Abstract
BackgroundThere have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and β-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories.FindingsIn this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech) of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations.ConclusionsQPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number.
Highlights
Characterization of genetic variants is fundamental in understanding human heterogeneity and susceptibility to disease.Regions where humans differ in diploid DNA dosage are known as copy number variations (CNV) and are an important component of genetic variation
quantitative PCR (QPCR) is very sensitive to quality of the template DNA, generating systematic biases that could produce falsepositive or negative disease associations
We have developed a version of the Paralogue Ratio Test (PRT) method that uses pyrosequencing and quantification of different sequence variants to distinguish test and reference amplicons
Summary
Regions where humans differ in diploid DNA dosage are known as copy number variations (CNV) and are an important component of genetic variation. CNVs are believed to encompass more nucleotide content than single nucleotide polymorphisms (SNPs) [1] and between 12% and 18% [2,3] of the euchromatic human genome is suggested to be copy number variable [4]. B-defensins induce the production of diverse chemokines and cytokines such as MCP-1, macrophage inflammatory protein. The beta-defensin cluster varies in copy number between 2 and copies per diploid genome with most people having 2–7 copies [28,38,39,40,41]. There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and b-defensins and Crohn’s disease. Real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, the Paralogue Ratio Test (PRT) is being used in more and more laboratories
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