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. 2011 Aug 18:12:418.
doi: 10.1186/1471-2164-12-418.

Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders

Danielle Carpenter  1 , Susan Walker, Natalie Prescott, Joost Schalkwijk, John Al Armour
Affiliations

Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders

Danielle Carpenter et al. BMC Genomics. .

Abstract

Background: 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. 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.

Results: We 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.

Conclusions: Differential 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.

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Figures

Figure 1
Figure 1
Distribution of the unrounded copy number values for 1581 European samples. The distribution shows a common range of 0-4 copies, with few high copy number outliers. The distribution shows peaks centred on the integer values with gaps between most clusters.
Figure 2
Figure 2
A cumulative frequency plot of unrounded copy number values measured by the three PRT systems, (CCL3C, CCL4A and LTR61A) among samples assigned a copy number of 2 for the random UK control samples (149 samples). All three systems show a similar distribution around the mean value, displaying no obvious heterogeneity between the systems.
Figure 3
Figure 3
A cumulative frequency plot for controls (solid black line) and cases (broken black line) for Crohn's disease samples (a), rheumatoid arthritis (b), and psoriasis (c) with copy number of 2. There is no significant difference between the means of the distributions for the Crohn's disease and rheumatoid arthritis samples, whereas for the psoriasis samples the values for cases are significantly shifted towards lower values relative to the controls. Pre-digestion with the enzyme BccI, prior to PRT (d) shown here on a subset of 20 Dutch samples, resolves the significant difference between case and control samples.
See this image and copyright information in PMC

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