DL2O1R 2DL2O2F 2DL2O2R 2DL3R1F 2DL3R1R 2DL3R2F 2DL3R2R 2DL3R3F 2DL3R3R 2DL3R4F 2DL3R4R 2DL3R5F 2DL3R5R 2DL3O1F 2DL3O1R 2DL3O2F 2DL3O2R 2DL3O3F 2DL3O3R 2DL3O4F 2DL3O4R

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Results

Conclusion