Open Access Methodology article

8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations

Xianghong Zhang123*, Sebastian Müller4, Michael Möller2, Klaus Huse2, Stefan Taudien2, Malte Book1, Frank Stuber1, Matthias Platzer2 and Marco Groth2

Author Affiliations

1 University Department of Anaesthesiology and Pain Medicine, Bern University Hospital, Inselspital, Bern, Switzerland

2 Genome Analysis, Leibniz Institute for Age Research – Fritz Lipmann Institute, Jena, Germany

3 Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland

4 Systems Biology/Bioinformatics Group, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany

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BMC Genomics 2014, 15:64  doi:10.1186/1471-2164-15-64

Published: 24 January 2014

Abstract

Background

The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination.

Results

We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes.

Conclusion

MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

Keywords:
Beta-defensin; Copy number variation; Quantitative real-time PCR; Paralog ratio tests; Multiplex ligation-dependent probe amplification; Clustering; Pseudogene; Low frequency sequence variations