A hybrid qPCR/SNP array approach allows cost efficient assessment of KIR gene copy numbers in large samples.
BACKGROUND
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). 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.


RESULTS
We 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.


CONCLUSIONS
We 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.
Nikolas
N
Pontikos
Deborah J
DJ
Smyth
Helen
H
Schuilenburg
Joanna M M
JM
Howson
Neil M
NM
Walker
Oliver S
OS
Burren
Hui
H
Guo
Suna
S
Onengut-Gumuscu
Wei-Min
WM
Chen
Patrick
P
Concannon
Stephen S
SS
Rich
Jyothi
J
Jayaraman
Wei
W
Jiang
James A
JA
Traherne
John
J
Trowsdale
John A
JA
Todd
JDRF/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, CB2 0XY, Cambridge, UK. chris.wallace@cimr.cam.ac.uk.
Chris
C
Wallace
D000483:Alleles; D016022:Case-Control Studies; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics; D018628:Gene Dosage; D020022:Genetic Predisposition to Disease; D015234:HLA-A Antigens / Q000235:genetics; D015235:HLA-B Antigens / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D054340:Receptors, KIR / Q000235:genetics*; D054341:Receptors, KIR3DL1 / Q000235:genetics; D054349:Receptors, KIR3DS1 / Q000235:genetics
D015234:HLA-A Antigens; D015235:HLA-B Antigens; C515083:KIR3DL1 protein, human; D054340:Receptors, KIR; D054341:Receptors, KIR3DL1; D054349:Receptors, KIR3DS1