Real-time PCR-based genotyping assay for CXCR2 polymorphisms

Clinica Chimica Acta 341 (2004) 93 – 100 www.elsevier.com/locate/clinchim

Real-time PCR-based genotyping assay for CXCR2 polymorphisms Manish Gupta, Pengfei Song, Charles R. Yates, Bernd Meibohm * Department of Pharmaceutical Sciences, College of Pharmacy, The University of Tennessee Health Science Center, 874 Union Avenue, Suite 5p, Memphis, TN 38163, USA Received 11 September 2003; received in revised form 12 November 2003; accepted 13 November 2003

Abstract Background: The human chemokine receptor CXCR2 (IL8RB) is a high affinity receptor for interleukin-8 as well as other CXC chemokines, and is involved in the chemotaxis of immune cells. Genetic variants of CXCR2 have potential relevance in various inflammatory human disorders. We developed a real-time polymerase chain reaction (PCR)-based allelic discrimination assay for the detection of the CXCR2 single nucleotide polymorphisms (SNPs) C785T, T1208C and G1440A. Methods: Polymorphisms were delineated using PCR amplification of specific alleles (PASA). Allele-specific primers were developed for both wild-type and mutant alleles. An additional nucleotide mismatch at the third position from the 3Vend of each primer was used to improve amplification specificity and to prevent generation of nonspecific products. Genotypes were assigned based on PCR growth curves and melt curve analysis performed on a SmartCyclerR using SYBR Green I chemistry. Results: Genotyping assignments were successfully performed in a set of 20 human DNA samples, and were validated by comparison with results from direct DNA sequencing and agarose gel electrophoresis of PCR products. Conclusions: Due to its rapid and relatively inexpensive performance and accuracy, the presented allelic discrimination assay for CXCR2 polymorphisms has wide applicability, especially for high-throughput sample analysis in large population genotyping studies. D 2004 Elsevier B.V. All rights reserved. Keywords: CXCR2; IL8RB; Genotyping; Real-time polymerase chain reaction; Allele-specific amplification

1. Introduction Interleukin-8 (IL-8), a member of the CXC chemokine family, acts as a potent activator and chemoattractant for neutrophils [1– 5]. Cellular activities of IL-8 are mediated by two receptors, CXCR1 (IL8RA) and CXCR2 (IL8RB), which are encoded by genes located on chromosomes 2q34 – q35 [1,2]. The IL-8Rs * Corresponding auhtor. Tel.: +1-901-448-1206; fax: +1-901448-6940. E-mail address: [email protected] (B. Meibohm). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.11.017

are members of the G-protein-coupled family of receptors, which feature seven transmembrane domains. The two receptors have 78% homology in protein amino acid sequence and bind IL-8 with similar affinity [3]. While CXCR1 binds only two CXC chemokines, IL-8 and granulocyte chemotactic protein (GCP)-2, CXCR2 also binds with high affinity other CXC chemokines, such as GROa, GROh, GROg and neutrophil-activating peptide 2 [6]. The CXCR2 gene consists of 11 exons. The open reading frame is encoded entirely by a single exon, exon 11. The remaining exons, however, are alternatively

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Table 1 Primer sequence for genotyping assay based on CXCR2 sequence accession no. M99412a Polymorphism

Annealing position

Primer sequence

Product size (bp)

C785T

10,638 – 10,657

208

T1208C

11,061 – 11,080

G1440A

11,338 – 11,312

FW-TCGTCCTCATCTTCCTGGT C FM-TCGTCCTCATCTTCCTGGT T R-AGTCCATGGCGAAACTTCTG FW-CCATTGTGGTCACAGGATG T FM-CCATTGTGGTCACAGGATG C R-TGCAGAGCTGTCTCACTGGA RW-GTATTTTTAGTAGAGACAGGGTTTGA C RM-GTATTTTTAGTAGAGACAGGGTTTGA T F-CCTCACCCCTTGCCATAAT

198

200

a F indicates forward primer, R reverse primer. FW and FM indicate forward primers for wild-type and mutant allele, respectively. RW and RM indicate reverse primers for wild-type and mutant allele, respectively. Underlined nucleotides indicate the site of polymorphism and corresponding wild-type and mutant nucleotides. Nucleotides in bold indicate nucleotide mismatches three bases from the 3V termini of the published sequence, which had little effect on specific PCR product yield but nonspecific PCR product yield was drastically reduced to undetectable levels.

spliced, giving rise to seven distinct messenger RNA (mRNA) variants [4,5]. The mapped susceptibility loci for several human disorders, such as rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, and juvenile amyotrophic lateral sclerosis include chromosome 2q35 [7]. Thus, polymorphisms in this region may have relevance for susceptibility to and pathogenesis of these disorders. Renzoni et al. [8] recently reported three novel single nucleotide polymorphisms (SNPs) at nucleotide + 785 (C ! T), + 1208 (T ! C) and + 1440 (G ! A) (corresponding to positions 10,657, 11,080 and 11,312 of sequence M99412, respectively). The CXCR2 C785T polymorphism is located in exon 11 and results in a silent mutation with codon change from CTC (leucine) to CTT (leucine), whereas the T1208C and the G1440A polymorphisms are both in the noncoding region of the CXCR2 gene. The authors investigated the distribution of these polymorphisms in systemic sclerosis and cryptogenic fibrosing alveolitis. They observed a strong linkage between the 785C, 1208T and 1440G alleles. The authors identified a significant increase in the frequency of homozygous 785T and 1208C genotypes in systemic sclerosis patients compared to a control group. The allele frequency for 785C, 1208T and

1440G in Caucasian control subjects was 0.48, 0.44 and 0.44, respectively. There have been several reports indicating that CXCR2 variants might provide valuable information for the pathogenesis of and susceptibility to chronic inflammatory conditions involving neutrophil recruitment, especially rheumatoid and respiratory diseases [7 –10]. The CXCR2 polymorphism detection methods previously described involve multiple steps and are time-consuming [7,8]. The development of rapid genotyping techniques to assess the CXCR2 polymorphisms is thus desirable for clinical and epidemiologic studies, especially if a large number of samples has to be processed. The recent advent of a real-time polymerase chain reaction (PCR) technique has proven useful as a fast, inexpensive methodology for allelic discrimination assays. We report the development of a real-time PCR-based genotyping assay to detect the CXCR2 receptor SNPs C785T, T1208C and G1440A. This method takes advantage of the SYBR Green I fluorescent dye for real-time detection of PCR products and, based on the length and nucleotide contents, for the melting curve analysis of PCR products. The reliability and discriminating power of this technique were documented by comparison of the results of our real-time PCR-based assay with direct

Fig. 1. Plot of fluorescence versus cycle number using human genomic DNA obtained from individuals with TT (Panel A), TC (Panel B) and CC (Panel C) genotypes for the T1208C SNP. Two separate tubes were used to discriminate between T and C allele using the common reverse primer 1208R coupled with either the wild-type specific primer 1208FW (black line) or the mutant specific primer 1208FM (gray line). PCR growth curves that exceed the threshold fluorescence (Ct) indicate specific product formation.

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DNA sequencing, and have previously been shown by our laboratory for genetic variants of other genes [11,12].

2. Materials and methods 2.1. Primer design Oligonucleotide primers were designed for both wild-type and mutant alleles based upon the published human CXCR2 receptor sequence (sequence accession nos. M99412 and M73969) using the Primer3 program (Whitehead Institute for Biomedical Research, http://www.genome.wi.mit.edu/cgi-bin/ primer/primer3_www.cgi). Discrimination between wild-type and mutant alleles was achieved using PCR amplification of allele-specific primers to prevent non-Watson Crick base pairing [13 – 15]. Two forward primers and a common reverse primer were designed based on the nucleotide difference at the 3Vterminal base for allelic discrimination of C785T and T1208C. For allelic discrimination of G1440A, however, we designed two reverse primers and a common forward primer due to a high GC content in the region upstream of the SNP location. Since Taq DNA polymerase lacks 3V to 5V exonuclease activity, a primer with a mismatch in the 3V terminal with regard to the template will be amplified with reduced efficiency, allowing discrimination between matched and mismatched templates. Briefly, the allelic discrimination for T1208C was achieved by designing two sense primers (1208FW and 1208FM) based on the nucleotide difference (T or C) located at the 3V terminal base. In order to prevent the amplification of the nonmatching primer, an additional nucleotide mismatch (A to T) located three bases from the 3V termini of the allelespecific primers (1208FW and 1208FM) was incorporated [16] (Table 1). These changes were made to improve the amplification specificity and to prevent the generation of nonspecific products, which could otherwise occur by the annealing and extension of the

1208FW primer to the nonspecific template. A similar strategy was used to achieve allelic discrimination for C785T and G1440A (Table 1). Oligo toolkit (http:// www.operon.com) was used to detect hairpin structures and primer – dimers. Primers were synthesized by Integrated DNA Technologies (Coralville, IA). Expected amplicon lengths were 208, 198 and 200 bp for C785T, T1208C and G1440A, respectively. 2.2. Real-time PCR amplification Genomic DNA was obtained for 10 African-Americans and 10 Northern Europeans from the Human Genetic Cell Repository, sponsored by the National Institute of General Medical Sciences (http://locus. umdnj.edu/nigms). This DNA had been isolated from EBV-transformed lymphoblasts of female donors. The optical absorbance ratio at 260 and 280 nm as a measure of purity was 1.8 F 0.1 for all samples. The use of Human Genetic Cell Repository samples was approved by the University of Tennessee Institutional Review Board. Polymorphisms were detected using PCR amplification of specific alleles (PASA) [12,15] on a SmartCyclerR (Cepheid, Sunnyvale, CA). PCR was performed in two separate tubes for normal and variant alleles. All reactions were carried out in a total volume of 25 Al. Each reaction mixture contained a 1:12,500 dilution SYBR Green I nucleic acid gel stain 10,000  in dimethyl sulfoxide (DMSO) (Molecular Probes, Eugene, OR), 0.2 mmol/l dNTP mixture, 200 nmol/ l of both forward and reverse primer, 1U Taq DNA Polymerase (Promega, Madison, WI), 6% DMSO, 1  SmartCycler additive reagent (a 5  additive reagent containing BSA at 1 mg/ml, Trehalose at 750 nmol/l, and Tween-20 at 1% v/v) (Cepheid), and 10 ng genomic DNA in 1  PCR buffer (pH 8.3, 10  solution containing 100 mmol/l Tris – HCl, 500 mmol/l KCl, and 15 nmol/l MgCl2 and 0.01% Gelatin) (Sigma, St. Louis, MO). The amplification program consisted of initial denaturation of 95 jC (5 min) followed by 27 cycles of 95 jC (15 s), annealing at 60 jC (30 s), and

Fig. 2. Melt curve plots obtained after amplification of TT (Panel A), TC (Panel B) and CC (Panel C) genomic DNA using the common reverse primer 1208R coupled with either the wild-type specific primer 1208FW (black line) or the mutant specific primer 1208FM (gray line). Melt curves were generated by plotting the negative first derivative of the fluorescence ([  dF/dT]) versus temperature. The melt temperature (86 jC) was identical for PCR products formed using either wild-type or mutant specific primers.

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extension at 72 jC (45 s). After amplification, melt analysis was performed by heating the reaction mixture from 60 to 95 jC at a rate of 0.2 jC/s. PCR products for sequencing the 785 locus were generated using the sense primer (5V-ATGCGGGTCATCTTTGCTGT-3V) and the antisense primer (5VTTGAGGCAGCTGTGAAGGAT-3V). PCR products for sequencing the 1208 and 1440 loci were generated using the sense primer (5V-GGGTTCCTCCCTTCTCTTCA-3V) and the antisense primer (5V-TTACAGGCACTCACCACCAC-3V). 2.3. Product analysis The real-time fluorescence signal generated by the nonspecific double-stranded DNA binding dye SYBR Green I was analyzed using the SmartCyclerR software. A threshold cycle Ct was determined for each sample using the exponential growth phase and the baseline signal from fluorescence versus cycle number plots. Melting curve analysis was performed by slowly heating DNA fragments in the presence of the dsDNA-specific fluorescent dye SYBR Green I. As the sample is heated, fluorescence ( F) rapidly decreases when the melting temperature of a particular fragment is reached. The negative first derivative peaks ([  dF/dT] versus temperature) were used to identify specific PCR products. The presence of an allele in a DNA sample was concluded for a PCR reaction with an allele-specific primer based on the assignment of a threshold cycle Ct and the presence of a melting curve peak at a primer-specific temperature.

Amplification reactions were also routinely checked for the presence of nonspecific products by agarose gel electrophoresis. The genotyping method was validated using direct sequencing (ABI PrismR 3100, Applied Biosystems, Foster City, CA) after the PCR products were isolated by QIAquick (Qiagen, Valencia, CA).

3. Results The exponential growth phase and melt peak of the specific PCR product were monitored for a set of positive controls. Fig. 1 illustrates the results of the CXCR2 T1208C allelic discrimination assay using homozygous 1208T genomic DNA amplified with a common primer 1208R and either the wild-type specific primer 1208FW or the mutant specific primer 1208FM. Specific product formation is indicated by the PCR growth curve exceeding the threshold fluorescence (Ct). When primers 1208R and 1208FW were used to amplify homozygous 1208T genomic DNA, the PCR growth curve exceeded the Ct value at approximately 21 cycles (Fig. 1A), and the melt analysis (negative first derivative) yielded a characteristic sharp peak at approximately 86 jC (Fig. 2A). However, PCR growth curves remained at approximately background fluorescence, and no distinct melt analysis peak was noted when primers 1208R and 1208FM were used to amplify homozygous 1208T genomic DNA (Figs. 1A and 2A). Agarose gel electrophoresis yielded the expected 198-bp product

Fig. 3. Gel electrophoresis for PCR products obtained from the CXCR2 T1208C genotyping assay using ethidium bromide-stained 1% agarose gel. PCR products from DNA homozygous for the 1208T allele are in lanes 1 and 2, from heterozygous DNA in lanes 3 and 4, and from nullizygous DNA in lanes 5 and 6. Lanes 1, 3 and 5 represent PCR products from reactions with wild-type specific primer 1208FW, lanes 2, 4 and 6 represent products using the mutant specific primer 1208FM. M = Molecular marker.

M. Gupta et al. / Clinica Chimica Acta 341 (2004) 93–100 Table 2 Allele frequency for the CXCR2-SNPs C785T, T1208C and G1440A in 10 Northern Europeans and 10 African-Americans SNP

Population Allele frequency

C785T

Northern European AfricanAmerican T1208C Northern European AfricanAmerican G1440A Northern European AfricanAmerican

Genotype frequency

wt

mut

wt/wt wt/mut mut/mut

0.40

0.60

0.1

0.6

0.3

0.25

0.75

0

0.5

0.5

0.35

0.65

0.1

0.5

0.4

0.15

0.85

0

0.3

0.7

0.50

0.50

0.2

0.6

0.2

0.50

0.50

0.3

0.4

0.3

when homozygous 1208T DNA was amplified with primers 1208R and 1208FW (Fig. 3). However, no bands were visualized after homozygous 1208T DNA was amplified using primers 1208R and 1208FM (Fig. 3). Similarly, allelic discrimination was achieved after amplification of homozygous 1208C DNA using primers 1208R, 1208FW and 1208FM (Figs. 1C, 2C and 3). Heterozygous T1208C yielded amplification with both wild-type and mutant specific primers and a distinct melt peak was observed after amplification with both wild-type and mutant specific primers (Figs. 1B, 2B and 3). The results were further confirmed with direct sequencing. Results from direct sequencing of selected tested individuals were in perfect agreement with the real-time PCR-based genotyping results. Similarly, allelic discrimination was successfully achieved for C785T and G1440A after amplification using their respective primers listed in Table 1. Genotype frequencies of our sample population of African-Americans and Northern Europeans follow Hardy – Weinberg Equilibrium and are presented in Table 2. The observed allele frequencies are similar to those previously reported for Caucasians [8].

4. Discussion Genotyping of CXCR2 variants may provide valuable information on disease susceptibility and pathogenesis of common complex disorders that involve

99

neutrophilic inflammatory processes. The CXCR2 C785T polymorphism does not result in an amino acid substitution in the protein sequence, and CXCR2 T1208C and G1440A lie in the noncoding region. All of them, however, have the potential of altering mRNA processing, stability or translation. Most of the current methods for CXCR2 genotyping require post-PCR handling, such as agarose or microtiter array diagonal gel electrophoresis, and thus are time-consuming and may increase the risk of PCR contamination [7,8]. Thus, we developed and validated a rapid, inexpensive and robust PCR-based screening methodology for CXCR2 polymorphisms which may provide significant advantages over current genotyping techniques, e.g. sequencing of PCR products or restriction fragment length polymorphism assays (RFLP). The presented method takes advantage of the fluorescent property of SYBR Green I dye and the melting curve analysis that allows the detection and distinction of PCR products of different length. The use of SYBR Green I is relatively inexpensive and thus may make the presented approach more costeffective compared to other fluorescence-based PCR techniques for SNP detection. In addition, the use of fluorescence chemistry is known to increase the sensitivity for PCR-based assays [12]. This is reflected by a decreased amount of genetic starting material required for the presented assay compared to traditional genotyping approaches like RFLP [17,18]. The use of allele-specific primers containing an additional mismatch eliminates the need for extensive optimization of PCR conditions, a fact that reduces time and effort during assay setup and increases assay robustness during sample analysis. With no need for post-PCR handling, the real-time PCR assay provides a very rapid genotyping approach. Approximately one hour is needed to complete setting, performance and analysis of the 27 PCR cycles required by the presented assay. High sensitivity, high specificity, relatively low cost, rapid performance, and lack of extensive post amplification manipulation make this method amenable to high-throughput sample processing. Indeed, we tested and validated the methodology for achieving high throughput on an ABI PrismR 7000 Sequence Detection System (Applied Biosystems) using a 96-

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well format with the exactly same conditions as for the SmartCyclerR (Cepheid). While the presented genotyping approach by allele-specific amplification was primarily developed for use on real-time PCR analyzers, it can also be performed with traditional thermal cyclers followed by agarose gel electrophoresis and thus has universal applicability. By virtue of its simplicity and versatility, the method has potential for use in industrial scale genetic studies or in clinical diagnostic settings. Our laboratory currently uses it to assess the allelic frequency of the respective CXCR2 variants and determine their clinical significance in different populations with various chronic inflammatory diseases.

Acknowledgements This work was supported by the University of Tennessee Center of Excellence for Genomics and Bioinformatics.

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