Determination of Beta-Defensin Genomic Copy Number in Different Populations: A Comparison of Three Methods

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.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.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

Genetic variability in beta-defensins is not associated with susceptibility to Staphylococcus aureus bacteremia.

Human beta-defensins are key components of human innate immunity to a variety of pathogens, including Staphylococcus aureus. The aim of the present study was to investigate a potential association between gene variations in DEFB1 and DEFB103/DEFB4 and the development of S. aureus bacteremia (SAB) employing a case-control design.Cases were unique patients with documented SAB, identified with the National S. aureus Bacteremia Register, a comprehensive dataset of all episodes of community associated-SABs (CA-SAB) occurring in children (≤20 yrs) in Denmark from 1990 to 2006. Controls were age-matched healthy individuals with no history of SAB. DNA obtained from cases and controls using the Danish Newborn Screening Biobank were genotyped for functional polymorphisms of DEFB1 by Sanger sequencing and copy number variation of the DEFB103 and DEFB4 genes using Pyrosequencing-based Paralogue Ratio Test (P-PRT).193 ethnic Danish SAB cases with 382 age-matched controls were used for this study. S. aureus isolates represented a variety of bacterial (i.e., different spa types) types similar to SAB isolates in general. DEFB1 minor allele frequencies of rs11362 (cases vs. controls 0.47/0.44), rs1800972 (0.21/0.24), and rs1799946 (0.32/0.33) were not significantly different in cases compared with controls. Also, DEFB4/DEFB103 gene copy numbers (means 4.83/4.92) were not significantly different in cases compared with controls.Using a large, unique cohort of pediatric CA-SAB, we found no significant association between DEFB1 genetic variation or DEFB4/DEFB103 gene copy number and susceptibility for SAB

<i>DEFB1</i> Genotype and minor allele frequencies in CA-SAB cases and controls.

<p><i>DEFB1</i> Genotype and minor allele frequencies in CA-SAB cases and controls.</p

Phage distribution among SAB cases 1990–2006.

<p>Phage distribution among SAB cases 1990–2006.</p

Power curves for an unpaired t-test with 193 cases and 382 controls, assuming equal variance in cases and controls and a significance level of 0.05.

<p>True difference in mean copy number is shown on the x-axis, and the power to detect such differences is shown on the y-axis. Curves are shown for three different copy number standard deviations: 1.1 (solid), 1.2 (dashed), and 1.3 (dotted).</p

Frequency distribution of <i>DEFB4/DEFB103</i> GCN for SAB cases and controls.

<p>Frequency distribution of <i>DEFB4/DEFB103</i> GCN for SAB cases and controls.</p

Scatterplots showing reproducibility of positive controls between different experiments.

<p>These figures plot known copy number from control samples against actual raw copy number values, for each experiment. Note that all five populations are not represented: several experiments tested samples from more than one population, which was an additional control against systematic bias. The legend on the graphs indicates the population from which the majority of samples in that particular experiment derived. a) QPCR assay, b) PPRT assay, c) Triplex PRT assay.</p