J Gastroenterol (2009) 44:774–780 DOI 10.1007/s00535-009-0071-5
ORIGINAL ARTICLE—LIVER, PANCREAS, AND BILIARY TRACT
Functional polymorphisms of the cyclooxygenase (PTGS2) gene and risk for gallbladder cancer in a North Indian population Kshitij Srivastava Æ Anvesha Srivastava Æ Sachchida Nand Pandey Æ Ashok Kumar Æ Balraj Mittal
Received: 15 January 2009 / Accepted: 22 March 2009 / Published online: 20 May 2009 Ó Springer 2009
Abstract Background Cyclooxygenase-2 (PTGS2) overexpression has been implicated in various cancers. We aimed to evaluate the role of PTGS2 -1195G[A [reference sequence (rs) 689466], -765G[C (rs20417) and ?8473T[C (rs5275) polymorphisms in conferring interindividual susceptibility to gallbladder cancer. Materials and methods The study included 167 gallbladder cancer cases and 184 controls. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism. Risk was estimated using unconditional logistic regression. Results Significant risk was observed in the presence of PTGS2 -1195GA (P = 0.006; odds ratio = 2.00; 95% confidence interval = 1.2–3.3) and AA genotypes (P = 0.050; odds ratio = 2.98; 95% confidence interval = 1.0–8.9). Combined risk due to GA ? AA genotypes was 2.12 (P = 0.002; 95% confidence interval = 1.3–3.3; P-trend = 0.001). Sub-grouping showed a risk due to the PTGS2 -1195(GA ? AA) genotype in males (P = 0.007; odds ratio = 2.97; 95% confidence interval = 1.3–6.5), patients without gallstones (P = 0.001; odds ratio = 2.53; 95% confidence interval = 1.4–4.7) and with late-onset
K. Srivastava and A. Srivastava contributed equally to the manuscript. K. Srivastava A. Srivastava S. N. Pandey B. Mittal (&) Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow 226014, Uttar Pradesh, India e-mail:
[email protected];
[email protected] A. Kumar Department of Surgical Gastroenterology, SGPGIMS, Lucknow 226014, Uttar Pradesh, India
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gallbladder cancer (P = 0.012; odds ratio = 1.99; 95% confidence interval = 1.1–3.4). Gallbladder cancer patients who used tobacco were at increased risk in the presence of the PTGS2 -765GC genotype (P = 0.018; odds ratio = 2.96; 95% confidence interval = 1.2–7.2). Discussion An association of PTGS2 -1195G[A polymorphism with gallbladder cancer, particularly in patients without gallstones, suggests a direct role of PTGS2 in cancer development. Keywords GBC (gallbladder cancer) Haplotype PCR-RFLP Polymorphism PTGS2
Introduction Gallbladder cancer (GBC) is an aggressive tumor that extends early and results in rapid death. The incidence of GBC shows a marked regional specificity, with the highest incidence rate reported for women in Delhi, India (21.5/ 100,000) [1]. Although gallstones and primary sclerosing cholangitis have been associated with GBC, the exact mechanism of its etiopathogenesis remains unexplored [2]. Various evidence suggests that the causal pathway for GBC pathogenesis involves chronic inflammation of the biliary epithelium [3, 4]. Use of non-steroidal anti-inflammatory drugs (NSAIDs) has been shown to lower GBC risk [5, 6] by suppressing prostaglandin production through inhibition of the enzyme prostaglandin-endoperoxide synthase [PTGS, also commonly called cyclooxygenase (COX)]. PTGS2-derived PGE2 is the major pro-inflammatory prostaglandin produced in many human solid tumors [7]. Over-expression of PTGS2 may lead to disproportionate levels of PGE2, which plays a crucial role in cancer development, including hyper-proliferation, transformation,
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tumor growth, invasion and metastasis, resulting in an increased risk for cancer [8, 9]. It is possible that naturally occurring sequence variations affecting transcriptional and post-transcriptional regulation of PTGS2 gene expression might contribute to a substantial degree of inter-individual variability in susceptibility to cancer, as well as response to the treatment. Currently, three PTGS isoenzymes are known—PTGS1, PTGS2 and PTGS3. PTGS1 is constitutive in nature, while PTGS2 codes for an inducible enzyme that is expressed in response to tissue inflammation [10]. PTGS3 isoenzyme is a splice variant of PTGS1 that retains intron one and has a frameshift mutation. PTGS2 over-expression has been observed in malignancies of various organs, such as the colorectum, lung, breast, prostate, bladder, stomach and esophagus [11]. The human PTGS2 gene is located on chromosome 1q25.2–q25.3 and is about 8.3 kbp in size. The PTGS2 promoter region contains binding sites for several key cis-acting regulatory elements [12]. Several single nucleotide polymorphisms in the PTGS2 gene have been described so far, but only a few have functional significance. The -1195G[A (rs689466) polymorphism creates a c-MYB binding site, which results in higher transcriptional activity of the PTGS2 gene [13]. The -765G[C (rs20417) polymorphism is located within a putative SP1 binding site that lowers promoter activity [14]. Another genetic variation, located at the 30 -untranslated region (UTR) of the PTGS2 gene, ?8473T[C (rs5275), has been suggested to affect gene expression through altered messenger RNA stability and translational efficiency [15]. So far, there has only been one report exploring the role of a common genetic variant of PTGS2 in susceptibility to gallbladder cancer [16]. However, even after close association with various cancers, the role of genetic variants present in the promoter and 30 -UTR region of PTGS2 remains unexplored concerning susceptibility to GBC. Therefore, this study was designed to evaluate the association of PTGS2 -1195G[A, -765G[C and ?8473T[C polymorphisms with the risk of GBC in a North Indian population.
Materials and methods Study population This case control study comprised 167 consecutive newly diagnosed GBC patients [fine-needle aspirated cell cytology (FNAC) and histopathologically proven] recruited from the clinics of the Department of Gastroenterology and Gastro-surgery of Sanjay Gandhi Post Graduate Institute of
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Medical Sciences (SGPGIMS) in Lucknow, India, from March 2005 to March 2008. Staging of cancer was documented according to the American Joint Committee on Cancer. The clinical profile of GBC patients was based on hospital investigations. A total of 184 healthy controls were randomly selected from the general population and were age, gender and ethnicity matched to the patients. The inclusion criteria for controls were the 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 use. Both patients and controls had similar ethnicity. To test the possibility for population stratification, we used a genomic control method as described by Devlin et al. [17]. Tobacco usage in any form, such as smoking cigarettes, bidi (leaf-rolled unrefined tobacco) or chewing (non-smoking tobacco), was recorded. The majority of the female patients were housewives, and the male patients were not engaged in any hazardous occupations. The study was approved by the local ethics committee of the Institute. Sample DNA extraction A venous blood sample (5 ml) was collected from each subject and was kept frozen till DNA extraction. Genomic DNA was isolated from peripheral blood leukocytes by the salting-out protocol [18]. Extracted DNA was quantified using a Nanodrop Analyzer (ND-1000) spectrophotometer (Nano Drop Technologies, Inc., Wilmington, DE). Genotyping The PTGS2 polymorphisms were selected on the basis of their functional role, a reported prevalence of at least 5% for the variant allele and published evidence of an association with cancer. The genotypes were determined by the PCR-RFLP method, and the primers, annealing temperature, length of amplified fragments, restriction pattern and restriction enzymes used are listed in Table 1. The PCR was done using 50 ng of genomic DNA, 10 pmol of each primer and 1 9 PCR Master Mix (MBI Fermentas, USA) in a 10-ll reaction volume. The PCR reaction conditions used were as described previously [13]. As a negative control, the PCR mix without the DNA sample was used to ensure a contamination-free PCR product. Reaction products were digested with the respective restriction endonuclease (MBI Fermentas, USA) at 37°C for 16 h and resolved on 15% polyacrylamide gel. The digested PCR products were observed using UV image system (Vilber Lourmat, France).
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Table 1 Primer sequences, amplicon size, restriction enzyme and restriction pattern for PTGS2 polymorphisms Polymorphism
Primer sequences
Length (bp)
Restriction enzyme
-1195G[A (rs689466)
F: 50 -GCCCTTCATAGGAGATACTGG-30
272 bp
Pvu II
R: 50 -CCCTGAGCACTACCCATGAT-30
Restriction pattern (bp) G/G: 272 G/A: 272, 220, 52 A/A: 220, 52
-765G[C (rs20417)
0
0
F: 5 -GCTAAGTTGCTTTCAACAGAAGAAAT-3
100 bp
Hha I
R: 50 -TATTATGACGAGAATTTACCTTTCGC-30
G/G: 100 G/C: 25, 75, 100 C/C: 25, 75
?8473T[C (rs5275)
F: 50 -GTTTGAAATTTTAAAGTACTTTTGAT-30
147 bp
Bcl I
R: 50 -TTTCAAATTATTGTTTCATTGC-30
T/T: 147 T/C: 147, 123, 24 C/C: 123, 24
Quality control Twenty percent of samples from patients and controls, including samples of each genotype, were re-genotyped by other laboratory personnel. No discrepancy was found after sequencing randomly selected 10% samples. Subgroup stratifications To explore its effect, subjects were stratified according to gender. Stratification was also done on the basis of gallstone status and analyzed separately. GBC patients were also stratified on the basis of age of onset. Patients up to 49 years of age were denoted as early onset (\50 years), and patients above 50 years were denoted as late-onset patients (C50 years).
Table 2 Characteristic profile of controls and GBC patients Characteristic
Healthy controls n GBC case patients n (%) (%)
Total (N)
184
167
Male
63 (34.2)
65 (38.9)
Female
121 (65.8)
102 (61.1)
54.87 ± 7.341
53.57 ± 10.562
Gender
Age at interview, years ± SD Stages 0, I
None
II
9 (5.4)
III
55 (32.9)
IV
103 (61.7)
Gallstone present
None
83 (49.7)
Gallstone absent
All
84 (50.3)
Statistical analysis
Tobacco users
–
51 (30.5)
The sample size was calculated using the QUANTO 1.1 program (hydra.usc.edu/gxe). The desired power of our study was set at 80%. Descriptive statistics of patients and controls were presented as the mean and SDs for continuous measures, while frequencies and percentages were used for categorical measures. Genotype frequencies for each PTGS2 marker in controls were examined for deviation from Hardy–Weinberg equilibrium (HWE) using the v2 test. Chi-square analysis was used to compare categorical variables, using a 5% level of significance. Binary logistic regression analysis was used to estimate the odds ratio (OR) and its 95% confidence interval (CI) with age and gender adjusted. All statistical analyses were performed using SPSS software version 15.0 (SPSS, Chicago, IL). A trend test for each genotype was performed according to the number of copies of the variant allele (0, 1 or 2). Statistical analysis of the PTGS2 haplotypes estimation and linkage disequilibrium was conducted using the Arlequin software version 2.00 by expectation-maximization (EM) algorithm [19].
Results
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Population characteristics Baseline characteristics of GBC patients and their age- and gender-matched controls are presented in Table 2. The genomic control method ruled out the possibility of population stratification in our study. The mean age was not significantly different in GBC patients and controls. Gallstones were present in 49.7% of GBC patients, and most of the GBC patients were in advanced stages of cancer (stage III and stage IV). About 30.5% of the GBC patients were associated with tobacco usage in some form (smoking, chewing or both) (Table 2). All cancer patients were incident cases, and none of the controls had a family history of cancer. Genotype distribution of PTGS2 polymorphisms The distribution of -1195G[A, -765G[C and ?8473T[C PTGS2 genotypes is shown in Table 3. Distribution of
J Gastroenterol (2009) 44:774–780 Table 3 Case control polymorphisms Genotype
distribution
Control/case (N)
777 of
genotypes
of
PTGS2
OR (95% CI)a
P-value
-1195G[A (rs689466) GG
142/104
Reference
–
GA
37/52
2.00 (1.2–3.3)
0.006b
AA
5/11
2.98 (1.0–8.9)
0.051
GA ? AA
42/63
2.12 (1.3–3.4)
0.002b
P-trend = 0.001 -765G[C (rs20417) GG 131/127
Reference
–
GC
45/32
0.73 (0.4–1.2)
0.239
CC
8/8
1.06 (0.4–2.9)
0.906
GC ? CC
53/40
0.78 (0.4–1.2)
0.317
P-trend = 0.455 ?8473T[C (rs5275) TT
67/51
Reference
–
TC
88/91
1.36 (0.8–2.2)
0.196
CC
29/25
1.21 (0.6–2.1)
0.730
TC ? CC
117/116
1.30 (0.8–2.0)
0.247
P-trend = 0.481 Combined effects 0–2 variant alleles
158/136
Reference
–
[2 variant alleles
26/31
1.38 (0.7–2.4)
0.266
a
Adjusted for age and gender
increased risk for GBC (P = 0.025; OR = 11.67; 95%CI = 1.4–100.0). The combined variant-containing genotypes (GA ? AA) also showed statistically significant increased risk for GBC in males when compared to the wildtype genotype (P = 0.007; OR = 2.97; 95%CI = 1.3–6.5), which was significant even after Bonferroni adjustment for multiple comparisons showing an additive effect (Table 4). None of the genotypes of PTGS2 -765G[C and ?8473T[C were significantly associated with GBC even after stratification on the basis of gender (data not shown). PTGS2 polymorphisms and modulation of risk in presence of gallstone To explore the modulation of risk by the gallstone status, GBC patients were further stratified on the basis of presence or absence of gallstones and compared separately with controls. The PTGS2 -1195GA, AA and variant-containing (GA plus AA) genotypes showed statistical significance (P = 0.007, 0.026 and 0.001) and conferred substantial risk for GBC (OR = 2.32; 95%CI = 1.3–4.2), (OR = 4.03; 95%CI = 1.2–13.7) and (OR = 2.53; 95%CI = 1.4–4.7), respectively, in patients without gallstones (Table 4). The PTGS2 -765G[C and ?8473T[C genotypes did not confer any risk for GBC even after this stratification (data not shown).
b
Remained significant after Bonferroni adjustment for multiple comparisons
genotypes of all the polymorphisms in controls was in accordance with Hardy–Weinberg equilibrium (P [ 0.05). Association of PTGS2 polymorphisms with GBC On comparing the genotype frequency distribution in gallbladder cancer patients with that of controls, the frequency of the variant PTGS2 -1195GA genotype was associated with a significant increased risk (P = 0.006; OR = 2.00; 95%CI = 1.2–3.3) of GBC (remained significant after Bonferroni adjustment for multiple comparisons) (Table 3). The trend test was also significant (P = 0.001). The risk due to variant-containing genotypes (GA ? AA) was significant (P = 0.002; OR = 2.12; 95%CI = 1.3–3.3) when compared with homozygous wild-type GG genotype. However, none of the genotypes of PTGS2 -765G[C and ?8473T[C polymorphisms were significantly associated with susceptibility to gallbladder cancer (Table 3). To further explore the role of these polymorphisms in susceptibility to gallbladder cancer, GBC patients and controls were stratified on the basis of various host characteristics. In male GBC patients the variant genotype of PTGS2 -1195 polymorphism (AA) showed a significantly
PTGS2 polymorphisms and age of onset In order to further delineate the role of these polymorphisms, all the patients were divided in two groups on the basis of age of diagnosis. The patients were termed as early onset (aged 0–49 years) and late onset (aged 50 onwards) (Table 4). In the GBC patients having late onset of GBC, PTGS2 -1195 GA, AA and combined variant-containing genotypes (GA ? AA) were significantly associated with high risk of disease (P = 0.036; OR = 1.86; 95%CI = 1.0–3.3), (P = 0.041;OR = 3.57;95%CI = 1.0–12.1) and (P = 0.012; OR = 1.99; 95%CI = 1.1–3.4), respectively (Table 4). Haplotype analysis of PTGS2 polymorphisms There was significant linkage disequilibrium between PTGS2 -1195 and -765 loci (v2 = 50.12, D’ = 0.8502); -765 and 8473 loci (v2 = 19.42, D’ = 0.6277); -1195 and 8473 loci (v2 = 11.57, D’ = 0.8933). A total of seven haplotypes were observed in subjects (Table 5). Compared with the most common haplotype G-1195G-765T?8473, the A-1195G-765T?8473 haplotype was associated with a significantly increased risk of GBC (P = 0.027; OR = 2.20; 95%CI = 1.1–4.4). The P-value was also calculated by the log-likelihood ratio test (global haplotype association), which was also significant (P = 0.02). The frequency
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Table 4 Association of PTGS2 -1195G[A polymorphism with GBC risk stratified by host characteristics Host characteristic
Control/case
OR (95% CI)a
P-value
Control/case
OR (95% CI)a
P-value
Gallstone
N = 184/84
Gallstone absent
N = 184/83
Gallstone present
GG
142/49
Reference
142/55
Reference
GA
37/28
2.32 (1.3–4.2)
P = 0.007b
37/24
1.73 (0.9–3.2)
P = 0.078
AA
5/7
4.03 (1.2–13.7)
P = 0.026
5/4
2.05 (0.5–7.9)
P = 0.300
GA ? AA
42/35
2.53 (1.4–4.7)
P = 0.001b
42/28
1.77 (0.9–3.1)
P = 0.052
Gender
N = 121/102
Female
N = 63/65
Male
GG
92/67
Reference
50/37
Reference
GA
25/32
1.84 (1.0–3.4)
P = 0.054
12/20
2.28 (0.9–5.3)
P = 0.054
AA
4/3
1.04 (0.2–4.8)
P = 0.958
1/8
11.67 (1.4–100.0)
P = 0.025
GA ? AA
29/35
1.73 (0.9–3.1)
P = 0.071
13/28
2.97 (1.3–6.5)
P = 0.007b
Age of onset GG
N = 43/55 35/37
Early (\50 years) Reference
N = 141/112 107/67
Late (C50 years) Reference
GA
7/16
2.09 (0.7–5.8)
P = 0.157
30/36
1.86 (1.0–3.3)
P = 0.036
AA
1/2
1.28 (0.1–17.2)
P = 0.854
4/9
3.57 (1.0–12.1)
P = 0.041
GA ? AA
8/18
2.39 (0.8–6.6)
P = 0.097
34/45
1.99 (1.1–3.4)
P = 0.012b
a
Adjusted for age and gender
b
Remained significant after Bonferroni adjustment for multiple comparisons
Table 5 Frequency distribution of PTGS2 -1195G[A, -765G[C and ?8473T[C haplotypes in GBC patients and controls
a
Adjusted for age and gender
Haplotype
Haplotype frequencies (control)
Haplotype frequencies (GBC patients)
P-value
OR (95%CI)a
G-1195G-765T?8473
0.46
0.41
–
1 (Reference)
G-1195G-765C?8473
0.26
0.25
0.825
1.04 (0.7–1.5)
A-1195G-765T?8473 G-1195C-765C?8473
0.06 0.07
0.11 0.09
0.027 0.470
2.20 (1.1–4.4) 1.31 (0.6–2.8)
A-1195G-765C?8473
0.05
0.08
0.479
1.17 (0.8–1.8)
G-1195C-765T?8473
0.07
0.03
0.050
0.36 (0.1–1.0)
A-1195C-765T?8473
0.01
0.02
0.224
2.81 (0.5–14.3)
Global haplotype association P-value: 0.02
distributions of all other haplotypes between GBC patients and controls were almost similar and not associated with GBC risk.
Discussion Various factors appear to be responsible for the development of the multifactorial etiology of GBC, either as initiators (endo- and exogeneous mutagens) or promoters (chronic inflammation) [20]. Recent reports have shown elevated PTGS2 expression in up to 85% of colon carcinomas and 50% of colon adenoma [21]. Over-expression of PTGS2 was found to be a sufficient cause for tumorigenesis in animal models, and deletion of the PTGS2 gene suppresses tumor progression in mice predisposed to intestinal neoplasia [22]. These findings provide compelling evidence that PTGS2 is an obligatory player in human cancers.
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This study investigated the role of all three PTGS2 polymorphisms known to modulate the transcriptional activity and stability of mRNA in the susceptibility to GBC in a case control design. The worldwide frequency distribution of the polymorphisms analyzed in our study is provided in Table 6. The frequency distribution of the studied polymorphisms in our study was comparable to the European (CEU) population. We found that combined A allele carriers of -1195 G[A polymorphisms were significantly associated with risk of GBC, especially in males. The -1195AA genotype has also been associated with an increased risk of esophageal squamous cell carcinoma (ESCC) [13] and gastric cancer [23]. Studies have also shown that -1195A allele is able to bind c-MYB, one of the important transcription factors that activates PTGS2 expression. c-MYB is required to keep a balance among cell division, differentiation and survival [24]. Luciferase assays have also shown significantly increased transcriptional activity of the PTGS2 gene in
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Table 6 Worldwide allele frequency distribution of PTGS2 polymorphisms analyzed Population
Wild allele
Variant allele
European (CEU)a
0.858
0.142
Asian (HCB)a
0.489
0.511
Asian (JPT)a
0.500
0.500
Sub-Saharan African (YRI)a
0.925
0.075
Chinese [16]
0.49
0.51
0.87
0.13
European (CEU)a
0.819
0.181
Asian (HCB)a
0.933
0.067
Asian (JPT)a
0.943
0.057
-1195G[A (rs689466)
Current study -765G[C (rs20417)
Sub-Saharan African (YRI)
a
0.593
0.407
Chinese[13]
0.98
0.02
Current study
0.83
0.17
European (CEU)a
0.629
0.371
Asian (HCB)a
0.833
0.167
Asian (JPT)
0.818
0.182
Sub-Saharan African (YRI)a
0.331
0.669
Chinese [13]
0.83
0.17
Current study
0.60
0.40
?8473T[C (rs5275)
a
a
Hapmap data
individuals carrying the -1195A allele [13, 25]. The elevated levels of PTGS2 lead to overproduction of prostaglandins (PGE2), which, being procarcinogenic, can support tumor growth by various signaling pathways controlling angiogenesis, cell proliferation, suppression of immune responses, invasiveness and also inhibiting tumor cells apoptosis [7, 8, 10]. A previous study by Sakoda [16] focusing on some exonic and intronic genetic variants of the PTGS2 gene in susceptibility to GBC found no association in GBC, though some of their SNPs were found to be associated with other biliary tract cancers. This may be due to SNP selection as the SNPs in the promoter region have a larger influence and their observed association may be the reflection of other risk alleles in linkage disequilibrium. The risk for GBC due to increased PTGS2 expression, which results in increased inflammation, is the major pathway through which PTGS2 affects GBC susceptibility. The risk due to PTGS2 was not modulated by the presence of gallstones, suggesting a direct transcriptional up-regulation in the presence of the -1195 variant allele. Here it is worth mentioning that only a small fraction of patients with cholelithiasis (*1–3%) develop GBC, and also 50.3% of patients with GBC in our study were free of gallstones. Therefore, factors other than cholelithiasis may also significantly contribute to the etiopathogenesis of GBC. Risk
was also significantly associated with patients who manifest the disease in the late stage of life. Moreover, GBC is a late-onset cancer, and the majority of our patients are in this group. It appears that prolonged inflammation results in accumulation of DNA damage, which results in a late manifestation of the disease. In our population the -765G[C polymorphism was not associated with susceptibility to GBC, overall as well as in any of the subgroups. Similarly, no association was observed between the -765G[C polymorphism and risk of colorectal cancer [26]. In the present study, no association of ?8473T[C polymorphism and gallbladder cancer susceptibility was observed. Earlier, no association was observed with increased risk of breast cancer [27]. Although it has been suggested that the expression of PTGS2 depends on the complex interaction of multiple signals derived from the 30 -UTR, the role of the ?8473T[C polymorphism in susceptibility to GBC appears to be small. In various cancers, it has been shown that combined genetic variability at the PTGS2 locus plays a role in carcinogenic processes under specific conditions [28]. Out of the three studied polymorphisms, only PTGS2 -1195G[A was significantly associated with GBC risk in the single locus analyses, but the combined effect of variant genotypes of all the three loci (-1195G[A, -765G[C and 8473C[T) were found to be associated with a significantly increased risk of GBC development. Specifically, the haplotype A-1195G-765T8473 was associated with a significantly increased risk of gallbladder cancer. These data indicate that functional polymorphisms in the regulatory region of the PTGS2 gene may contribute to the etiology of GBC. In summary, this study adds to the importance of PTGS2-mediated inflammation and progression to gallbladder cancer. This is the first report exploring the association of -1195G[A, -765G[C and 30 -UTR ?8473T[C polymorphisms in GBC susceptibility. However, carcinogenesis is a complex process, and interrelated pathways of immunological response exist. Thus, the role of these polymorphisms along with other genes and the underlying gene-environment interaction needs to be explored in different populations. Acknowledgment The study was supported by fellowship grants from the Counsel of Scientific and Industrial Research (CSIR), Government of India. Conflict of interest statement no competing interests.
The author(s) declare that they have
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