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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Human oxoguanine glycosylase 1 (hOGG1) is a DNA repair enzyme, which plays important roles in the base excision repair (BER) pathway. Several studies reported a common polymorphism Ser326Cys (rs1052133) in hOGG1, which conferred the susceptibility of bladder cancer. We hypothesized that the polymorphism is associated with risk of bladder cancer in a Chinese population. In a case-control study of 1050 histologically confirmed bladder cancer patients and 1404 age and sex matched healthy controls, we genotyped the hOGG1 Ser326Cys polymorphism using TaqMan technology and assessed its association with bladder cancer risk. We found that the hOGG1 Ser/Cys + Ser/Ser genotypes were associated with a significantly increased risk of bladder cancer (adjusted odds ratio [OR] = 1.19, 95% confidence interval [CI] = 1.01–1.41), compared with the Cys/Cys genotype. Furthermore, the increased risk was more pronounced among subjects over age 65 years (OR = 1.31, 95% CI = 1.04–1.66), male subjects (OR = 1.21, 95% CI = 1.00–1.47), ever smokers (OR = 1.29, 95% CI = 1.00–1.68) and heavy smokers (>20 pack-years) (OR = 1.45, 95% CI = 1.03–2.04). No significant association was observed in the stratification of tumor grade and tumor stage for bladder cancer. In conclusion, our results suggest that hOGG1 Ser326Cys polymorphism may contribute to the susceptibility to bladder cancer in a Chinese population. (Cancer Sci 2012; 103: 1215–1220)

Bladder cancer is the 7th most common cancer in men and the 17th most common in women in the world.[1] It has a higher incidence in Western countries compared with Asian countries.[2] Accumulative epidemiologic studies have suggested that the occupational exposures, environmental pollutions, dietary habits and cigarette smoking are associated with bladder cancer.[2] Cigarette smoking is a predominant risk factor for bladder cancer, accounting for nearly 50% of the cases in men and 30% in women,[3] yet only a part of smokers develop bladder cancer, suggesting that inter-individual differences including genetic susceptibility in critical genes may play an important role in bladder carcinogenesis.

Base excision repair (BER) is a multiprotein pathway, which operates on small lesions such as oxidized or reduced bases, fragmented or nonbulky adducts, or those produced by methylating agents.[4, 5] The human 8-oxoguanine glycosylase 1 (hOGG1) is a major base-excision repairing enzyme for oxidative DNA damage.[6] It encoded by the hOGG1 gene can directly remove 8-hydroxyguanine (8-OH-G), one of the major constituents in DNA damage, as part of the base excision repair pathway.[7, 8] High levels of 8-OH-G have been reported in several kinds of human tumor tissues, including breast, lung, renal and colorectal carcinomas[9-12] compared with their non-tumor counterparts. Furthermore, one report on the ability of hOGG1 to suppress GC to TA transversions in human cells in vivo strengthened that hOGG1 played a critical role in preventing mutations in human cells.[13] Defects in hOGG1 that affected its DNA repair capability might have an involvement in carcinogenesis.[13] There was a C[RIGHTWARDS ARROW]G transversion in exon 7 of the hOGG1gene, resulting in an amino acid substitution from serine to cysteine in codon 326 (Ser326Cys, rs1052133). Bravard et al.[14] indicated that the 326Ser allele rendered the protein extremely sensitive to oxidation, leading to an impairment of its DNA glycosylase activity, even under normal cell growth conditions.

Recently, some studies reported that the hOGG1 Ser326Cys polymorphism was associated with the risk of lung,[15] esophageal,[16] stomach,[17] prostate,[18] and orolaryngeal cancer.[19, 20] Karahalil et al.[21] identified that hOGG1 Cys326Cys was a risk factor for bladder cancer, while Kim et al.[22] found that the hOGG1 326Ser allele was associated with recurrence in non-muscle invasive bladder cancer (NMIBC).

In the present study, we hypothesized that the hOGG1 Ser326Cys polymorphism is associated with risk of bladder cancer. To validate this hypothesis, we genotyped the polymorphism and tested its association with risk of bladder cancer in our ongoing, hospital-based, case-control study in a Chinese population.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Study subjects

This ongoing case-control study consisted of 1050 bladder cancer patients and 1404 cancer-free controls, which has been reported previously.[23] Briefly, consecutive cases with histopathologically confirmed transitional cell carcinoma of bladder cancer were recruited from January 2003. Patients who had previous cancer, metastasized cancer from other or unknown origin, previous radiotherapy or chemotherapy were excluded. Approximately 95% of the eligible patients contacted chose to participate. All cases of pathological diagnosis for tumor stage were according to the 2002 International Union Against Cancer (UICC) tumor-nodes-metastasis (TNM) classification, and for grade as the World Health Organization (WHO) 1973 grading of urothelial papilloma: well differentiated (grade 1, G1), moderately differentiated (grade 2, G2), or poorly differentiated (grade 3, G3). The cancer-free controls were genetically unrelated to the cases. Controls were frequency matched to cases by age (±5 years) and sex, and were excluded from the study if they had any cancer history or clinical symptoms of bladder cancer. The response rate of controls in the study was > 85%. All subjects were interviewed by a guided questionnaire on demographic and lifestyle factors. People who smoked daily for > 1 year were defined as ever smokers. Ever smokers who had quit smoking for > 1 year were defined as former smokers, and the others as current smokers. Pack-years ([cigarettes/day/20] × years smoked) were calculated to indicate the cumulative smoking dose and the smokers were further categorized into three groups: never smokers (pack-years = 0), light smokers (0 < pack-years ≤ 20) and heavy smokers (pack-years > 20). After the interview, a 5-mL venous blood sample was obtained from each one. The study was approved by the institutional review board of Nanjing Medical University.

Genotyping

The hOGG1 Ser326Cys polymorphism was genotyped using the 384 well ABI 7900HT Real Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers, probes and reaction conditions for each single nucleotide polymorphism are available upon request. The polymorphism analysis was performed by two people independently in a blinded manner. Ten percent of random samples were selected for confirmation, and the results were 100% concordant. Two DNA samples of the controls failed to generate reliable results, only 1402 controls were included in the final analysis.

Measurement of 8-OHdG levels

The serum values of 8-OHdG could be determined from 50 bladder cancer patients and 50 healthy controls. Serum samples were stored at −80°C until the time of analysis. To determine serum 8-OHdG levels, Highly Sensitive 8-OHdG Check ELISA kit (R&D Systems, Minneapolis, MN, USA) was used. The assays were performed following the instructions of the manufacturer.

Statistical analysis

Differences in the distributions of demographic characteristics, selected variables and genotype frequencies of hOGG1 Ser326Cys polymorphism between the cases and controls were evaluated using χ2-test (for categorical variables). Hardy–Weinberg equilibrium was tested using a goodness-of-fit χ2-test. Unconditional univariate and multivariate logistic regression analyses were used to obtain the crude and adjusted odds ratios (ORs) for bladder cancer risk and their 95% confidence intervals (CIs). To explore potential interaction between the polymorphism and tobacco smoking, we assessed a multiplicative gene-environment interaction by logistic regression analysis including main effect variables and their product terms. Mann–Whitney test and analysis of variance (anova) were used to compare levels of serum 8-OHdG. < 0.05 was considered statistically significant, and all statistical tests were two-sided. All the statistical analyses were performed with Statistical Analysis System software (9.1; SAS Institute, Cary, NC, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Characteristics of the study population

The characteristics of the 1050 bladder cancer and 1404 controls are described in Table 1. There were no significant differences between the cases and controls on age and sex (= 0.356 for age, and 0.550 for sex). However, there were more smokers (47.3%) among the cases than the controls (38.2%), and the difference was statistically significant (< 0.001). Of the 1050 bladder cancer patients, the tumor grades from G1 to G3 were 49.2%, 35.2% and 15.6%, respectively. In addition, 65.5% of patients were in superficial stage, whereas 34.5% of patients were in invasive stage.

Table 1. Frequency distributions of selected variables between the bladder cancer cases and cancer-free controls
VariablesCases (= 1050)

Controls

(n = 1404)

P-valuea
n % n %
  1. a

    Two-sided χ2 test for the frequency distributions of selected variables between the cases and controls.

Age
≤6550047.669549.5 0.356
>6555052.470950.5
Sex
Male83979.9110878.9 0.550
Female21120.129621.1
Smoking status
Never55352.786761.8<0.001
Ever49747.353738.2
Former22521.417912.7
Current27225.935825.5
Pack-years smoked
055352.786761.8<0.001
~2019218.325918.4
>2030529.027819.8
Tumor grade
G151749.2
G237035.2
G316315.6
Tumor stage
Superficial (pTa–pT1)68865.5
Invasive (pT2–pT4)36234.5

Association between the hOGG1 Ser326Cys polymorphism and bladder cancer risk

The genotype and allele distributions of the hOGG1 Ser326Cys polymorphism in the cases and controls are shown in Table 2. The observed genotype frequencies for this polymorphism among the controls were in agreement with the Hardy–Weinberg equilibrium (= 0.673). Logistic regression analysis revealed that the hOGG1 Ser/Cys genotype was associated with a significantly increased risk of bladder cancer, compared with the Cys/Cys genotype (adjusted OR = 1.23, 95% CI = 1.03–1.46). Furthermore, we observed that individuals carrying Ser/Cys and Ser/Ser genotypes had an increased risk of bladder cancer, compared with the Cys/Cys genotype (adjusted OR = 1.19, 95% CI = 1.01–1.41).

Table 2. Genotype and allele frequencies of the hOGG1 Ser326Cys polymorphism among the cases and controls and their associations with risk of bladder cancer
VariablesCases (= 1050)Controls (= 1402)Crude OR (95% CI)Adjusted OR (95% CI)a P-valuea
n % n %
  1. a

    Adjusted for age, sex, and pack-years smoked in logistic regression model.

Cys/Cys34432.851436.71.00 (reference)1.00 (reference)
Ser/Cys55152.567648.21.22 (1.02–1.45)1.23 (1.03–1.46)0.025
Ser/Ser15514.721215.11.09 (0.85–1.40)1.09 (0.85–1.40)0.509
Ser/Cys + Ser/Ser70667.288863.31.19 (1.00–1.41)1.19 (1.01–1.41)0.042
Ser allele0.410 0.392 0.214

We further assessed the effect of hOGG1 Ser326Cys polymorphism on bladder cancer risk by stratifying by age, sex, smoking status and pack-years smoked. As shown in Table 3, we found that the increased risk was more pronounced among older patients (adjusted OR = 1.31, 95% CI = 1.04–1.66), male patients (adjusted OR = 1.21, 95% CI = 1.00–1.47), ever smokers (adjusted OR = 1.29, 95% CI = 1.00–1.68) and heavy smokers (>20 pack-years) (adjusted OR = 1.45, 95% CI = 1.03–2.04). In the stratification of tumor grade and tumor stage of bladder cancer cases, no significant association was observed (Table 4).

Table 3. Stratification analyses between the genotypes of the hOGG1 Ser326Cys polymorphism and risk of bladder cancer
VariablesCases/ControlsGenotypes (Cases/Controls)Crude OR (95% CI)Adjusted OR (95% CI)a
Cys/CysSer/Cys + Ser/Ser
  1. a

    Adjusted for age, sex, and pack-years smoked in logistic regression model.

Age (years)
≤65500/694 166/242334/4521.08 (0.85–1.37)1.07 (0.84–1.37)
>65550/708 178/272372/4361.30 (1.03–1.65)1.31 (1.04–1.66)
Sex
Male839/1106266/397573/7091.21 (1.00–1.46)1.21 (1.00–1.47)
Female211/296 78/117133/1791.12 (0.78–1.60)1.13 (0.78–1.63)
Smoking status
Never553/866 191/319362/5471.11 (0.88–1.38)1.11 (0.89–1.39)
Ever497/536 153/195344/3411.29 (0.99–1.67)1.29 (1.00–1.68)
Pack-years smoked
0553/866 191/318362/5481.10 (0.88–1.38)1.11 (0.88–1.38)
~20192/258 56/84 136/1741.17 (0.78–1.76)1.14 (0.76–1.72)
>20305/278 97/112208/1661.45 (1.03–2.03)1.45 (1.03–2.04)
Table 4. Associations between the hOGG1 Ser326Cys polymorphism and progression of bladder cancer
Variables hOGG1 genotype (n %)Crude OR (95% CI)Adjusted OR (95% CI)a
Cys/Cys

Ser/Cys + 

Ser/Ser

  1. a

    Adjusted for age, sex, and pack-years smoked in logistic regression model.

Controls

(= 1402)

514 (36.7)888 (63.3)1.00 (Reference)1.00 (Reference)

Cases

(= 1050)

Tumor grade
  G1168 (32.5)349 (67.5)1.20 (0.97–1.49)1.21 (0.98–1.50)
  G2120 (32.4)250 (67.6)1.21 (0.95–1.54)1.22 (0.96–1.56)
  G356 (34.4)107 (65.6)1.11 (0.79–1.56)1.13 (0.80–1.60)
Tumor stage
Superficial228 (33.1)460 (66.9)1.17 (0.96–1.42)1.17 (0.97–1.42)
Invasive116 (32.0)246 (68.0)1.23 (0.96–1.57)1.25 (0.97–1.60)

Interaction between hOGG1 Ser326Cys polymorphism and tobacco smoking

Compared with non-smokers with the Cys/Cys genotype, a statistically significantly increased risk was observed in smokers with Cys/Cys and Ser/Cys + Ser/Ser genotypes (adjusted OR = 1.37, 95% CI = 1.01–1.86 and adjusted OR = 1.77, 95% CI = 1.36–2.31, respectively) (Table 5). Because Ser allele exerted a higher risk effect in smokers, we evaluated whether there was the existence of an interaction between smoking status and the hOGG1 Ser326Cys polymorphism. Nevertheless, we did not observe a multiplicative interaction between the polymorphism and smoking status (= 0.406) (Table 5).

Table 5. Interaction analyses of the hOGG1 Ser326Cys polymorphism and tobacco smoking
Smoking statusGenotypesCases n (%)Controls n (%)Adjusted OR (95% CI)a P-valuea
  1. a

    Adjusted for age and sex in logistic regression model.

Non-smokersCys/Cys1913191.00 (reference)
Non-smokersSer/Cys + Ser/Ser3625471.11 (0.89–1.39)0.362
SmokersCys/Cys1531951.37 (1.01–1.86)0.040
SmokersSer/Cys + Ser/Ser3443411.77 (1.36–2.31)<0.001
P interaction (multiplicative) 0.406

Measurement of 8-OHdG levels by ELISA

The potential relationship between 8-OHdG and the Ser326Cys polymorphism in the hOGG1 gene was examined in the pooled population of cases and controls. Among all the objects, the cases showed significantly higher levels as compared with controls (= 0.024) (Fig. 1a). Then we investigated whether the levels of 8-OHdG were modified by hOGG1 gene polymorphism. As shown in Figure 1(b), there was no statistically significant association between levels of 8-OHdG in DNA and hOGG1 Ser326Cys polymorphism among the controls (= 0.284).

image

Figure 1. Measurement of 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels by enzyme linked immunosorbent assay (ELISA). (a) Levels of serum 8-OHdG between bladder cancer patients and control subjects. (b) Levels of 8-OHdG according to human oxoguanine glycosylase 1 ( hOGG1 ) genotypes among controls.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

In the present study, we investigated the association between the hOGG1 Ser326Cys polymorphism and bladder cancer risk in a Chinese population. We observed that the hOGG1 Ser/Cys + Ser/Ser genotypes were associated with an increased risk of bladder cancer, compared with the Cys/Cys genotype, which was more evident for subgroups of older subjects, males, ever smokers and heavy smokers.

Several genes encoding for DNA repair molecules implicated in maintaining genomic integrity have been proposed as cancer-susceptibility genes.[24] Wang et al.[25] found an increased risk of bladder cancer associated with the XRCC1 194Trp/Trp and 280Arg/His genotypes (adjusted odds ratio = 3.90, 95% confidence interval = 1.69–8.98 for 194Trp/Trp and 2.53, 1.67–3.83 for 280Arg/His) compared with the 194Arg/Arg and 280Arg/Arg genotypes, respectively. In contrast, the APE1-656GG genotype was associated with a decreased risk of bladder cancer (0.57, 0.33–0.98) compared with the TT genotype. While CCNH V270A, ERCC6 M1097V, RAD23B A249V and XPD D312N were thought to have the best ability to predict bladder cancer risk in a study of Chen et al.[26] Base excision repair (BER) is a very important mechanism for repairing DNA damage and is believed to be related to human cancers.[27] It plays a key role in removing DNA damage from oxidation, deamination, and ring fragmentation. HOGG1 is an essential enzyme for BER, which encodes a DNA glycosylase that catalyzes the excision of 8-oxoguanine from oxidative damaged DNA.[28]

Recent studies have provided confirmed evidence on the functional role of hOGG1. A functional assay revealed that the hOGG1 protein encoded by the Ser allele exhibited substantially higher DNA repair activity than the Cys variant.[8] Bravard et al.[14] found that individuals with the Cys variant could more readily accumulate mutations under conditions of oxidative stress. Furthermore, Yamane et al.[7] showed that the hOGG1 Cys326 protein has a lower ability to suppress mutations than the Ser protein in human cells in vivo. While Janssen et al.[29] showed that DNA repair activity of hOGG1 in human lymphocytes is not dependent on the genetic Ser326Cys polymorphism. Ambiguous results have been obtained on the effect of such an amino acid change on the DNA repair activity of the hOGG1-Cys326 protein compared with the wild-type hOGG1 protein.[13] The level of 8-hydroxy-2′-deoxyguanosine (8-OHdG), products of oxidative DNA damage, is generally regarded as a biomarker of mutagenesis consequent to oxidative stress.[30] The relation between 8-OHdG formation and carcinogenesis has been studied. In bladder cancer, increased levels of 8-OHdG have been detected in the urine and bladder tissues.[31, 32] In our current study, we elucidated the association between serum levels of 8-OHdG and the OGG1 repair capacity among bladder cancer cases and controls. Our results demonstrated the cases showed significantly higher levels as compared with controls. The finding was consistent with other studies showing higher levels of DNA damage among patients compared to controls.[33, 34] However, the small number of samples did not allow us to make a comparison of the genotype distribution among controls in order to determine whether the hOGG1 Ser allele contributed to the risk of bladder cancer.

Molecular epidemiological studies of hOGG1 Ser326Cys in susceptibility of bladder cancer have reported disparate findings. Kim et al.[22] were the first to link hOGG1 Ser326Cys to bladder cancer and they found that the hOGG1 Ser/Cys + Ser/Ser genotypes were associated with the recurrence of superficial bladder cancer. While several studies showed that Cys/Cys genotype appeared to be a risk factor for bladder cancer.[21, 22, 35-39] In this study, we found that the Ser/Cys + Ser/Ser genotypes were associated with an increased risk in bladder cancer patients, which was consistent with the results from Kim et al.[22] It also suggested that the hOGG1 genotype may be a useful prognostic genetic marker for NMIBC.[28] But our findings indicated that no significant difference was observed between NMIBC and hOGG1 Ser326Cys polymorphism. The conflicting results between studies might be explained by the difference of allele frequency in different ethnic populations, because the hOGG1 Ser allele was found to be enriched in the Europeans (minor allele frequency, MAF = 0.224), while the Cys allele had a predominant percentage in Asians (MAF = 0.500).

In addition, we observed that individuals carrying Ser/Cys + Ser/Ser genotypes have a significantly increased risk for bladder cancer in ever smokers, especially in heavy smokers (>20 pack-years), which was consistent with previous studies showing that levels of 8-OH-G and repair activity for 8-OH-G are higher in smokers.[34, 40] Actually, cigarette smoking is a predominant risk factor for bladder cancer and account for nearly 50% of the cases in men and 30% in women.[3] Exposure to tobacco smoking can increase production of reactive oxygen species (ROS), which have the potential to induce oxidative damage, and variation in BER ability may be associated with bladder cancer risk.[36] In this study, the frequency of the Cys allele among smokers of healthy controls was 30.8%, of bladder cancers was 36.4%. But we did not find multiplicative joint effect between the hOGG1 rs1052133 polymorphism and risk of bladder cancer among smokers, which may be due to the relatively small sample size, especially for the examination of gene-environment interactions in stratified analyses.[41] We also observed that individuals carrying Ser/Cys + Ser/Ser genotypes have a significantly increased risk for bladder cancer in older subjects (>65 year). This observation is well supported by many studies,[42-44] which link DNA damage accumulation with age. Furthermore, our results indicated this risk was more pronounced in males who were thought to be under a long-term exposure to smoking or unknown risk factors involved in the etiology of bladder cancer, suggesting that men are inherently more susceptible to certain carcinogens than women. Unfortunately, we did not have detailed information on these factors, and further studies are warranted.

Several potential limitations of the present study should be considerate. First, our study was a hospital-based case-control study. Thus, we cannot exclude the chance of selection bias of subjects that may have been associated with a particular genotype. Nevertheless, the genotype distributions of hOGG1 polymorphism in our study population were similar to distributions reported in a Chinese population. For instance, the frequencies of the Ser/Ser, Ser/Cys, and Cys/Cys genotypes of the hOGG1 Ser326Cys polymorphism in our controls were 15.1%, 48.2%, and 36.7%, respectively, compared with 16.0%, 49.0%, and 35.0%, respectively, in a study reported in Chinese population.[45] Second, in addition to tobacco smoking and alcohol use, we did not obtain enough information on occupational exposure that may interact with the hOGG1 gene. Third, it is well recognized that urothelial tumors develop along two major pathways such as papillary and non-papillary or solid. As information about pathological variables such as papillary/non-papillary are lacking in our study, we cannot include papillary/non-papillary in pathological variables and do advanced analysis. Finally, enzyme-linked immunosorbent assay (ELISA), the method used in this article, still has some shortcomings. It is better to verify the results of serum 8-OHdG by high-performance liquid chromatography with electrochemical detection (HPLC-EC) or gas chromatography-mass spectrometry (GC-MS) in the future.

In conclusion, we found that the hOGG1 Ser326Cys polymorphism was associated with bladder cancer risk in a Chinese population, which further indicated that this polymorphism might be useful as a biomarker for genetic susceptibility in bladder cancer. Large studies and functional characterizations are warranted to validate and expand our findings.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This study was partly supported by National Natural Science Foundation of China (30972444, and 81102089), the Key Program of Natural Science Foundation of Jiangsu Province (BK2010080), Natural Science Foundation of Jiangsu Province (BK2011773 and BK2011775), the Key Program for Basic Research of Jiangsu Provincial Department of Education (11KJB330002), the Qin Lan Project of Jiangsu Provincial Department of Education, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).

Abbreviations
8-OHdG

8-hydroxy-2′-deoxyguanosine

8-OH-G

8-hydroxyguanine

BER

base excision repair

hOGG1

human oxoguanine glycosylase 1

NMIBC

non-muscle invasive bladder cancer

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References