Abstract
This study aimed at evaluating the association and interaction among human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) genotypic polymorphism, smoking status and oral cancer risk in Taiwan. For this purpose, the well-known polymorphic variants of hOGG1, codon 326, was analyzed for its association with oral cancer susceptibility, and its joint effect with individual smoking habits on oral cancer susceptibility. In total, 620 patients with oral cancer and 620 healthy controls were recruited from the China Medical Hospital and genotyped. The results showed that the hOGG1 codon 326 genotypes were differently distributed between the oral cancer and control groups (p=0.0266), with the C allele of hOGG1 codon 326 being significantly (p=0.0046) more frequently found in cancer patients than in controls. We further analyzed the genetic-smoking joint effects on oral cancer risk and found an interaction between hOGG1 codon 326 genotypes and smoking status. The hOGG1 codon 326 CC genotype was associated with oral cancer risk only in the smoker group (p=0.0198), but not in the non-chewer group (p=0.8357). Our results provide evidence that the C allele of hOGG1 codon 326 may have a joint effect with smoking on the development of oral cancer.
Oral cancer is ranked as the fourth most common type of cancer among Taiwanese males, with a peak at 55-59 years old, and is the leading type of cancer causing death in the 40-50-year-old age group (1). Three main factors, smoking, alcohol drinking, and betel quid chewing, are identified as being closely related to oral carcinogenesis. Smoking may generate reactive oxygen species (ROS) including superoxide anion radicals and hydrogen peroxide, which may further induce DNA single- and double-strand breaks. Sustained oxidative stress, such as the one occuring on smoking and betel quid exposure, induce oxidative DNA adducts in the human genome, and 8-hydroxy-2-deoxyguanine (8-OH-dG) seems to be the major form (2, 3). 8-OH-dG is mutagenic and, if not repaired on time, can cause severe transversions of GC to TA in several oncogenes and tumor-suppressor genes, in turn leading to carcinogenesis (2, 3). 8-OH-dG and other oxidative DNA adducts are repaired by the base excision repair pathway (4). As part of this pathway, the human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) gene encodes a DNA glycosylase which catalyzes the cleavage of the glycosylic bond between an oxidized base and the sugar moiety, leaving an abasic apurinic/apyrumidinic site in DNA. The resulting apurinic/apyrumidinic site is then incised, and the repair is completed by successive actions of a phosphodiesterase, a DNA polymerase, and a DNA ligase (5).
Among the common single-nucleotide polymorphisms (SNPs) of hOGG1 gene, the one located in the exon 7, resulting in an amino acid substitution of serine (Ser) with cysteine (Cys) at codon 326 (Ser326Cys, rs1052133), has been demonstrated to affect hOGG1 function (6). Those cells with a Cys-encoding allele (G) exhibited a reduced DNA repair activity (6), which has been reported to be associated with the risk of many types of cancer (7). In 2002, a study investigating the role of hOGG1 Ser326Cys genotypes in a Caucasian population was performed, but their sample size was rather small (cases/controls=169/338), and the samples of tumor were collected from the tonsil, tongue, floor of mouth, larynx and esophagus of the patients, not specifically oral cancer.
In the present study, we aimed at analyzing the hOGG1 Ser326Cys genotypes in a Taiwanese oral cancer population (controls/cases=620/620), and investigated the interaction of hOGG1 Ser326Cys genotypes and smoking habits in this specific Taiwanese population.
The primer sequences, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) conditions for hOGG1 gene polymorphism determinations.
Materials and Methods
Study population and sample collection. Six-hundred and twenty patients diagnosed with oral cancer were recruited at the outpatient clinics of general surgery during 1998-2010 at the China Medical University Hospital in central Taiwan. The clinical characteristics of patients, including histological details, were all graded and defined by expert surgeons (Dr. Tsai M., Tsou Y., Shih L. and Tseng H.). All patients voluntarily participated and provided their peripheral blood for this research. The same numbers of healthy volunteers without oral cancer as the controls were selected by matching for their age, gender and habits after initial random sampling from the Health Examination Cohort of the hospital. The exclusion criteria for the control group included previous malignancy, metastasized cancer from other or unknown origin, and any familial diseases. Both groups completed a well-informed questionnaire, which included their individual habits. Smokers were defined as daily or almost daily smokers who had smoked at least five packs of cigarettes in their lifetime. Smokers were asked for the age of initiation, whether they were currently smoking or had already quit, and if so, when they had quit, and on average, how many cigarettes they smoked or had smoked daily. Our study was approved by the Institutional Review Board of the China Medical University Hospital and written-informed consent was obtained from all participants.
Genotyping assays. Genomic DNA was prepared from peripheral blood leukocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and was further processed according to previous studies (8-17). The polymerase chain reaction (PCR) cycling conditions were: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min. Pairs of PCR primer sequences and restriction enzyme for each DNA product are all listed in Table I.
Statistical analyses. Only those with both genotypic and clinical data (controls/cases=620/620) were included for the final analysis. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotypic frequencies of hOGG1 codon 326 in the controls from those expected under the Hardy–Weinberg equilibrium was examined by the goodness-of-fit test. Pearson's chi-square test was performed to compare the distributions of the genotypes between case and control groups. Data were recognized as being statistically significant when the p-value was less than 0.05.
Results
The demographic distribution of characteristics of all the patients and healthy controls are summarized in Table II. These characteristics are all well-matched in the two groups and none of the differences between the two groups was statistically significant (p>0.05) (Table II).
The frequencies of the genotypes for hOGG1 codon 326 in controls and patients with oral cancer are listed and analyzed in Table III. The genotype distributions of hOGG1 codon 326 were significantly different between oral cancer and control groups (p=0.0266) (Table III). The frequencies of the alleles for hOGG1 codon 326 in controls and oral cancer patients are also shown in Table III, and the trend is more obvious. The data showed that the C allele of the hOGG1 codon 326 polymorphism was significantly associated with higher oral cancer risk (p=0.0046). It is more convincing to provide the results from multiple approaches, so we have also performed an analysis of odds ratios for oral cancer risk among the variant genotypes. The odds ratio analysis showed that those who carry homologous CC have a 1.53-fold higher oral cancer risk (95% confidence interval (CI)=1.12-2.08), compared with those with homologous GG. Combining the CC with CG group vs. the GG group has a similar higher risk compared with those with homologous GG (odds ratio=1.26, 95% CI=1.00-1.58). The conclusive finding deduced from the data in Tables III and IV is that the C allele of hOGG1 codon 326 seems to be associated with a higher risk for oral cancer in Taiwanese.
The interaction of the genotype of hOGG1 codon 326 and smoking habit was of interest. The genotype distribution of hOGG1 codon 326 polymorphism was significantly different between oral cancer and control groups who had a smoking habit (p=0.0198), but was not significant (p=0.8357) in non-smokers (Table IV). Consistent with the findings in Table III, the C allele frequency was still significantly higher in patients with cancer with a smoking habit than in smoking controls. There was no such difference between the non-smoking groups.
Discussion
In order to examine the role of hOGG1 in oral cancer, in this study, we selected the most important SNP of the hOGG1 gene, that of codon 326, and clarified its association with the susceptibility for oral cancer in a Taiwanese population where oral cancer density is the highest worldwide. We found that the C variant genotypes of hOGG1 codon 326 were significantly associated with a higher susceptibility for oral cancer (Table III).
Characteristics of patients with oral cancer and controls.
Distribution of human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) codon 326 genetic and allelic frequencies among oral cancer patients and control groups.
Previous studies have implicated the hOGG1 codon 326 polymorphism in risk for smoking- and/or alcohol-related orolaryngeal cancer in the U.S.A. (18). Significant increases in risk were found for the homozygous G/G genotype and orolaryngeal cancer in the Caucasian study (18), in contract to our finding. In addition, the risky G allele had obvious joint effects with both smoking and alcohol drinking on orolaryngeal carcinogenesis (18). Furthermore, a significant positive association between hOGG1 genotype and cancer risk was also observed for esophageal cancer (19). To compare the difference between Caucasian and oriental populations, we have also analyzed the association between hOGG1 codon 326 genotypes and oral cancer risk in patients and controls who have a smoking habit in Taiwan, an oriental population (Table IV). Interestingly, the interaction between hOGG1 codon 326 and smoking habit is obvious, and the C allele appears indeed to be risky (Table IV). Compared with the previous study, we have a larger population of both controls and cases for further stratification, with higher power of analysis. In addition, during sampling, we recruited almost the same proportion of cases and controls taking their individual habits into consideration (Table I). Furthermore, all the individuals investigated in this study are Taiwanese, and much more genetically conserved population than the population collected in the U.S.A. However, firm conclusions about oral carcinogenesis may be confounded with variations among ethnicities with different levels of exposure to environmental lifestyle factors, such as smoking, and multi-institutional studies including different ethnic group; more careful matching between cases and controls should be performed in future studies. We have also stratified the population by gender, and the findings in the overall population were consistent in Taiwanese males, but no significance was found in Taiwanese females (data not shown). This may be due to the fact that the gender ratio is about nine to one for male to female, and the female sample size was limited.
Distribution of human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) codon 326 genotypes in oral cancer patients after stratification by individual smoking habit.
We propose that the different genotypes of codon 326 may affect hOGG1 enzymatic activity, slightly altering its normal function. Generally speaking, oxidative insults to genomic DNA are continuously happening, and may be caused by both endogenous oxidative stress and exogenous carcinogens. If hOGG1 does not function well, DNA adducts could be left unrepaired, leading to mutations or even carcinogenesis. In the Taiwanese population, as individuals with the C allele(s) become older, the alterations towards carcinogenesis may accumulate via the decreasing functions of hOGG1. There are several studies that suggest that the amino acid change in hOGG1 may affect the catalytic properties of the enzyme (20, 21). An indirect explanation for the functional relevance of the polymorphism is that the variant allele may be linked to other functional polymorphisms in hOGG1 involved in the removal of oxidative DNA damage. A direct explanation is that the variant genotype may be deficient in repair of oxidative DNA damage only under conditions of excessive cellular oxidative stress (20). However, it is strange that our hypothesis is against the finding that cells with a Cys-encoding allele (G) exhibited reduced DNA repair activity (6), and individual DNA repair capacity in our patients should be investigated more carefully to re-examine this hypothesis.
In conclusion, oral cancer is highly smoking-related and this is so far the largest study which focuses on the codon 326 polymorphism of hOGG1 and on its joint effect with smoking habit regarding oral cancer risk. Our results indicate that the effect of the C allele of hOGG1 codon 326 interacts with smoking habits and may play an important role in oral carcinogenesis.
Acknowledgements
We thank Meng-Hsuan Lee, Fang-Jing Li, Hong-Xue Ji, Yi-Ting Chang, and the Tissue Bank at the China Medical University for their technical assistance. This study was supported by research grants from the Terry Fox Cancer Research Foundation, National Science Council (NSC101-2320-B-039-045), Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence (DOH101-TD-C-111-005) and China Medical University and Hospital (DMR-101-031).
Footnotes
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↵* These Authors contribute equally to this study.
- Received May 8, 2012.
- Revision received July 26, 2012.
- Accepted July 30, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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