The Clinical Respiratory Journal
ORIGINAL ARTICLE
Association between genetic polymorphisms in XPD and XRCC1 genes and risks of non-small cell lung cancer in East Chinese Han population Yong Du, Yanchao He, Zhoufang Mei, Ling Qian, Jindong Shi and Zhijun Jie Department of Respiratory Medicine, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China
Abstract Background and Aims: Lung cancer is a multifactorial disease. Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing 1 (XRCC1) genes are 2 important susceptibility genes related to lung cancer. In this study, we explored the correlation between genetic polymorphisms in XPD and XRCC1 and the risk of non-small cell lung cancer (NSCLC) in the East Chinese Han population. We also investigated risk factors associated with non-small cell lung cancer in this population. Methods: We conducted a case control study in 120 NSCLC patients and 120 healthy controls. The NSCLC patients were further divided into three subgroups, squamous carcinoma, adenocarcinoma and other type of cancer, according to tumor histology. No patients had undergone any treatment. Polymerase chain reaction and restriction fragment length polymorphism technologies were applied to detect the distribution of XPD-751, XRCC1-194 and XRCC1-399 genes in all patients. Results: The results showed significant gene frequency differences for all three genes between patients with NSCLC and control patients. Significantly different frequencies of XPD-751-Gln, XRCC-194-Trp and XRCC1-399-Gln mutant alleles were observed between the two groups. XPD-751SNP and XRCC1-194SNP frequencies varied among the three lung cancer groups, while the frequency of XRCC1-399SNP did not differ significantly. Conclusions: The results suggested that genetic polymorphisms in XPD-751, XRCC1-194 and XRCC1-399 were related to the risk of NSCLC, among which XPD-751SNP was responsible for adenocarcinoma, while XRCC1-194SNP was closely linked to squamous carcinoma. Smoking and XRCC1-194SNP were risk factors of NSCLC. Please cite this paper as: Du Y, He Y, Mei Z, Qian L, Shi J and Jie Z. Association between genetic polymorphisms in XPD and XRCC1 genes and risks of non-small cell lung cancer in East Chinese Han population. Clin Respir J 2014; ••: ••–••. DOI:10.1111/crj.12218.
Key words non-small-cell cancer – single-nucleotide polymorphism – xeroderma pigmentosum group D – X-ray repair cross-complementing 1 Correspondence Zhijun Jie, MD, PhD, Department of Respiratory Medicine, The Fifth People’s Hospital of Shanghai, Fudan University, No. 801 Heqing Road, Minhang District, 200240 Shanghai, China. Tel: +86 189 181 68602 Fax: +86 21 640 85875 email: [email protected] Received: 17 April 2014 Revision requested: 09 August 2014 Accepted: 29 September 2014 DOI:10.1111/crj.12218 Authorship and contributorship Yong Du is responsible for study design, data analysis and writing the article. Yanchao He and Zhoufang Mei have contributed in collecting samples and in the extraction of genomic DNA. Ling Qian is in charge of PCR amplification and agarose gel electrophoresis. Jindong Shi has helped in RFLP technology and polyacrylamide gel electrophoresis. Zhijun Jie is responsible for the supervision of performance of study, data analysis and reporting work. Ethics The study protocol was approved by the ethics committee of the Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China. All subjects in this study gave signed informed consent before the research. Conflict of interest The authors have stated explicitly that there are no conflicts of interest in connection with this article.
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
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XPD.XRCC1 genetic polymorphisms and NSCLC
Introduction Lung cancer is a multifactorial, multistage process (1), and smoking is its most important risk factor. However, only 10%–15% of smokers develop lung cancer, while 10%–15% of lung cancer patients never smoke at all, suggesting that there are individual differences in lung cancer genetic susceptibility. Among the numerous genetic studies on lung cancer pathogenesis, those on DNA repair genes have become a major focus in lung cancer research. DNA injury repair is a complicated process involving many enzymes and proteins. Once the genes related to DNA repair are mutated, the genomic DNA repair capacity (DRC) may be impaired (2), leading to increased genomic instability. Single-nucleotide polymorphisms (SNPs) (3, 4) in DNA repair genes are single-base mutations that may change the encoded amino acid, causing individual differences in DRC (5), and finally leading to adverse consequences such as cancer. Xeroderma pigmentosum (XP) is a type of DNA repair deficiency disease caused by deficiency in nucleotide excision repair (NER) capacity (6). XPD, an adenosine triphosphate-dependent helicase, is involved in transcription as the core component of the transcription factor IIH. The XPD protein helicase activity, the XPD gene expression product, can unlock the DNA’s double helix structure and cut or remove damaged DNA. Once the XPD gene is mutated, DNA repair deficiency may result from decreased XPD protein expression (7, 8). To date, seven SNP sites have been identified in the XPD gene, among which the genetic mutations Asp312Asn and Lys751Gln are the most common (9) and are related to lung cancer. X-ray repair cross-complementing 1 (XRCC1) is one of the important genes involved in mammal DNA repair. It participates in the cell cycle checkpoint function and responds to DNA damage, playing a critical role in base excision repair (BER) and single-strand break repair (10, 11). Recent studies indicated that there are three amino acid mutation sites in the XRCC1 gene, namely 194 (Arg→Trp), 280 (Arg→His) and 399 (Arg→Gln) (12). These mutations in encoding regions can change the function of XRCC1 and increase individual susceptibility to lung cancer. In recent years, lung cancer incidence and mortality rates have increased in the East Chinese Han population. Genetic susceptibility studies of lung cancer help to screen high-risk populations and provide guidance for reducing lung cancer’s incidence. Most studies on DNA repair genes have focused on the relationships between lung cancer and the sensitivities of radiotherapy and chemotherapy (13–15). They neglect the
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fact that mutations in DNA repair genes can also lead to decreased DRC in the susceptible population, thereby causing lung cancer. In this study, we performed a case control study in patients with newly diagnosed non-small cell lung cancer (NSCLC) who had not undergone surgery, radiotherapy, or chemotherapy. We used polymerase chain reaction (PCR) and restriction fragment length polymorphism technologies to analyze the relationships between NSCLC and three SNPs, including XPD751, XRCC1-194 and XRCC1-399. This may be an effective theoretical basis for high-risk NSCLC population screenings and the development of molecular targeted therapies.
Materials and methods Patient selection We enrolled 120 NSCLC patients who were seen at our hospital from March 2010 to March 2012. All patients were diagnosed via bronchoscopic biopsy or cytology examinations and were divided into three groups according to pathology: squamous carcinoma, adenocarcinoma and other cancer type (such as large cell lung cancer, carcinoid and undifferentiated carcinoma). No patients had received surgery, radiotherapy, or chemotherapy. Additionally, 120 healthy individuals were enrolled in the control group. These individuals underwent physical examinations at our hospital during the same time frame. No abnormalities were found in chest imaging and serum tumor marker tests (including CEA.AFP.NSE.SCC.CA211.CA125.CA199) for patients in the control group. All patients were from the East China Han population and did not have a history of hereditary diseases such as diabetes, hypertension, coronal heart disease, or other respiratory diseases. Informed consent was obtained from all patients before participation in the study. The study protocol was approved by the Fifth People’s Hospital of Shanghai, Fudan University, and all participants provided written informed consent.
Collection of blood samples and extraction of DNA Peripheral venous blood (4 mL) was collected from all patients after a fasting period, with ethylenediaminetetraacetic acid as an anticoagulant. Blood samples were kept frozen at −20°C before genomic DNA extraction using a DNA extraction kit from TIANGEN Science and Technology Co., Ltd. (Beijing, China). The DNA was dissolved and frozen at −20°C.
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
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Gene amplification and digestion Primer sequences, amplification and digestion conditions were chosen based on related literature. Genomic DNA was amplified using PCR. Reactions contained Taq DNA polymerase (Dalian Takara Biotechnology Co., Ltd., Liaoning, China), Deoxynucleotide triphosphates and primers. The PCR products were digested with corresponding enzymes. The amplification procedure of the XPD-751 and XRCC1-194 genes were all as follows: predenaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 61°C for 1 min and 72°C for 1 min, and a final elongation at 72°C for 5 min. The primer sequences for XPD-751 were F: 5′-GCCCGCTCTGGATTATACG-3′ and R: 5′-CTATC ATCTCCTGGCCCCC-3′. Its products were digested with Pst I (Shanghai SANGON Biological Engineering Co., Ltd., Shanghai, China) at 37°C for 1 h. The primer sequences of XRCC1-194 were F: 5′-GCCAGGG CCCCTCCTTCAA-3′ and R: 5′-TACCCTCAGACC CACGAGT-3′. Its products were digested with Pvu II (Shanghai SANGON Biological Engineering Co., Ltd.) at 37°C for 1 h. The amplification procedure of XRCC1-399 gene was as follows: predenaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 3 min and a final elongation at 72°C for 5 min. The primer sequences were F: 5′-TTGTGCTTTCTCTGTGTCCA-3′and R: 5′-TCC TCCAGCCTTTTCTGATA-3′. Its products were digested with MspI (Shanghai SANGON Biological Engineering Co., Ltd.) at 37°C for 1 h. All the digestion products were subjected to 2% agarose gel electrophoresis. The electrophoretic bands were observed with an electrophoretic display device to determine the genotype of each specimen.
Statistical analysis SPSS 17.0 software (IBM, Chicago, IL, USA) was used for statistical analysis. All alleles complied with the
XPD.XRCC1 genetic polymorphisms and NSCLC
Hardy–Weinberg genetic equilibrium. Frequency of alleles = (number of homozygous individuals × 2 + number of heterozygous individuals)/(total number of individuals × 2). All measurements were analyzed with Student’s t-tests, and count data were analyzed using chi-squared tests. The relative risk of mutated genes was estimated with 95% dependent intervals. Screening of risk factors and their interactions were analyzed with the logistic regression method. Differences with P values less than 0.05 were considered statistically significant. All statistical analyses were conducted with two-tailed probability tests.
Results Demographic data (i.e. patient age, gender, smoking history and smoking index) are listed in Table 1. There was no significant difference in ages or genders between the two groups (P > 0.05). The smoking history and smoking index were significantly higher in the three NSCLC subgroups than in the control group (P < 0.05). The genotype and allele frequencies of the three selected genes in the patient and control groups are listed in Tables 2 and 3. There were significant differences in genotype frequencies at XPD-751, XRCC1-194 and XRCC1-399 polymorphic sites between the two groups (P = 0.026, 0.025 and 0.044, respectively). The allele frequencies of these three SNPs significantly differed between the two groups (P = 0.001, 0.003 and 0.002, respectively). NSCLC patients were stratified by histopathology: squamous carcinoma, adenocarcinoma and other type of cancer. The genetic allele frequencies of the selected genes in these groups are listed in Table 4. There were significant differences in XPD-751SNP and XRCC1194SNP among the three groups (P = 0.023 and 0.003, respectively), among which the mutated gene frequencies of XPD-751 in the squamous carcinoma group and of XRCC1-194 in the adenocarcinoma group were
Table 1. Characteristics of the study population Lung cancer Characteristics
Control
Squamous cell carcinoma
Adenocarcinoma
Other types
Number of cases Sex (male/female) Age (years) Smoking (yes/no) Smoking index (smoking years × smoking number per day)
120 64/56 64.3 ± 13.8 61/59 446 ± 142
52 28/24 65.8 ± 13.3 36/16* 624 ± 128*
48 25/23 67.2 ± 15.1 38/14* 638 ± 135*
20 11/9 66.5 ± 14.2 15/5* 629 ± 141*
*P < 0.05, compared with the control.
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
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Table 2. Genotype frequencies of the three selected genes in the two groups Gene
Polymorphism
Lung cancer
Control
Model
XPD gene Lys751Gln
Lys/Lys Gln/Lys Gln/Gln Arg/Arg Trp/Arg Trp/Trp Arg/Arg Gln/Arg Gln/Gln
78 24 18 68 33 19 81 16 23
96 16 8 88 21 11 95 15 10
Additive Dominant Recessive Additive Dominant Recessive Additive Dominant Recessive
XRCC1 gene Arg194Trp XRCC1 gene Arg399Gln
OR (95% CI) 2.154 (1.201–3.861) 2.471 (1.030–5.926) 2.103 (1.223–3.617) 1.864 (0.846–4.109) 1.830 (1.021–3.278) 2.608 (1.183–5.753)
P 0.026 0.009 0.038 0.025 0.007 0.118 0.044 0.041 0.015
CI, confidence interval; OR, odds ratio.
higher than in the other two groups. There was no significant difference in the frequency of XRCC1399SNP among the three groups (P = 0.598). Results of multifactor logistic regression analysis of NSCLC-related risk factors are listed in Table 5. Compared with the control group, the odds ratio (OR) of NSCLC in XPD-751SNP and XRCC1-399SNP carriers
were 1.325 [95% confidence interval (CI): 0.673–2.238, P = 0.134] and 0.805 (95% CI: 0.735–3.418, P = 0.323), which had no statistical difference. However, the OR of NSCLC in smokers and XRCC1-194SNP carriers were 2.786 (95% CI: 1.238–6.565, P = 0.026) and 4.389 (95% CI: 1.788–16.331, P = 0.009), which were significantly different.
Table 3. The allele frequencies of the three selected genes in the two groups Gene
Allele
Lung cancer
Control
OR (95% CI)
P
XPD gene Lys751Gln XRCC1 gene Arg194Trp XRCC1 gene Arg399Gln
Lys Gln Arg Trp Arg Gln/
180 60 169 71 178 62
208 32 197 43 205 35
2.167 (1.350–3.478)
0.001
1.925 (1.251–2.961)
0.003
2.040 (1.287–3.233)
0.002
CI, confidence interval; OR, odds ratio.
Table 4. Allele frequencies in patients with different pathological types of non-small cell lung cancer Gene
Allele
Squamous cell carcinoma
Adenocarcinoma
Other types
XPD gene Lys751Gln XRCC1 gene Arg194Trp XRCC1 gene Arg399Gln
Lys Gln Arg Trp Arg Gln/
71 33 80 24 76 28
81 15 56 40 76 20
32 8 33 7 30 10
P 0.023 0.003 0.598
Table 5. Logistic regression analysis of risk factors Factor
OR
P
β
SE
95% CI
Smoking XPD-751SNP XRCC1-194SNP XRCC1-399SNP
2.786 1.325 4.389 0.805
0.026 0.134 0.009 0.323
0.957 0.309 1.915 −0.486
0.372 0.394 0.683 0.351
1.238–6.565 0.673–2.238 1.788–16.331 0.735–3.418
CI, confidence interval; OR, odds ratio; SE, standard error.
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XPD.XRCC1 genetic polymorphisms and NSCLC
Table 6. Analysis of the interaction between smoking and XRCC1-194SNP Factors
OR
P
β
95% CI
Smoking XRCC1-194SNP Smoking and XRCC1-194SNP
1.937 1.628 9.732
0.146 0.578 0.008
0.587 0.684 2.631
0.762–6.895 0.587–5.449 1.561–11.392
CI, confidence interval; OR, odds ratio; SNP, single-nucleotide polymorphism.
The interactions between the two high-risk factors (smoking and XRCC1-194SNP) can be seen in Table 6. The risk of NSCLC in smokers without XRCC1194SNP was 1.937 times higher than that of the control group, while that of non-smokers with XRCC1194SNP was 1.628 times higher than that of the control group. However, in smokers with XRCC1194SNP, the risk was as much as 9.732-fold higher than that in the control group (P < 0.05, 95% CI: 1.561–11.392), and the OR value of which was much more than the product of the former two conditions (9.732 > 1.937 × 1.628).
Discussion Our study found significant differences in XPD-751 mutation and allele frequencies between patients with NSCLC and healthy controls, suggesting a close relationship between XPD-751 SNP and NSCLC. These data are consistent with other scientists’ conclusions both in China and abroad. In a meta-analysis of several case control studies, Chinese scholar Zhan Ping (16) found that the XPD-751 SNP was related to NSCLC occurrence, with an increased association observed in Caucasians and smokers. European scholar LopezCima (17) also found that XPD-751SNP was related to NSCLC in a study of multiple genes in a population from Northern Spain. Additionally, De Ruyck (18) came to the same conclusion in a population of Belgian Caucasians, with a greater association observed in non-smokers than in smokers. The significant difference in the allele frequency of XRCC1-194 between NSCLC patients and controls suggested a close correlation between XRCC1-194SNP and NSCLC. Studies in China and abroad have not been consistent in this regard. Chinese scholar Huang Jin (19) found that XRCC1-194SNP was a susceptibility factor for NSCLC using a meta-analysis of the XRCC1-194 gene in the Chinese mainland population. However, Chinese scholar Sun Yan (20) found no obvious correlation between the XRCC1-194 polymorphism and the occurrence of NSCLC in a study of
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
non-smoking Han individuals. European scholar Lopez-Cima (17) also found no significant correlation between the two factors in a study of genetic polymorphisms in XRCC1 in the Spanish population. Furthermore, the significant difference in mutated allele frequencies of XRCC1-399 between the two groups also supported the correlation between XRCC1399SNP and the occurrence of NSCLC. Studies in China and abroad have not been conclusive. Chinese scholar Li Zheng (21) found a correlation between XRCC1-399SNP and NSCLC through a genetic polymorphism study of BER in the Chinese population. Similarly, Korean scholar Kim (22) found that XRCC1399SNP had an important role in the development of NSCLC through studying the 3 XRCC1 genetic sites in the Korean population. Nevertheless, foreign scholar Improta (23) found no association between XRCC1399SNP and NSCLC through a polymorphism study of XRCC1 in a southern Italian population. Our study investigated genetic susceptibility to NSCLC directly rather than the relationship between genetic polymorphisms in lung cancer and sensitivity to radiotherapy and chemotherapy. The results of this research were not completely consistent with those of other studies, which could be attributed to different regions, racial differences and environmental exposures, in addition to differences in experimental design and data analysis. Among those reasons above, we imply that the region difference may influence our results because NSCLC incidence in this area was obviously rising. We also speculate that the three gene mutations in local residents are higher than those in immigrants. Thus, we plan to expand the sample size of NSCLC to further determine whether gene polymorphisms are associated with regional distribution and to select the population displaying the gene mutation as a high-risk factor of NSCLC in the East Chinese Han population. Using an allele frequency study of different lung cancers, we found that genetic polymorphisms in XPD-751 were closely linked to squamous carcinoma in NSCLC. This conclusion is consistent with that of northern Chinese scholar Xing De Yin (24), but not consistent with that of southern Chinese scholar Ouyang (25) who found that the XPD-751 polymorphism was correlated with adenocarcinoma rather than squamous carcinoma. Moreover, we found a correlation between genetic polymorphisms in XRCC1-194 and adenocarcinoma in patients with NSCLC by comparing this gene’s frequency in different lung cancer types. Our conclusion was consistent with those of some Asian scholars. Japanese scholar Tanaka (26) found a correlation between 5
XPD.XRCC1 genetic polymorphisms and NSCLC
XRCC1-194 polymorphisms and NSCLC, especially adenocarcinoma, by studying the synonymous single nucleotide mutation site in the DNA repair pathway. Afterward, we did not find obvious differences in the genetic mutation frequency of XRCC1-399 between patients with different types of lung cancer through a genetic frequency distribution study. But Chinese scholar Ouyang (25) found that polymorphisms in XRCC1-399 were also associated with the occurrence of adenocarcinoma through a study of lung cancer cases in southern China. We analyzed three gene SNPs above in different types of lung cancer and discovered that the XPD-751 SNP and XRCC1-194 SNP were related to squamous carcinoma and adenocarcinoma, respectively. Many previous research had shown that squamous cell carcinoma and NER system injuries were closely interrelated so we hypothesized that XPD was an important element of the NER system, and XPD-751 mutation could easily induce squamous cell carcinoma. In the same way, the BER system damage was relevant to adenocarcinoma development. Therefore, as it is a significant repair gene of the BER system, a XRCC1-194 mutation might likely promote adenocarcinoma, but XRCC1-399 mutation was not related to NSCLC histological classification. Those diverse results might reflect differences in etiology of histological types of lung cancer. Finally, we found that XRCC1-194SNP and smoking were two risk factors for NSCLC through multifactor logistic regression analysis. Furthermore, there was a positive interaction between XRCC1-194SNP and smoking, which was consistent with the conclusions of Korean scholar Kim (22) and Chinese scholar Su Jia (27). Our research indicated that XRCC1194SNP and smoking increased the risk of NSCLC, but also that the interaction between both of them appeared as super multiplicative model. Under normal circumstances, DNA repair systems could clear the DNA additive product to maintain the DNA’s normal structure. However, smoking could produce large amounts of DNA adducts to cause carcinogenesis in vivo. If a relevant DNA repair gene like XRCC1-194 mutated at the same time, the individual risk of lung cancer would increase as a ‘waterfall’ reaction. Therefore, the XRCC1-194 mutation in the smoking population needs careful attention. In conclusion, we inferred that genetic polymorphisms in XPD-751, XRCC1-194 and XRCC1-399 were all correlated with NSCLC occurrence. Genetic polymorphisms of XPD-751 and XRCC1-194 were related to squamous cell carcinoma and adenocarcinoma, respectively. Smoking and XRCC1-194 SNP
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were two high-risk factors of NSCLC, which existed as a super multiplicative interactive effect. Through this genetic research, the high-risk population for NSCLC can be screened, followed regularly, given follow-up chest computed tomography and related NSCLC tumor marker tests, and be diagnosed as soon as possible. This could cure a higher proportion of patients and provide an effective theoretical basis for targeted gene therapy. Nevertheless, this study presented some limitations. For example, the restricted sample size in NSCLC subgroups may produce some biases on test results. Consequently, further reasonable experiments with a larger sample size are necessary.
Acknowledgments This research was supported by grants from the natural science foundation of Shanghai Minhang District (2013MHZ022), the Shanghai Committee of Science and Technology (No. 134119b1200) and Outstanding Academic Leader of Health System in Shanghai (No. XBR2013078).
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10. Karahalil B, Bohr VA, Wilson DM 3rd. Impact of DNA polymorphisms in key DNA base excision repair proteins on cancer risk. Hum Exp Toxicol. 2012;31: 981–1005. 11. Zhou Y, Che G. Analysis of the relationship between DNA repair gene polymorphisms and lung cancer susceptibility. Chin J Clin Oncol. 2013;40: 551–4. 12. Wang N, Wu YJ, Zhou XL, Wu YM. [The polymorphisms of XRCC1 gene and susceptibility to pulmonary cancer]. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2012;30: 41–4. 13. Li D, Zhou Q, Liu Y, Yang Y, Li Q. DNA repair gene polymorphism associated with sensitivity of lung cancer to therapy. Med Oncol. 2012;29: 1622–8. 14. Butkiewicz D, Drosik A, Suwinski R, Krzes´niak M, Rusin M, Kosarewicz A, Rachtan J, Matuszczyk I, Gawkowska-Suwin´ska M. Influence of DNA repair gene polymorphisms on prognosis in inoperable non-small cell lung cancer patients treated with radiotherapy and platinum-based chemotherapy. Int J Cancer. 2012;131: E1100–8. 15. Vaezi A, Feldman CH, Niedernhofer LJ. ERCC1 and XRCC1 as biomarkers for lung and head and neck cancer. Pharmgenomics Pers Med. 2011;4: 47–63. 16. Zhan P, Wang Q, Wei SZ, Wang J, Qian Q, Yu LK, Song Y. ERCC2/XPD Lys751Gln and Asp312Asn gene polymorphism and lung cancer risk: a meta-analysis involving 22 case-control studies. J Thorac Oncol. 2010;5: 1337–45. 17. Lopez-Cima MF, Gonzalez-Arriaga P, Garcia-Castro L, Pascual T, Marrón MG, Puente XS, Tardón A. Polymorphisms in XPC, XPD, XRCC1, and XRCC3 DNA repair genes and lung cancer risk in a population of northern Spain. BMC Cancer. 2007;7: 162. 18. De Ruyck K, Szaumkessel M, De Rudder I, Dehoorne A, Vral A, Claes K, Velghe A, Van Meerbeeck J, Thierens H. Polymorphisms in base-excision repair and nucleotideexcision repair genes in relation to lung cancer risk. Mutat Res. 2007;631: 101–10. 19. Huang J, Zhang J, Zhao Y, Liao B, Liu J, Li L, Liao M, Wang L. The Arg194Trp polymorphism in the XRCC1
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ORIGINAL ARTICLE
Association between genetic polymorphisms in XPD and XRCC1 genes and risks of non-small cell lung cancer in East Chinese Han population Yong Du, Yanchao He, Zhoufang Mei, Ling Qian, Jindong Shi and Zhijun Jie Department of Respiratory Medicine, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China
Abstract Background and Aims: Lung cancer is a multifactorial disease. Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing 1 (XRCC1) genes are 2 important susceptibility genes related to lung cancer. In this study, we explored the correlation between genetic polymorphisms in XPD and XRCC1 and the risk of non-small cell lung cancer (NSCLC) in the East Chinese Han population. We also investigated risk factors associated with non-small cell lung cancer in this population. Methods: We conducted a case control study in 120 NSCLC patients and 120 healthy controls. The NSCLC patients were further divided into three subgroups, squamous carcinoma, adenocarcinoma and other type of cancer, according to tumor histology. No patients had undergone any treatment. Polymerase chain reaction and restriction fragment length polymorphism technologies were applied to detect the distribution of XPD-751, XRCC1-194 and XRCC1-399 genes in all patients. Results: The results showed significant gene frequency differences for all three genes between patients with NSCLC and control patients. Significantly different frequencies of XPD-751-Gln, XRCC-194-Trp and XRCC1-399-Gln mutant alleles were observed between the two groups. XPD-751SNP and XRCC1-194SNP frequencies varied among the three lung cancer groups, while the frequency of XRCC1-399SNP did not differ significantly. Conclusions: The results suggested that genetic polymorphisms in XPD-751, XRCC1-194 and XRCC1-399 were related to the risk of NSCLC, among which XPD-751SNP was responsible for adenocarcinoma, while XRCC1-194SNP was closely linked to squamous carcinoma. Smoking and XRCC1-194SNP were risk factors of NSCLC. Please cite this paper as: Du Y, He Y, Mei Z, Qian L, Shi J and Jie Z. Association between genetic polymorphisms in XPD and XRCC1 genes and risks of non-small cell lung cancer in East Chinese Han population. Clin Respir J 2014; ••: ••–••. DOI:10.1111/crj.12218.
Key words non-small-cell cancer – single-nucleotide polymorphism – xeroderma pigmentosum group D – X-ray repair cross-complementing 1 Correspondence Zhijun Jie, MD, PhD, Department of Respiratory Medicine, The Fifth People’s Hospital of Shanghai, Fudan University, No. 801 Heqing Road, Minhang District, 200240 Shanghai, China. Tel: +86 189 181 68602 Fax: +86 21 640 85875 email: [email protected] Received: 17 April 2014 Revision requested: 09 August 2014 Accepted: 29 September 2014 DOI:10.1111/crj.12218 Authorship and contributorship Yong Du is responsible for study design, data analysis and writing the article. Yanchao He and Zhoufang Mei have contributed in collecting samples and in the extraction of genomic DNA. Ling Qian is in charge of PCR amplification and agarose gel electrophoresis. Jindong Shi has helped in RFLP technology and polyacrylamide gel electrophoresis. Zhijun Jie is responsible for the supervision of performance of study, data analysis and reporting work. Ethics The study protocol was approved by the ethics committee of the Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China. All subjects in this study gave signed informed consent before the research. Conflict of interest The authors have stated explicitly that there are no conflicts of interest in connection with this article.
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
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XPD.XRCC1 genetic polymorphisms and NSCLC
Introduction Lung cancer is a multifactorial, multistage process (1), and smoking is its most important risk factor. However, only 10%–15% of smokers develop lung cancer, while 10%–15% of lung cancer patients never smoke at all, suggesting that there are individual differences in lung cancer genetic susceptibility. Among the numerous genetic studies on lung cancer pathogenesis, those on DNA repair genes have become a major focus in lung cancer research. DNA injury repair is a complicated process involving many enzymes and proteins. Once the genes related to DNA repair are mutated, the genomic DNA repair capacity (DRC) may be impaired (2), leading to increased genomic instability. Single-nucleotide polymorphisms (SNPs) (3, 4) in DNA repair genes are single-base mutations that may change the encoded amino acid, causing individual differences in DRC (5), and finally leading to adverse consequences such as cancer. Xeroderma pigmentosum (XP) is a type of DNA repair deficiency disease caused by deficiency in nucleotide excision repair (NER) capacity (6). XPD, an adenosine triphosphate-dependent helicase, is involved in transcription as the core component of the transcription factor IIH. The XPD protein helicase activity, the XPD gene expression product, can unlock the DNA’s double helix structure and cut or remove damaged DNA. Once the XPD gene is mutated, DNA repair deficiency may result from decreased XPD protein expression (7, 8). To date, seven SNP sites have been identified in the XPD gene, among which the genetic mutations Asp312Asn and Lys751Gln are the most common (9) and are related to lung cancer. X-ray repair cross-complementing 1 (XRCC1) is one of the important genes involved in mammal DNA repair. It participates in the cell cycle checkpoint function and responds to DNA damage, playing a critical role in base excision repair (BER) and single-strand break repair (10, 11). Recent studies indicated that there are three amino acid mutation sites in the XRCC1 gene, namely 194 (Arg→Trp), 280 (Arg→His) and 399 (Arg→Gln) (12). These mutations in encoding regions can change the function of XRCC1 and increase individual susceptibility to lung cancer. In recent years, lung cancer incidence and mortality rates have increased in the East Chinese Han population. Genetic susceptibility studies of lung cancer help to screen high-risk populations and provide guidance for reducing lung cancer’s incidence. Most studies on DNA repair genes have focused on the relationships between lung cancer and the sensitivities of radiotherapy and chemotherapy (13–15). They neglect the
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fact that mutations in DNA repair genes can also lead to decreased DRC in the susceptible population, thereby causing lung cancer. In this study, we performed a case control study in patients with newly diagnosed non-small cell lung cancer (NSCLC) who had not undergone surgery, radiotherapy, or chemotherapy. We used polymerase chain reaction (PCR) and restriction fragment length polymorphism technologies to analyze the relationships between NSCLC and three SNPs, including XPD751, XRCC1-194 and XRCC1-399. This may be an effective theoretical basis for high-risk NSCLC population screenings and the development of molecular targeted therapies.
Materials and methods Patient selection We enrolled 120 NSCLC patients who were seen at our hospital from March 2010 to March 2012. All patients were diagnosed via bronchoscopic biopsy or cytology examinations and were divided into three groups according to pathology: squamous carcinoma, adenocarcinoma and other cancer type (such as large cell lung cancer, carcinoid and undifferentiated carcinoma). No patients had received surgery, radiotherapy, or chemotherapy. Additionally, 120 healthy individuals were enrolled in the control group. These individuals underwent physical examinations at our hospital during the same time frame. No abnormalities were found in chest imaging and serum tumor marker tests (including CEA.AFP.NSE.SCC.CA211.CA125.CA199) for patients in the control group. All patients were from the East China Han population and did not have a history of hereditary diseases such as diabetes, hypertension, coronal heart disease, or other respiratory diseases. Informed consent was obtained from all patients before participation in the study. The study protocol was approved by the Fifth People’s Hospital of Shanghai, Fudan University, and all participants provided written informed consent.
Collection of blood samples and extraction of DNA Peripheral venous blood (4 mL) was collected from all patients after a fasting period, with ethylenediaminetetraacetic acid as an anticoagulant. Blood samples were kept frozen at −20°C before genomic DNA extraction using a DNA extraction kit from TIANGEN Science and Technology Co., Ltd. (Beijing, China). The DNA was dissolved and frozen at −20°C.
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Gene amplification and digestion Primer sequences, amplification and digestion conditions were chosen based on related literature. Genomic DNA was amplified using PCR. Reactions contained Taq DNA polymerase (Dalian Takara Biotechnology Co., Ltd., Liaoning, China), Deoxynucleotide triphosphates and primers. The PCR products were digested with corresponding enzymes. The amplification procedure of the XPD-751 and XRCC1-194 genes were all as follows: predenaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 61°C for 1 min and 72°C for 1 min, and a final elongation at 72°C for 5 min. The primer sequences for XPD-751 were F: 5′-GCCCGCTCTGGATTATACG-3′ and R: 5′-CTATC ATCTCCTGGCCCCC-3′. Its products were digested with Pst I (Shanghai SANGON Biological Engineering Co., Ltd., Shanghai, China) at 37°C for 1 h. The primer sequences of XRCC1-194 were F: 5′-GCCAGGG CCCCTCCTTCAA-3′ and R: 5′-TACCCTCAGACC CACGAGT-3′. Its products were digested with Pvu II (Shanghai SANGON Biological Engineering Co., Ltd.) at 37°C for 1 h. The amplification procedure of XRCC1-399 gene was as follows: predenaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 3 min and a final elongation at 72°C for 5 min. The primer sequences were F: 5′-TTGTGCTTTCTCTGTGTCCA-3′and R: 5′-TCC TCCAGCCTTTTCTGATA-3′. Its products were digested with MspI (Shanghai SANGON Biological Engineering Co., Ltd.) at 37°C for 1 h. All the digestion products were subjected to 2% agarose gel electrophoresis. The electrophoretic bands were observed with an electrophoretic display device to determine the genotype of each specimen.
Statistical analysis SPSS 17.0 software (IBM, Chicago, IL, USA) was used for statistical analysis. All alleles complied with the
XPD.XRCC1 genetic polymorphisms and NSCLC
Hardy–Weinberg genetic equilibrium. Frequency of alleles = (number of homozygous individuals × 2 + number of heterozygous individuals)/(total number of individuals × 2). All measurements were analyzed with Student’s t-tests, and count data were analyzed using chi-squared tests. The relative risk of mutated genes was estimated with 95% dependent intervals. Screening of risk factors and their interactions were analyzed with the logistic regression method. Differences with P values less than 0.05 were considered statistically significant. All statistical analyses were conducted with two-tailed probability tests.
Results Demographic data (i.e. patient age, gender, smoking history and smoking index) are listed in Table 1. There was no significant difference in ages or genders between the two groups (P > 0.05). The smoking history and smoking index were significantly higher in the three NSCLC subgroups than in the control group (P < 0.05). The genotype and allele frequencies of the three selected genes in the patient and control groups are listed in Tables 2 and 3. There were significant differences in genotype frequencies at XPD-751, XRCC1-194 and XRCC1-399 polymorphic sites between the two groups (P = 0.026, 0.025 and 0.044, respectively). The allele frequencies of these three SNPs significantly differed between the two groups (P = 0.001, 0.003 and 0.002, respectively). NSCLC patients were stratified by histopathology: squamous carcinoma, adenocarcinoma and other type of cancer. The genetic allele frequencies of the selected genes in these groups are listed in Table 4. There were significant differences in XPD-751SNP and XRCC1194SNP among the three groups (P = 0.023 and 0.003, respectively), among which the mutated gene frequencies of XPD-751 in the squamous carcinoma group and of XRCC1-194 in the adenocarcinoma group were
Table 1. Characteristics of the study population Lung cancer Characteristics
Control
Squamous cell carcinoma
Adenocarcinoma
Other types
Number of cases Sex (male/female) Age (years) Smoking (yes/no) Smoking index (smoking years × smoking number per day)
120 64/56 64.3 ± 13.8 61/59 446 ± 142
52 28/24 65.8 ± 13.3 36/16* 624 ± 128*
48 25/23 67.2 ± 15.1 38/14* 638 ± 135*
20 11/9 66.5 ± 14.2 15/5* 629 ± 141*
*P < 0.05, compared with the control.
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
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Table 2. Genotype frequencies of the three selected genes in the two groups Gene
Polymorphism
Lung cancer
Control
Model
XPD gene Lys751Gln
Lys/Lys Gln/Lys Gln/Gln Arg/Arg Trp/Arg Trp/Trp Arg/Arg Gln/Arg Gln/Gln
78 24 18 68 33 19 81 16 23
96 16 8 88 21 11 95 15 10
Additive Dominant Recessive Additive Dominant Recessive Additive Dominant Recessive
XRCC1 gene Arg194Trp XRCC1 gene Arg399Gln
OR (95% CI) 2.154 (1.201–3.861) 2.471 (1.030–5.926) 2.103 (1.223–3.617) 1.864 (0.846–4.109) 1.830 (1.021–3.278) 2.608 (1.183–5.753)
P 0.026 0.009 0.038 0.025 0.007 0.118 0.044 0.041 0.015
CI, confidence interval; OR, odds ratio.
higher than in the other two groups. There was no significant difference in the frequency of XRCC1399SNP among the three groups (P = 0.598). Results of multifactor logistic regression analysis of NSCLC-related risk factors are listed in Table 5. Compared with the control group, the odds ratio (OR) of NSCLC in XPD-751SNP and XRCC1-399SNP carriers
were 1.325 [95% confidence interval (CI): 0.673–2.238, P = 0.134] and 0.805 (95% CI: 0.735–3.418, P = 0.323), which had no statistical difference. However, the OR of NSCLC in smokers and XRCC1-194SNP carriers were 2.786 (95% CI: 1.238–6.565, P = 0.026) and 4.389 (95% CI: 1.788–16.331, P = 0.009), which were significantly different.
Table 3. The allele frequencies of the three selected genes in the two groups Gene
Allele
Lung cancer
Control
OR (95% CI)
P
XPD gene Lys751Gln XRCC1 gene Arg194Trp XRCC1 gene Arg399Gln
Lys Gln Arg Trp Arg Gln/
180 60 169 71 178 62
208 32 197 43 205 35
2.167 (1.350–3.478)
0.001
1.925 (1.251–2.961)
0.003
2.040 (1.287–3.233)
0.002
CI, confidence interval; OR, odds ratio.
Table 4. Allele frequencies in patients with different pathological types of non-small cell lung cancer Gene
Allele
Squamous cell carcinoma
Adenocarcinoma
Other types
XPD gene Lys751Gln XRCC1 gene Arg194Trp XRCC1 gene Arg399Gln
Lys Gln Arg Trp Arg Gln/
71 33 80 24 76 28
81 15 56 40 76 20
32 8 33 7 30 10
P 0.023 0.003 0.598
Table 5. Logistic regression analysis of risk factors Factor
OR
P
β
SE
95% CI
Smoking XPD-751SNP XRCC1-194SNP XRCC1-399SNP
2.786 1.325 4.389 0.805
0.026 0.134 0.009 0.323
0.957 0.309 1.915 −0.486
0.372 0.394 0.683 0.351
1.238–6.565 0.673–2.238 1.788–16.331 0.735–3.418
CI, confidence interval; OR, odds ratio; SE, standard error.
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XPD.XRCC1 genetic polymorphisms and NSCLC
Table 6. Analysis of the interaction between smoking and XRCC1-194SNP Factors
OR
P
β
95% CI
Smoking XRCC1-194SNP Smoking and XRCC1-194SNP
1.937 1.628 9.732
0.146 0.578 0.008
0.587 0.684 2.631
0.762–6.895 0.587–5.449 1.561–11.392
CI, confidence interval; OR, odds ratio; SNP, single-nucleotide polymorphism.
The interactions between the two high-risk factors (smoking and XRCC1-194SNP) can be seen in Table 6. The risk of NSCLC in smokers without XRCC1194SNP was 1.937 times higher than that of the control group, while that of non-smokers with XRCC1194SNP was 1.628 times higher than that of the control group. However, in smokers with XRCC1194SNP, the risk was as much as 9.732-fold higher than that in the control group (P < 0.05, 95% CI: 1.561–11.392), and the OR value of which was much more than the product of the former two conditions (9.732 > 1.937 × 1.628).
Discussion Our study found significant differences in XPD-751 mutation and allele frequencies between patients with NSCLC and healthy controls, suggesting a close relationship between XPD-751 SNP and NSCLC. These data are consistent with other scientists’ conclusions both in China and abroad. In a meta-analysis of several case control studies, Chinese scholar Zhan Ping (16) found that the XPD-751 SNP was related to NSCLC occurrence, with an increased association observed in Caucasians and smokers. European scholar LopezCima (17) also found that XPD-751SNP was related to NSCLC in a study of multiple genes in a population from Northern Spain. Additionally, De Ruyck (18) came to the same conclusion in a population of Belgian Caucasians, with a greater association observed in non-smokers than in smokers. The significant difference in the allele frequency of XRCC1-194 between NSCLC patients and controls suggested a close correlation between XRCC1-194SNP and NSCLC. Studies in China and abroad have not been consistent in this regard. Chinese scholar Huang Jin (19) found that XRCC1-194SNP was a susceptibility factor for NSCLC using a meta-analysis of the XRCC1-194 gene in the Chinese mainland population. However, Chinese scholar Sun Yan (20) found no obvious correlation between the XRCC1-194 polymorphism and the occurrence of NSCLC in a study of
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
non-smoking Han individuals. European scholar Lopez-Cima (17) also found no significant correlation between the two factors in a study of genetic polymorphisms in XRCC1 in the Spanish population. Furthermore, the significant difference in mutated allele frequencies of XRCC1-399 between the two groups also supported the correlation between XRCC1399SNP and the occurrence of NSCLC. Studies in China and abroad have not been conclusive. Chinese scholar Li Zheng (21) found a correlation between XRCC1-399SNP and NSCLC through a genetic polymorphism study of BER in the Chinese population. Similarly, Korean scholar Kim (22) found that XRCC1399SNP had an important role in the development of NSCLC through studying the 3 XRCC1 genetic sites in the Korean population. Nevertheless, foreign scholar Improta (23) found no association between XRCC1399SNP and NSCLC through a polymorphism study of XRCC1 in a southern Italian population. Our study investigated genetic susceptibility to NSCLC directly rather than the relationship between genetic polymorphisms in lung cancer and sensitivity to radiotherapy and chemotherapy. The results of this research were not completely consistent with those of other studies, which could be attributed to different regions, racial differences and environmental exposures, in addition to differences in experimental design and data analysis. Among those reasons above, we imply that the region difference may influence our results because NSCLC incidence in this area was obviously rising. We also speculate that the three gene mutations in local residents are higher than those in immigrants. Thus, we plan to expand the sample size of NSCLC to further determine whether gene polymorphisms are associated with regional distribution and to select the population displaying the gene mutation as a high-risk factor of NSCLC in the East Chinese Han population. Using an allele frequency study of different lung cancers, we found that genetic polymorphisms in XPD-751 were closely linked to squamous carcinoma in NSCLC. This conclusion is consistent with that of northern Chinese scholar Xing De Yin (24), but not consistent with that of southern Chinese scholar Ouyang (25) who found that the XPD-751 polymorphism was correlated with adenocarcinoma rather than squamous carcinoma. Moreover, we found a correlation between genetic polymorphisms in XRCC1-194 and adenocarcinoma in patients with NSCLC by comparing this gene’s frequency in different lung cancer types. Our conclusion was consistent with those of some Asian scholars. Japanese scholar Tanaka (26) found a correlation between 5
XPD.XRCC1 genetic polymorphisms and NSCLC
XRCC1-194 polymorphisms and NSCLC, especially adenocarcinoma, by studying the synonymous single nucleotide mutation site in the DNA repair pathway. Afterward, we did not find obvious differences in the genetic mutation frequency of XRCC1-399 between patients with different types of lung cancer through a genetic frequency distribution study. But Chinese scholar Ouyang (25) found that polymorphisms in XRCC1-399 were also associated with the occurrence of adenocarcinoma through a study of lung cancer cases in southern China. We analyzed three gene SNPs above in different types of lung cancer and discovered that the XPD-751 SNP and XRCC1-194 SNP were related to squamous carcinoma and adenocarcinoma, respectively. Many previous research had shown that squamous cell carcinoma and NER system injuries were closely interrelated so we hypothesized that XPD was an important element of the NER system, and XPD-751 mutation could easily induce squamous cell carcinoma. In the same way, the BER system damage was relevant to adenocarcinoma development. Therefore, as it is a significant repair gene of the BER system, a XRCC1-194 mutation might likely promote adenocarcinoma, but XRCC1-399 mutation was not related to NSCLC histological classification. Those diverse results might reflect differences in etiology of histological types of lung cancer. Finally, we found that XRCC1-194SNP and smoking were two risk factors for NSCLC through multifactor logistic regression analysis. Furthermore, there was a positive interaction between XRCC1-194SNP and smoking, which was consistent with the conclusions of Korean scholar Kim (22) and Chinese scholar Su Jia (27). Our research indicated that XRCC1194SNP and smoking increased the risk of NSCLC, but also that the interaction between both of them appeared as super multiplicative model. Under normal circumstances, DNA repair systems could clear the DNA additive product to maintain the DNA’s normal structure. However, smoking could produce large amounts of DNA adducts to cause carcinogenesis in vivo. If a relevant DNA repair gene like XRCC1-194 mutated at the same time, the individual risk of lung cancer would increase as a ‘waterfall’ reaction. Therefore, the XRCC1-194 mutation in the smoking population needs careful attention. In conclusion, we inferred that genetic polymorphisms in XPD-751, XRCC1-194 and XRCC1-399 were all correlated with NSCLC occurrence. Genetic polymorphisms of XPD-751 and XRCC1-194 were related to squamous cell carcinoma and adenocarcinoma, respectively. Smoking and XRCC1-194 SNP
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were two high-risk factors of NSCLC, which existed as a super multiplicative interactive effect. Through this genetic research, the high-risk population for NSCLC can be screened, followed regularly, given follow-up chest computed tomography and related NSCLC tumor marker tests, and be diagnosed as soon as possible. This could cure a higher proportion of patients and provide an effective theoretical basis for targeted gene therapy. Nevertheless, this study presented some limitations. For example, the restricted sample size in NSCLC subgroups may produce some biases on test results. Consequently, further reasonable experiments with a larger sample size are necessary.
Acknowledgments This research was supported by grants from the natural science foundation of Shanghai Minhang District (2013MHZ022), the Shanghai Committee of Science and Technology (No. 134119b1200) and Outstanding Academic Leader of Health System in Shanghai (No. XBR2013078).
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