Abstract
BackgroundKiller Immunoglobulin-like Receptors (KIRs) are surface receptors of natural killer cells that bind to their corresponding Human Leukocyte Antigen (HLA) class I ligands, making them interesting candidate genes for HLA-associated autoimmune diseases, including type 1 diabetes (T1D). However, allelic and copy number variation in the KIR region effectively mask it from standard genome-wide association studies: single nucleotide polymorphism (SNP) probes targeting the region are often discarded by standard genotype callers since they exhibit variable cluster numbers. Quantitative Polymerase Chain Reaction (qPCR) assays address this issue. However, their cost is prohibitive at the sample sizes required for detecting effects typically observed in complex genetic diseases.ResultsWe propose a more powerful and cost-effective alternative, which combines signals from SNPs with more than three clusters found in existing datasets, with qPCR on a subset of samples. First, we showed that noise and batch effects in multiplexed qPCR assays are addressed through normalisation and simultaneous copy number calling of multiple genes. Then, we used supervised classification to impute copy numbers of specific KIR genes from SNP signals. We applied this method to assess copy number variation in two KIR genes, KIR3DL1 and KIR3DS1, which are suitable candidates for T1D susceptibility since they encode the only KIR molecules known to bind with HLA-Bw4 epitopes. We find no association between KIR3DL1/3DS1 copy number and T1D in 6744 cases and 5362 controls; a sample size twenty-fold larger than in any previous KIR association study. Due to our sample size, we can exclude odds ratios larger than 1.1 for the common KIR3DL1/3DS1 copy number groups at the 5% significance level.ConclusionWe found no evidence of association of KIR3DL1/3DS1 copy number with T1D, either overall or dependent on HLA-Bw4 epitope. Five other KIR genes, KIR2DS4, KIR2DL3, KIR2DL5, KIR2DS5 and KIR2DS1, in high linkage disequilibrium with KIR3DL1 and KIR3DS1, are also unlikely to be significantly associated. Our approach could potentially be applied to other KIR genes to allow cost effective assaying of gene copy number in large samples.
Highlights
Killer Immunoglobulin-like Receptors (KIRs) are surface receptors of natural killer cells that bind to their corresponding Human Leukocyte Antigen (HLA) class I ligands, making them interesting candidate genes for HLA-associated autoimmune diseases, including type 1 diabetes (T1D)
The KIR3DL1 protein is known to interact with the HLA class I allotypes that contain the HLA-Bw4 serological epitope [2,3], whereas the protein encoded by KIR3DS1, which shares 97% sequence similarity to KIR3DL1, is thought to bind the more restrictive HLABw4-80I epitope subset [4]
This has resulted in low single nucleotide polymorphism (SNP) coverage in the KIR region for the common SNP arrays
Summary
Killer Immunoglobulin-like Receptors (KIRs) are surface receptors of natural killer cells that bind to their corresponding Human Leukocyte Antigen (HLA) class I ligands, making them interesting candidate genes for HLA-associated autoimmune diseases, including type 1 diabetes (T1D). Targeted quantitative Polymerase Chain Reaction (qPCR) assays have been used to detect presence or absence of individual KIR genes and more recently, determine copy numbers [1]. These remain expensive and labour intensive compared to SNP arrays. We are able to use qPCR results in a modest number of samples to impute copy numbers into a larger sample for which SNP array signals are available We illustrate this method by applying it to two genes in the KIR complex, KIR3DL1 and KIR3DS1, which are suitable candidates for T1D association due to their interaction with HLA class I molecules. KIR3DL1/3DS1 association with T1D has only been studied using qPCR assays in limited sample sizes, which assess presence or absence of each KIR gene [8]
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