Introduction

Gene copy number variations (CNVs) contribute significantly to interindividual genetic variability (1). One important site of genomic rearrangement is the defensin gene locus located at chromosome 8p23.1 (2). Defensins are antimicrobial and immunomodulating polypeptides that play an important role in the innate immune system of mammals, insects, and plants (3-8). Genes for human β-defensin 2 (hBD-2), β-defensin 3 (hBD-3), and β-defensin 4 (hBD-4) were named DEFB4, DEFB103A, and DEFB104A, respectively. Recent studies have shown functional implications of variation in DEFB4, DEFB103A, and DEFB104A gene copy numbers (2,9). Since defensins play an important role in the innate immune response, quantitative variations in the gene copy number might contribute to susceptibility to infectious and inflammatory diseases (10).

In principle, CNVs are detectable using screening techniques such as DNA arrays or sequencing. Redon et al. compared the single-nucleotide polymorphism genotyping array method and the comparative genomic hybridization array method for CNV detection (1), finding that both high-throughput methods detected deletions and duplications qualitatively, but failed to generate definitive gene copy numbers. Recently, parallel sequencing emerged as a method for quantitative identification of genomic structural variance, including deletions, duplications, and rearrangements (11,12). However, this method is limited by its use of random amplification primers and lack of definitive copy number quantification. Although genome-wide CNV mapping has been simplified by these different direct screening methods, none of these approaches currently offers reliable single gene quantification.

Consequently, defensin CNVs are analyzed with methods that offer single gene quantification, such as MLPA, PPRT, fluorescence in situ hybridization (FISH), or real-time PCR. To date, a gold standard method for defensin gene quantification has not been established (2,9, 13-15). Hollox et al. reported concordant gene copy numbers of DEFB4, DEFB103A, and DEFB104A by multiplex amplifiable probe hybridization and semiquantitative fluorescence in situ hybridization (2). Our own multiplex ligation-dependent probe amplification (MLPA) results confirmed this concordance (14), whereas PCR analysis detected discordance in intra-individual β-defensin copy numbers (13). Other groups have reported inter-method quantification comparisons that showed inconsistent results for absolute copy number (16,17). Therefore, a thorough reevaluation of different quantification methods is required. The aim of this study was to compare MLPA and real-time PCR for β-defensin gene quantification through the analysis of 80 genomic DNA samples.

Materials and methods

Genomic DNA

DNA samples from cell lines and whole blood samples were included. 42 genomic DNA samples, derived from immortalized B-lymphoblastoid cells lines, were purchased from the Coriell Institute for Medical Research (Camden, NJ, USA) and the European Collection of Cell Cultures (ECACC; Wiltshire, UK). Samples were named NAxxxxx (Coriell) and Cxxxx (ECACC) and have been designated with a superscripted ‘HM’ if they were used in the HapMap Project (18). In addition, 38 DNA samples (code RC) from a white population extracted from whole blood were also analyzed. Written informed consent from healthy volunteers was obtained, as requested by the local ethics committee. This procedure complied with all relevant laws, guidelines, and policies. For simplicity, all specimens will be referred to hereafter as “DNA samples.” The concentration and purity of DNA was determined using a NanoDrop device (NanoDrop Technologies, Wilmington, DE, USA). MLPA and PPRT data from the Coriell and ECACC samples were part of a previous report (14).

Real-time PCR assay

The primers and hybridization probes were synthesized according to our previously published approach (13). The albumin gene (ALB) served as a single copy per chromosome reference gene in this study. The method of real-time quantitative PCR using LightCycler Relative Quantification Software 1.0 (Roche, Mannheim, Germany) for analysis requires an appropriate calibrator. Genomic DNA sample NA18608^HM, with one copy of target gene per haploid genome as determined by different methods (14,19) and one copy of reference gene per haploid genome, was used as calibrator. The PCR reaction was performed according to the protocol established by our group (13). In brief, this method of relative real-time quantitative PCR requires a coefficiency file from which the efficiency of PCR is calculated by the software. To create a coefficiency file for the ALB gene and the DEFB4, DEFB103A, or DEFB104A gene, an unknown genomic DNA sample was prepared in a 10-step dilution series. Then, for each dilution, the β-defensin and the ALB genes were amplified in a single capillary to acquire the relative quantification standard curves for both of these genes. Using the LightCycler software, a coefficiency file can be prepared from the two relative quantification standard curves. Data analysis of the real-time PCR method has been described in detail by our group in a previous publication (13).