Screening of copy number polymorphisms in human β-defensin genes using modified real-time quantitative PCR

Journal of Immunological Methods 308 (2006) 231 – 240 www.elsevier.com/locate/jim

Research paper

Screening of copy number polymorphisms in human h-defensin genes using modified real-time quantitative PCR QiXing Chen a,b, Malte Book a, XiangMing Fang b, Andreas Hoeft a, Frank Stuber a,* a

Department of Anaesthesiology and Intensive Care Medicine, University of Bonn, 53105 Bonn, Germany b School of Medicine, Zhejiang University, Hangzhou, P. R. China Received 26 August 2005; received in revised form 3 November 2005; accepted 14 November 2005 Available online 15 December 2005

Abstract Defensins are cationic antimicrobial peptides, which play an important role in host immune defense to some infectious diseases as well as immune disease and skin disease. Recent studies identified that the genes coding for human h-defensin 2 (DEFB4), human h-defensin 3 (DEFB103) and human h-defensin 4 (DEFB104) showed variation in copy numbers. This variation may have an impact on gene expression levels. Here, we have demonstrated a real-time PCR-based method to measure h-defensin gene copy number. Using this relative real-time quantitative PCR, we developed a new rapid and reliable approach, which involves amplification of the target locus (DEFB4 or DEFB103 or DEFB104) and the single-copy reference locus (human serum albumin, ALB) in a single PCR reaction. A calibrator was prepared by recombining one copy of the target gene and one copy of the reference gene into a plasmid. After correcting the PCR amplification efficiency, which differed between the defensin gene and ALB gene, and normalization by the calibrator, the ratio of the copy number of the target gene to that of the reference gene in an unknown sample was determined. This normalized ratio directly related to the gene copy number. The assay was validated using previously genotyped samples, which demonstrated high accuracy and reliability of the method. Furthermore, this method was used to screen the copy number variations of these three h-defensin genes in healthy blood donors. This method proved to be a reliable and fast tool to genotype gene copy number variations in projects associating genomic variations with gene expression or with population phenotypes in epidemiologic studies. D 2005 Elsevier B.V. All rights reserved. Keywords: Copy number polymorphisms; Human h-defensin genes; DEFB4; DEFB103; DEFB104; Real-time quantitative PCR

1. Introduction Abbreviations: HNP, human neutrophil peptide; hBD, human hdefensin; DEFB4, human h-defensin 2; DEFB103, human h-defensin 3; DEFB104, human h-defensin 4; FISH, fluorescence in situ hybridization; CGH, comparative genome hybridization; MAPH, multiplex amplifiable probe hybridization; MLPA, multiplex ligation-dependent probe amplification; CNP, copy number polymorphism; ALB, human serum albumin. * Corresponding author. Tel.: +49 228 2874114; fax: +49 228 2874125. E-mail address: [email protected] (F. Stuber). 0022-1759/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2005.11.001

Defensins are cysteine-rich cationic antimicrobial polypeptides with three or four disulfide bridges, which constitute an important part of the immune system in mammals and insects as well as plants (Lehrer and Ganz, 2002; Raj and Dentino, 2002; Ganz, 2003). In humans, based on the organization of three intramolecular disulfide bonds, two families of defensins have been characterized and termed as adefensins and h-defensins. In a-defensins, human neu-

232

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

trophil peptides 1–4 (HNP1–4) are mainly expressed in neutrophils, whereas human defensins 5–6 (HD 5– 6) are located in intestinal Paneth cells and reproductive tract epithelia (Ganz, 2003). Human h-defensins (hBDs) are epithelium-derived antimicrobial peptides, in which DEFB4, DEFB103 and DEFB104 show stronger and more specific microbicidal activity than a-defensins in ex vivo studies (Bensch et al., 1995; Harder et al., 1997, 2001; Garcı´a et al., 2001). Furthermore, the concentration of DEFB4 in body fluid was elevated in infected individuals (Ashitani et al., 2001; Schaller-Bals et al., 2002; Hiratsuka et al., 2003). Enhanced mRNA expression levels of DEFB4, DEFB103 and DEFB104 were observed in biopsies from patients with ulcerative colitis (Wehkamp et al., 2003). However, inactivated DEFB4 in the respiratory tract has been associated with the incidence of infections in cystic fibrosis patients (Singh et al., 1998) and the inducibility of DEFB4 and DEFB103 expression was diminished in Crohn’s disease (Wehkamp et al., 2003). These findings indicate that defensins may have pronounced tasks in the immune response to infection. Besides their bactericidal properties, DEFB4 exerts chemotactic activity to T cells, dendritic cells and neutrophils. DEFB103 and DEFB104 are chemoattractants for monocytes, which suggest that these defensins may act as a multi-functional mediator in the immune system (Yang et al., 1999, 2002). The genes coding for DEFB4, DEFB103 and DEFB104 have been localized to 8p22–23, which is a frequent site of chromosomal rearrangements (Giglio et al., 2001; Lehrer and Ganz, 2002; Hollox et al., 2003). Recent studies showed existence of various copy numbers of DEFB4, DEFB103 and DEFB104 in this region (Hollox et al., 2003; Boniotto et al., 2004; Taudien et al., 2004; Linzmeier and Ganz, 2005). Furthermore, the copy number variation of DEFB4 was associated with the mRNA expression level of DEFB4 (Hollox et al., 2003). Since defensins play an important role in the innate immune response, this finding may help to uncover the genetic predisposition of these components for susceptibility to some infectious diseases. Molecular genetic methods for measurement of gene copy number variations include Southern blot (PetrijBosch et al., 1997), chromosomal comparative genome hybridization (CGH) (Kallioniemi et al., 1992), fluorescence in situ hybridization (FISH) (Kato et al., 2004), multiplex amplifiable probe hybridization (MAPH) and multiplex ligation-dependent probe amplification (MLPA) (Armour et al., 2004). Recently, quantitative

real-time PCR served as an alternative approach to detect gene copy number variations (Ginzinger, 2002). Quantitative real-time PCR-genotyping depends on the principle that the fractional cycle number (Ct or Cp), at which the amount of an amplified target reaches a fixed threshold, is related to the starting amount of target. A higher or lower starting copy number of genomic DNA target will result in a significant earlier or later increase in fluorescence, respectively. Using a comparative Ct method, the relative gene number can be quantified (Ginzinger, 2002). This method has been successfully applied for gene dosage detection (Wilke et al., 2000; Anhuf et al., 2003; Schaeffeler et al., 2003; Laccone et al., 2004). In this study, we developed a highly sensitive and specific method to detect the copy number variations of defensin genes using real-time quantitative PCR. The method was robust and rapid, and represents a powerful method to screen copy number polymorphisms (CNP) of defensins in gene expression projects or in population-based association studies. 2. Materials and methods 2.1. Objects Forty-four healthy Caucasian blood donors were enrolled to investigate the gene copy number variations of DEFB4, DEFB103 and DEFB104 with this method. This study was approved by the local ethics committee and informed written consent was obtained from all the participants. 2.2. Cell culture Human lymphocyte cell lines BO0183, AF0103, AF0105 and TT0296 were purchased from the European Collection of Animal Cell Culture (ECACC, Wiltshire, UK). The cells were grown in RMPI 1640 medium (Biochrom, Berlin, Germany) supplied with 10% FBS (Biochrom, Berlin, Germany), 2 mM l-glutamine as well as 100 U/ml penicillin and 100 Ag/ml streptomycin (Invitrogen, Karlsruhe, Germany) at 37 8C in a 5% CO2 atmosphere. 2.3. DNA extraction DNA was extracted from peripheral blood and culture cells using QIAamp DNA blood kits (Qiagen, Hilden, Germany). Compared with other DNA extraction kits, this kit was recommended for acquiring a high quality DNA template, which is very important

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

233

Table 1 Sequence of primers and probes Gene (GenBank accession no.)

Primer/probe

Sequence of primers and fluorescence labeling probes

DEFB4 (AF071216)

DEFB4_F DEFB4_R

5V-AgTTTTgAgTTCTTACACgCTg-3V 5V-gCATCAgCCACAgCAgCT-3V

DEFB4_FL DEFB4_LC DEFB103_A DEFB103_S DEFB103_FL DEFB103_LC DEFB104_A DEFB104_S DEFB104_FL DEFB104_LC ALB_F ALB_R ALB_FL ALB_705

5V-ggTATAAACAAATTggCACCTgTggTC-FL 5V-LC 640-CCCTggAACAAAATgCTgCAAAA-PH 5V-CACACTTTACAACACTCTCgTCATgT-3V 5V-CCTTCCTAAAACCTTTCCgTg-3V 5V-gCAgCTgAgCACAgCACACCg-FL 5V-LC 640-CCgCCTCTgACTCTgCAATAATATTTCTgT-PH 5V-CAgCgACTCTAgggACC-3V 5V-ACAgTgCCATATCCTgTTATCT-3V 5V-CATgCTgTTTgAgAAAATgggAT-FL 5V-LC 640-AgAgCTTACTgAATCgTACAAAACCCT-PH 5V-ggTCCTgAACCAgTTATgTg-3V 5V-TAAgggCAACACTCCAATAC-3V 5V-CATggTCgCCTgTTCACCAAggAT-FL 5V-LC 705-CTgTgCAgCATTTggTgACTCTgTCA-PH

DEFB103 (AC130360)

DEFB104 (AJ314835)

ALB (M12523)

for the reliability of the experiment (Aarskog and Vedeler, 2000). The concentration of DNA was determined by a spectrophotometer. DNA was diluted in 10 mM pH 8.5 Tris-buffer to a concentration of ~ 50 ng/ Al and stored at 20 8C. 2.4. Primers and probes The primers and hybridization probes were synthesized by TIB-MOLBIOL (TIB-MOLBIOL, Berlin, Germany). The melting temperature of probes was 5–10 8C higher than those of the matching primers. In each pair of probes, the donor probe was labeled with fluorescein (FITC) at the 3V end; the 5V end of the acceptor probe was connected to LightCycler-red-640 (LC-640; for DEFB4, DEFB103, DEFB104) or LightCycler-red705 (LC-705; for human serum albumin which served as a single copy reference gene in this study); and the 3V end was blocked by phosphorylation to prevent the acceptor probe from elongation during PCR. Primers and probes were diluted in PCR-grade ddH2O and stored at 20 8C in aliquots for single use. The sequence of all the primers and probes used in this study is shown in Table 1.

Nucleotide position 4377–4398 4596–4579 4509–4535 4537–4559 117,055–117,080 117,278–117,258 117,160–117,180 117,182–117,211 5989–5973 5774–5795 5906–5928 5930–5956 15,542–15,561 15,884–15,865 15,637–15,614 15,612–15,587

Genomic organization Intron 1 Downstream of exon 2 Exon 2 Exon 2 Exon 2 Intron 1 Exon 2 Exon 2 Exon 2 Intron 1 Exon 2 Exon 2 Exon 12 Intron 12 Exon 12 Exon 12

2.5. Calibrator and control The method of relative real-time quantitative PCR using LightCycler Relative Quantification Software 1.0 (Roche, Mannheim, Germany) for analysis (see below) requires an appropriate calibrator. To construct a calibrator for real-time PCR analysis, the PCR products of DEFB4 (or DEFB103 or DEFB104) and human serum albumin (ALB) gene segments were cloned into pGEM-T-easy vector (Promega, Mannheim, Germany) by primer adapters containing endonuclease sites. Meanwhile, plasmids containing two fragments of the target gene (DEFB4 or DEFB103 or DEFB104) and one fragment of the ALB gene within a pGEM-T-easy vector were prepared as controls. The constructs of calibrator and control are illustrated in Fig. 1. Plasmid DNA was successfully used as a calibrator for quantification of gene copy number (Tse et al., 2005; Hijri and Sanders, 2005). Amplification of recombinant plasmid DNA and target genomic DNA has been demonstrated to occur with the same efficiency by optimization of the real-time PCR technique (Teo et al., 2002; Hijri and Sanders, 2005). In the present study, the PCR primers and hybridyzation probes were designed

Fig. 1. Calibrator (above) and control (below) from multiple cloning site of recombinant pGEM-T Easy vector containing fragments of DEFB4 and albumin (ALB) genes. The size of the DEFB4 and ALB fragments are 220 bp and 343 bp, respectively. The calibrator contains one copy of DEFB4 and one copy of ALB, while the control includes two copies of DEFB4 and one copy of ALB.

234

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

for high specificity. The PCR reaction components for calibrator (plasmid DNA) and samples (chromosomal DNA) were prepared from a same reaction mix. The Cp value for calibrator and samples was detected between 26 and 28 cycles of the PCR reaction. 2.6. Real-time quantitative PCR PCR was performed using the LightCycler system (Roche, Mannheim, Germany), the LightCycler FastStart DNA Master Hybridization Probes kit (Roche, Mannheim, Germany) and the LightCycler Sample Capillaries (Roche, Mannheim, Germany) in reaction volumes of 20 Al. All reactions of the same run were prepared from the same master mix and contained 1 LightCycler FastStart DNA Master Hybridization Probes, 4 mM MgCl2, 1000 nM ALB forward primer and 1000 mM ALB reverse primer, 150 nM ALB sensor probe and 150 nM ALB anchor probe, 100 nM sensor probe and 100 nM anchor probe for DEFB4 or DEFB103 or DEFB104, 200 nM forward primer and 200 nM reverse primer for DEFB4 or DEFB104, or 250 nM forward primer and 250 nM reverse primer for DEFB103, respectively. A total of 10 ng genomic DNA was dispensed into each capillary. Each sample was amplified in duplicate. The thermal cycling conditions were as follows: a pre-run at 95 8C for 10 min, 45 cycles with a 5 s denaturation step at 95 8C followed by a 59 8C annealing step for 8 s and a 72 8C extension step for 8 s according to the LightCycler manual. The calibrator was amplified in each run in parallel with the samples. In addition, each PCR run was monitored by the control. A no-template control (negative control) was also included in each assay. 2.7. Data analysis Data evaluation was carried out using the LightCycler Software 3.5 and LightCycler Relative Quantification Software 1.0. The crossing cycle number (Cross Point or CP, same as Ct mentioned above) was determined for all PCR reactions using Second Derivative Maximum Method in which the Cp value is achieved by a software algorithm, which identifies the first turning point of the fluorescence curve. Separate relative standard curves were generated for the targets and reference loci to conduct a coefficiency file using the LightCycler Relative Quantification Software 1.0. After the PCR run, the LCDA data were exported into a *.txt file. Then, the *.txt files of target gene (DEFB4 or DEFB103 or DEFB104) and reference gene (ALB)

from relative samples were imported into the LightCycler Relative Quantification Software 1.0. Based on the coefficiency file, the ratio of the copy number of the h-defensin gene relative to that of the ALB gene in each sample was quantified by the software algorithm and expressed as a normalized ratio compared to the calibrator. Normalized ratio = E TCpT(C) CpT(S)  E RCpR(S) CpR(C) (E: efficiency of PCR amplification, T: target gene, R: reference gene, S: unknown sample, C: calibrator). Since the ratio of defensin gene copy number to ALB gene copy number in the calibrator was 1, the absolute copy number of DEFB4 or DEFB103 or DEFB104 in a diploid genome is twice that of the normalized ratio. 2.8. Statistical analysis Data means were expressed with standard deviations (S.D.). For group comparisons of the normalized ratio, normality of the data distribution was evaluated by the Kolmogorov–Smirnov test. Data were analyzed using one-way ANOVA or t-test. The Bonferroni correction was applied when necessary. p b 0.05 was considered to be a significant difference. Statistical analysis was performed using GraphPad PRISM 3.00 (Graph Pad Inc., San Diego, CA). 3. Results 3.1. Assay development To ensure PCR product amplification from genomic DNA only, the up-stream and down-stream primers for each tested gene were designed in exons and introns, respectively. Initially, separate and multiplex pre-runs at various concentrations of primers and probes for hdefensin and ALB genes were tested. In this study, the primer pairs, which resulted in the highest amplification rate both for h-defensin and ALB, were used. Performing the amplification of each sample in duplicate runs showed almost complete overlap in the exponential and plateau phases of parallel amplification plots. Calculation of the Cp value using the Second Derivative Maximum Method resulted in low Cp S.D. values (mean 0.062, range 0.016–0.138 vs. mean 0.130, range 0.037–0.309, using the fit point method in which a threshold was set manually). To create a coefficiency file for the ALB gene and the DEFB4, DEFB103 or DEFB104 genes, an unknown genomic DNA sample was diluted in 10-time series. Then, the h-defensin gene and the ALB gene

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

were amplified in one capillary to acquire the relative standard curves for both of these genes. The two relative standard curves would constitute a coefficiency file, as shown in Fig. 2. When the DEFB4 and the ALB gene were amplified simultaneously, the amplifi-

235

cation efficiency of each gene was different and even the difference from high template concentration to low template concentration was detected (Fig. 2). Using the fit coefficient function, the LightCycler Relative Quantification Software calculated the amplification

A

DEFB4 Coefficient A 5.9447e+000 Coefficient B -2.7909e-001 Coefficient C -2.3267e+001 Coefficient D 1.7752e+000 Coefficient E -3.6116e-002 Coefficient F -2.6774e-021 Coefficient G 2.8440e+001 Normalized MSE 7.7985e-005

ALB Coefficient A 6.8270e+000 Coefficient B -2.9770e-001 Coefficient C -1.7050e+001 Coefficient D 1.3086e+000 Coefficient E -2.7015e-002 Coefficient F -6.0473e-021 Coefficient G 2.9730e+001 Normalized MSE 9.8132e-004

DEFB103 Coefficient A 1.8230e+000 Coefficient B -2.8410e-001 Coefficient C -1.1021e+002 Coefficient D 6.9403e+000 Coefficient E -1.1647e-001 Coefficient F -1.5940e-020 Coefficient G 3.1015e+001 Normalized MSE 1.1006e-004

ALB Coefficient A 2.7820e+000 Coefficient B -2.9921e-001 Coefficient C -1.3594e+002 Coefficient D 8.2074e+000 Coefficient E -1.3041e-001 Coefficient F -4.1218e-020 Coefficient G 3.2615e+001 Normalized MSE 3.5545e-004

DEFB104 Coefficient A 1.1632e+000 Coefficient B -2.7733e-001 Coefficient C 1.4272e+000 Coefficient D -2.9526e-001 Coefficient E 3.0427e-004 Coefficient F -1.1233e-021 Coefficient G 2.9460e+001 Normalized MSE 9.1033e-005

ALB Coefficient A 1.7156e+000 Coefficient B -2.7359e-001 Coefficient C 5.7476e-001 Coefficient D -1.9272e-001 Coefficient E -1.4331e-003 Coefficient F -2.1321e-012 Coefficient G 2.8215e+001 Normalized MSE 3.1405e-005

B

C

Fig. 2. Coefficiency file for amplification of h-defensins and albumin. A relative standard curve is fitted through the data points to minimize the fit error. The fit function is mathematically defined by fit coefficients: coefficients A and B for calculation of the linear part; coefficients C, D, E and F for calculation of the linear part; coefficient G for calculation of the transition-point; normalized MSE refers to normalized mean squared error. (A) DEFB4, (B) DEFB103 and (C) DEFB104.

236

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

Table 2 Validation of copy number of DEFB4 on the basis of three independent runs DNA

Copy number

Expected normalized ratio

Mean

Range

S.D.

CV (%)

BO0183 AF0103 AF0105 TT0296

3 4 4 5

1.5 2 2 2.5

1.42 1.98 1.81 2.40

1.40–1.44 1.92–2.07 1.80–1.82 2.34–2.48

0.02 0.08 0.01 0.07

1.73 3.98 0.58 3.04

S.D., standard deviation; CV, coefficient of variation.

efficiency of each gene automatically and introduced a correction for different amplification efficiencies in the sample quantification process. This gave a result that was more accurate than that obtained using the conventional linear fit (Sagner and Goldstein, 2001). Another prerequisite for analysis with the LightCycler Relative Quantification Software 1.0 is a calibrator in which the copy number ratio of the target gene to the reference gene is known. Since the CNP in individuals is unknown to the investigator, a plasmid DNA containing the fragments of DEFB4 or DEFB103 or DEFB104 and ALB gene was constructed. Since only a single copy of DEFB4 or DEFB103 or DEFB104 and one copy of the ALB gene were inserted in this plasmid DNA, the copy number ratio of DEFB4 or DEFB103 or DEFB104 to the ALB gene was 1. In addition, to monitor the PCR runs, a control plasmid DNA, which included two copies of DEFB4 or DEFB103 or DEFB104 fragment and one copy of the ALB gene fragment, was tested with other samples in every run. Only when the normalized ratio of the control was 2 were the PCR results deemed to be reliable. 3.2. Evaluation of the method For validation of the method, four DNA samples (from the lymphocyte cell line) with a known number of three, four and five copies of DEFB4 determined

previously (Hollox et al., 2003, personal communication) were initially analyzed in duplicates in 3 different runs to estimate the precision and reproducibility of the assay. As shown in Table 2, the mean of the normalized ratio as a measure of the haploid DEFB4 gene copy number matched well with the known gene copy number in each sample. When the four samples were categorized into three groups (three copies, four copies and five copies), the mean normalized ratio (F S.D.) was 1.42 F 0.05, 1.89 F 0.11 and 2.40 F 0.12 for each group, respectively ( p b 0.001, one-way ANOVA). Importantly, the normalized ratios did not overlap between the different groups. This indicates that this method is sensitive enough to discriminate one-copy differences between samples. Meanwhile, both the intraassay and interassay variability of the method were evaluated by measuring the copy numbers of DEFB4 in six samples. The intraassay variability was determined from duplicates within the same run. The coefficient of variation (CV) in 12 measurements ranged between 0.33% and 3.50%. The interassay variability was assessed separately on six different samples on three different days. The CV ranged between 1.33% and 10.16%. Taken together, these analysis demonstrated that the copy number polymorphisms could be correctly and reproducibly determined using real-time quantitative PCR.

Table 3 Copy number polymorphisms of DEFB4, DEFB103 and DEFB104 detected by real-time quantitative PCR Gene

Copy number

Mean

Range

S.D.

95% CI

CV (%)

Genotype frequency

DEFB4

2 3 4 5 6 7 2 3 4 2 3

1.02 1.54 1.98 2.45 2.93 3.49 1.12 1.48 1.94 1.02 1.43

– 1.34–1.69 1.78–2.19 2.33–2.77 – – 0.92–1.19 1.31–1.65 1.77–2.08 0.79–1.18 1.30–1.56

– 0.11 0.13 0.14 – – 0.07 0.09 0.10 0.10 0.07

– 1.48–1.59 1.93–2.03 2.38–2.52 – – 1.09–1.16 1.46–1.51 1.88–2.01 1.00–1.05 1.40–1.46

–

2.27% 20.45% 52.27% 20.45% 2.27% 2.27% 20.45% 65.91% 13.64% 70.45% 29.55%

DEFB103

DEFB104

S.D., standard deviation; CI, confidence interval; CV, coefficient of variation.

7.22 6.72 5.71 – – 6.28 5.98 5.11 10.23 4.97

(1/44) (9/44) (23/44) (9/44) (1/44) (1/44) (9/44) (29/44) (6/44) (31/44) (13/44)

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

3.3. Genotyping copy number variation in DEFB4, DEFB103 and DEFB104 The CNPs of DEFB4, DEFB103 and DEFB104 were screened in 44 healthy blood donors using realtime quantitative PCR. As shown in Tables 3 and 4, the CNP of these three genes showed intraindividual differences. In DEFB4, the three-, four- and five-copy numTable 4 Copy number of DEFB4, DEFB103 and DEFB104 per diploid genome Sample number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Copy number of h-defensin genes DEFB4

DEFB103

DEFB104

4 4 4 4 4 6 4 5 4 4 4 3 3 4 3 4 5 3 5 5 5 5 3 5 2 3 5 5 3 4 4 4 4 4 4 4 3 4 4 4 3 4 7 4

3 3 3 3 3 4 3 4 3 3 3 2 2 3 2 3 3 2 4 3 3 3 2 4 2 3 3 3 2 3 3 3 3 3 4 3 2 3 3 3 3 2 4 3

3 2 2 2 3 3 3 3 2 2 3 2 2 2 2 2 3 2 3 2 2 3 2 3 2 2 2 3 2 2 2 2 2 3 2 2 2 2 2 2 2 2 3 2

237

ber variants were the main genotypes (20.45%, 52.27% and 20.45%, respectively); the other copy variants were less frequent. For DEFB103, the three-copy number variant was the most frequent genotype (65.91%), with two-copy and four-copy variants occurring with frequencies of 20.45% and 13.64%, respectively. In contrast, in DEFB104, the two-copy variant was the dominant genotype (70.45%). There were 29.55% individuals showing three copy numbers of DEFB104. No other copy numbers of DEFB104 were detected in these samples. Importantly, there was no overlap between the groups of different copy numbers for all three defensin genes. 4. Discussion In this study, a method of real-time quantitative PCR to detect copy number polymorphisms of human hdefensin genes is demonstrated. Furthermore, using this method, the CNPs of DEFB4, DEFB103 and DEFB104 in 44 DNA samples were determined, which presented the first comprehensive genotype data of the CNPs in these three genes. With the ability to measure the PCR products as they are accumulating, or in breal timeQ, real-time quantitative PCR is widely used for quantification of bacterial or viral pathogens (Szuhai et al., 2001; He et al., 2002) as well as for the analysis of minimal residual disease (Elmaagacli, 2002) and gene expression (Gallagher et al., 2003). Recently, this method has become an attractive method for gene copy number measurements, which has been proven to be fast, precise, reproducible and high-throughput (Aarskog and Vedeler, 2000; Laccone et al., 2004; Suo et al., 2004; Kindich et al., 2005). In contrast, traditional methods for measuring DNA copy numbers, such as FISH, are difficult to perform in high throughput and fail to detect small deletions/duplications (Ginzinger, 2002; Rooms et al., 2005). Southern blot and chromosomal CGH require relatively large amounts of genomic DNA; in addition, the method of Southern blot is time- and labor-consuming (Ginzinger, 2002; Rooms et al., 2005). MAPH and MLPA are promising newcomers to detect dosage changes. However, neither of these two methods is able to detect translocations in balanced form (Rooms et al., 2005). Furthermore, MAPH is sensitive and able to detect a large relative change in copy number, such as one copy instead of two, but not sensitive enough to discriminate between high copy numbers (Hollox et al., 2003). Using the LightCycler Software 3.5 and LightCycler Relative Quantification Software 1.0, the copy number

238

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

polymorphisms in DEFB4, DEFB103 and DEFB104 genes were distinguished accurately and sensitively. Compared to other real-time quantitative PCR methods reported previously (Aarskog and Vedeler, 2000; Wilke et al., 2000; Anhuf et al., 2003; Schaeffeler et al., 2003; Chain et al., 2005; Linzmeier and Ganz, 2005), the method introduced here has several advantages: (1) cross point determination using the Second Derivative Maximum Method. Minor deviations in the PCR amplification and the fluorescence detection within the logarithmic phase can lead to significant variation in the Cp value, which may lead to misinterpretation of the gene copy number. Since any single nucleotide polymorphism within the primer region may affect PCR amplification, primer sequences were chosen from areas of total alignment when comparing published sequences of the same target gene. Meanwhile, optimizing the method for detection of threshold fluorescence to calculate the optimal Cp value is very important. In the Second Derivative Maximum Method, Cp determination is only defined by curve shape, not by fluorescence background or a manually defined crossing line, which avoids setting thresholds in each experiment and makes the results more precise and reproducible (see Results). (2) Fit correction of PCR efficiency (Fig. 2). So far, for relative quantification of gene copy numbers, two methods are well characterized: the DDCt method and the standard curve method. However, in the DDCt method, the PCR efficiency for both target gene and reference gene should be identical, which imposes a major task on the investigator to optimize the PCR condition. Regarding amplification of the same gene, the PCR efficiency may be different because of template concentration variation (Fig. 2), which impairs comparison of the PCR efficiency between the target and reference genes. For the standard curve method, the standard curves both for the target and reference genes are included in each PCR run, which lowers the throughput of the method. In the method described here, the standard curves for the target and reference genes were determined once and stored as a coefficient file, which could be used for each analysis. Meanwhile, using fit coefficient function, the PCR efficiency of the detected gene was accurately calculated, especially in low concentration samples (Sagner and Goldstein, 2001). Furthermore, based on the algorithm deposited in the LightCycler Relative Quantification Software 1.0, the method calculates the final result (normalized ratio) only depending on the Cp value and PCR efficiency of both genes in the calibrator and the sample, without requiring identical PCR efficiency between

these genes. These contributed to the simple and accurate approach (Suo et al., 2004). (3) Plasmid DNA as calibrator and control. Having a calibrator including only one copy of the h-defensin gene and one copy of the ALB gene is a requirement for analysis with the LightCycler Relative Quantification Software 1.0. Using this plasmid as calibrator, together with another plasmid containing two copies of the h-defensin gene and one copy of the ALB gene as control, facilitated the development of this method. This eliminates the need for calibrator and control from genomic DNA samples, which should be detected using other additional methods. As demonstrated by analysis of the DNA samples from four lymphocyte cell lines with known DEFB4 copy number (Hollox et al., 2003, personal communication), the method showed correct and reproducible results. The mean of the normalized ratios from duplicates in the run agreed well with the expected value (Table 2). In addition, the intraassay variability ranged between 0.33% and 3.50%, while the interassay variability was between 1.33% and 10.16%. Furthermore, the assay was completed within 1 h. Using this method, the CNPs of the three defensin genes were detected in 44 Caucasians. The coefficient of variation of all the assays was between 4.97% and 10.23%. Importantly, the present study has shown that the copy numbers of DEFB4, DEFB103 and DEFB104 differ within the same individual. For DEFB4, three copies, four copies and five copies are the main genotypes and this is consistent with a previous report (Hollox et al., 2003). Since Hollox et al. used only one pair of probes located in intron 1 and exon 2 of DEFB4 to test the copy number of these three defensin genes, this may compromise the copy number of the other two genes in individual donors as the DEFB103 and DEFB104 copy numbers were not detected directly. Since these three h-defensins display diverse biological activity to different bacteria, our findings may underline different tasks of defensins in the immune response to invading microorganisms. However, Linzmeier and Ganz (2005) utilized a SYBR green-based DDCt method to investigate CNPs of the defensin family in different ethnic populations. They reported a mean of five-copy numbers per diploid genome, in contrast to the results of Hollox and this report. The difference may be explained by the different strategies adopted and the different sensitivity of these two methods. Ethnic differences may also contribute to the discrepancy. Recently, copy number variations in human a-defensin genes have also been identified (Aldred et al., 2005; Linzmeier and Ganz, 2005). Defensins are antimicrobi-

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

al peptides and are important components in innate immunity for protecting the host from invading microorganisms (Fang et al., 2003; Ganz, 2003). In the light of their broad bactericidal spectrum as well as their mediating role between innate immunity and adaptive immunity, these findings may indicate a genetic role of defensins in immune defense. Further clinical association studies will help to discover the role of the copy number polymorphism of the h-defensin genes in certain infectious or skin diseases. Real-time quantitative PCR will provide a powerful tool for screening copy number polymorphisms in these studies. Acknowledgment This study is supported by German Research Foundation BO1929/2-1 (M.B.). We thank Dr. Hollox for providing the information on the cell lines and the copy numbers in these cell lines. References Aarskog, N.K., Vedeler, C.A., 2000. Real-time quantitative polymerase chain reaction. A new method that detects both the peripheral myelin protein 22 duplication in Charcot-Marie-Tooth type 1A disease and the peripheral myelin protein 22 deletion in hereditary neuropathy with liability to pressure palsies. Hum. Genet. 107, 494. Aldred, P.M., Hollox, E.J., Armour, J.A., 2005. Copy number polymorphism and expression level variation of the human alphadefensin genes DEFA1 and DEFA3. Hum. Mol. Genet. 14, 2045. Anhuf, D., Eggermann, T., Rudnik-Schoneborn, S., Zerres, K., 2003. Determination of SMN1 and SMN2 copy number using TaqMan technology. Hum. Mutat. 22, 74. Armour, J.A., Rad, I.A., Hollox, E.J., Akrami, S.M., Cross, G.S., 2004. Gene dosage analysis by multiplex amplifiable probe hybridization. Methods Mol. Med. 92, 125. Ashitani, J.I., Mukae, H., Hiratsuka, T., Nakazato, M., Kumamoto, K., Matsukura, S., 2001. Plasma and BAL fluid concentrations of antimicrobial peptides in patients with Mycobacterium avium– intracellulare infection. Chest 119, 1131. Bensch, K.W., Raida, M., Magert, H.J., Schulz-Knappe, P., Forssmann, W.G., 1995. hBD-1: a novel h-defensin from human plasma. FEBS Lett. 368, 331. Boniotto, M., Ventura, M., Eskdale, J., Crovella, S., Gallagher, G., 2004. Evidence for duplication of the human defensin gene DEFB4 in chromosomal region 8p22–23 and implications for the analysis of SNP allele distribution. Genet. Test 8, 325. Chain, J.L., Joachims, M.L., Hooker, S.W., Laurent, A.B., KnottCraig, C.K., Thompson, L.F., 2005. Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements. J. Immunol. Methods 300, 12. Elmaagacli, A.H., 2002. Real-time PCR for monitoring minimal residual disease and chimerism in patients after allogeneic transplantation. Int. J. Hematol. 76 (Suppl 2), 204.

239

Fang, X.M., Shu, Q., Chen, Q.X., Book, M., Sahl, H.G., Hoeft, A., Stuber, F., 2003. Differential expression of alpha- and beta-defensins in human peripheral blood. Eur. J. Clin. Invest. 33, 82. Gallagher, R.E., Yeap, B.Y., Bi, W., Livak, K.J., Beaubier, N., Rao, S., Bloomfield, C.D., Appelbaum, F.R., Tallman, M.S., Slack, J.L., Willman, C.L., 2003. Quantitative real-time RT-PCR analysis of PML-RAR alpha mRNA in acute promyelocytic leukemia: assessment of prognostic significance in adult patients from intergroup protocol 0129. Blood 101, 2521. Ganz, T., 2003. Defensins: antimicrodial peptides of innate immunity. Nat. Rev. Immunol. 3, 710. Garcı´a, J.R., Jaumann, F., Schulz, S., Krause, A., Rodrı´guez-Jime´nez, J., Forssmann, U., Adermann, K., Klu¨ver, E., Vogelmeier, C., Becker, D., Hedrich, R., Forssmann, W.G., Bals, R., 2001. Identification of a novel, multifunctional h-defensin (human h-defensin 3) with specific antimicrobial activity: its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction. Cell Tissue Res. 306, 257. Giglio, S., Broman, K.W., Matsumoto, N., Calvari, V., Gimelli, G., Neumann, T., Ohashi, H., Voullaire, L., Larizza, D., Giorda, R., Weber, J.L., Ledbetter, D.H, Zuffardi, O., 2001. Olfactory receptor-gene clusters, genomic-inversion polymorphisms, and common chromosome rearrangements. Am. J. Hum. Genet. 68, 874. Ginzinger, D.G., 2002. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503. Harder, J., Bartels, J., Christophers, E., Schro¨der, J.M., 1997. A peptide antibiotic from human skin. Nature 387, 861. Harder, J., Bartels, J., Christophers, E., Schro¨der, J.M., 2001. Isolation and characterization of human h-defensin-3, a novel human inducible peptide antibiotic. J. Biol. Chem. 276, 5707. He, Q., Wang, J.P., Osato, M., Lachman, L.B., 2002. Real-time quantitative PCR for detection of Helicobacter pylori. J. Clin. Microbiol. 40, 3720. Hijri, M., Sanders, I.R., 2005. Low gene copy number shows that arbuscular mycorrhizal fungi inherit genetically different nuclei. Nature 433, 160. Hiratsuka, R., Mukae, H., Iiboshi, H., Ashitani, J., Nabeshima, K., Minematsu, T., Chino, N., Ihi, T., Kohno, S., Nakazato, M., 2003. Increased concentrations of human beta-defensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis. Thorax 58, 425. Hollox, E.J., Armour, J.A., Barber, J.C., 2003. Extensive normal copy number variation of a beta-defensin antimicrobial-gene cluster. Am. J. Hum. Genet. 73, 591. Kallioniemi, A., Kallioniemi, O.P., Sudar, D., Rutovitz, D., Gray, J.W., Waldman, F., Pinkel, D., 1992. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258, 818. Kato, H., Arakawa, A., Suzumori, K., Kataoka, N., Young, S.R., 2004. FISH analysis of BRCA1 copy number in paraffin-embedded ovarian cancer tissue samples. Exp. Mol. Pathol. 76, 138. Kindich, R., Florl, A.R., Jung, V., Engers, R., Muller, M., Schulz, W.A., Wullich, B., 2005. Application of a modified real-time PCR technique for relative gene copy number quantification to the determination of the relationship between NKX3.1 loss and MYC gain in prostate cancer. Clin. Chem. 51, 649. Laccone, F., Junemann, I., Whatley, S., Morgan, R., Butler, R., Huppke, P., Ravine, D., 2004. Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome. Hum. Mutat. 23, 234.

240

Q.X. Chen et al. / Journal of Immunological Methods 308 (2006) 231–240

Lehrer, R.I., Ganz, T., 2002. Defensins of vertebrate animals. Curr. Opin. Immunol. 14, 96. Linzmeier, R.M., Ganz, T., 2005. Human defensin gene copy number polymorphisms: comprehensive analysis of independent variation in alpha- and beta-defensin regions at 8p22–p23. Genomics 86, 423. Petrij-Bosch, A., Peelen, T., van Vliet, M., van Eijk, R., Olmer, R., Drusedau, M., Hogervorst, F.B., Hageman, S., Arts, P.J., Ligtenberg, M.J., Meijers-Heijboer, H., Klijn, J.G., Vasen, H.F., Cornelisse, C.J., van ’t Veer, L.J., Bakker, E., van Ommen, G.J., Devilee, P., 1997. BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients. Nat. Genet. 17, 341. Raj, P.A., Dentino, A.R., 2002. Current status of defensins and their role in innate and adaptive immunity. FEMS Microbiol. Lett. 206, 9. Rooms, L., Reyniers, E., Kooy, R.F., 2005. Subtelomeric rearrangements in the mentally retarded: a comparison of detection methods. Hum. Mutat. 25, 513. Sagner, G., Goldstein, C., 2001. Principles, workflows and advantages of the new LightCycler Relative Quantification Software. Biochemica 3, 15. Schaeffeler, E., Schwab, M., Eichelbaum, M., Zanger, U.M., 2003. CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-time PCR. Hum. Mutat. 22, 476. Schaller-Bals, S., Schulze, A., Bals, R., 2002. Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection. Am. J. Respir. Crit. Care Med. 165, 992. Singh, P.K., Jia, H.P., Wiles, K., Hesselberth, J., Liu, L., Conway, B.A., Greenberg, E.P., Valore, E.V., Welsh, M.J., Ganz, T., Tack, B.F., McCray Jr., P.B., 1998. Production of beta-defensins by human airway epithelia. Proc. Natl. Acad. Sci. U. S. A. 95, 14961. Suo, Z., Daehli, K.U., Lindboe, C.F., Borgen, E., Bassarova, A., Nesland, J.M., 2004. Real-time PCR quantification of c-erbB-2 gene is an alternative for FISH in the clinical management of breast carcinoma patients. Int. J. Surg. Pathol. 12, 311. Szuhai, K., Sandhausm, E., Kolkman-Uljee, S.M., Lemaitre, M., Truffert, J.C., Dirks, R.W., Tanke, H.J., Fleuren, G.J., Schuuring,

E., Raap, A.K., 2001. A novel strategy for human papillomavirus detection and genotyping with SybrGreen and molecular beacon polymerase chain reaction. Am. J. Pathol. 159, 1651. Taudien, S., Galgoczy, P., Huse, K., Reichwald, K., Schilhabel, M., Szafranski, K., Shimizu, A., Asakawa, S., Frankish, A., Loncarevic, I.F., Shimizu, N., Siddiqui, R., Platzer, M., 2004. Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence. BMC Genomics 5, 92. Teo, I., Choi, J., Morlese, J., Taylor, G., Shaunak, S., 2002. LightCycler qPCR optimisation for low copy number target DNA. J. Immunol. Methods 270, 119. Tse, C., Brault, D., Gligorov, J., Antoine, M., Neumann, R., Lotz, J.-P., Capeau, J., 2005. Evaluation of the quantitative analytical methods Real-Time PCR for HER-2 gene quantification and ELISA of serum HER-2 protein and comparison with fluorescence in situ hybridization and immunohistochemistry for determining HER-2 status in breast cancer patients. Clin. Chem. 51, 1093. Wehkamp, J., Harder, J., Weichenthal, M., Mueller, O., Herrlinger, K.R., Fellermann, K., Schroeder, J.M., Stange, E.F., 2003. Inducible and constitutive beta-defensins are differentially expressed in Crohn’s disease and ulcerative colitis. Inflamm. Bowel Dis. 9, 215. Wilke, K., Duman, B., Horst, J., 2000. Diagnosis of haploidy and triploidy based on measurement of gene copy number by real-time PCR. Hum. Mutat. 16, 431. Yang, D., Chertov, O., Bykovskaia, S.N., Chen, Q., Buffo, M.J., Shogan, J., Andeerson, M., Schro¨der, J.M., Wang, J.M., Howard, O.M., Oppenheim, J.J., 1999. h-Defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286, 525. Yang, D., Biragyn, A., Kwak, L.W., Oppenheim, J.J., 2002. Mammalian defensins in immunity: more than just microbicidal. Trends Immunol. 23, 291.