Abstract
Aims. Nasopharyngeal cancer (NPC) is a multi-factorial disease, and the genetic background may be a crucial etiologic factor. The xeroderma pigmentosum complementation group C (XPC) is mainly involved in DNA damage repair, and the sequence variants in XPC gene may modulate DNA repair capacity and consequently lead to an individual's susceptibility to NPC. The aims of this study were to examine the association between XPC Val499Ala, Lys939Gln, PAT polymorphisms and the genetic susceptibility of nasopharyngeal carcinoma (NPC) in Chinese population. Methods. We analyzed the three XPC gene polymorphisms in 153 patients with NPC and 168 age- and sex-matched controls in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure. Results. There were significant differences in the genotype and allele distribution of XPC Val499Ala among cases and controls. The 499Val allele carriers were associated with a significantly increased risk of NPC compared with the non-carriers (OR=1.603; 95%CI, 1.160∼2.216, p=0.005). Consistent with the results of the genotype analysis, the 499Val/939Lys/PAT–haplotype was associated with a significantly increased risk of NPC as compared with the 499Ala/939Lys/PAT–haplotype (OR=1.901; 95% CI, 1.284∼2.814, p=0.002).The interaction between the Val499Ala polymorphism and gender or smoking status did not been found in NPC risk. Conclusions. Our data demonstrated that XPC 499Val allele and its haplotype were strongly associated with NPC, which indicated that Val499Ala polymorphism may be a contributing factor in the NPC development.
Nasopharyngeal carcinoma (NPC) has a marked geographic and ethnic distribution. It is rare among Caucasians in Western Europe and North America, with the incidence usually less than 1/1 000 000 Citation[1]. The disease is predominantly endemic in Southeast Asia and southern China. In these high-risk areas, the incidence is 10–50/100 000 Citation[2]. Previous studies have demonstrated that NPC is a multi-factorial disease. Epstein-Barr virus (EBV) infection, environmental factor and individual genetic susceptibility are widely recognized as the risk factors in the development of NPC. But, the molecular mechanism of NPC carcinogenesis remains unclear.
The XPC protein is one of the DNA-repairing enzymes, which is absolutely necessary in nucleotide excision repair (NER) pathway that is responsible for bulky adducts and UV-induced DNA damage repair. It binds tightly with HR23B (one of 2 human homologs of Saccharomyces cerevisiae NER factor RAD23) to form XPC–HR23B complex Citation[3], which acts as an early damage detector and a molecular matchmaker for recruitment of other components of the repair apparatus to the damaged DNA in global genomic NER Citation[4], Citation[5]. The normal function of the XPC is essential in maintaining genomic integrity and preventing cellular neoplastic transformation Citation[6]. DNA repair gene polymorphisms have been reported that could lead to the deficiency of DNA repair capacity, and subsequently contribute to genomic instability and an individual's susceptibility to cancer. The most extensively polymorphisms studies of XPC are Val499Ala (a T -to- C transition in exon8), Lys939Gln (a A -to- C transition in exon15) and Poly-AT (PAT, an insertion of 83 bases of A and T with a concurrent 5 base deletion in intron 9). Recently, genetic polymorphisms of the XPC gene have been implicated in the susceptibility to a range of cancers, including bladder cancer Citation[7], squamous cell carcinoma of the head and neck Citation[8] and lung cancer Citation[9]. However to our knowledge, there are no studies about XPC gene polymorphisms and NPC susceptibility. In the current study, we evaluated whether XPC Val499Ala, Lys939Gln, PAT polymorphisms contribute to NPC risk in a Chinese population.
Materials and methods
Subjects
The case group consisted of 153 diagnosed patients with histologically confirmed NPC in Sichuan Province, China, from January 2005 to May 2006. Patients with secondary and recurrent tumors were excluded. The control group comprised 168 healthy volunteers who visited the general health check-up and they matched to the cases by age, gender and smoking status in frequency. These cancer-free controls were genetically unrelated to the cases and all subjects were of the Han ethnic group. Data on age, gender, smoking status and amount were derived from questionnaires through personal interviews by two trained investigators. For each subject, the results of the questionnaires from the two investigators were required to be consistent. After informed consent obtained, and with the approval of the ethics committee of Chinese Human Genome, each study participant donated 2–5 ml of blood in EDTA tubes and stored at −20°C. Genomic DNA was extracted from peripheral blood by proteinase K digestion and phenol/chloroform extraction.
Genotyping
Genotyping for polymorphisms of XPC Val499Ala, Lys939Gln, PAT were detected by using PCR-RFLP technique. Polymerase chain reaction (PCR) assays were used to amplify exon8, exon15 and intron9 of XPC containing the polymorphisms interested. The primers used to identify the polymorphism were as follows: 5′-TAA GGA CCC AAG CTT GCC CG-3′and 5′-CCC ACT TTT CCT CCT GCT CAC AG-3′for exon8 Citation[9]; 5′-GAT GCA GGA GGT GGA CTC TCT -3′And 5′-GTA GTG GGG CAG CAG CAA CT-3′for exon15 Citation[7]; 5′-TAG CAC CCA GCA GTC AAA G-3′and 5′-TGT GAA TGT GCT TAA TGC TG-3′ for intron9 Citation[8]. Each PCR (25 µl vol) generally comprised 50–100 ng genomic DNA, 0.4 pmol/L of each primer, 0.1 mmol/L of dNTP mix, 0.2 U Taq DNA polymerase (Invitrogen, Shanghai, China), 1.5 mmol/L MgCl2 and 1.0×PCR buffer. Thermal cycling conditions were an initial denaturation at 94°C for 4 min, 35 cycles of denaturation at 94°C for 35 s, 45 s of annealing (64°C for Val499Ala and Lys939Gln, 67°C for PAT) and 55 s of elongation at 72°C, followed by a final extension at 72°C for 7 min. The various PCR products were digested with specific restriction enzymes. Ten microliters of PCR products were digested respectively with 5 units of Pvu II (for codon 939), Sac II (for codon 499) [FERMENTAS, Chengdu, China] at 37°C for 4 h. The products were then resolved on 8% polyacrylamide gel. For quality control, the three PCR-RFLP assays were repeated on 20% of the samples and the replicates were 100% concordant.
Statistical analysis
Differences in genotype distributions and allele frequencies in the NPC cases and the controls were compared for statistical significance using the Pearson χ2 test or Fisher's Exact Test. The odds ratios (OR) with 95% confidence intervals (CI) were used to estimate the association of certain polymorphism and NPC. Hardy-Weinberg equilibrium was tested by a goodness-of-fit χ2 to compare the observed genotype frequencies within the case-control groups to the anticipated genotype frequencies calculated from the observed allele frequency. Statistical significant was set at p < 0.05, all analysis were conducted using the SPSS 11.5 statistical package. The linkage disequilibrium (LD) of the three loci, haplotypes and their frequencies were estimated by using the SHEsis software Citation[10] (available at http://202.120.7.14/analysis/myAnalysis.php).
Results
Characteristics of the study population
Select characteristics of the 153 NPC cases and 168 cancer-free controls are summarized in . The mean ages of cases and controls (mean±SD) were almost identical (49.8±10.5 versus 48.3±11.2). There were slightly more males among the cases (70.4%) than in controls (68.3%), but this difference was not statistically significant (p > 0.05). As expected, the smoking history and current smoking status among patients and controls were quite similar.
Table I. Characteristics of the study population.
Polymorphisms of XPC Val499Ala, Lys939Gln, PAT
The association between XPC Val499Ala, Lys939Gln, PAT polymorphisms and NPC risk were summarized in . The genotype distributions of all the polymorphisms studied were in accordance with Hardy-Weinberg equilibrium (HWE). The genotype frequencies of Val499Ala were 18.3% (Val/Val), 47.7% (Val/Ala) and 34.0% (Ala/Ala) among cases and 7.7% (Val/Val), 47.1% (Val/Ala), 45.2% (Ala/Ala) among control subjects. There were significant differences in the genotype and allele frequencies of the Val499Ala polymorphism between NPC patients and control groups (χ2=9.871, p = 0.007 and χ2=8.225, p = 0.004 respectively). The frequency of 499Val allele was markedly higher in the cases than in the controls (42.2% versus 31.2%), subjects with the 499Val allele were at statistically significant increased risk of NPC relative to subjects with the 499 Ala allele (OR = 1.603; 5%CI, 1.160∼2.216, p = 0.005). The genotype frequencies and allelic frequencies of Lys939Gln and PAT polymorphisms among cases were similar to those of controls in this investigation, and any significant association of the two polymorphisms and NPC risk was not found.
Table II. Frequency distributions of XPC polymorphisms among cases and controls and their associations with NPC.
Frequency distributions of XPC haplotypes and their associations with NPC
The three polymorphisms were in strong LD. The parameter of linkage disequilibrium D’ was 0.980(Val499Ala and Lys939Gln), 0.942(Val499Ala and PAT), 0.971(Lys939Gln and PAT). Eight haplotypes were observed in this study, and their frequency distributions among cases and controls were shown in . To avoid possible errors in either haplotype analysis or the estimation process, five haplotypes that had a frequency of <2% were combined for further analysis. The frequency of 499Val/939Lys/PAT−(TA − ) was significantly higher in the cases than in the controls (41.5% versus 30.4%), which was associated with a significantly increased risk of NPC as compared with the 499Ala/939Lys/PAT−(CA − ) haplotype (OR = 1.901; 95%CI, 1.284∼2.814, p = 0.002).
Table III. Frequency distributions of XPC haplotypes and their associations with NPC.
Stratification analysis of XPC Val499Ala polymorphism and NPC risk
To estimate the interaction between XPC Val499Ala polymorphism genotypes and smoking status or gender, stratified OR was calculated. The risk for XPC 499 genotypes among various smoking status (current, former and never) or gender (male and female) were presented in . The XPC 499Val/Ala and Val/Val genotypes were not associated with a significantly increased risk of NPC in male or current smoker groups compared to Ala/Ala genotype (OR = 1.545, 95%CI, 0.891∼2.676; p = 0.129 and OR = 1.670, 95%CI, 0.825∼3.381; p = 0.161 respectively), and the interaction between XPC Val499Ala genotypes and gender or smoking status did not been found in the NPC risk.
Table IV. Stratification analysis of XPC Val499Ala polymorphism in relation to NPC by gender and smoking.
Discussion
To our knowledge, the present study is the first to examine the Val499Ala, Lys939Gln, PAT polymorphisms of the XPC gene in patients with NPC in a Chinese population. In this study, we have demonstrated that the Val499Ala polymorphism and the XPC haplotype were significantly associated with the NPC risk, and no statistically significant association between XPC Val499Ala polymorphism and NPC risk was found when stratifying the gender and smoking status. These findings indicated that Val499Ala may be a contributing factor in the NPC development.
In the current study, the frequency of 499Val allele among the healthy Chinese population was 0.312, which was comparable to those in healthy Korean Citation[11] and SNP500 Cancer database (http://snp500cancer.nci.nih.gov/home.cfm; dbSNP ID: rs2228000, 0.289 and 0.235 respectively). The allele frequencies of XPC 939Gln and PAT+ in Korean and American population were 0.394, 0.372 and 0.383, 0.408 respectively Citation[11], Citation[12], which were similar to those frequencies observed in this study (0.345 and 0.357 respectively). We also found that the Val499Ala, Lys939Gln, PAT polymorphisms were in LD, the magnitude of the LD among these three polymorphisms in our study subjects were in agreement with previous studies Citation[9], Citation[11], Citation[13]. The frequencies of three common haplotypes 499Val/939Lys/PAT−, 499Ala/939Lys/PAT− and 499Ala/939Gln/PAT+ respectively in controls were 0.304, 0.336 and 0.342 respectively, which were similar to those in Korean (0.271, 0.370 and 0.359 respectively) Citation[14].
XPC is a part of the XPC-HR23B complex that identifies target lesions in the NER repair pathway Citation[4]. It is suggested that the normal XPC gene is critical for the cells to complete excision repair of bulky DNA lesions Citation[15]. Mice defective in the XPC gene are highly prone to cancers of lung and liver, when exposed to chemical carcinogens, and highly vulnerable to skin cancer following exposure to UV radiation Citation[16], Citation[17], which suggested that XPC plays an important role to preventing carcinogenesis. The XPC gene polymorphisms may alter an individual's capacity of repairing damaged DNA and therefore may lead to genetic instability and carcinogenesis. Qiao et al. (2002a) and Qiao et al. (2002b) Citation[18], Citation[19]. showed that the XPC PAT + allele reduced repair the UV-DNA damage in the host cell reactivation assay. Vodicka et al. Citation[20] observed that XPC 939Gln variant elevated chromatid aberrations and might influence base excision repair activity in an irradiation-specific DNA repair.
A number of molecular epidemiologic studies on the XPC genotypes and cancer susceptibility have been reported. Lee et al. Citation[11] and Citation[14] showed that the XPC Val499Ala was not associated with lung cancer risk in Korean populations, in contrast to these Hu et al. Citation[9] and our current study revealed that the XPC 499Val allele was associated with a significantly increased risk of lung cancer and NPC respectively in Chinese populations. Consistent with our results, Hirata et al. Citation[21] showed that Lys939Gln may not be linked to susceptibility for renal cell carcinoma in a Japanese population, Hu et al. Citation[9] and Son et al. Citation[14], showed that Lys939Gln was not associated with the lung cancer development in a Chinese and Korean population respectively. However, Vogel et al. Citation[22], showed that 939 Gln allele had significantly increased the risk of lung cancer in a Danish population. Sanyal et al. Citation[7] suggested that the homozygote individuals for the 939Gln allele were at an almost 2-fold increased risk of bladder cancer in a Swedish population. In contrast to two previous studies Citation[12], Citation[14] and the current study, Marin et al. Citation[23], found that the PAT + /+ subjects were at significantly increased risk for lung cancer in a Spanish population, and Blankenburg et al. Citation[13] demonstrated that PAT+ was associated with an increased risk of melanoma a German population.
DNA repair protein XPC is a major player in the NER pathway and essential for genomic integrity. It is plausible that variations in DNA repair genes may result in individuals with a low repair capacity, which may lead to individuals more sensitive to DNA damage and therefore more prone to cancer. However, the association of DNA repair gene XPC Val499Ala, Lys939Gln, PAT polymorphisms and cancer risk was not uniform, even conflicting in specific population and various case-control studies. Although it is hard to decipher the reasons for these discrepancies, but several possibilities should be considered. First, it may be due to the genetic trait differences, DNA repair gene polymorphisms were distinct in specific population, various ethnicity and geographic region. For example, a protective haplotype in one population may increase the cancer risk in another population due to other linked polymorphisms that exhibit a stronger effect on the susceptibility to cancer Citation[17]. Furthermore, the presence of variants in metabolism and other critical DNA repair genes may alter the influence of variants in the XPC genes, and the presence of an allelic variant that induces subtle differences in protein activity may result in different cancer risks depending on the exposure or the metabolic pathways available in different tissues Citation[24]. In addition, cancer is a multi-factorial disease, and the individuals’ exposure to various environmental factors and genetic susceptibility may result in different DNA damage and influence the DNA repair protein activity, as, Lunn et al. Citation[25] suggested that the XPD Lys751Gln polymorphism Lys allele may have different effects in different types of DNA damage in the initiation of different cancers. In the end, the inadequate study design such as non-random sampling and a limited sample size should be also considered. The possible selection bias that might have been present in the hospital-based, case-control study is a relevant issue.
In conclusion, to our knowledge, this is the first study of the association of XPC Val499Ala, Lys939Gln, PAT polymorphisms, their haplotypes and NPC risk. Our results showed that the XPC codon 499Val allele and 499Val/939Lys/PAT−(TA-) haplotype were associated with a significantly increased risk of NPC, which suggested that the XPC gene polymorphisms may be involved in the development of NPC. However, the molecular mechanism by which the XPC gene polymorphisms are associated with NPC is unclear, so additional studies will be needed to explore further, whether XPC polymorphisms are an independent risk factor or an indirect marker of different genetic and environmental factors. Further studies on the XPC sequence variants and their biologic functions are also needed to characterize the molecular mechanisms by which XPC is involved in susceptibility to NPC in diverse ethnic populations.
Acknowledgements
This study was supported by Sichuan Key Science and Technologic Programs (No. 06Z09-005) and Chengdu Technologic Programs (No. 06GGYB349SF-034).
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