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A novel single nucleotide polymorphism in XRCC4 gene is associated with oral cancer susceptibility in Taiwanese patients
Oral Oncology (2008) 44, 898–902
available at www.sciencedirect.com
journal homepage: http://intl.elsevierhealth.com/journals/oron/
A novel single nucleotide polymorphism in XRCC4 gene is associated with oral cancer susceptibility in Taiwanese patients Chang-Fang Chiu a,e, Ming-Hsui Tsai b,e, Hsien-Chang Tseng b, Cheng-Li Wang e, Chung-Hsing Wang c, Cheng-Nan Wu g, Cheng-Chieh Lin d, Da-Tian Bau e,f,* a
Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan, ROC Department of Otolaryngology, China Medical University Hospital, Taichung, Taiwan, ROC c Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan, ROC d Department of Family Medicine, China Medical University Hospital, Taichung, Taiwan, ROC e Terry Fox Cancer Research Lab, China Medical University Hospital, 2 Yuh-Der Road, Taichung 404, Taiwan, ROC f Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan, ROC g Institute of Medical Bioscience, Central-Taiwan University of Science and Technology, Taichung, Taiwan, ROC b
Received 7 September 2007; received in revised form 16 November 2007; accepted 19 November 2007 Available online 4 March 2008
KEYWORDS XRCC4; Polymorphism; Oral cancer; Carcinogenesis
Summary The DNA double strand break repair gene XRCC4, an important caretaker of genome stability, is suggested to play a role in the development of human carcinogenesis. However, no evidence has been provided showing that XRCC4 was associated with oral oncology. In this hospital-based case-control study, the association of XRCC4 G-1394T (rs6869366), intron 3 (rs28360071), intron 7 (rs28360317), and intron 7 (rs1805377) polymorphisms with oral cancer risk in a Taiwanese population was first investigated. In total, 318 patients with oral cancer and 318 age- and gender-matched healthy controls were genotyped. We found a significant different distribution in the frequency of the XRCC4 intron 3 genotype, but not the XRCC4 G-1394T or intron 7 genotypes, between the oral cancer and control groups. Those who had heterozygous del/ins at XRCC4 intron 3 showed a 1.57-fold (95% confidence interval = 1.12–2.21) increased risk of oral cancer compared to those with ins/ins. As for XRCC4 G-1394T or intron 7 polymorphisms, there was no difference in the distribution between the oral cancer and control groups. There were significant gene–environment interactions between XRCC4 intron 3 genotype with
* Corresponding author. Address: Terry Fox Cancer Research Lab, China Medical University Hospital, 2 Yuh-Der Road, Taichung, 404, Taiwan, ROC. Tel.: +886 422053366x3312; fax: +886 422053366x1511. 1368-8375/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2007.11.007
XRCC4 Polymorphism in oral cancer
899
smoking and with betel quid chewing, but not with alcoholism. In smoker and betel quid chewer groups, the XRCC4 intron 3 del variants exhibited 2.57- and 3.03-fold higher risks than the ins genotype, respectively. Our results firstly suggest that the XRCC4 intron 3 del genotype may be associated with oral oncology and may be a novel useful marker for primary prevention and anticancer intervention.
ª 2007 Elsevier Ltd. All rights reserved.
Introduction
Materials and methods
Oral cancer has been identified as a significant public health threat all over the world.1–4 With continuously increasing incidence and mortality for the past two decades, oral cancer has become the fourth most common cause of male cancer death in Taiwan.5 However, the genomic etiology of oral cancer is of great interest but largely unknown. Human DNA repair mechanisms protect the genome from DNA damage caused by endogenous and environmental agents.6 Mutations or defects in the DNA repairing system are essential for tumorigenesis.7–9 Therefore, it is logical to suspect that some genetic variants of DNA repair genes might contribute to oral cancer pathogenesis. Sequence variants in DNA repair genes also are thought to modulate DNA repair capacity and consequently may be associated with altered cancer risk.10 X-ray cross-complementing group 4 (XRCC4) gene, which is important in non-homologous end-joining repair pathway, is found to restore DNA double strand break repair and the ability to support V(D)J recombination of transiently introduced substrates in the XR-1 CHO cell line.11 The XRCC4 protein interacts directly with Ku70/Ku80,12 and it is hypothesized that XRCC4 serves as a flexible tether between Ku70/Ku80 and its associated protein Ligase4.12 XRCC4 is required for precise end-joining of blunt DNA double strand breaks in mammalian fibroblasts.13 In the gene-targeting mutation mice model, XRCC4 gene inactivation leads to late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated post-mitotic neuronal cells.14,15 These findings demonstrated that differentiating lymphocytes and neurons strictly require the XRCC4 endjoining proteins. Thus, it is reasonable that polymorphisms of XRCC4 gene can be sustained in the genome for such a long time of carcinogenesis. Up to now, there are few papers studying the role of XRCC4 gene in cancer, not to mention XRCC4 polymorphisms in oral cancer susceptibility. Since DNA repair gene alterations have been shown to cause a reduction in DNA repair capacity, we hypothesized that DNA repair gene polymorphisms may be risk factors for oral cancer. To test this hypothesis, we determined the genotypic frequency of four polymorphisms of the XRCC4 gene at G-1394T (rs6869366), intron 3 (rs 28360071), intron 7 (rs28360317) and intron 7 (rs1805377), which will hereafter been referred to as XRCC4-1, XRCC4– 2, XRCC4–3 and XRCC4–4, respectively, using a polymerase chain reaction-based restriction fragment length polymorphism method. To the best of our knowledge, this is the first study carried out to evaluate the contribution of XRCC4 polymorphisms in oral oncology all over the world.
Study population and sample collection Three hundred and eighteen patients diagnosed with oral cancer were recruited at the outpatient clinics of general surgery between 1998 and 2007 at the China Medical University Hospital, Taichung, Taiwan, Republic of China. All patients voluntarily participated, completed a self-administered questionnaire and provided peripheral blood samples. The same amounts of non-oral cancer healthy people as controls were selected by matching for age and gender after initial random sampling from the Health Examination Cohort of the hospital. The questionnaire administered to the subjects included questions on history and frequency of alcohol consumption, betel quid chewing and smoking habits. Self-reported alcohol consumption, betel quid chewing and smoking habits were evaluated and classified as categorical variables. Information on these factors was obtained as more than twice a week for five years as ‘‘ever’’. 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 leucocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and further processed according to PCR–RFLP methods. Briefly, the following primers were used for XRCC4–1: 50 -GATGCGAACTCAAAGATACTGA-30 and 50 -TGTAAAGCCAGTACTCAAACTT-30 ; for XRCC4–2: 50 -TCCTGTTACCATTTCAGTGTTAT30 and 50 -CACCTGTGTTCAATTCCAGCTT-30 ; for XRCC4–3: 50 -ATACTGTGTTTGGAACTCCT-30 for CCT-positive forward primer, 50 -ATACTGTGTTTGGAACTAGA-30 for CCT-negative forward primer, and 50 -TATCCTATCATCTCTGGATA-30 as reverse common primer; and for XRCC4–4: 50 -TTCACTTATGTGTCTCTTCA-30 and 50 -AACATAGTCTAGTGAACATC-30 . The following cycling conditions were performed: 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. The PCR products of XRCC4–2 were 109 bp for the del alleletype and 139 bp for the ins alleletype, respectively. The PCR products of XRCC4–3 were 239 bp for CCT-positive form and no product for CCT-negative form. The PCR products were studied after digestion with Hinc II, and Tsp509I restriction enzymes for XRCC4–1 (cut from 300 bp T type into 200 + 100 bp G type) and XRCC4–4 (cut from 237 bp G type into 79 + 158 bp A type), respectively. Ten percent of DNA samples were genotyped for a second time and the concordance rate was 100%.
900
Statistical analyses 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 XRCC4 single nucleotide polymorphisms in the control subjects from those expected under the Hardy–Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s v2 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 XRCC4 genotypes between cases and controls. Cancer risk associated with the genotypes was estimated as odds ratio (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. Data was recognized as significant when the statistical p-value was less than 0.05.
Results Distributions of the demographics and clinical variables were presented in Table 1. The mean ages of the oral cancer patients and the controls were 48.66 (SD = 8.62) and 53.26 (SD = 10.83) years, respectively. The proportion of men and women were the same ratio in the patient and control groups (i.e., male/female = 282/36). The most common sites of primary tumor were tongue and buccal mucosa in this patient population. Approximately 40% of the study cohort consisted of patients diagnosed with TNM late stage (III and IV) oral cancer. Among the characteristics compared, only the smoking statuses were differently distributed between oral cancer patient and control groups (Table 1). The frequencies of the alleles for the XRCC4–1, XRCC4–2, XRCC4–3, and XRCC4–4, between oral cancer and control groups are shown in Table 2. The distributions of all these polymorphisms were in Hardy–Weinberg equilibrium. Obviously, the D allele at XRCC4–2 was significantly associated with oral cancer risk (p = 0.0193). In contrast, G or T allele at XRCC4–1, I or D allele at XRCC4–3, A or G allele at XRCC4–4, were not differently distributed in the oral cancer patient and control groups (p > 0.05). The representative PCR–RFLP analyses for the XRCC4–2 polymorphisms are shown in Fig. 1. The frequencies of the genotypes of XRCC4–1, XRCC4–2, XRCC4–3 and XRCC4–4 polymorphisms in the oral cancer and control groups are shown in Table 3. Using I/I as the reference group, there was an obvious association between the heterozygotes and homozygotes of D at XRCC4–2 and oral cancer risk (Table 3). A combination of the homozygotes and heterozygotes of D (with D) showed that the D allele at XRCC4–2 conferred a 1.55-fold risk factor for oral cancer (Table 3). Neither heterozygotes of G at XRCC4–1, heteroor homozygotes of D at XRCC4–3, hetero- nor homozygotes of G at XRCC4–4, seemed to be risky genotypes for oral cancer (Table 3). Thus, among the four XRCC4 polymorphisms investigated, only the D allele at XRCC4–2 seems to be positively associated with oral cancer risk. The joint effects of XRCC4–2 genotypes and environmental factors on oral cancer risk are shown in Table 4. Smokers, alcoholics and betel quid chewers were classified as described in ‘Materials and methods’ into two groups, never and ever. Among the three oral cancer-related habits evaluated, the XRCC4–2 was shown to have interactions with smoking and betel quid chewing, but not with alcohol
C.-F. Chiu et al. Table 1 Demographics and clinical characteristics of oral cancer patients from Central Taiwan Characteristics Age, year <40 40–49 50–59 60–69 P70 Gender Male Female
Control number (%) 33 90 78 70 47
(10.38) (28.30) (24.53) (22.01) (14.78)
282 (88.68) 36 (11.32)
Cases number (%) 35 94 76 65 48
(11.01) (29.56) (23.90) (20.44) (15.09)
282 (88.68) 36 (11.32)
Site of primary tumora Tongue Buccal mucosa Others
157 (49.37) 90 (28.30) 71 (22.33)
Clinical stagea Stage I Stage II Stage III Stage IV
71 125 76 46
Smoking habit Never Ever
p-Value
0.9849
1.00
(22.33) (39.31) (23.90) (14.47)
208 (65.41) 110 (34.59)
173 (54.40) 145 (45.60)
0.0059b
Alcohol drinking habit Never 146 (45.91) Ever 172 (54.09)
143 (44.97) 175 (55.03)
0.8735
Betel chewing habit Never 253 (79.56) Ever 65 (20.44)
232 (72.96) 86 (27.04)
0.0623
a b
Surgical cases only. p-Value significant at <0.05.
Table 2 Allele frequencies for XRCC4 polymorphisms in the control and oral cancer groups Allele
Controls (%)
Cases (%)
N = 636
N = 636
p-Valuea
XRCC4–1 Allele T Allele G
617 (97.0) 19 (3.0)
609 (95.8) 27 (4.2)
0.2296
XRCC4–2 Allele I Allele D
538 (84.6) 98 (15.4)
506 (79.6) 130 (20.4)
0.0193
XRCC4–3 Allele I Allele D
478 (75.2) 158 (24.8)
492 (77.4) 144 (22.6)
0.3563
XRCC4–4 Allele A Allele G
464 (73.0) 172 (27.0)
473 (74.4) 163 (25.6)
0.5667
a
p-Value based on v2 test.
XRCC4 Polymorphism in oral cancer
901 Table 4 Joint effects of XRCC4–2 polymorphisms and environmental factors on oral cancer risk Habits
Figure 1 PCR-based restriction analysis of the XRCC4–2 polymorphism shown on 3% agarose electrophoresis. M: 100 bp DNA size marker, I/I: insertion homozygote, D/I: heterozygote, and D/D: deletion homozygote.
Controls (n)
Cases (n)
Odds ratio (95% CI)a
Smoking Never I/I I/D + D/D Ever I/I I/D + D/D
144 64 83 27
117 56 79 66
1.00 (ref) 1.08 (0.70–1.66) 1.00 (ref) 2.57 (1.49–4.42)b
Alcohol Never I/I I/D + D/D Ever I/I I/D + D/D
103 43 124 48
87 56 109 66
1.00 1.54 1.00 1.56
Alcohol Never I/I I/D + D/D Ever I/I I/D + D/D
174 79 53 12
145 87 51 35
1.00 (ref) 1.32 (0.91–1.93) 1.00 (ref) 3.03 (1.42–6.48)b
a
Table 3 Association of XRCC4 polymorphisms and oral cancer risk Controls (%)
XRCC4–1 T/T G/T G/G
299 (94.0) 19 (6.0) 0 (0)
291 (91.5) 27 (8.5) 0 (0)
1.00 (ref) 1.46 (0.79–2.68) 1.03 (0.02–51.96)
XRCC4–2 I/I I/D D/D With D
227 (71.4) 84 (26.4) 7 (2.2) 91 (28.6)
196 (61.6) 114 (35.8) 8 (2.5) 122 (38.4)
1.00 (ref) 1.57 (1.12–2.21)b 1.32 (0.47–3.72) 1.55 (1.11–2.16)b
XRCC4–2 I/I I/D D/D With D
195 88 35 123
(61.3) (27.7) (11.0) (38.7)
196 (61.6) 100 (31.4) 22 (6.9) 122 (38.4)
1.00 1.13 0.63 0.99
(ref) (0.80–1.60) (0.35–1.11) (0.72–1.36)
XRCC4–2 A/A A/G G/G With G
167 (52.5) 130 (40.9) 21 (6.6) 151 (47.5)
173 (54.4) 127 (39.9) 18 (5.7) 145 (45.6)
1.00 0.94 0.83 0.93
(ref) (0.68–1.30) (0.43–1.61) (0.68–1.27)
a
(ref) (0.95–2.52) (ref) (0.99–2.46)
CI, confidence interval. Statistically identified significant.
Odds ratio (95% CI)a
Genotype
b
Cases (%)
b
Genotype
CI, confidence interval. Statistically identified significant.
drinking. In smoking and betel quid chewing groups, the XRCC4–2 D variants conferred 2.57-fold (95% CI = 1.49– 4.42) and 3.03-fold (95% CI = 1.42–6.48) increased risks compared to the I genotype, respectively (Table 4).
Discussion The present study has investigated the role of XRCC4 gene polymorphisms in oral cancer susceptibility. To the best of our knowledge, there were very few reports studying XRCC4 polymorphisms in carcinogenesis, including oral cancer, not
to mention the joint effects of them with environmental factors. The large sample size and concise data analysis strengthen the accuracy and reliability of our finding. The frequencies of XRCC4 polymorphisms variant alleles were similar to those reported in the NCBI website in the Asian population studies, which suggest no selection bias for the subject’s enrolments in terms of genotypes. Therefore, the need for the present results to be verified in even larger studies is not so urgent. In this study, the distribution of the D allele at XRCC4–2 (20.4%) was significantly higher in the oral cancer group than in the control group (15.4%) (Table 2). It was also found that participants with heterozygous D at XRCC4–2 had a 1.57-fold higher risk of oral cancer (Table 3). After combining the heterozygous and homozygous participants in both groups, there was still an obvious increased risk of 1.55-fold (Table 3). All these data suggested that D allele at XRCC4–2 was a novel biomarker for oral oncology, and as long as D allele was detected, no matter whether is hetero- or homozygotes, the carriers were more susceptible to oral cancer. As for other XRCC4 polymorphisms, our results indicated that their genetic differences were not associated with oral cancer risk. Three potential gene–environment interactions about oral carcinogenesis, XRCC4 with cigarette smoking, alcohol consumption and betel quid chewing, were also investigated and evaluated in our study (Table 4). Betel quid chewing is reported to induce free radical-induced DNA damage and strand breaks, and tobacco extracts contain some potential carcinogens may also cause DNA strand breaks, both of which should be repaired via XRCC4 involved pathway. Similar to previous findings about double strand break repair genes with environmental factors, such as NBS1 with smoking in lung cancer,16 and Ku70/K80/DNA-PKcs/Ligase4/ XRCC4 with estrogen in breast cancer,17 our study provides
902 evidence for possible gene–environment joint effects in carcinogenesis. It is interesting that there were joint effects between risky XRCC4 genotypes, smoking and betel quid chewing, but not alcohol drinking (Table 4). In smoker and betel quid chewer groups, the XRCC4–2 D variants conferred 2.57-fold (95% CI = 1.49–4.42) and 3.03-fold (95% CI = 1.42–6.48) than the I genotype, respectively (Table 4). As the previous findings,18–23 our results suggest that genetic variants involved in DNA repair genes indeed involved in cancer etiology. Some of the famous oral cancer-related environmental risk factors, such as consumption of betel quid, and smoking here, may modulate oral cancer risk in combination with those genetic susceptibilities in different repair pathways. Therefore, combinational investigations of different genes in various DNA repair systems, and genotype–phenotype correlations among these systems, in addition to gene–environment joint effects here, may be looked forward in the future to clarify the detail mechanisms involved in oral oncology. It is agreed that the complex environmental carcinogens may generate various types of DNA damage, activate their responsible DNA proteins in different DNA repair pathways to remove them. In this case, further investigations of other SNPs in DNA repair genes, and the repair capacity determined in oral cancer patients’ cells can help us to the integrative picture about the overall oral oncology. In conclusion, this is the first study to investigate the association between XRCC4 gene polymorphisms and oral cancer. Our findings suggest that D allele of XRCC4–2 was associated with higher susceptibility to oral cancer, and the D allele of XRCC4–2 (rs28360071) may be a novel useful biomarker for oral oncology primary prevention and anticancer intervention.
Conflict of Interest Statement We declare that we did not hold any financial and personal relationship with other people or organization that could inappropriately influence our work.
Acknowledgements We appreciate Dr. C.Y. Shen, Dr. Y.L. Lo, and Dr. W.K. Yang for constructive reviewing of this manuscript, and Chia-Wen Tsai, Hsiu-Yun Liang, and Chiao-Lin Lin for their technical assistance. This study was supported by research grants from the China Medical University and Hospital (DMR-97-061), Terry Fox Cancer Research Foundation, and the National Science Council (NSC 95-2320-B-039-014-MY3, second year).
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C.-F. Chiu et al. 4. Moore RJ, Doherty DA, Do KA, Chamberlain RM, Khuri FR. Racial disparity in survival of patients with squamous cell carcinoma of the oral cavity and pharynx. Ethn Health 2001;6: 165–77. 5. Department of Health, Taiwan: Cancer registration system annual report. Taiwan, Department of Health; 2006. 6. Sugimura T, Kumimoto H, Tohnai I, Fukui T, Matsuo K, Tsurusako S, et al. Gene–environment interaction involved in oral carcinogenesis: molecular epidemiological study for metabolic and DNA repair gene polymorphisms. J Oral Pathol Med 2006;35:11–8. 7. Vogelstein B, Alberts B, Shine K. Genetics. Please don’t call it cloning! Science 2002;295:1237. 8. Gu J, Zhao H, Dinney CP, Zhu Y, Leibovici D, Bermejo CE, et al. Nucleotide excision repair gene polymorphisms and recurrence after treatment for superficial bladder cancer. Clin Cancer Res 2005;11:1408–15. 9. Miller KL, Karagas MR, Kraft P, Hunter DJ, Catalano PJ, Byler SH, et al. XPA, haplotypes, and risk of basal and squamous cell carcinoma. Carcinogenesis 2006;27:1670–5. 10. Hung RJ, Hall J, Brennan P, Boffetta P. Genetic polymorphisms in the base excision repair pathway and cancer risk: a huge review. Am J Epidemiol 2005;162:925–42. 11. Li Z, Otevrel T, Gao Y, Cheng HL, Seed B, Stamato TD, et al. Cell 1995;83:1079–89. 12. Mari PO, Florea BI, Persengiev SP, Verkaik NS, Bruggenwirth HT, Modesti M, et al. Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4. Proc Natl Acad Sci USA 2006;103:18597–602. 13. van Heemst D, Brugmans L, Verkaik NS, van Gent DC. Endjoining of blunt DNA double-strand breaks in mammalian fibroblasts is precise and requires DNA-PK and XRCC4. DNA Repair (Amst) 2004;3:43–50. 14. Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, et al. A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell 1998;95:891–902. 15. Gao Y, Ferguson DO, Xie W, Manis JP, Sekiguchi J, Frank KM, et al. Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development. Nature 2000;404:897–900. 16. Medina PP, Ahrendt SA, Pollan M, Fernandez P, Sidransky D, Sanchez-Cespedes M. Screening of homologous recombination gene polymorphisms in lung cancer patients reveals an association of the NBS1-185Gln variant and p53 gene mutations. Cancer Epidemiol Biomark Prevent 2003;12:699–704. 17. Fu YP, Yu JC, Cheng TC, Lou MA, Hsu GC, Wu CY, et al. Breast cancer risk associated with genotypic polymorphism of the nonhomologous end-joining genes: a multigenic study on cancer susceptibility. Cancer Res 2003;63:2440–6. 18. Bau DT, Fu YP, Chen ST, Cheng TC, Yu JC, Wu PE, et al. Breast cancer risk and the DNA double-strand break end-joining capacity of non-homologous end-joining genes are affected by BRCA1. Cancer Res 2004;64:5013–9. 19. Bau DT, Mau YC, Shen CY. Role of BRCA1 in non-homologous end-joining. Cancer Lett 2006;240:1–8. 20. Bau DT, Wu HC, Chiu CF, Lin CC, Hsu CM, Wang CL, et al. Association of XPD polymorphisms with prostate cancer in Taiwanese patients. Anticancer Res 2007;27:2893–6. 21. Bau DT, Tsai MH, Huang CY, Lee CC, Tseng HC, Lo YL, et al. Relationship between polymorphisms of nucleotide excision repair genes and oral cancer risk in Taiwan: evidence for modification of smoking habit. Chin J Physiol 2007;50:1–7. 22. Chiu CF, et al. A novel single nucleotide polymorphism in ERCC6 gene is associated with oral cancer susceptibility in taiwanese patients. Oral Oncol 2008;44:582–6. 23. Kietthubthew S, Sriplung H, Au WW, Ishida T. Polymorphism in DNA repair genes and oral squamous cell carcinoma in Thailand. Int J Hyg Environ Health 2006;209:21–9.
available at www.sciencedirect.com
journal homepage: http://intl.elsevierhealth.com/journals/oron/
A novel single nucleotide polymorphism in XRCC4 gene is associated with oral cancer susceptibility in Taiwanese patients Chang-Fang Chiu a,e, Ming-Hsui Tsai b,e, Hsien-Chang Tseng b, Cheng-Li Wang e, Chung-Hsing Wang c, Cheng-Nan Wu g, Cheng-Chieh Lin d, Da-Tian Bau e,f,* a
Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan, ROC Department of Otolaryngology, China Medical University Hospital, Taichung, Taiwan, ROC c Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan, ROC d Department of Family Medicine, China Medical University Hospital, Taichung, Taiwan, ROC e Terry Fox Cancer Research Lab, China Medical University Hospital, 2 Yuh-Der Road, Taichung 404, Taiwan, ROC f Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan, ROC g Institute of Medical Bioscience, Central-Taiwan University of Science and Technology, Taichung, Taiwan, ROC b
Received 7 September 2007; received in revised form 16 November 2007; accepted 19 November 2007 Available online 4 March 2008
KEYWORDS XRCC4; Polymorphism; Oral cancer; Carcinogenesis
Summary The DNA double strand break repair gene XRCC4, an important caretaker of genome stability, is suggested to play a role in the development of human carcinogenesis. However, no evidence has been provided showing that XRCC4 was associated with oral oncology. In this hospital-based case-control study, the association of XRCC4 G-1394T (rs6869366), intron 3 (rs28360071), intron 7 (rs28360317), and intron 7 (rs1805377) polymorphisms with oral cancer risk in a Taiwanese population was first investigated. In total, 318 patients with oral cancer and 318 age- and gender-matched healthy controls were genotyped. We found a significant different distribution in the frequency of the XRCC4 intron 3 genotype, but not the XRCC4 G-1394T or intron 7 genotypes, between the oral cancer and control groups. Those who had heterozygous del/ins at XRCC4 intron 3 showed a 1.57-fold (95% confidence interval = 1.12–2.21) increased risk of oral cancer compared to those with ins/ins. As for XRCC4 G-1394T or intron 7 polymorphisms, there was no difference in the distribution between the oral cancer and control groups. There were significant gene–environment interactions between XRCC4 intron 3 genotype with
* Corresponding author. Address: Terry Fox Cancer Research Lab, China Medical University Hospital, 2 Yuh-Der Road, Taichung, 404, Taiwan, ROC. Tel.: +886 422053366x3312; fax: +886 422053366x1511. 1368-8375/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2007.11.007
XRCC4 Polymorphism in oral cancer
899
smoking and with betel quid chewing, but not with alcoholism. In smoker and betel quid chewer groups, the XRCC4 intron 3 del variants exhibited 2.57- and 3.03-fold higher risks than the ins genotype, respectively. Our results firstly suggest that the XRCC4 intron 3 del genotype may be associated with oral oncology and may be a novel useful marker for primary prevention and anticancer intervention.
ª 2007 Elsevier Ltd. All rights reserved.
Introduction
Materials and methods
Oral cancer has been identified as a significant public health threat all over the world.1–4 With continuously increasing incidence and mortality for the past two decades, oral cancer has become the fourth most common cause of male cancer death in Taiwan.5 However, the genomic etiology of oral cancer is of great interest but largely unknown. Human DNA repair mechanisms protect the genome from DNA damage caused by endogenous and environmental agents.6 Mutations or defects in the DNA repairing system are essential for tumorigenesis.7–9 Therefore, it is logical to suspect that some genetic variants of DNA repair genes might contribute to oral cancer pathogenesis. Sequence variants in DNA repair genes also are thought to modulate DNA repair capacity and consequently may be associated with altered cancer risk.10 X-ray cross-complementing group 4 (XRCC4) gene, which is important in non-homologous end-joining repair pathway, is found to restore DNA double strand break repair and the ability to support V(D)J recombination of transiently introduced substrates in the XR-1 CHO cell line.11 The XRCC4 protein interacts directly with Ku70/Ku80,12 and it is hypothesized that XRCC4 serves as a flexible tether between Ku70/Ku80 and its associated protein Ligase4.12 XRCC4 is required for precise end-joining of blunt DNA double strand breaks in mammalian fibroblasts.13 In the gene-targeting mutation mice model, XRCC4 gene inactivation leads to late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated post-mitotic neuronal cells.14,15 These findings demonstrated that differentiating lymphocytes and neurons strictly require the XRCC4 endjoining proteins. Thus, it is reasonable that polymorphisms of XRCC4 gene can be sustained in the genome for such a long time of carcinogenesis. Up to now, there are few papers studying the role of XRCC4 gene in cancer, not to mention XRCC4 polymorphisms in oral cancer susceptibility. Since DNA repair gene alterations have been shown to cause a reduction in DNA repair capacity, we hypothesized that DNA repair gene polymorphisms may be risk factors for oral cancer. To test this hypothesis, we determined the genotypic frequency of four polymorphisms of the XRCC4 gene at G-1394T (rs6869366), intron 3 (rs 28360071), intron 7 (rs28360317) and intron 7 (rs1805377), which will hereafter been referred to as XRCC4-1, XRCC4– 2, XRCC4–3 and XRCC4–4, respectively, using a polymerase chain reaction-based restriction fragment length polymorphism method. To the best of our knowledge, this is the first study carried out to evaluate the contribution of XRCC4 polymorphisms in oral oncology all over the world.
Study population and sample collection Three hundred and eighteen patients diagnosed with oral cancer were recruited at the outpatient clinics of general surgery between 1998 and 2007 at the China Medical University Hospital, Taichung, Taiwan, Republic of China. All patients voluntarily participated, completed a self-administered questionnaire and provided peripheral blood samples. The same amounts of non-oral cancer healthy people as controls were selected by matching for age and gender after initial random sampling from the Health Examination Cohort of the hospital. The questionnaire administered to the subjects included questions on history and frequency of alcohol consumption, betel quid chewing and smoking habits. Self-reported alcohol consumption, betel quid chewing and smoking habits were evaluated and classified as categorical variables. Information on these factors was obtained as more than twice a week for five years as ‘‘ever’’. 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 leucocytes using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and further processed according to PCR–RFLP methods. Briefly, the following primers were used for XRCC4–1: 50 -GATGCGAACTCAAAGATACTGA-30 and 50 -TGTAAAGCCAGTACTCAAACTT-30 ; for XRCC4–2: 50 -TCCTGTTACCATTTCAGTGTTAT30 and 50 -CACCTGTGTTCAATTCCAGCTT-30 ; for XRCC4–3: 50 -ATACTGTGTTTGGAACTCCT-30 for CCT-positive forward primer, 50 -ATACTGTGTTTGGAACTAGA-30 for CCT-negative forward primer, and 50 -TATCCTATCATCTCTGGATA-30 as reverse common primer; and for XRCC4–4: 50 -TTCACTTATGTGTCTCTTCA-30 and 50 -AACATAGTCTAGTGAACATC-30 . The following cycling conditions were performed: 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. The PCR products of XRCC4–2 were 109 bp for the del alleletype and 139 bp for the ins alleletype, respectively. The PCR products of XRCC4–3 were 239 bp for CCT-positive form and no product for CCT-negative form. The PCR products were studied after digestion with Hinc II, and Tsp509I restriction enzymes for XRCC4–1 (cut from 300 bp T type into 200 + 100 bp G type) and XRCC4–4 (cut from 237 bp G type into 79 + 158 bp A type), respectively. Ten percent of DNA samples were genotyped for a second time and the concordance rate was 100%.
900
Statistical analyses 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 XRCC4 single nucleotide polymorphisms in the control subjects from those expected under the Hardy–Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s v2 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 XRCC4 genotypes between cases and controls. Cancer risk associated with the genotypes was estimated as odds ratio (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. Data was recognized as significant when the statistical p-value was less than 0.05.
Results Distributions of the demographics and clinical variables were presented in Table 1. The mean ages of the oral cancer patients and the controls were 48.66 (SD = 8.62) and 53.26 (SD = 10.83) years, respectively. The proportion of men and women were the same ratio in the patient and control groups (i.e., male/female = 282/36). The most common sites of primary tumor were tongue and buccal mucosa in this patient population. Approximately 40% of the study cohort consisted of patients diagnosed with TNM late stage (III and IV) oral cancer. Among the characteristics compared, only the smoking statuses were differently distributed between oral cancer patient and control groups (Table 1). The frequencies of the alleles for the XRCC4–1, XRCC4–2, XRCC4–3, and XRCC4–4, between oral cancer and control groups are shown in Table 2. The distributions of all these polymorphisms were in Hardy–Weinberg equilibrium. Obviously, the D allele at XRCC4–2 was significantly associated with oral cancer risk (p = 0.0193). In contrast, G or T allele at XRCC4–1, I or D allele at XRCC4–3, A or G allele at XRCC4–4, were not differently distributed in the oral cancer patient and control groups (p > 0.05). The representative PCR–RFLP analyses for the XRCC4–2 polymorphisms are shown in Fig. 1. The frequencies of the genotypes of XRCC4–1, XRCC4–2, XRCC4–3 and XRCC4–4 polymorphisms in the oral cancer and control groups are shown in Table 3. Using I/I as the reference group, there was an obvious association between the heterozygotes and homozygotes of D at XRCC4–2 and oral cancer risk (Table 3). A combination of the homozygotes and heterozygotes of D (with D) showed that the D allele at XRCC4–2 conferred a 1.55-fold risk factor for oral cancer (Table 3). Neither heterozygotes of G at XRCC4–1, heteroor homozygotes of D at XRCC4–3, hetero- nor homozygotes of G at XRCC4–4, seemed to be risky genotypes for oral cancer (Table 3). Thus, among the four XRCC4 polymorphisms investigated, only the D allele at XRCC4–2 seems to be positively associated with oral cancer risk. The joint effects of XRCC4–2 genotypes and environmental factors on oral cancer risk are shown in Table 4. Smokers, alcoholics and betel quid chewers were classified as described in ‘Materials and methods’ into two groups, never and ever. Among the three oral cancer-related habits evaluated, the XRCC4–2 was shown to have interactions with smoking and betel quid chewing, but not with alcohol
C.-F. Chiu et al. Table 1 Demographics and clinical characteristics of oral cancer patients from Central Taiwan Characteristics Age, year <40 40–49 50–59 60–69 P70 Gender Male Female
Control number (%) 33 90 78 70 47
(10.38) (28.30) (24.53) (22.01) (14.78)
282 (88.68) 36 (11.32)
Cases number (%) 35 94 76 65 48
(11.01) (29.56) (23.90) (20.44) (15.09)
282 (88.68) 36 (11.32)
Site of primary tumora Tongue Buccal mucosa Others
157 (49.37) 90 (28.30) 71 (22.33)
Clinical stagea Stage I Stage II Stage III Stage IV
71 125 76 46
Smoking habit Never Ever
p-Value
0.9849
1.00
(22.33) (39.31) (23.90) (14.47)
208 (65.41) 110 (34.59)
173 (54.40) 145 (45.60)
0.0059b
Alcohol drinking habit Never 146 (45.91) Ever 172 (54.09)
143 (44.97) 175 (55.03)
0.8735
Betel chewing habit Never 253 (79.56) Ever 65 (20.44)
232 (72.96) 86 (27.04)
0.0623
a b
Surgical cases only. p-Value significant at <0.05.
Table 2 Allele frequencies for XRCC4 polymorphisms in the control and oral cancer groups Allele
Controls (%)
Cases (%)
N = 636
N = 636
p-Valuea
XRCC4–1 Allele T Allele G
617 (97.0) 19 (3.0)
609 (95.8) 27 (4.2)
0.2296
XRCC4–2 Allele I Allele D
538 (84.6) 98 (15.4)
506 (79.6) 130 (20.4)
0.0193
XRCC4–3 Allele I Allele D
478 (75.2) 158 (24.8)
492 (77.4) 144 (22.6)
0.3563
XRCC4–4 Allele A Allele G
464 (73.0) 172 (27.0)
473 (74.4) 163 (25.6)
0.5667
a
p-Value based on v2 test.
XRCC4 Polymorphism in oral cancer
901 Table 4 Joint effects of XRCC4–2 polymorphisms and environmental factors on oral cancer risk Habits
Figure 1 PCR-based restriction analysis of the XRCC4–2 polymorphism shown on 3% agarose electrophoresis. M: 100 bp DNA size marker, I/I: insertion homozygote, D/I: heterozygote, and D/D: deletion homozygote.
Controls (n)
Cases (n)
Odds ratio (95% CI)a
Smoking Never I/I I/D + D/D Ever I/I I/D + D/D
144 64 83 27
117 56 79 66
1.00 (ref) 1.08 (0.70–1.66) 1.00 (ref) 2.57 (1.49–4.42)b
Alcohol Never I/I I/D + D/D Ever I/I I/D + D/D
103 43 124 48
87 56 109 66
1.00 1.54 1.00 1.56
Alcohol Never I/I I/D + D/D Ever I/I I/D + D/D
174 79 53 12
145 87 51 35
1.00 (ref) 1.32 (0.91–1.93) 1.00 (ref) 3.03 (1.42–6.48)b
a
Table 3 Association of XRCC4 polymorphisms and oral cancer risk Controls (%)
XRCC4–1 T/T G/T G/G
299 (94.0) 19 (6.0) 0 (0)
291 (91.5) 27 (8.5) 0 (0)
1.00 (ref) 1.46 (0.79–2.68) 1.03 (0.02–51.96)
XRCC4–2 I/I I/D D/D With D
227 (71.4) 84 (26.4) 7 (2.2) 91 (28.6)
196 (61.6) 114 (35.8) 8 (2.5) 122 (38.4)
1.00 (ref) 1.57 (1.12–2.21)b 1.32 (0.47–3.72) 1.55 (1.11–2.16)b
XRCC4–2 I/I I/D D/D With D
195 88 35 123
(61.3) (27.7) (11.0) (38.7)
196 (61.6) 100 (31.4) 22 (6.9) 122 (38.4)
1.00 1.13 0.63 0.99
(ref) (0.80–1.60) (0.35–1.11) (0.72–1.36)
XRCC4–2 A/A A/G G/G With G
167 (52.5) 130 (40.9) 21 (6.6) 151 (47.5)
173 (54.4) 127 (39.9) 18 (5.7) 145 (45.6)
1.00 0.94 0.83 0.93
(ref) (0.68–1.30) (0.43–1.61) (0.68–1.27)
a
(ref) (0.95–2.52) (ref) (0.99–2.46)
CI, confidence interval. Statistically identified significant.
Odds ratio (95% CI)a
Genotype
b
Cases (%)
b
Genotype
CI, confidence interval. Statistically identified significant.
drinking. In smoking and betel quid chewing groups, the XRCC4–2 D variants conferred 2.57-fold (95% CI = 1.49– 4.42) and 3.03-fold (95% CI = 1.42–6.48) increased risks compared to the I genotype, respectively (Table 4).
Discussion The present study has investigated the role of XRCC4 gene polymorphisms in oral cancer susceptibility. To the best of our knowledge, there were very few reports studying XRCC4 polymorphisms in carcinogenesis, including oral cancer, not
to mention the joint effects of them with environmental factors. The large sample size and concise data analysis strengthen the accuracy and reliability of our finding. The frequencies of XRCC4 polymorphisms variant alleles were similar to those reported in the NCBI website in the Asian population studies, which suggest no selection bias for the subject’s enrolments in terms of genotypes. Therefore, the need for the present results to be verified in even larger studies is not so urgent. In this study, the distribution of the D allele at XRCC4–2 (20.4%) was significantly higher in the oral cancer group than in the control group (15.4%) (Table 2). It was also found that participants with heterozygous D at XRCC4–2 had a 1.57-fold higher risk of oral cancer (Table 3). After combining the heterozygous and homozygous participants in both groups, there was still an obvious increased risk of 1.55-fold (Table 3). All these data suggested that D allele at XRCC4–2 was a novel biomarker for oral oncology, and as long as D allele was detected, no matter whether is hetero- or homozygotes, the carriers were more susceptible to oral cancer. As for other XRCC4 polymorphisms, our results indicated that their genetic differences were not associated with oral cancer risk. Three potential gene–environment interactions about oral carcinogenesis, XRCC4 with cigarette smoking, alcohol consumption and betel quid chewing, were also investigated and evaluated in our study (Table 4). Betel quid chewing is reported to induce free radical-induced DNA damage and strand breaks, and tobacco extracts contain some potential carcinogens may also cause DNA strand breaks, both of which should be repaired via XRCC4 involved pathway. Similar to previous findings about double strand break repair genes with environmental factors, such as NBS1 with smoking in lung cancer,16 and Ku70/K80/DNA-PKcs/Ligase4/ XRCC4 with estrogen in breast cancer,17 our study provides
902 evidence for possible gene–environment joint effects in carcinogenesis. It is interesting that there were joint effects between risky XRCC4 genotypes, smoking and betel quid chewing, but not alcohol drinking (Table 4). In smoker and betel quid chewer groups, the XRCC4–2 D variants conferred 2.57-fold (95% CI = 1.49–4.42) and 3.03-fold (95% CI = 1.42–6.48) than the I genotype, respectively (Table 4). As the previous findings,18–23 our results suggest that genetic variants involved in DNA repair genes indeed involved in cancer etiology. Some of the famous oral cancer-related environmental risk factors, such as consumption of betel quid, and smoking here, may modulate oral cancer risk in combination with those genetic susceptibilities in different repair pathways. Therefore, combinational investigations of different genes in various DNA repair systems, and genotype–phenotype correlations among these systems, in addition to gene–environment joint effects here, may be looked forward in the future to clarify the detail mechanisms involved in oral oncology. It is agreed that the complex environmental carcinogens may generate various types of DNA damage, activate their responsible DNA proteins in different DNA repair pathways to remove them. In this case, further investigations of other SNPs in DNA repair genes, and the repair capacity determined in oral cancer patients’ cells can help us to the integrative picture about the overall oral oncology. In conclusion, this is the first study to investigate the association between XRCC4 gene polymorphisms and oral cancer. Our findings suggest that D allele of XRCC4–2 was associated with higher susceptibility to oral cancer, and the D allele of XRCC4–2 (rs28360071) may be a novel useful biomarker for oral oncology primary prevention and anticancer intervention.
Conflict of Interest Statement We declare that we did not hold any financial and personal relationship with other people or organization that could inappropriately influence our work.
Acknowledgements We appreciate Dr. C.Y. Shen, Dr. Y.L. Lo, and Dr. W.K. Yang for constructive reviewing of this manuscript, and Chia-Wen Tsai, Hsiu-Yun Liang, and Chiao-Lin Lin for their technical assistance. This study was supported by research grants from the China Medical University and Hospital (DMR-97-061), Terry Fox Cancer Research Foundation, and the National Science Council (NSC 95-2320-B-039-014-MY3, second year).
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