Abstract
Aim: To evaluate the association between the polymorphisms of the Exo1 gene and the risk of lung cancer in central Taiwan. Patients and Methods: In this hospital-based study, the association of Exo1 A-1419G (rs3754093), C-908G (rs10802996), A238G (rs1776177), C498T (rs1635517), K589E (rs1047840), G670E (rs1776148), C723R (rs1635498), L757P (rs9350) and C3114T (rs851797) polymorphisms with lung cancer risk in a central Taiwanese population was investigated. In total, 358 patients with lung cancer and 358 age- and gender-matched healthy controls recruited from the China Medical Hospital in central Taiwan were genotyped. Results: A significantly different distribution was found in the frequency of the Exo1 K589E genotype, but not the other genotypes, between the lung cancer and control groups. The A allele Exo1 K589E conferred a significantly (p=0.0097) increased risk of lung cancer. As for the rest of the polymorphisms, there was no difference in distribution between the lung cancer and control groups. Gene environment interactions with smoking were significant for Exo1 K589E polymorphism. The Exo1 K589E AG and AA genotype in association with smoking conferred an increased risk of 1.7208 (95% confidence interval=1.2188-2.4295) for lung cancer. Conclusion: Our results provide the first evidence that the A allele of Exo1 K589E may be associated with the development of lung cancer and may be a novel useful marker for primary prevention and anticancer intervention.
Lung cancer has become one of the most common malignancies worldwide (1). In Taiwan, lung cancer is a common cancer and the leading cause of cancer-related deaths (2). Lung cancer is important for its high incidence, high mortality and low 5-year survival rate (3). Among the well-known causes of lung cancer, smoking is considered to be the most important risk factor (4-6). More than 60 carcinogens have been detected in tobacco smoke and these carcinogens could form adducts and induce oxidative damage to DNA, resulting in genome instability (7). DNA damage and genome instability are thought of as the first steps of carcinogenesis. The DNA repair system is responsible for removing DNA damage and maintaining genome stability, and each type of DNA injury is repaired via its specific repair pathway.
One of the major DNA repair pathways in human cells is that of the mismatch repair (MMR) pathway, which maintains genomic stability, modulates DNA recombination, and mediates cell cycle arrest (8). This system is important in preventing malignancies, and former reports indicated that deficient mutations of the mismatch repair system lead to various types of cancer, including lung cancer (9-11). The gene exonuclease 1 (Exo1; MIM #606063) belongs to the MMR system, and to the RAD2 nuclease family. It is located at chromosome 1q42-q43, contains one untranslated exon followed by 13 coding exons and encodes an 846 amino acid protein (12-14). Exo1 can interact physically with the MMR proteins MSH2 and MLH1 in both yeast and human cells, and with MSH3 in human cells (14-19). Recent findings indicated that mammalian Exo1 is responsible for mutation prevention and plays a role in normal meiosis. They also indicated that mice with Exo1 inactivation have reduced survival time and increased risk for tumor development, specifically lymphoma (20).
Single nucleotide polymorphisms (SNPs) of DNA repair genes have been associated with susceptibility to several types of cancer, including oral, gastric, breast, prostate, colorectal and lung cancer (21-28). These reports indicated that SNPs of the DNA repair system may affect the genes' functions or expression levels, and the capacity of those gene-related systems. Therefore, cancer susceptibility will be higher in people who carry risky genotypes. There are already several SNPs of Exo1 which have been reported as genetic risk factors of cancer. In 2005, a study investigating a Japanese population found that two polymorphisms of Exo1 gene, T439M and P757L, are associated with colorectal cancer risk (27). In 2008, the association between SNPs of Exo1 and lung cancer susceptibility was also examined in a Chinese population, indicating that K589E is associated with lung cancer risk (28). In order to understand and prevent local lung cancer, we have chosen up to nine SNPs of Exo1 and investigated their frequencies in a Taiwanese population.
The primer sequences and polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) conditions for Exo1 gene polymorphisms.
Patients and Methods
Study population and sample collection. Three hundred and sixty-seven cancer patients diagnosed with lung cancer were recruited at the outpatient clinics of general surgery between 2005-2008 at the China Medical University Hospital, Taichung, Taiwan, Republic of China. The clinical characteristics of patients including histological details were all graded and defined by expert surgeons. All patients voluntarily participated, completed a self-administered questionnaire and provided peripheral blood samples. An equal number of non-lung cancer healthy volunteers as controls were selected by matching for age, gender and habits after initial random sampling from the Health Examination Cohort of the hospital. The exclusion criteria of the control group included previous malignancy, metastasized cancer from other or unknown origin, and any familial or genetic diseases. Both groups completed a short questionnaire which included habits. 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, ROC) and further processed according to previous studies (21-26). The 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, and 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 listed in Table I.
Statistical analyses. Only those matches with all SNP data (case/control=358/358) were selected for 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 genotype frequencies of Exo1 SNPs in the controls from those expected under the Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson's χ2 test or Fisher's exact test (when the expected number in any cell was less than five) was used to compare the distribution of the Exo1 genotypes between cases and controls. Data were recognized as significant when the statistical p was less than 0.05.
Results
The frequency distributions of selected characteristics of 358 lung cancer patients and controls are shown in Table II. These characteristics of patients and controls are all well matched. None of these differences between groups were statistically significant (p>0.05) (Table II).
PCR-based restriction analysis of the Exo1 K589E rs1047840 polymorphism shown on 2.5% agarose electrophoresis. M: 100 bp DNA size marker, GG: enzyme indigestible homozygote, AG: heterozygote, and AA: enzyme digestible homozygote.
Characteristics of lung cancer patients and controls.
The frequency of the genotypes for the Exo1 A-1419G, C-908G, A238G, C498T, K589E, G670E, C723R, L757P and C3114T in controls and lung cancer patients is shown in Table III. The genotype distribution of the genetic polymorphisms of Exo1 K589E was significantly different between lung cancer and control groups (p=0.00097), while those for all the other polymorphisms were not significant (p>0.05) (Table III). Exo1 K589E was significantly associated with lung cancer. Representative PCR-based restriction analyses for the Exo1 K589E polymorphisms are shown in Figure 1.
The frequency of the alleles for Exo1 A-1419G, C-908G, A238G, C498T, K589E, G670E, C723R, L757P and C3114T in controls and lung cancer patients is shown in Table IV. The distributions of all these polymorphisms were in Hardy-Weinberg equilibrium and were similar between controls and lung cancer patients. The A allele of the Exo1 K589E polymorphism was significantly associated with lung cancer (p=0.0022).
The genotype distribution of various genetic polymorphisms of Exo1 K589E was significantly different between lung cancer and control groups with a smoking habit (p=0.0019) (Table V), while those for the rest of the polymorphisms were not significant (p>0.05) (data not shown). The A allele frequency was significantly higher in cancer patients who smoked than in controls, and patients who did not smoke. Individuals with Exo1 K589E AA or AG who smoked were approximately 1.7-fold more likely to have lung cancer than those who did not smoke.
Distribution of Exo1 genotypes among lung cancer patients and controls.
Discussion
In order to find potential biomarkers of lung cancer, in this study, we selected nine SNPs of the Exo1 gene and investigated their associations with susceptibility for lung cancer in the population of central Taiwan. Among these nine polymorphisms, we found that variant genotypes of Exo1 K589E were significantly associated with a higher susceptibility for lung cancer (Tables III and IV).
Distribution of Exo1 alleles among lung cancer patients and controls.
Exo1 K589E rs1047840 genotype and lung cancer after stratification by smoking.
Among the DNA repair systems, one of the major roles is played by the MMR system. The MMR system is responsible for correcting the mismatch between bases and the small insertion/deletion loops. Thus, it is essential in maintaining the integrity of the genome (29, 30). Exo1 is the only exonuclease involved in the human MMR system, playing a critical role as both 5′-3′ and 3′-5′ nucleases and contributing to the overall integrity of the MMR complex (31). Because Exo1 plays a distinctive role in the MMR system, the Exo1 gene has become a significant target gene and has been widely investigated for its association with risks of various malignancies (32-34).
In this study, we found that Exo1 K589E was associated with lung cancer susceptibility in Taiwan, and the only polymorphism which has a definite positive association is located on exon12 of the Exo1 gene and its change causes the 589th amino acid of the Exo1 protein product to be altered from lysine to glutamic acid. The amino acid change at codon 589 might influence the products of Exo1 mRNA, for K589E was located at an exonic splicing enhancer (ESE) region (28). Our results in Taiwan are consistent with the work in mainland China (28). On the contrary, Zienolddiny et al. have found no significant association of Exo1 K589E polymorphism and risk of non-small cell lung cancer in a Caucasian Norwegian population (35). The similarity between our results and these of Jin et al. (28) may be caused by ethnicity; this polymorphism may associate with Mongolian lung cancer, but not that of Caucasians.
We further analyzed the association between K589E genotype and lung cancer risk in patients and controls who have cigarette smoking habits. Interestingly, the interaction between Exo1 K589E and cigarette smoking habit is obvious: people with the AA or AG genotype have a 1.72-fold higher risk of lung cancer than people with the GG genotype (Table V). We propose that the A allele of K589E may affect Exo1 activity, slightly influencing its normal function. As people with A allele(s) get older, the alteration towards carcinogenesis may accumulate via increasing unremoved DNA adducts. Cigarette smoking, a well-known cause of DNA damage, will release many DNA damage inducers into the respiratory system and cause DNA damage to the cells. Therefore, in people who have a risky genetic variant, such as those carrying the A allele of K589E, and also have a smoking habit, the joint effect of these factors will synergistically increase their lung cancer susceptibility.
To sum up, this is the first study which focuses on the SNPs of Exo1 and lung cancer in Taiwan, and the presence of the A allele of K589E was associated with a higher risk of lung cancer. The A allele of K589E may be a useful marker in lung oncology for anticancer application, and early cancer detection.
Acknowledgements
We thank Yung-Shun Kuo, Hua-Shiang Chen, Chiao-Lin Lin, and the tissue bank at China Medical University for their technical assistance. This study was supported by research grants from the Terry Fox Cancer Research Foundation and the National Science Council (NSC 95-2320-B-039-014-MY3).
Footnotes
-
↵* These authors contributed equally to this study.
- Received July 28, 2008.
- Revision received November 20, 2008.
- Accepted December 2, 2008.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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