Determination of beta-defensin genomic copy number in different populations: a comparison of three methods

TY - JOUR

T1 - Determination of beta-defensin genomic copy number in different populations

T2 - a comparison of three methods

AU - Fode, Peder

AU - Jespersgaard, Cathrine

AU - Hardwick, Robert J

AU - Bogle, Helen

AU - Theisen, Michael

AU - Dodoo, Daniel

AU - Lenicek, Martin

AU - Vitek, Libor

AU - Vieira, Ana

AU - Freitas, Joao

AU - Andersen, Paal Skytt

AU - Hollox, Edward J

PY - 2011

Y1 - 2011

N2 - Background: There 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. Findings: In 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. Conclusions: QPCR 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.

AB - Background: There 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. Findings: In 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. Conclusions: QPCR 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.

KW - Base Sequence

KW - Chromosome Mapping

KW - Cohort Studies

KW - Czech Republic

KW - Denmark

KW - Gene Dosage

KW - Genetic Predisposition to Disease

KW - Genetics, Population

KW - Genome, Human

KW - Ghana

KW - Great Britain

KW - Humans

KW - Molecular Sequence Data

KW - Population

KW - Portugal

KW - beta-Defensins

U2 - 10.1371/journal.pone.0016768

DO - 10.1371/journal.pone.0016768

M3 - Journal article

C2 - 21364933

SN - 1932-6203

VL - 6

SP - e16768

JO - P L o S One

JF - P L o S One

IS - 2

ER -