Association with Gallbladder Cancer in North Indian ... - Springer Link

Ann Surg Oncol (2009) 16:1695–1703 DOI 10.1245/s10434-009-0354-3

ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

Single-Nucleotide Polymorphisms of DNA Repair Genes OGG1 and XRCC1: Association with Gallbladder Cancer in North Indian Population Anvesha Srivastava, MSc1, Kshitij Srivastava, MSc1, Sachchida Nand Pandey, PhD1, G. Choudhuri, MD2, and B. Mittal, PhD1 Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, UP, India; 2Department of Gastroenterolgy, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, UP, India

1

ABSTRACT Background. DNA damage by endogenous or exogenous source of reactive oxygen species (ROS) plays an important role in induction and progression of various cancers. Physiologically, gallbladder is likely to be exposed to various ROS which leads to extensive DNA damage. Cells overcome the DNA damage by repair mechanisms. Genetic variants of OGG1 and XRCC1, important enzymes participating in base excision repair pathway, may confer interindividual variations in susceptibility to gallbladder cancer (GBC). This study was aimed to examine the role of OGG1 Ser326Cys (rs1052133) and XRCC1 Arg194Trp (C [ T) (rs25487) and Arg399Gln (G [ A) (rs1799782) polymorphisms in GBC susceptibility. Methods. The study included 173 GBC patients and 204 controls. Genotyping was done by polymerase chain reaction restriction fragment length polymorphism (PCRRFLP) method. Differences in the frequencies were estimated by chi-square test and risk was estimated by using unconditional logistic regression after adjusting for age and gender. Results. OGG1 Cys/Cys genotype frequency was significantly higher in GBC patients [odds ratio (OR) = 2.93; 95% confidence interval (CI) = 1.14–7.51]. The increased risk was more pronounced in female GBC patients (OR = 5.92; 95%CI = 1.20–29.13), patients with gallstone (OR = 5.50; 95%CI = 1.99–15.16), female gender, and late onset of disease (OR = 4.72, 95%CI = 1.43–15.53).

Ó Society of Surgical Oncology 2009 First Received: 29 September 2008; Published Online: 6 March 2009 B. Mittal, PhD e-mail: [email protected]

In XRCC1 Arg399Gln polymorphism, significant differences in frequencies of Gln/Gln and Arg/Gln genotypes conferred significantly low risk for GBC (OR = 0.62; 95%CI = 0.39–0.97 and OR = 0.37; 95%CI = 0.19–0.71 respectively). However, XRCC1 Arg194Trp polymorphism was not associated with GBC. The carriers of Arg-Gln haplotype consisting of 194Arg and 399Gln alleles of XRCC1 were also at significant low risk for GBC (OR = 0.59, 95%CI = 0.42–0.82). Interaction of genotypes and tobacco usage did not modulate the risk. Conclusion. Results suggest that Cys/Cys genotype of OGG1 Ser326Cys polymorphism is associated with increased risk of GBC. However, Arg399Gln polymorphism and Arg-Gln haplotype comprising XRCC1 Arg194Trp and Arg399Gln polymorphisms conferred low risk for GBC susceptibility. Gallbladder cancer (GBC) is an aggressive tumor with highest incidence rate for women in Delhi, India (21.5/ 100,000).1 Diverse genetic and environmental factors might be responsible for such geographic inconsistencies.2 GBC affects females up to six times more frequently and risk factors recognized include cholelithiasis, obesity, reproductive factors, chronic infection, and environmental exposure.3,4 Worldwide involvement of GBC with cholelithiasis and chronic inflammation of gallbladder has been established.5 Oxidative stress increases damage to cellular components, including DNA. The cell either repairs this damage or activates the apoptotic pathway. An important mechanism to protect cell against mutagenic effect of reactive oxygen species (ROS), which plays a critical role in various diseases including cancers, is base excision repair pathway (BER).6–9 A major product of ROS attack is 7,8-dihydro-8-oxoguanine (8-oxoG), which causes incorporation of deoxyadenosine

1696

triphosphate (dATP) at its complementary strand and results in G-to-T transversion.10,11 The enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) initiates the BER pathway, implicated in elimination of 8oxoG. The human OGG1 protein catalyzes the excision of 8oxoG from DNA.12,13 The OGG1 gene is located at 3p25, a region which is found to be frequently missing in various tumors, particularly lung and kidney tumors, which show loss of heterozygosity of markers.14 The 1245C [ G polymorphism (Ser326Cys) (rs1052133) is a well-known OGG1 gene polymorphism which results in substitution from serine to cysteine at codon 326.7,14 The OGG1-Cys326 protein has lower resistance to restrain spontaneous mutations in an Escherichia coli strain defective in 8-oxoG repair.14 Furthermore, the OGG1 Ser326Cys variant has been consistently associated with increased risk of cancers including GBC.15 Another enzyme of BER system is X-ray repair cross complementing group 1 (XRCC1), which encodes a scaffold protein implicated in both single-strand break repair and BER.16 XRCC1 protein participates in the removal of ‘‘nonbulky’’ DNA adducts, repair of oxidative DNA damage, and DNA damage attributed to ionizing radiations.17 It interacts with DNA polymerase ß, polyadenosine diphosphate-ribose polymerases 1 and 2, APE1/APEX1, OGG1, and proliferating cell nuclear antigen (PCNA). The XRCC1 is located at 19q13.2 and consists of 17 exons encoding 633 amino acids protein. A common genetic variation located in interacting binding region of XRCC1 (C [ T) in exon 6 results in Arg194Trp (rs25487) substitution in codon 194 while another substitution (G [ A) in exon 10, changes amino acid Arg to Glu (rs1799782) in codon 399. The Arg399Gln polymorphism resides in BRCA1 C-terminal domain which interacts with poly (ADP-ribose) polymerase (PARP) and variant allele 194Trp results in a nonconservative substitution in hydrophobic region of XRCC1.18 Variations in DNA repair efficiency may be the result of combinations of multiple alleles conferring differences in biological function of various DNA repair proteins. Thus, we sought to investigate the association of OGG1 Ser326Cys, XRCC1 Arg194Trp, and Arg399Gln polymorphisms in North Indian population, which is known to have a very high incidence rate of GBC. We also evaluated the influence of these polymorphisms in modulating risk of GBC due to various confounding factors such as gallstones, sex, age of onset of the disease, and tobacco usage. MATERIALS AND METHODS Patients The present case–control study comprised 173 cases of GBC patients [fine-needle-aspirated cell cytology (FNAC) and histopathologically proven] recruited among patients

A. Srivastava et al.

attending the clinics of Department of Gastroenterology and Gastro-surgery of Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS) Lucknow, UP, India from March 2005 to March 2008. Staging of cancer was documented according to American Joint Committee on Cancer.19 The clinical profile of GBC patients was based on hospital investigations. A total of 204 healthy controls were recruited from unrelated individuals of north India. The mean age and sex ratio of controls was matched to that of GBC patients. The inclusion criteria for controls were absence of prior history of cancer, precancerous lesions, asthma, coronary artery disease, diabetes mellitus, and gallstones proven by ultrasonography. After obtaining the informed consent, all the individuals were personally interviewed for information on ethnicity, food habits, occupation, and tobacco usage. Both patients and control groups had similar ethnicity. Tobacco usage in any form such as smoking cigarette, bidi (leaf-rolled unrefined tobacco) or chewing (nonsmoking) tobacco was recorded. Majority of the female patients were housewives and the male patients were not engaged in any hazardous occupations. The study was approved by local ethics committee of the institute. Blood samples from all the subjects were collected in ethylene diamine tetraacetic acid (EDTA) vials and stored at -70°C. Genotyping Laboratory personnel were blinded to the case–control status of the subjects. The genomic DNA was extracted from peripheral blood leucocytes pellet using the standard salting-out method.20 Genetic variant of OGG1 gene loci was determined by using PCR-RFLP. OGG1 Ser326Cys polymorphic site containing fragment was amplified by PCR. The reaction was conducted in total volume of 10 lL containing 100 ng genomic DNA and 15 pmoles of each primer. Primers used were as described by Jiao et al., which amplified a 200-bp fragment.15 The C [ G transversion at the 1245 polymorphic site of OGG1 gene creates a Fnu4HI restriction site. Digestion product was analyzed on 2% agarose gel. The 326Cys allele was characterized by the presence of two digested fragments of 100 bp and was compared with genotype standards (confirmed by direct sequencing). The primers set used to amplify XRCC1 Arg194Trp and Arg399Gln polymorphic sites amplicons were as described by Ramachandran et al.21 The XRCC1 Arg194Trp polymorphism containing amplicon (138 bp) was digested with 5 units of PvuII enzyme for 14 h. The presence of two bands of 75 and 63 bp showed the presence of Trp allele. The XRCC1 Arg399Gln polymorphic site containing amplicon (236 bp) was digested by 5 units of Hpa II

DNA Repair Gene Polymorphisms and GBC

enzyme at 37°C for 12 h. The digested product with Arg399Arg genotype showed two bands of 177 and 59 bp. To improve the genotyping quality and validation, 20% of samples were regenotyped by other laboratory personnel and results were reproducible with no discrepancy in genotyping. Genotyping of 10% of samples were confirmed by DNA sequencing.

1697 TABLE 1 Characteristic profile of controls and GBC patients GBC patients

Controls

Total

173

204

Female

107 (63.0%)

121 (59.3%)

Male

66 (37.0%)

83 (40.7%)

Age ± SD

53.6 ± 10.7

51.4 ± 9.8

0, I

None

–

II

12 (7.5%)

–

III

61 (38.1%)

–

IV

87 (54.4%)

–

83 (48.0) 90 (52.0)

None All

54 (31.2)

–

P value

0.673 0.136

Stagesa

Subgroup Stratifications To explore the effect of gender, subjects were stratified according to gender. Stratification was also done on the basis of gallstone status and was analyzed separately. GBC patients were also stratified on the basis of age of onset. Patients up to 50 years of age were denoted as early-onset (\50 years) and patients above 50 years were denoted as late-onset patients ([50 years).

Gallstone status Gallstone present Gallstone absent Tobacco usage Tobacco user a

Stage (n = 160)

Statistical Analysis The sample size was calculated using QUANTO 1.1 program (http://hydra.usc.edu/gxe). Desired power of our study was set at 80%. Descriptive statistics of patients and controls were presented as mean and standard deviation (SD) for continuous measures while frequencies and percentages were used for categorical measures. The chisquared goodness of fit test was used for any deviation from Hardy–Weinberg equilibrium in controls. Differences in genotype and allele frequencies between study groups were estimated by chi-square. Gallbladder cancer risk in relation to OGG1 genotypes was estimated by using unconditional logistic regression to calculate odds ratios (OR) and 95% confidence intervals. The ORs were adjusted for several confounding factors such as age and gender. A trend test for each genotype was performed according to the number of copies of the variant allele (0, 1 or 2). All statistical analyses were performed using SPSS software version 11.5 (SPSS, Chicago, IL, USA) and tests of statistical significance were two-sided and differences were taken as significant when P-value was \ 0.05. RESULTS Demographic characteristics of GBC patient and controls are summarized in Table 1. Mean age was not significantly different in GBC patients and controls. Gallstones were present in 48.0% of GBC patients and most of the GBC patients were in advanced stages of cancer (stages III and IV). About 31.2% of the GBC patients were associated with tobacco usage (Table 1). All cancer patients were incident cases and none of the controls had family history of cancer. It was evident from the interview that all

the cancer patients and controls belonged to same ethnic population, i.e., North Indian. DNA Repair Gene Polymorphisms and Gallbladder Cancer OGG1 Ser326Cys, XRCC1 Arg194Trp and Arg399Gln polymorphisms in controls were consistent with Hardy– Weinberg equilibrium. On comparing the genotype frequency distribution in gallbladder cancer patients with that of controls, the frequency of variant OGG1 Cys/Cys genotype was significantly associated with increased risk of GBC (P = 0.025; OR = 2.93; 95%CI = 1.14–7.51). The trend test was not significant (P = 0.062 for trend). The risk of the variant containing genotypes (Ser/Cys ? Cys/Cys) was 1.16 (95% CI 0.77–1.74) compared with the risk associated with the homozygous wild-type genotype. However, it did not reach statistical significance (P = 0.480) (Table 2). None of the genotypes and alleles of XRCC1 Arg194Trp were significantly associated with susceptibility of gallbladder cancer. The trend test and risk due to presence of variant containing genotype was also not significant (Table 2). In case of XRCC1 Arg399Gln polymorphism, frequencies of genotypes Arg/Gln and Gln/Gln were considerably higher in controls as compared with GBC patients. These frequency differences were statistically significant (P = 0.039 and P = 0.003, respectively) and conferred low risk for GBC (OR = 0.62 and OR = 0.37, respectively) (Table 2). The trend test was also significant (P = 0.001 for trend). The protective effect was also significant (P = 0.006) when variant containing genotypes (Arg/Gln ? Gln/Gln) were

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TABLE 2 Frequency distribution of OGG1 Ser326Cys, XRCC1 Arg194Trp, and Arg399Gln genotypes and allele in GBC patients and controls Genotype

Control/case (N) OR (95% CI) 204/173 [%]

P-value

genotypes the significant protection persisted in males only (P = 0.004; OR = 0.36; 95%CI = 0.18–0.72) (Table 4). DNA Repair Gene Polymorphism and Modulation of Risk in Presence of Gallstone

OGG1 Ser 326Cys (rs1052133) Ser/Ser Ser/Cys

112/88 [55/51] 85/69 [42/40]

Reference – 1.01 (0.65–1.54) 0.969

Cys/Cys

7/16 [3/9]

2.93 (1.14–7.51) 0.025

Ser/Cys ? Cys/Cys 92/85 [45/49]

1.16 (0.77–1.74) 0.480 ptrend = 0.062

XRCC1 Arg194Trp (rs25487) Arg/Arg

162/145 [79/84] Reference

–

Arg/Trp

40/26 [20/15]

0.74 (0.42–1.27) 0.280

Trp/Trp

2/2 [1/1]

1.01 (0.14–7.79) 0.943

Arg/Trp ?Trp/Trp

42/28 [21/16]

1.02 (1.00–1.04) 0.304 ptrend = 0.333

XRCC1 Arg399Gln (rs1799782) Arg/Arg

65/80 [32/46]

Reference

Arg/Gln

99/74 [48/43]

0.62 (0.39–0.97) 0.039

Gln/Gln

40/19 [20/11]

Arg/Gln ? Gln/Gln 139/93 [68/54]

–

0.37 (0.19–0.71) 0.003 0.55 (0.36–0.84) 0.006 ptrend = 0.001

Bold values indicate significant difference

compared with homozygous wild-type genotype (OR = 0.55; 95%CI = 0.36–0.84) (Table 2). The haplotypes of XRCC1 Arg194Trp and Arg399Gln were constructed by maximum-likelihood method. Three major haplotypes were observed, of which frequency of Arg-Gln haplotype was significantly lower in GBC patients in comparison with controls and was associated with significantly low risk (P = 0.002; OR = 0.59; 95%CI = 0.42–0.82) (data not shown). To further explore the role of these polymorphisms in susceptibility to gallbladder cancer, GBC patients and controls were stratified on the basis of gender. The frequency of OGG1 Cys326Cys genotype in female GBC patients was significantly higher in comparison with controls (P = 0.029; OR = 5.92; 95%CI = 1.20–29.13), conferring approximately sixfold high risk for cancer. However, this significance vanished (P = 0.232) when variant containing genotypes were combined (Ser/ Cys ? Cys/Cys) and compared with homozygous wild type (Table 3). Even after gender stratification, none of the genotypes of XRCC1 Arg194Trp were significantly associated with gallbladder cancer (data not shown). However, significant low risk for XRCC1 Arg399Gln Arg/Gln and Gln/Gln genotypes (P = 0.029 and P = 0.031) was observed in both genders. On combining the variant containing

To examine the role of these DNA repair gene polymorphisms in pathogenic mechanism of cancer, GBC patients were further stratified on the basis of presence or absence of gallstones. GBC patients with and without gallstone (absence of gallstones) were compared separately with controls. The frequency distribution of OGG1 Cys/ Cys genotype in GBC patients with stone was significantly high (P = 0.001) and conferred very high risk (OR = 5.50; 95%CI = 1.99–15.16). On combining the variant containing genotypes (Ser/Cys ? Cys/Cys) and comparing with wild-type genotype, the significance vanished while moderate risk persisted. (Table 3) On the other hand, significant decrease in the frequency of XRCC1 Arg399Gln, Gln/Gln genotype was observed in GBC patients without gallstones as compared with controls (P = 0.003; OR = 0.27; 95%CI = 0.11–0.64) (Table 4). This significance was not much affected after combining the variant containing genotypes (P = 0.002; OR = 0.44; 95%CI = 0.26–0.74) (Table 4). The low risk due to presence of Arg-Gln haplotype XRCC1 was also limited to the GBC patients without stone when compared with controls (P = 0.0002; OR = 0.44; 95%CI = 0.29–0.69) (data not shown). DNA Repair Gene Polymorphism and Age of Onset In order to further delineate the role of these polymorphisms, all the patients were divided into two groups on the basis of age of diagnosis. The patients were termed early onset (aged 0–49 years) and late onset (aged 50 years onwards). In the GBC patients having late onset of GBC, OGG1 Cys/Cys genotype was significantly associated with high risk of disease (P = 0.010; OR = 4.72; 95%CI = 1.43–15.53). This significance vanished on combining variant containing genotypes and comparing it with homozygous wild type (Table 3). However, no significant difference was found in case of XRCC1 Arg194Trp polymorphisms after this stratification. On the contrary, XRCC1 399 Gln/Gln genotype was significantly associated with low risk for patients with early onset of GBC (P = 0.020; OR = 0.23; 95%CI = 0.06– 0.79) and showed borderline significance with the lateonset group (P = 0.056; OR = 0.47; 95%CI = 0.18– 0.88). On combining the variant containing genotypes and comparing with homozygous wild type, significant low risk was observed for both groups (Table 4).


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TABLE 3 Frequency distribution of OGG1 Ser 326Cys (rs1052133) genotypes in GBC patients and controls after subdividing on the basis of gallstone status, gender, and age of onset Host characteristic Gender

Control/case 121/107 [%]

OR (95% CI) P-value Female

Control/case 83/66 [%]

OR (95% CI) P-value Male

Ser/Ser

68/52 [56/48]

Reference

44/36 [53/54]

Reference

Ser/Cys

51/46 [42/43]

1.16 (0.67– 2.00)

34/23 [41/35]

0.81 (0.40–1.62)

2/9 [2/9]

P = 0.545 5.92 (1.20–29.13)

5/7 [6/11]

P = 0.558 1.69 (0.49–5.80)

Cys/Cys

P = 0.029 Ser/Cys ? Cys/Cys

53/56 [44/52]

P = 0.401

1.38 (0.81–2.34)

39/29 [47/44]

P = 0.232 Gallstone

0.88 (0.46–1.69) P = 0.704

204/83 [%]

Gallstone present

204/90 [%]

Gallstone absent

Ser/Ser

112/36 [55/43]

Reference

112/52 [55/58]

Reference

Ser/Cys

85/35 [42/42]

1.28 (0.74–2.21)

85/34 [42/38]

P = 0.370 Cys/Cys

7/12 [3/15]

5.50 (1.99–15.16)

7/4 [3/4]


92/47 [45/57]

1.18 (0.32–4.46) P = 0.797

1.59 (0.95–2.67)

92/38 [45/42]

P = 0.076 Age at onset

0.83 (0.49–1.41) P = 0.505

0.86 (0.52–1.44) P = 0.569

60/55 [%]

Early (\50 years)

184/118 [%]

Late ([50 years)

Ser/Ser

30/32 [50/58]

Reference

82/56 [46/48]

Reference

Ser/Cys

27/19 [45/35]

0.67 (0.31–1.46) P = 0.292

58/50 [32/42]

1.26 (0.75–2.09) P = 0.375

Cys/Cys

3/4 [5/7]

1.24 (0.25–6.04)

4/12 [2/10]


30/24 [50/44]

4.72 (1.43–15.53) P = 0.010

0.77 (0.37–1.63) P = 0.497

62/61 [34/52]

1.37 (0.83–2.26) P = 0.220

Bold values indicate significant difference

DNA Repair Gene Polymorphism and Interaction with Tobacco Usage Interactions of tobacco usage with genotypes of OGG1 Ser326Cys, XRCC1 Arg399Gln, and XRCC1 Arg194Gln genes were analyzed in case-only analysis to explore the modulation of risk for gallbladder cancer. GBC risk modulation due to tobacco usage was not associated with any of the genotypes of the studied polymorphisms. On combining the variant containing genotypes no significant modulation of risk was observed when compared with homozygous wild type (data not shown). Gene–Gene Interaction of DNA Repair Gene Polymorphism and Gallbladder Cancer In order to look for the interaction among these gene variants, various genotype combinations of OGG1 Ser326Cys, XRCC1 Arg194Gln, and Arg399Gln polymorphisms were examined using logistic regression. An overall significant decrease in the risk for gallbladder cancer was observed due to interaction between OGG1 Ser326Cys and XRCC1 Arg399Gln genes in the presence of Ser/Cys 9

Gln/Gln model (P = 0.024; OR = 0.30; 95%CI = 0.10– 0.85) (Table 5). However, other interaction models of these polymorphisms were not significantly associated with GBC (Table 5). DISCUSSION Oxidative DNA damage, by either endogenous or exogenous sources of ROS, has been linked to carcinogenesis.22 Physiologically gallbladder is more prone to the exposure of carcinogens and ROS generated in various normal cellular metabolisms. Endogenous production of ROS in gallbladder might be due to lipid peroxidation and biliary stasis due to gallstones. Threefold increased risk for GBC in the subjects with Cys/Cys genotype of OGG1 Ser326Cys polymorphism was observed. Studies showed that protein encoded by OGG1 326Ser allele (wild type) demonstrates substantially higher DNA repair activity than 326Cys variant.23–25 Significant high frequency of 326Cys variant in GBC patients suggests reduced 8-oxoG repair activity by OGG1 resulting in increased GC to TA transversion.11 High levels of 8-oxoG have been reported in several human cancers, including

1700


TABLE 4 Frequency distribution of XRCC1 Arg399Gln (rs1799782) genotypes in GBC patients and controls after subdividing on the basis of gallstone status, gender, and age of onset Host Characteristic Gender

Control/Case 121/107 [%]

OR (95% CI) P-value Female

Control/Case 83/66 [%]

OR (95% CI) P-value Male

Arg/Arg

45/49 [37/46]

Reference

20/31 [24/47]

Reference

Arg/Gln

54/49 [45/46]

0.86 (0.49–1.51)

45/25 [54/38]

0.36 (0.17–0.76)

22/9 [18/8]

P = 0.600 0.37 (0.15–0.90)

18/10 [22/15]

P = 0.008 0.35 (0.13–0.90)

Gln/Gln

P = 0.029 Arg/Gln ? Gln/Gln

76/58

P = 0.031

0.72 (0.42–1.22)

63/35 [76/53]

P = 0.222 Gallstone

0.36 (0.18–0.72) P = 0.004

204/83 [%]

Gallstone present

204/90 [%]

Gallstone absent

Arg/Arg

65/33 [31/40]

Reference

65/47 [32/52]

Reference

Arg/Gln

99/39 [49/47]

0.78 (0.44–1.36)

99/35 [48/39]

P = 0.378 Gln/Gln

40/11 [20/13]

0.52 (0.23–1.15)

40/8 [20/9]


139/50 [68/60]

0.27 (0.11–0.64) P = 0.003

0.70 (0.41–1.19)

139/43 [6/48]

P = 0.192 Age at onset

0.51 (0.29–0.88) P = 0.016

0.44(0.26–0.74) P = 0.002

60/55 [%]

Early(\50 years)

144/118 [%]

Late ([50 years)

Arg/Arg

16/25 [27/45]

Reference

49/55 [34/46]

Reference

Arg/Gln

31/25 [51/45]

0.49 (0.2–1.1) P = 0.093

68/49 [47/42]

0.64 (0.39–1.17) P = 0.109

Gln/Gln

13/5 [22/10]

0.23(0.06–0.79)

27/14 [19/12]


44/30 [73/55]

0.47 (0.18–0.88) P = 0.056

0.40(0.18–0.89)

95/63 [52/53]

P = 0.024

0.59 (0.35–0.99) P = 0.046

Bold values indicate significant difference TABLE 5 Gene–gene interaction among OGG1 Ser326Cys, XRCC1 Arg194Trp, and Arg399Gln genotypes in GBC patients Gene OGG1 Ser 326 Cys 9 XRCC1 codon 399 OGG1 Ser 326 Cys 9 XRCC1 Codon 194 XRCC1 Codon 194 9 XRCC1 Codon 399 OGG1 Ser 326 Cys 9 XRCC1 codon 194 9 XRCC1 codon 399

Interaction model

P-value

Odds ratio

95% C I

Ser/Cys 9 Gln/Gln

0.024

0.305

0.10–0.85

Ser/Cys 9 Arg/Gln

0.339

1.999

0.48–8.28

Cys/Cys 9 Arg/Trp

0.481

2.387

0.21–26.8

Arg/Trp 9 Arg/Gln

0.179

0.578

0.26–1.28

Arg/Trp 9 Gln/Gln

0.060

0.134

0.02–1.09

Ser/Cys 9 Arg/Trp 9 Arg/Gln

0.689

0.787

0.24–2.54

Ser/Cys 9 Arg/Trp 9 Gln/Gln

0.198

0.242

0.02–2.10

In interaction model Ser/Ser genotype of OGG1, Arg/Arg genotype of XRCC1 Codon 194, and Arg/Arg genotype of XRCC1 Codon 399 were taken as reference for regression analysis. Bold values indicate significant difference

lung, esophageal, prostate, head and neck, nasopharyngeal, and orolaryngeal cancers.8,14,26–29 Accumulation of up to sevenfold higher levels of 8-oxoG and increased spontaneous mutation frequency has also been reported for two independently obtained OGG1-/- mice.25 The trend analysis was not significant, suggesting that there may not be necessarily a linear trend of variant genotype and GBC risk. No significant risk of combined variant containing genotypes with GBC (in subgroup analysis also) also indicates that Cys allele exerts a recessive effect and its

homozygote carriers would have potentially high risk for GBC. Our findings of increased risk due to OGG1 polymorphic variant in GBC patients with gallstones is in concordance with the only report of OGG1 Ser326Cys polymorphism in GBC.15 Increased risk of cancer due to long-standing gallstone may be due to physical assault or irritation of gallbladder epithelium along with biliary stasis. These may result in increased ROS and inflammation of gallbladder epithelium, which may further proceed


towards carcinoma in situ in the presence of compromised DNA repair system due to OGG1 326Cys genotype. In the present study, we compared GBC patients having gallstones with healthy controls without gallstones. As gallstones themselves can cause mechanical injury and DNA damage, the comparison with patients with gallstone disease may also be desired. The obtained results provide a compelling evidence for the role of gallstone status in GBC susceptibility with respect to studied DNA repair genes which needs to be explored further. Significant risk due to OGG1 polymorphism in female GBC patients may be due to either gallstone disease which is more predominant in females or may be the reflection of larger sample size in this group. Risk was also significantly associated with patients who manifest the disease in late stage of life. It appears that low OGG1 activity may cause accumulation of damaged DNA through cell divisions, leading to late manifestation. Here, it is also worth mentioning that GBC is a lateonset cancer which is more pronounced in females. Although increased 8-hydroxy-2-deoxyguanosine (8OH-dG) levels correlated with number of cigarettes smoked, have been observed in smokers’ lung DNA, we did not find any association with OGG1 Ser326Cys polymorphism and tobacco status in GBC patients.8 Majority of the tobacco user GBC patients take smokeless tobacco, which is not attributed as a significant risk factor in GBC patients from India. In addition, overall number of tobacco users in patient group was also low. Therefore, to firmly conclude the role of OGG1 polymorphism in modulation of tobacco-related risk of GBC is difficult. Recently, XRCC1 Arg399Gln polymorphism has drawn considerable attention due to its location in BRCT-I interaction domain of XRCC1 with poly (ADP-ribose) polymerase.30 Associations have been reported between 399Gln allele and higher DNA adduct levels, higher sister chromatid exchange frequency, and radiation-induced G2phase delay.21,31,32 However, in vitro experiment showed that both the alleles equally complement single-strand break repair defect in EM9 cells, suggesting that 399Gln variation does not affect the function.16 In XRCC1, 399Gln allele conferred significantly reduced risk but Arg194Trp polymorphism did not affect GBC susceptibility. On comparing the homozygous wild type with variant containing genotypes, increased significance was observed at all the levels showing an additive effect of the variant genotype. Low risk due to Arg-Gln haplotype shows that Arg194Trp polymorphism does not influence GBC susceptibility alone or in association with Arg399Gln polymorphism. Although XRCC1 polymorphisms is studied extensively in various cancers, results are conflicting. For variant 194Trp allele, marginal reduced risk among 194Trp carriers was observed in cutaneous melanoma, bladder, breast,

1701

lung, and stomach cancers.33 Similar to our study, in a recent report by Huang et al. Trp/Trp genotype was not associated with GBC susceptibility.34 The 399Gln allele has been linked to an increased risk of lung, colorectal, breast, stomach, and oral cancers.35–38 On the contrary, it was associated with reduced risk of bladder, esophageal and cutaneous melanomas, and nonmelanoma skin cancers.39,40 However, null association was found with GBC and lung cancer.34,38,41 According to low-risk model for cancer, 399Gln protein would be associated with reduced repair and increased cancer risk in both nondividing cells and apoptosis-abrogated cells but would be associated with reduced cancer risk in dividing and apoptotic mechanism in intact cells.40 However, along with main gene effect, role of gene–environment interaction and linkage to other important polymorphisms may not be ruled out. No effect of tobacco usage in modulation of GBC risk due to 194Trp or 399Gln variants was observed. Most epidemiological studies associated with 399Gln variant also did not find elevated risk of tobacco-related cancers, including lung or head and neck cancers.30 Moderate protection for bladder cancer was reported in moderate smokers and heavy smokers.39,42 However, no interaction of Arg194Trp polymorphism and cigarette smoking was reported for breast cancer risk.16 Gene–gene interaction of the studied three polymorphisms showed significant protection for Ser/Cys 9 Gln/ Gln interaction combination. Although Cys/Cys 9 Arg/Trp model showed high risk for GBC but was not significant. This again emphasizes strong protective role of Gln/Gln genotype even in presence of high risk conferring OGG1 326Cys allele. Our study strengthens the role of OGG1 Ser326Cys polymorphism as a genetic basis for poor antioxidant status in gallbladder and also reinforces the role of oxidative damage in gallbladder carcinogenesis. It was also observed that the there was low risk in the presence of Arg399Gln polymorphism while no role of Arg194Trp polymorphism was observed in our population. However, carcinogenesis is a complex process, and there exist interrelated pathways of biological response to DNA damage in which multiple genetic mechanisms are involved. Thus, role of these polymorphisms along with other genes in repair pathway needs to be explored in different populations. ACKNOWLEDGEMENTS The study was supported by research and fellowship grants from Indian Council of Medical Research and Department of Science and Technology, Govt. of India.

REFERENCES 1. Randi G, Franceschi S, La Vecchia C. Gallbladder cancer worldwide: Geographical distribution and risk factors. Int J Cancer. 2006;118:1591–1602.

1702 2. Coleman MP, Esteve J, Damiecki P, Arslan A, Renard H. Trends in cancer incidence and mortality, Lyon, IARC 1993. 3. Wistuba II, Gazdar AF. Gallbladder cancer: lessons from a rare tumor. Nat Rev Cancer. 2004;4:695–706. 4. Lazcano-Ponce EC, Miquel JF, Munoz N. Epidemiology and molecular pathology of gallbladder cancer. CA Cancer J Clin. 2001;51:349–64. 5. Takahashi T, Shivapurkar N, Riquelme E. Aberrant promoter hypermethylation of multiple genes in gallbladder carcinoma and chronic cholecystitis. Clin Cancer Res. 2004;10:6126–33. 6. Chatterjee A, Mambo E, Zhang Y, De Weese T, Sidransky D. Targeting of mutant hogg1 in mammalian mitochondria and nucleus: effect on cellular survival upon oxidative stress. BMC Cancer. 2006;6:235. 7. Sakamoto T, Higaki Y, Hara M, Ichiba M, Horita M, Mizuta T, et al. Ser326Cys Polymorphism and risk of hepatocellular carcinoma among Japanese. J Epidemiol. 2006;16:233–9. 8. Elahi A, Zheng Z, Park J, Eyring K, McCaffrey T, Lazarus P. The human OGG1 DNA repair enzyme and its association with orolaryngeal cancer risk. Carcinogenesis. 2002;23:1229–34. 9. Speina E, Arczewska KD, Gackowski D, Zieli n´ ska M, Siomek A, Kowalewski J, et al. Contribution of hMTH1 to the maintenance of 8-oxoguanine levels in lung DNA of non-small-cell lung cancer patients. J Natl Cancer Inst. 2005;97:384–95. 10. Bravard A, Vacher M, Gouget B, Coutant A, Hillairet de Boisferon F, Marsin S, et al. Redox regulation of human OGG1 activity in response to cellular oxidative stress. Mol Cell Biol. 2006;26:7430–6. 11. Nyaga SG, Lohani A, Jaruga P, Trzeciak AR, Dizdaroglu M, Evans MK. Reduced repair of 8-hydroxyguanine in the human breast cancer cell line, HCC1937. BMC Cancer. 2006;6:297. 12. Ide H, Kotera M. Human DNA Glycosylases involved in the repair of oxidatively damaged DNA. Biol Pharm Bull. 2004;27:480–5. 13. Zhang H, Mizumachi T, Carcel-Trullols J, Li L, Naito A, Spencer HJ, et al. Targeting human 8-oxoguanine DNA glycosylase (hOGG1) to mitochondria enhances cisplatin cytotoxicity in hepatoma cells. Carcinogenesis. 2007;28:1629–37. 14. Park HW, Kim IJ, Kang HC, Jang SG, Ahn SA, Lee JS, et al. The hOGG1 Ser326Cys polymorphism is not associated with colorectal cancer. Risk J Epidemiol. 2007;17:156–60. 15. Jiao X, Huang J, Wu S, Lv M, Hu Y, Jianfu, et al. hOGG1 Ser326Cys polymorphism and susceptibility to gallbladder cancer in a Chinese population. Int J Cancer. 2007;121:501–5. 16. Han J, Hankinson SE, De Vivo I, Spiegelman D, Tamimi RM, Mohrenweiser HW, et al. A prospective study of XRCC1 haplotypes and their interaction with plasma carotenoids on breast cancer risk. Cancer Res. 2003;63:8536–41. 17. Chang-Claude J, Popanda O, Tan XL, Kropp S, Helmbold I, von Fournier D, et al. Associations between polymorphisms in the DNA repair genes, XRCC1, APE1, and XPD and acute side effects of radiotherapy in breast cancer patients. Clin Cancer Res. 2005;11:4802–9. 18. Jennifer JH, Tasha RS, Mark SM, Harvey WM, Andrea G, Douglas Case L. Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity. Carcinogenesis. 2001;22:917–22. 19. Bearhs O, Henson D, Hutter R (1988) Manual for staging of cancer. Philadelphia: Lippincott; 1988. 20. Miller SA, Dykes DD, Polesky HF. A salting procedure for extracting DNA from nucleated cells. Nucl Acid Res. 1988;16:1215. 21. Ramachandran S, Ramadas K, Hariharan R, Kumar RR, Pillai MR. Single nucleotide polymorphisms of DNA repair genes XRCC1 and XPD and its molecular mapping in Indian oral cancer. Oral Oncol. 2006;42:350–62.

A. Srivastava et al. 22. Loı¨c Le M, Timothy D, Annette LJ, Ann S, Lynne R. Association of the hOGG1 Ser326Cys polymorphism with lung cancer risk. Cancer Epidemiol Biomarkers Prev. 2002;11:409–12. 23. Kohno T, Kunitoh H, Toyama K, Yamamoto S, Kuchiba A, Saito D, et al. Association of the OGG1-Ser326Cys polymorphism with lung adenocarcinoma risk. Cancer Sci. 2006;97:724–8. 24. Shen J, Deininger P, Hunt JD, Zhao H. 8-Hydroxy–20-deoxyguanosine (8-OH-dG) as a potential survival biomarker in patients with nonsmall-cell lung cancer. Cancer. 2007;109:574–80. 25. Luna L, Rolseth V, Hildrestrand GA, Otterlei M, Dantzer F, Bjøra˚s M, et al. Dynamic relocalization of hOGG1 during the cell cycle is disrupted in cells harboring the hOGG1-Cys326 polymorphic variant. Nucl Acids Res. 2005;33:1813–24. 26. Sugimura H, Kohno T, Wakai K. hOGG1 Ser326Cys polymorphism and lung cancer susceptibility. Cancer Epidemiol Biomarkers Prev. 1999;8:669–74. 27. Marchand L Le, Donlon T, Lum-Jones A, Seifried A, Wilkens LR. Association of the hOGG1 Ser326Cys polymorphism with lung cancer risk. Cancer Epidemiol Biomarkers Prev. 2002;11:409–12. 28. Xing DY, Tan W, Song N, Lin DX. Ser326Cys polymorphism in hOGG1 gene and risk of esophageal cancer in a Chinese population. Int J Cancer. 2001;95:140–3. 29. Sheehan AM, McGregor DK, Patel A, Shidham V, Fan CY, Chang CC. Expression of human 8-oxoguanine DNA glycosylase (hOGG1) in follicular lymphoma. Mod Pathol. 2006;18:1512–8. 30. Shen M, Hung RJ, Brennan P, Malaveille C, Donato F, Placidi D, et al. Polymorphisms of the DNA repair genes XRCC1, XRCC3, XPD, interaction with environmental exposures, and bladder cancer risk in a case-control study in northern Italy. Cancer Epidemiol Biomark Prev. 2003;12:1234–40. 31. Duell EJ, Wiencke JW, Cheng TJ, Varkonyi A, Zuo ZF, Ashok TDS. Polymorphisms in the DNA repair genes XRCC1and ERCC2 and biomarkers of DNA damage in human blood mononuclear cells. Carcinogenesis. 2000;21:965–71. 32. Hu JJ, Smith TR, Miller MS, Mohrenweiser HW, Golden A, Case LD. Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity. Carcinogenesis. 2001;22:917–22. 33. Goode EL, Ulrich CM, Potter JD. Polymorphisms in DNA repair genes and associations with cancer risk. Cancer Epidemiol Biomark Prev. 2002;11:1513–30. 34. Huang WY, Gao YT, Rashid A, Sakoda LC, Deng J, Shen MC, et al. Selected base excision repair gene polymorphisms and susceptibility to biliary tract cancer and biliary stones: a population-based case-control study in China. Carcinogenesis. 2008;29:100–5. 35. Park JY, Lee SY, Jeon HS, Bae NC, Chae SC, Joo S, et al. Polymorphism of the DNA repair gene XRCC1 and risk of primary lung cancer. Cancer Epidemiol Biomark Prev. 2002;11:23–7. 36. Zhou W, Liu G, Miller DP, Thurston SW, Xu LL, Wain JC, et al. Polymorphisms in the DNA repair genes XRCC1 and ERCC2, smoking, and lung cancer risk. Cancer Epidemiol Biomark Prev. 2003;12:359–65. 37. Sturgis EM, Castillo EJ, Li L, Zheng R, Eicher SA, Clayman GL, et al. Polymorphisms of DNA repair gene XRCC1 in squamous cell carcinoma of the head and neck. Carcinogenesis. 1999;20:2125–9. 38. Shu XO, Cai Q, Gao YT, Wen W, Jin F, Zheng W. A Populationbased case-control study of the Arg399Gln polymorphism in DNA repair gene XRCC1 and risk of breast cancer. Cancer Epidemiol Biomark Prev. 2003;12:1462–7. 39. Stern MC, Umbachvan DM, Gils CH, Lunn RM, Taylor JA. DNA repair gene XRCC1 polymorphisms, smoking, and bladder cancer risk. Cancer Epidemiol. Biomark Prev. 2001;10:125–31.

DNA Repair Gene Polymorphisms and GBC 40. Nelson HH, Kelsey KT, Mott LA, Karagas MR. The XRCC1 Arg399Gln polymorphism, sunburn, and non-melanoma skin cancer: evidence of gene–environment interaction. Cancer Res. 2002;62:152–5. 41. Misra RR, Ratnasinghe D, Tangrea JA, Virtamo J, Andersen MR, Barrett M, et al. Polymorphisms in the DNA repair genes XPD,

1703 XRCC1, XRCC3, and APE/ref–1, and the risk of lung cancer among male smokers in Finland. Cancer Lett. 2003;191:171–8. 42. Matullo G, Guarrera S, Carturan S, Peluso M, Malaveille C, Davico L, et al. DNA repair gene polymorphisms, bulky DNA adducts in white blood cells and bladder cancer in a case-control study. Int J Cancer. 2001;92:562–7.