High-throughput killer cell immunoglobulin-like receptor genotyping by MALDI-TOF mass spectrometry with discovery of novel alleles
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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.
KeywordsKIR HLA MALDI-TOF Genotyping SNP
We wish to express our gratitude to Drs. Derek Middleton and Carlos Vilches and their laboratories for their generosity and gracious sharing of expertise. Finally, we are grateful for the technical and administrative contributions from J Agraz, K Saeteurn, N Bose, E Sanseau, K Guttierrez, J-C Cossec, and M Vinson. This work was supported by grants R21 AI 65254-01A1, P01 CA 111412, and UO1 AI067068-01 from the National Institutes of Health and the My Brother Joey Foundation to Elizabeth Trachtenberg. The MALDI-TOF MassARRAY system was acquired through NIH-NCRR #1S10RR 16703-01. The investigation was conducted at the Children’s Hospital and Research Center Oakland in facilities constructed with support from Research Facilities Improvement Program Grant Number CO6RR-16226 from the National Center for Research Resources, National Institutes of Health.
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