Polymorphisms in ERCC2, MSH2, and OGG1 DNA repair genes and gallbladder cancer risk in a population of Northern India

Authors


Abstract

BACKGROUND:

Genetic variants of DNA repair enzymes may lead to genetic instability and contribute to gallbladder (GB) carcinogenesis.

METHODS:

A case-control study (230 GB carcinogenesis patients and 230 controls) was undertaken to evaluate whether genetic variations in 3 DNA repair genes ERCC2 (Asp312Asn [rs1799793] and Lys751Gln [rs13181]), MSH2 (−118T>C [rs2303425] and IVS1 + 9G>C [rs2303426]), and OGG1 (Ser326Cys [rs1052133] and 748-15C>G [rs2072668]) are associated with GB carcinogenesis risk in a North Indian population.

RESULTS:

The authors found that the ERCC2 Asp312Asn AA, MSH2 IVS1 + 9G>C CC, OGG1 Ser326Cys GG and CG + GG, and OGG1 748-15C>G GG and CG + GG genotypes were significantly associated with an increased risk of GB carcinogenesis (odds ratio [OR], 2.1, 1.8, 2.5, 1.8, 2.0, and 1.6, respectively). In contrast, ERCC2 Lys751Gln, and MSH2 −118T>C markers showed no significant associations with GB carcinogenesis risk, although because of the small sample size their effects cannot be ruled out. Female GB carcinogenesis patients with the OGG1 748-15C>G GG, OGG1 Ser326Cys GG, and ERCC2 Asp312Asn genotypes had a greater risk for developing the disease (OR, 3.6, 7.7, and 2.7, respectively). There was a significant interaction between MSH2 IVS1 + 9G>C and OGG1 748-15C>G polymorphisms (P = .001). Furthermore, individuals with >6 variant alleles of the studied polymorphisms were at 4-fold increased risk for developing GB carcinogenesis. Classification and Regression Tree analysis revealed potential higher-order gene-gene interactions and categorized a few higher-risk subgroups for GB carcinogenesis.

CONCLUSIONS:

These results suggest that genetic variants in the DNA repair pathways may be involved in GB carcinogenesis etiology. Cancer 2010. © 2010 American Cancer Society.

Carcinogenesis is a complex process involving interplay between many genes. The DNA repair pathway corrects the genomic damage generated because of various exogenous and endogenous processes (carcinogens), reactive oxygen species, free radicals, and peroxides. This pathway if defective may give rise to various pathophysiological processes such as aging, degenerative diseases, and carcinogenesis. 1, 2

Carcinoma of the gallbladder (GB) is an aggressive malignancy, and the most common biliary tract tumor in the world, affecting women 2 to 3× more commonly than men. 3 Several epidemiologic studies have indicated the role of genetic factors in the pathogenesis of GB carcinogenesis by modifying the risk involved.4-7 Inherited polymorphisms of DNA repair genes may contribute to interindividual variations in the DNA repair capacities contributing to cancer susceptibility. Therefore, it was hypothesized that functional polymorphisms of DNA repair genes may influence the risk for GB carcinogenesis by altering the amino acid sequence of encoded protein, mRNA splicing, or transcription efficiency of mRNA.

To elucidate the role of these DNA repair enzymes in the susceptibility to GB carcinogenesis, we studied the risk in relation to 6 variants in 3 DNA repair enzymes (ERCC2 Asp312Asn [rs1799793] and Lys751Gln [rs13181]; MSH2 IVS1 + 9G>C [rs2303426] and −118T>C [rs2303425]; and OGG1 Ser326Cys [rs1052133] and 748-15C>G [rs2072668]) in 230 GB carcinogenesis cases and 230 controls. These polymorphisms have been reported to alter the risk for developing various malignancies. 8-12

MATERIALS AND METHODS

Study Population

The present case-control study comprised 230 consecutive newly diagnosed GB carcinogenesis patients (fine needle aspirated cell cytology and histopathologically proven) recruited from the clinics of the Department of Gastroenterology of Sanjay Gandhi Postgraduate Institute of Medical Sciences, a tertiary care hospital in Lucknow, Uttar Pradesh, India. Staging of cancer was documented according to the American Joint Committee on Cancer. 13 A total of 230 healthy controls were also selected from the general population and were age, sex, and ethnicity matched to the patients. The inclusion criteria for controls were as described previously.14 We used the software as described by Devlin and Roeder15 to rule out the possibility of population stratification bias. Informed consent was taken from all subjects, and the study was approved by the local ethics committee of the Institute.

Genotyping

The genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism method. The PCR reaction conditions, primers used, restriction pattern, and restriction enzymes used for studied polymorphisms were as described previously. 16 A 292-bp fragment of the OGG1 gene 748-15C>G polymorphism was amplified by PCR using self-designed primers. Fragments were separated on polyacrylamide gel and stained with ethidium bromide and were observed using an ultraviolet imaging system (Vilber Lourmat, Marne-la-Vallée, France). Ten percent of samples from patients and controls including samples of each genotype were sequenced to evaluate the quality of genotyping, which showed 100% concordance.

Statistical Analysis

As described previously, 14 the sample size was calculated using QUANTO 1.1, considering the minor allele frequency of the single nucleotide polymorphisms (SNPs) analyzed. The sample size of 230 cases and controls was adequate to give us a power of 80% (probability of not making a type II error). Gene-gene and gene-environment interactions were estimated by the logistic regression model, which included an interaction term as well as variables for genotypes (ERCC2, MSH2, or OGG1) and potential confounders (age and sex). The false-positive report probability for statistically significant observations was estimated using the methods described by Wacholder et al.17 To enhance the accuracy of obtained results and clinical implication of the study, the nonparametric Classification and Regression Tree (CART) analysis was used along with the logistic regression for higher-order gene-gene interactions using CART Software (version 6.0, Salford Systems, San Diego, Calif).18, 19

Evaluation of Coding Single Nucleotide Polymorphisms

Two complementary algorithms, Sorting Intolerant From Tolerant (SIFT) (http://blocks.fhcrc.org/sift/SIFT.html) and Polymorphism Phenotyping (Polymorphism Phenotyping) (http://www.bork.embl-eidelberg.de/PolyPhen/), 20, 21 were chosen for functional impact prediction of nonsynonymous coding SNPs. Sorting Intolerant From Tolerant predicts whether an amino acid substitution in a protein will have a phenotypic effect.22 Polymorphism Phenotyping is a computational tool for identification of potentially functional nonsynonymous SNPs. Predictions are based on a combination of phylogenetic, structural, and sequence annotation information characterizing a substitution and its position in the protein.23

RESULTS

Population Characteristics

Baseline characteristics of GB carcinogenesis patients and their age- and sex-matched controls are presented in Table 1. Gallstones were present in 51.0% of GB carcinogenesis patients, and most of the GB carcinogenesis patients were in advanced stages of cancer (stages III and IV). About 33.0% of the GB carcinogenesis patients were associated with tobacco usage in some form (smoking, chewing, or both). All cancer patients were incident cases, and none of the controls had family history of cancer.

Table 1. Characteristic Profile of Controls and GB Cancer Patients
CharacteristicHealthy Controls [n = 230]GB Cancer Patients [n = 230]
  1. GB, gallbladder; SD, standard deviation.

Sex, No. (%)
 Men78 (33.9)79 (34.3)
 Women152 (66.1)151 (65.7)
Age, mean (SD)51.73 ± 6.2451.41 ± 8.18
Stage, No. (%)
 0, I None
 II 12 (5.3)
 III 76 (33.0)
 IV 142 (61.7)
Gallstone present, No. (%)None117 (51.0)
Gallstone absent, No. (%)All113 (49.0)
Tobacco users, No. (%)76 (33.0)

Allelic Distribution of Studied Polymorphisms

The distribution of ERCC2 Asp312Asn, ERCC2 Lys751Gln, MSH2 IVS1 + 9G>C, MSH2 −118T>C, OGG1 748-15C>G, and OGG1 Ser326Cys genotypes is shown in Table 2. Distributions of genotypes of all the polymorphisms in controls were in accordance with Hardy-Weinberg equilibrium (P > .05). The frequencies of the variant alleles of the polymorphisms in our study are compared with previous published reports.

Table 2. Case-Control Distribution of Genotypes of Polymorphisms Analyzed
GenotypeCases, No. (%)Controls, No. (%)OR (95% CI)aPLRT (2 df)
  • OR indicates odds ratio; CI, confidence interval; LRT, likelihood ratio test.

  • a

    Adjusted for age and sex.

ERCC2 Asp312Asn (rs1799793)
 GG108 (47.0)112 (48.7)Reference 
 GA87 (37.8)100 (43.5)0.9 (0.6-1.4).694 
 AA35 (15.2)18 (7.8)2.1 (1.1-4.0).022 
 GA + AA122 (53.0)118 (51.3)1.1 (0.7-1.6).6640.032
 Ptrend   .04 
ERCC2 Lys751Gln (rs13181)
 AA93 (40.4)113 (49.1)Reference 
 AC103 (44.8)90 (39.1)1.4 (0.9-2.0).115 
 CC34 (14.8)27 (11.7)1.5 (0.9-2.7).140 
 AC + CC137 (59.6)117 (50.9)1.4 (0.9-2.1).0670.162
 Ptrend   .16 
MSH2 −118T>C (rs2303425)
 TT160 (69.6)158 (68.7)Reference 
 TC62 (27.0)66 (28.7)0.9 (0.6-1.4).812 
 CC8 (3.5)6 (2.6)1.4 (0.5-4.0).584 
 TC + CC70 (30.4)72 (31.3)0.9 (0.6-1.5).9340.818
 Ptrend   .81 
MSH2 IVS1 + 9G>C (rs2303426)
 GG48 (20.9)59 (25.7)Reference 
 GC107 (46.5)119 (51.7)1.1 (0.7-1.8).631 
 CC75 (32.6)52 (22.6)1.8 (1.1-3.1).027 
 GC + CC182 (79.1)171 (74.3)1.3 (0.9-2.1).2050.045
 Ptrend   .05 
OGG1 Ser326Cys (rs1052133)
 CC117 (50.9)137 (59.6)Reference 
 CG92 (40.0)82 (35.7)1.3 (0.9-1.9).139 
 GG21 (9.1)11 (4.8)2.5 (1.1-5.4).026 
 CG + GG113 (49.1)93 (40.4)1.8 (1.2-2.6).0020.045
 Ptrend   .07 
OGG1 748-15C>G (rs2072668)
 CC83 (36.1)109 (47.4)Reference 
 CG103 (44.8)92 (40.0)1.5 (1.0-2.2).050 
 GG44 (19.1)29 (12.6)2.0 (1.2-3.5).012 
 CG + GG147 (63.9)121 (52.6)1.6 (1.1-2.4).0120.022
 Ptrend   .027 

Association of ERCC2, MSH2, and OGG1 Polymorphisms With GB Carcinogenesis

On comparing the genotype frequency distribution in GB carcinogenesis patients with that of controls, the homozygous variant genotypes of MSH2 IVS1 + 9G>C, ERCC2 Asp312Asn, and OGG1 Ser326Cys polymorphisms showed statistically significant increased risk for developing GB carcinogenesis (P = .027; odds ratio [OR], 1.8; 95% confidence interval [CI], 1.1-3.1; P = .022; OR, 2.1; 95% CI, 1.1-4.0; and P = .026; OR, 2.5; 95% CI, 1.1-5.4, respectively). The risk from variant-containing genotypes (CG + GG) of OGG1 Ser326Cys was also significant (P = .002; OR, 1.8; 95% CI, 1.2-2.6) when compared with homozygous wild-type CC genotype. The OGG1 748-15C>G intronic polymorphism was also significantly associated with the risk for GB carcinogenesis (P = .012; OR, 2.0; 95% CI, 1.2-3.5) (Table 2).

For the other SNPs in DNA repair genes (ERCC2 Asp312Asn and MSH2 −118T>C), no statistically significant associations were observed in the present study (Table 2). Overall risk was estimated for total variant alleles (3-6 and >6 compared with 0-2). Patients with >6 variant alleles were significantly associated with greater risk for developing GB carcinogenesis compared with the 0-2 allele group (P < .001; OR, 3.8; 95% CI, 2.1-7.1). The false-positive report probability for the statistically significant results was also calculated.

To further explore the role of these polymorphisms in susceptibility to GB carcinogenesis, GB carcinogenesis patients and controls were stratified on the basis of various host characteristics. In female GB carcinogenesis patients, the variant genotype (CC) of ERCC2 Asp312Asn polymorphism showed significantly increased risk for GB carcinogenesis (P = .007; OR, 2.7; 95% CI, 1.3-5.5). The variant genotypes of OGG1 748-15C>G polymorphism (GG) and OGG1 Ser326Cys polymorphism (GG) showed significantly increased risk for GB carcinogenesis (P = .020; OR, 3.6; 95% CI, 1.2-10.7 and P = .015; OR, 7.7; 95% CI, 1.5-40.2, respectively) in female GB carcinogenesis patients. The combined variant containing genotypes AC + CC of ERCC2 Lys751Gln and GC + CC of MSH2 IVS1 + 9G>C polymorphisms also showed statistically significant increased risk for GB carcinogenesis in men when compared with wild-type genotype (P = .022; OR, 2.1; 95% CI, 1.1-3.9 and P = .011; OR, 2.9; 95% CI, 1.3-6.7, respectively), following a dominant model.

ERCC2, MSH2, and OGG1 Polymorphisms and Modulation of Risk in the Presence of Gallstones

To explore the modulation of risk by gallstone status, GB carcinogenesis patients were further stratified on the basis of presence or absence of gallstones and compared separately with controls. GB carcinogenesis patients with gallstones were found to have an approximately 3-fold higher risk of developing the disease with OGG1 Ser326Cys GG genotype (P = .015; OR, 3.1; 95% CI, 1.2-7.7). The GG (P = .026; OR, 2.1; 95% CI, 1.1-4.1) and CG + GG (P = .019; OR, 1.7; 95% CI, 1.1-2.8) genotypes of OGG1 748-15C>G polymorphism with gallstone were also found to have significantly increased risk of developing GB carcinogenesis. However, the AC and AC + CC genotypes of ERCC2 Lys751Gln polymorphism were found to be significantly associated with GB carcinogenesis risk in patients without gallstones (P = .032; OR, 1.7; 95% CI, 1.0-2.8 and P = .024; OR, 1.7; 95% CI, 1.1-2.7, respectively).

ERCC2, MSH2, and OGG1 Polymorphisms and Interaction With Tobacco Usage

Because tobacco is a leading risk factor for most cancers, we also stratified our analyses by tobacco usage in a case-only analysis to explore the modulation of risk for GB carcinogenesis. None of the genotypes of the polymorphisms studied reached statistical significance for developing GB carcinogenesis. Also, when GB carcinogenesis patients with 3 to 6 and ≥6 variant alleles were compared with the referent group (<3 variant alleles), there was no evidence of statistically significantly increased risk for tobacco users (data not shown).

Interaction of Polymorphisms in DNA Repair Genes and GB Carcinogenesis Risk

Finally, to test the interaction between individual polymorphisms in DNA repair genes and the risk of developing GB carcinogenesis, ORs were estimated for each pair of the studied polymorphisms (ERCC2 Asp312Asn, ERCC2 Lys751Gln, MSH2 IVS1 + 9G>C, MSH2 −118T>C, OGG1 Ser326Cys, and OGG1 748-15C>G). We found that the cases with the MSH2 IVS1 + 9 CC genotype were also more likely to have the OGG1 748-15 GG genotype than the controls (13.0% vs 6.2%; P = .011). These polymorphisms exert an additive joint effect, because the OR for subjects with the MSH2 IVS1 + 9 CC and OGG1 748-15 GG genotypes versus subjects with the MSH2 IVS1 + 9 GG and OGG1 748-15 CC genotypes, 3.1 (Table 3), is equal to the addition of the OR for subjects with the MSH2 IVS1 + 9 CC genotype versus the MSH2 IVS1 + 9 GG genotype and the OR for subjects with the OGG1 748-15 GG genotype versus the OGG1 748-15 CC genotype (ie, 1.8 + 2.0 = 3.8; Table 2). Furthermore, despite the finding that the ERCC2 Lys751Gln polymorphism did not alter the overall risk of developing GB carcinogenesis when studied independently, when this polymorphism was combined with those studied in MSH2 or OGG1, we observed an interaction between these polymorphisms. Individuals with the MSH2 IVS1 + 9 CC/ERCC2 Lys751Gln AC genotypes showed a statistically significant higher risk for developing GB carcinogenesis (OR, 2.2; CI, 1.2-4.2; P = .008).

Table 3. Interaction of ERCC2, MSH2, and OGG1 Polymorphisms and Gallbladder Cancer Risk Estimates
Genotype 1Genotype 2Cases, No. (%)Controls, No. (%)OR (95% CI)aP
  • OR indicates odds ratio; CI, confidence interval.

  • a

    Adjusted for age and sex.

MSH2 IVS1 + 9G>COGG1 748-15C>G    
GGCC24 (16.3)33 (25.8)Reference
GCCG47 (32.0)56 (43.8)1.1 (0.7-1.7).737
GCGG20 (13.6)14 (10.9)1.8 (0.9-3.9).098
CCCG37 (25.2)17 (13.3)2.8 (1.5-5.3).001
CCGG19 (12.9)8 (6.2)3.1 (1.3-7.3).011
MSH2 IVS1 + 9G>CERCC2 Lys751Gln    
GGAA20 (16.0)24 (22.7)Reference
GCAC48 (38.4)47 (44.3)1.2 (0.8-1.9).367
GCCC35 (28.0)19 (17.9)2.1 (0.9-4.9).092
CCAC11 (8.8)9 (8.5)2.2 (1.2-4.2).008
CCCC11 (8.8)7 (6.6)1.9 (0.7-5.1).188
AllelesCombined Effect of Minor Alleles
Cases, No. (%)Controls, No. (%)OR (95% CI)aP
0-2 variant alleles64 (27.8)49 (21.3)Reference
3-6 variant alleles145 (63.0)120 (52.2)1.1 (0.7-1.7).758
>6 variant alleles21 (9.1)61 (26.5)3.8 (2.1-7.1)<.001

Main Effects of Combined ERCC2, MSH2, and OGG1 Genotypes on GB Carcinogenesis Risk

When the ERCC2, MSH2, and OGG1 SNPs were further combined separately based on the number of the observed at-risk genotypes, we found that GB carcinogenesis risk increased as the number of OGG1 at-risk genotypes increased (adjusted OR, 1.4; 95% CI, 0.7-2.6 for 1 at-risk genotype and adjusted OR, 2.2; 95% CI, 1.0-4.7 for 2 at-risk genotypes, Ptrend = .037). When the number of the observed at-risk genotypes was dichotomized, the subjects who carried 1 to 2 at-risk genotypes had a 1.7-fold increased risk of GB carcinogenesis (adjusted OR, 1.7; 95% CI, 1.0-2.8) compared with those who carried 0 at-risk genotypes (Table 4).

Table 4. Main Effects of Combined ERCC2 Asp312Asn and Lys751Gln; MSH2 IVS1 + 9G>C and −118T>C; and OGG1 Ser326Cys and 748-15C>G Genotypes on Gallbladder Cancer Risk
GenotypesCases [n = 230], No. (%)Controls [n = 230], No. (%)Adjusted OR (95% CI)a
  • a

    Adjusted for age and sex.

ERCC2 Asp312Asn and Lys751Gln
 No. at-risk genotype
  0195 (84.8)203 (88.3)Reference
  15 (2.2)5 (2.2)1.1 (0.3-3.7)
  230 (13.0)22 (9.6)1.4 (0.8-2.6)
  Ptrend  .24
 Dichotomized groups
  0195 (84.8)203 (88.3)Reference
  1-235 (15.2)27 (11.7)1.7 (1.0-2.8)
MSH2 IVS1 + 9G>C and −118T>C
 No. at-risk genotype
  0155 (67.4)178 (77.4)Reference
  167 (29.1)46 (20.0)1.7 (1.1-2.6)
  28 (3.5)6 (2.6)1.6 (0.5-4.6)
  Ptrend  .025
 Dichotomized groups
  0155 (67.4)178 (77.4)Reference
  1-275 (32.6)52 (22.6)1.7 (1.1-2.5)
OGG1 Ser326Cys and 748-15C>G
 No. at-risk genotype
  0186 (80.9)201 (87.4)Reference
  123 (10.0)18 (7.8)1.4 (0.7-2.6)
  221 (9.1)11 (4.8)2.2 (1.0-4.7)
  Ptrend  .037
 Dichotomized groups
  0186 (80.9)201 (87.4)Reference
  1-244 (19.1)29 (12.6)1.7 (1.0-2.8)

Haplotype Analysis of ERCC2, MSH2, and OGG1 in Case and Control Groups

Haplotype analysis revealed that the G-G haplotype of the OGG1 Ser326Cys and 748-15C>G polymorphisms was significantly associated with GB carcinogenesis risk (P = <.001; OR, 2.36; 95% CI, 1.5-3.7). Global haplotype analysis indicated a statistically significant difference between cases and controls based on the distribution pattern of the 4 haplotypes (P < .001). Haplotype interaction analysis with sex also showed nonsignificant increased risk in women for the G-G haplotype (Pinteraction = .37; OR, 2.82; 95% CI, 1.5-5.2). No statistically significant associations were found between the haplotypes estimated in the ERCC2 or MSH2 genes and GB carcinogenesis risk.

CART Analysis

Figure 1 depicts the tree structure generated using CART analysis, which included all investigated genetic variants of the DNA repair pathway. The final tree structure contained 6 terminal nodes as defined by SNPs of the DNA repair pathway. The OGG1 Ser 326 Cys genotype was singled out in the first splitting lymph node, separating individuals with the wild-type–containing genotypes (low risk) from subjects with the homozygous variant genotype (high risk). Individuals with the variant genotypes of OGG1 748-15 and ERCC2 Asp312Asn exhibited the lowest GB carcinogenesis risk, with a 43% case rate (Fig. 1). Table 5 shows the OR estimates generated for the 3 different risk groups determined on the basis of the case ratio of each CART terminal lymph node. Compared with the low-risk group combining terminal nodes with a case ratio <45%, the medium-risk (case ratio between 45% and 55%) and high-risk groups (case ratio >55%) were both associated with a significantly increased GB carcinogenesis risk with ORs of 7.6 (95% CI, 4.4-13.2) and 1.7 (95% CI, 1.1-2.6), respectively (P for trend <.001). The predictive accuracy of the logistic regression model was evaluated using the classification table, which showed that the overall percentage of statistically significant results was >60%.

Figure 1.

The tree structure generated using Classification and Regression Tree analysis is shown. GBC indicates gallbladder cancer.

Table 5. Combined Analysis of the Effects of Genetic Variants in the DNA Repair Pathway on Gallbladder Cancer Risk Based on the Results of the CART Analysis
Risk GroupaCase/ Control, No.OR (95% CI)bP
  • CART indicates Classification and Regression Tree; OR, odds ratio; CI, confidence interval.

  • a

    Low risk, case ratio <45; medium risk, 45 ≤ case ratio < 55; high risk, case ratio ≥55.

  • b

    Adjusted for age and sex.

Low risk92/24Reference
Medium risk72/827.6 (4.4-13.2)<.001
High risk66/1241.7 (1.1-2.6).020
Ptrend  <.001

Predictions of Deleterious and Damaging Nonsynonymous Coding SNPs

Protein conservation analysis was performed using the Sorting Intolerant From Tolerant algorithm, which predicts whether an amino acid substitution may have an impact on protein function. The 3 nonsynonymous SNPs analyzed in this study (ERCC2 Asp312Asn and Lys751Gln; OGG1 Ser326Cys) were submitted independently to the Sorting Intolerant From Tolerant program to check its tolerance index. All 3 nonsynonymous SNPs were classified as tolerant, having scores between 0.22 and 1.00.

The impact of an amino acid substitution on the structure and function of the DNA repair protein was predicted by the Polymorphism Phenotyping software. The 3 nonsynonymous SNPs analyzed in this study (ERCC2 Asp312Asn and Lys751Gln; OGG1 Ser326Cys) were found to be benign, which means that they most likely lack any phenotypic effect.

DISCUSSION

Genetic variations in DNA repair genes may have an effect on the DNA repair capacity, thereby causing interindividual differences in the propensity to GB carcinogenesis. Several proteins are involved in DNA repair pathways. Genetic polymorphisms in ERCC2, MSH2, and OGG1 genes have been shown to be involved in various malignancies. 12, 24-26 In this case-control study, we assessed the individual and joint effects of 6 potentially functional SNPs in 3 DNA repair genes on GB carcinogenesis predisposition.

Our results showed that ERCC2 Asp312Asn, MSH2 IVS1 + 9G>C, OGG1 Ser326Cys, and OGG1 748-15C>G polymorphisms were significantly associated with predisposition to GB carcinogenesis. Individuals homozygous for the ERCC2 312Asn, MSH2 IVS1 + 9C, OGG1 326Cys, and OGG1 748-15G alleles have a higher risk of developing GB carcinogenesis (ORs, 2.1, 1.8, 2.5, and 2.0, respectively). Conversely, no association was found between ERCC2 Lys751Gln and MSH2 −118T>C polymorphisms in conferring risk for GB carcinogenesis.

The DNA repair enzyme ERCC2 is a 5′ to 3′ DNA helicase, a subunit of transcription factor IIH that is essential for transcription and nucleotide excision repair. Host cell reactivation assays have shown that 312Asn and 751Gln alleles of the ERCC2 gene have a reduced capacity to repair benzo(a)pyrene diol epoxide (BPDE)- and ultraviolet-induced damage 27, 28 and also show lower repair efficiency. 29, 30 It is well known that the variant allele at codon 312 of the ERCC2 gene removes the acidic moiety of the aspartic acid, and that the variant allele at codon 751 changes the electronic configuration of the amino acid.31 Therefore, these polymorphisms could affect an individual's DNA repair capacity and thus a predisposition toward various malignancies. In the present study, higher risk was observed for the ERCC2 Asp312Asn polymorphism. In vitro studies by Spitz et al.27 have reported that ERCC2 variant alleles at amino acid residues 312 and 751 were associated with a reduced capacity to repair damage induced by benzo(a)pyrene diol epoxide in lung cancer patients. Moreover, this association is biologically plausible because the ERCC2 312Asn allele is associated with reduced DNA repair capacity,32 and meta-analysis studies in lung cancer have shown significant association with the Asn allele.33, 34 The ERCC2 Lys751Gln polymorphism did not reach statistical significance in our study. Although reports have shown that these 2 polymorphisms are in linkage disequilibrium,16, 27 similar results have also been obtained in other studies.35 From our analysis using structural-based (Polymorphism Phenotyping scores) and evolutionary-based (Sorting Intolerant From Tolerant index) approaches, we found that the ERCC2 Asp312Asn and Lys751Gln polymorphisms were benign and most likely lack any phenotypic effect. The haplotype analysis also did not show any significant association between ERCC2 haplotypes and GB carcinogenesis risk.

The DNA mismatch repair system consists of >10 genes, the major ones being MSH2, hMSH3, hMSH6, hMLH1, hPMS1, and hPMS2. 36 Inactivation of these genes through genetic or epigenetic mechanisms affects genome integrity and has been implicated in the etiology of various cancers such as hereditary nonpolyposis colon cancer, ovarian cancer, and endometrial cancer.37, 38 MSH2, a highly conserved mismatch repair protein, is primarily responsible for the correction of replication defects that are caused by DNA polymerase errors.39 Polymorphisms in this gene have previously been reported to be associated with various carcinomas such as lung,40 oral,10 and breast cancer.41 To test whether MSH2 genetic variants are associated with GB carcinogenesis risk, we evaluated the potential association of 2 MSH2 polymorphisms, −118T>C and IVS1 + 9G>C. Only the MSH2 IVS1 + 9G>C polymorphism was significantly associated with the risk of GB carcinogenesis, whereas the MSH2 −118T>C polymorphism was not found to be associated. A study by Marra et al.42 has shown a 20% decrease in repair efficiency in lymphoblastoid cell-line–harboring MSH2 IVS1 + 9G>C gene polymorphism. Similarly, an association was also observed with this polymorphism and oral squamous cell carcinoma recurrence.10 Previous studies have shown linkage disequilibrium between the MSH2 −118T>C and IVS1 + 9G>C polymorphisms.40 But in our study, haplotype analysis revealed that none of the MSH2 haplotypes was significantly associated with GB carcinogenesis risk.

The human DNA repair enzyme OGG1 is a DNA glycosylase/AP lyase that efficiently repairs 8-hydroxy-2′-deoxyguanosine, which is among the most abundant oxidative products in DNA. 8-Hydroxy-2′-deoxyguanosine is highly mutagenic in vitro and in vivo, giving rise to GC to TA transversions on DNA replication, which are frequent in several oncogenes and tumor suppressor genes. 43, 44 We earlier reported an association with OGG1 Ser326Cys in a case-control study of GB carcinogenesis.45 Previous reports have shown significant association between OGG1 Ser326Cys polymorphism and risk of developing various cancers, including esophageal,46 lung,26 and GB carcinogenesis.9 In addition to OGG1 Ser326Cys, we also evaluated the influence of another polymorphism of OGG1 gene present in intron 4 (748-15C>G). Similar to risk conferred by the OGG1 Cys/Cys genotype, 748-15C>G, also showed significant association with GB carcinogenesis risk. This is an intronic polymorphism residing in intron 4 of the OGG1 gene, and frequent allelic imbalance in this region has been shown to be involved in head and neck squamous carcinogenesis.47 It seems that this polymorphism is involved in the splicing of OGG1 mRNA. Moreover, both of these polymorphisms followed a dominant model in their effect on GB carcinogenesis risk. Haplotypes can increase the power to identify disease associations compared with single polymorphism studies. Haplotype analysis revealed that of the 4 possible haplotypes, the G-G haplotype of the OGG1 Ser326Cys and 748-15C>G polymorphism was significantly associated with >2-fold increased risk of GB carcinogenesis. Moreover, when OGG1 Ser326Cys and OGG1 748-15C>G SNPs were combined, risk of GB carcinogenesis amplified as the number of OGG1 at-risk genotypes increased, suggesting that these 2 OGG1 SNPs may interact to contribute collectively to risk of GB carcinogenesis. However, considering the borderline CI and the multiple comparison issue, we cannot rule out type I error for this association.

Interaction between ERCC2 and MSH2 polymorphisms showed an increased risk of GB carcinogenesis (OR, 2.2). Similar interaction was found between MSH2 and OGG1 polymorphisms, suggesting that coordination between the analyzed DNA repair genes might contribute to the susceptibility toward GB carcinogenesis. We found significantly increased risk with increasing numbers of variant alleles of the DNA repair gene pathway. It was observed that individuals with >6 variant alleles of the studied polymorphisms were associated with a significantly greater risk of GB carcinogenesis compared with individuals carrying 0-2 at-risk alleles by up to 4-fold. The difference in the pooled effects of multiple polymorphisms may reveal the differences in the contribution of each DNA repair polymorphism to GB carcinogenesis risk as found by Matullo et al. 48

Stratified analysis showed varied results in different polymorphisms for their ability in modifying the GB carcinogenesis risk. Gallstones have been shown to be a strong risk factor for GB carcinogenesis. 49 The prevalence of gallstones reported in GB carcinogenesis patients in Western countries ranges from 74% to 92%; the prevalence of gallstones in the Indian population is estimated to be 50% to 70%.50 The OGG1 748-15G and OGG1 326Cys alleles showed significant association for GB carcinogenesis risk in patients with gallstones. Similar results were also observed by Jiao et al.9 However, ERCC2 Lys751Gln polymorphism showed significant association only in GB carcinogenesis patients without gallstones.

Stratification of GB carcinogenesis patients on the basis of sex showed that most of our studied DNA repair gene polymorphisms were statistically significant in the female cohort. This is consistent with the finding that GB carcinogenesis is a female-predominant disease. 3 However, there is limited information regarding the role of sex-specific variation in DNA repair capacity.51

No association was found with tobacco usage in our gene-environment study. Although our study was designed to have 80% power, our sample size may not have been adequate to identify significant associations in different strata in some subgroups, or to detect gene-environment interactions effectively.

GB carcinogenesis is a multifactorial and multistep disease with complex interplay between various genetic and environmental factors; we therefore performed CART analysis to further reveal the high-order gene-gene interactions of the DNA repair pathway in GB carcinogenesis development. We found that OGG1 Ser326Cys SNP was the best polymorphic signature for discriminating between cases and controls, which is consistent with the finding from the main effect analysis. The CART analysis grouped the study subjects according to different risk levels on the basis of the DNA repair gene polymorphisms. This shows that the development of GB carcinogenesis involves complex genetic interactions depending on the specific environmental exposures of the subjects. Our results should be interpreted with caution because of the limited number of subjects in some of the CART terminal nodes.

Our study has several strengths, such as that all our control subjects were under Hardy-Weinberg equilibrium, all our cases were histopathologically confirmed, matched cases and controls, and strict quality control for genotyping was used. To limit potential confounding false-positive associations because of moderate sample size and population stratification, we used software as described by Wacholder et al. 17 to filter out false-positive associations by setting very rigorous prior probabilities. However, the inclusion of SNPs in our study was based on potential functional role in genes with higher potential of being associated with cancer risk; a more comprehensive approach including tagging SNPs would present more persuasive support for the associations. This study attempted to determine which biomarkers of DNA repair pathway are useful for screening high-risk individuals for prevention and early detection of GB carcinogenesis. To further evaluate these polymorphisms and GB carcinogenesis risk, a much larger sample size and various expression studies are required. Moreover, independent validations done in a larger sample size will also be required to confirm small associations and to determine complex inter-relationships between the polymorphisms along with gene-gene and gene-environment interactions. However, like most association studies, there is definitely a need to replicate the study in different populations.

CONFLICT OF INTEREST DISCLOSURES

Supported by research and fellowship grants from the Counsel of Scientific and Industrial Research, government of India.

The authors declare that there are no conflicts of interest.

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