High-throughput killer cell immunoglobulin-like receptor genotyping by MALDI-TOF mass spectrometry with discovery of novel alleles

Abstract

The killer cell immunoglobulin-like receptors (KIR) interact with major histocompatibility complex (MHC) class I ligands to regulate the functions of natural killer cells and T cells. Like human leukocyte antigens class I, human KIR are highly variable and correlated with infection, autoimmunity, pregnancy syndromes, and transplantation outcome. Limiting the scope of KIR analysis is the low resolution, sensitivity, and speed of the established methods of KIR typing. In this study, we describe a first-generation single nucleotide polymorphism (SNP)-based method for typing the 17 human KIR genes and pseudogenes that uses analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It is a high-throughput method that requires minute amounts of genomic DNA for discrimination of KIR genes with some allelic resolution. A study of 233 individuals shows that the results obtained by the SNP-based KIR/MALDI-TOF method are consistent with those obtained with the established sequence-specific oligonucleotide probe or sequence-specific polymerase chain reaction methods. The added sensitivity of the KIR/MALDI-TOF method allowed putative novel alleles of the KIR2DL1, KIR3DL1, KIR2DS5, and KIR2DL5 genes to be identified. Sequencing the KIR2DL5 variant proved it was a newly discovered allele, one that appears associated with Hispanic and Native American populations. This KIR/MALDI-TOF method of KIR typing should facilitate population and disease-association studies that improve knowledge of the immunological functions of KIR-MHC class I interactions.

Fig. 1

Spectral data (left) and cluster plots (right) illustrating single nucleotide and double nucleotide calls with the hME assay “3DS1.3DL1.D1.S,” which discriminates between KIR3DS1 and KIR3DL1. a and c show spectral data and cluster plots for the single nucleotide call T (3DL1) and G (3DS1), respectively. b shows data for the double nucleotide call T/G (KIR3DL1 and KIR3DS1 both present). Arrows indicate specific data points illustrated in spectral graphs. For all panels, the lowest mass peak (6447.2 Da) represents the unextended primer. The peak produced when the polymerase pauses leading to incorporation of an unexpected dNTP (pausing peak) is also shown—Cluster plots (right) illustrate the intensity of the high mass product (T) peak vs intensity of the low mass product (G) peak for a given sample. The separation between clusters and the tightness within a cluster are indicative of the accuracy and specificity of the assay

Fig. 2

A high-throughput SNP-based MS method for KIR genotyping that is based on the SEQUENOM™ MALDI-TOF MS platform and homogenous MassExtend (hME) chemistry

Fig. 3

Two tiers of specificity are built into the KIR/MALDI assay. a The first tier of specificity is created by the “capture” primers used in the initial PCR. In this example, allelic forms containing an “A” fail to amplify (⊘) while those containing “T” are captured (and amplified) by the primers during the PCR. One of the KIR genes that is present in all haplotypes (“framework” gene) or another common KIR gene will also be captured with the same set of primers, thereby serving as an internal control for the PCR. b The design and directionality of the extension primer, along with the chosen deoxy (d)/dideoxy (dd) mixture provide the second tier of specificity. In this example, ddG, ddA, dC, and ddT are used in the extension reaction. When ddG is incorporated during the primer extension reaction using the capture product from the targeted locus (the uppermost product shown in both a and b) and framework gene, the reaction is terminated by the incorporation of the dideoxynucleotide. When dC is incorporated during primer extension using the capture product from the targeted locus and framework gene, the extension reaction proceeds until a dideoxy nucleotide is incorporated. The choice of the extension primer and deoxy/dideoxy mixes allows the creation of extension products that differ by length as well as composition. The extension products are measured by the MALDI-TOF MS, which can easily differentiate between products that differ in length by one or more bases