Abstract
BackgroundCopy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT) method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts.ResultsWe evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis.ConclusionsDifferential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion.
Highlights
Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging
Inaccurate copy number measurements can lead to differential bias between cases and controls and result in false positive findings [3]
Samples showing a higher level of discordance between the integer copy numbers assigned by the paralogue ratio test (PRT) systems were assayed for two microsatellites that are present within the repeat unit [11]
Summary
Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. Three forms of CNVs can be defined; insertion, deletion and multi-allelic, but accurate and standardised measurement of individual copy numbers is technically challenging. In SNP based studies differential bias can lead to differences in allele frequency estimates between batches of samples. In the context of CNVs bias can be seen as a systematic shift in the raw measurement around integer values for cases and controls, leading to differential mis-classification of samples by either overor under-estimation of the integer copy number. The power to detect disease associations in case control studies is reduced with inaccurate genotyping of copy number [4]. Accurate determination of copy number is limited by the precision of the methodology used, which needs to be robust and replicable to give maximum power and reduce spurious disease associations
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