Introduction

Gallbladder cancer (GBC) is usually the most prevalent malignancy in the biliary tract, which represents 80–95 % associated with biliary tract cancer around the world [1, 2]. GBC is the fifth most commonly occurring gastrointestinal cancer in the USA [3]. The worldwide rate regarding GBC malignancy exhibits variations, achieving epidemic levels for a few regions and also ethnicities. GBC carries an especially high occurrence in Chile, Japan, and also north India. Incidence of GBC is higher in north India as compared to south India [4–6]. Exact cause of etiology of GBC is still obscure. Various associated risk factors have been identified. Limited information is available related to genetic changes involved in GBC. The precise sequence associated with molecular alteration underlying GBC pathogenesis stays ambiguous. These genetic determinants within gallbladder carcinogenesis are generally inadequately identified, irrespective of around 1281 gene mutations that have already been discovered up to now [2].

The human 5,10-methylenetetrahydrofolate reductase (MTHFR) gene is located at chromosomal region 1p36.3 and consists of 11 exons spanning 2.2 kb [7, 8]. MTHFRis an important regulatory enzyme in the metabolism of homocysteine and folate [9] which is essential for DNA synthesis. It works as a key enzyme in the methylation process catalyzing reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Reduction in MTHFR activity increases the accumulation of 5,10-methylenetetrahydrofolate; this change may lead to the overall level of DNA damage in the cell [10]. Thus, folate deficiency induces chromosomal damage, formation of fragile sites, and found to be associated with tumorigenesis [11]. Deficiency of folate has been reported in general Indian population which may be an additional dietary factor for high risk of GBC in this population [12]. Changes in its activity resulting from polymorphism in the MTHFR could modify interindividual susceptibility to cancer. MTHFR is highly polymorphic in the general population. The two most common single nucleotide polymorphisms (SNPs), C677T and A1298C, have been identified [7, 13]. It has been reported that two single nucleotide polymorphisms (SNPs) of MTHFR gene, C677T (rs1801133 and Ala222Val), which results in the amino acid product changing from alanine to valine [7, 13], and A1298C (rs1801131 and Glu429Ala), which results in the amino acid product changing from glutamic acid to alanine [14] have been linked to the reduced enzyme activity [15]. The second common MTHFR polymorphism, a glutamate to alanine (A3 C) change at position 1298, also influences the specific activity of the enzyme, homocysteine levels, and plasma folate concentration [14].

It has been suggested that MTHFR C677T polymorphism and A1298C modulated the risk of developing various cancers like colorectal, gastric, and endometrial as well as leukemia [16–21]. However, the association involving these two frequent occurring MTHFR polymorphisms and the risk of developing GBC has not been looked at to our best knowledge. Thus, the aim of this work is to study the C677T and A1298C polymorphisms of the 5,10-MTHFR gene in patients with gallbladder cancer and to co-relate with various clinicopathological parameters.

Material and Methods

Subjects

Thirty patients of histopathologically proven GBC as cases, 30 cases of gallstones, and 30 cases of normal healthy control were recruited for study; 5 ml blood was collected in heparinized syringes from cases and controls. Samples were collected from the department of general surgery, and work was performed in the department of surgical oncology laboratory. Patients having diabetes, cardiovascular, renal and venous thrombotic diseases were excluded in the study. This study was ethically approved by the institute’s ethics committee. Informed consent was obtained from all individual participants included in the study.

Polymerase Chain Reaction

DNA isolation from blood had been performed by using phenol-chloroform method [22]. Polymerase chain reaction had been performed by using primer for MTHFR A1298C [13] and C677T [23]. PCR program for C677T was 95 °C for 2 min, 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s followed by 35 cycles and final extension at 72 °C for 10 min. While PCR program for A1298C was initial denaturation at 92 °C for 5 min, denaturation at 92 °C for 60 s, annealing at 52 °C for 60 s, and extension at 72 °C for 30 s followed by 35 cycles and final extension at 72 °C for 7 min.

Restriction Fragment Length Polymorphism

Genotyping was performed by restriction fragment length polymorphism (RFLP) method. MTHFR C677T was detected by digestion with HinfI (NEB) restriction enzyme in a total reaction volume of 20 μl and incubated at 37 °C for 1 h. It produced 175 and 23 bp fragments for homozygous condition (TT) and 198, 175, and 23 bp fragments for heterozygous condition (CT). While there was no restriction site for wild type, a 198-bp band was retained.

MboII restriction enzyme was used for A1298C MTHFR polymorphism detection, and digestion was performed in a total reaction volume of 20 μl and incubated at 37 °C for 1 h. It produced six fragments of 84, 56, 31, 30, 28, and 18 bp for 1298AC heterozygous type, five fragments for 56, 31, 30, 28, and 18 bp for 1298CC homozygous type, and four fragments of 84, 31, 30, and 18 bp for 1298 AA genotype.

Statistical Analysis

Statistical analysis had been performed by using SPSS (version 16.0, Chicago, IL, USA) software. Allele frequency has been calculated for each genotype under the assumption of Hardy-Weinberg equilibrium. The association between GBC and MTHFR polymorphism were analyzed by determining the odds ratio (OR) and confidence interval (CI) by doing univariate logistic regression. Correlation of both polymorphisms with different clinicoparameters was calculated by Pearson’s correlation test.

Results

The clinical characteristics of all the groups are listed in Table 1. The mean age of GBC, cholelithiasis, and healthy control were 56 ± 14, 44 ± 13, and 44 ± 13 years, respectively. Most of the patients were females in our study group. Female:male ratio was 6.5:1 in GBC group; 93.7 % GBC patients were of adenocarcinoma histopathology and 6.7 % cases of squamous cell carcinoma. Gallstones were present in the 63.3 % cases of gallbladder cancer. Of those, 16.7% were single and 46.7% multiple stones (Table 1).

Table 1 Clinical characteristic of GBC patients, cholelithiasis and healthy control
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The distribution of C677T and A1298C genotypes in all the three groups is shown in Table 2. No statistically significant differences were noted among MTHFR 677CC, CT, and TT genotypes (p = 0.85). Genotype frequency of A1298C polymorphism with AC, CC, and AA has showed a statistically significant difference among the groups (p < 0.001). AC genotype was present in 60% of gallbladder cancer cases in which gallstones were present. While both CC and AA genotypes were present in only two cases of gallbladder cancer (Table 3).

Table 2 Comparison of MTHFR C677T and A1298C genotypes between GBC, cholelithiasis, and control
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Table 3 Distribution of A1298C MTHFR polymorphism with presence of gallstone in gallbladder cancer patients
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Univariate logistic regression analysis has been performed to check the association of C677T and A1298C polymorphism among the three groups (gallbladder cancer, cholelithiasis, and normal control). When GBC has been assumed as case and normal healthy volunteer as control, A1298C was found to be highly significant (p < 0.001) while C677T was not significant (p = 1.0). In another case, when cholelithiasis group has been considered as case and healthy volunteer as control, then no statistically significant difference was found (p = 1.0), whereas A198C have exhibited a statistically significant difference (AC—CI 6.5, p < 0.001). Similarly, when GBC was taken as case and cholelithiasis patients as control group, then again, C677T did not show any significant difference in all the genotypes (p = 1.0) and a statistically significant difference was observed in A1298C polymorphism (AC—CI 4.902, p < 0.05) (Table 4).

Table 4 Univariate logistic regression analysis to look at the association between C677T and A1298C polymorphism and gallbladder cancer
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Correlation between the MTHFR polymorphism of cases and controls with different factors has been evaluated. Overall, there was no correlation between the C677T with any of the parameters. A1298C MTHFR genotype was significantly correlated with grade (r = 0.337; p < 0.001) and histopathology (r = 0.466; p<0.001) of the patients (Table 5).

Table 5 Correlation of C677T and A1298C polymorphism with different clinicopathological factors
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Discussion

In this study, genotype frequencies for MTHFR C677T polymorphism did not differ between cancer cases and controls (χ 2 = 2.44; p = 0.85). We were not able to find any evidence for the existence of this gene effect. The overall genotype difference was not statistically significant. Thus, we did not find any association of C677T polymorphism in GBC with different parameters. We observed no major significant association of the MTHFR C677T polymorphism with GBC risk. A single study done by Srivastava et al. also concluded that genetic risk for GBC is not modulated by MTHFR C677T polymorphism [24]. They inferred from their results that MTHFR C677T polymorphism is neither a risk nor a protective factor for GBC, suggesting that MTHFR genetic variation does not affect the susceptibility for gallbladder tumorigenesis [23]. Other study also reported that the C677T MTHFR polymorphism did not significantly contribute to the inherited genetic susceptibility to breast and prostate cancer, while they got some evidence for possible genetic contribution of MTHFR polymorphism to the development of head and neck carcinoma [25]. It has already been reported that frequency of C677T polymorphism is low in Indian population [26]. A preliminary study has been done which suggests that individuals with the TT genotype have a decreased risk of adult acute leukemia and increased risk of endometrial cancer, cervical neoplasia, and breast cancer [27]. A recent meta-analysis also suggested a significant association between thyroid cancer and MTHFR C677T polymorphism [28]. Another study performed by Yilmaz et al. suggested no association of MTHFR C677T with lung cancer among the Turkish population [29]. Some other case control studies found to have increased risk for other cancers including breast [30, 31], colorectal [32], lung [33], and head and neck [25].

To the best of our knowledge, this is the first report to examine the association between the MTHFR A1298C gene polymorphisms and risk of gallbladder cancer. In our study, the frequencies of the MTHFR 1298CC, 1298AC, and 1298AT genotypes were 6.7, 83.3, and 10 % in GBC group (χ 2 = 28.87; p < 0.001). AC genotype was found unusually high in comparison to control and was highly significant. Only 20 % distribution of AC was observed in control while it was elevated to 60 % in cholelithiasis and even higher in GBC (83.3%). Moreover, the distribution of AC genotype was also prominent in GBC cases in which gallstones were present. Out of 19 GBC samples with gallstones, 15 samples had AC genotype suggesting an overlapping role of AC genotype in GBC and development of gallstones. To further understand the role of this polymorphism in bridging GBC and gallstone development, univariate logistic regression analysis was performed. Firstly, when GBC was taken as cases and normal gallbladder as control, AC genotype in A1298C MTHFR polymorphism showed almost 33 times higher risk of development of GBC than control (p < 0.001; OR = 31.944) while CC genotype had 15 times higher risk (p < 0.05; OR = 15.333). Secondly, when cholelithiasis have taken as cases and normal gallbladder as control group, AC genotype showed a significantly higher risk of development of cholelithiasis (p < 0.001; OR = 6.516). Thereby, it signifies the role of this genotype in this polymorphism both in the development of GBC and cholelithiasis. Finally, when GBC was taken as case and cholelithiasis as control, then AC genotype of this polymorphism showed significantly related to the risk of development of GBC than cholelithiasis (p < 0.05; OR = 4.902). Thus, we can conclude that cholelithiasis sample with AC genotype of A1298C polymorphism is at a significantly higher risk of development of GBC. In addition, A1298C polymorphism was significantly correlated with the grade (r = 0.337, p < 0.001) and histopathology (r = 0.446, p < 0.001). Thus, we can also suggest here that A1298C polymorphism is contributing some risk to the development of gallbladder cancer. Nair et al. (2013) conducted a prospective case-control study to see the genetic association between methylenetetrahydrofolate reductase (MTHFR) A1298C polymorphism and recurrent pregnancy loss (RPL). In conclusion, they suggested that a significant risk of pregnancy loss associated with MTHFR A1298C polymorphism such that the presence of rare allele C and genotypes AC and CC significantly increases the risk of pregnancy loss [34, 35]. Zhang et al. also tried to see whether MTHFR C677T and A1298C polymorphisms could be associated with the sex-specific overall survival (OS) in patients with metastatic colon cancer treated with FU-based chemotherapy. They suggested the role of A1298C polymorphism in MTHFR as a prognostic marker in female patients with metastatic colon cancer [36]. Interestingly, others also concluded that A1298C polymorphism may be a predictor of colon cancer risk and have functional relevance [37]. A report also deduced that C677T and A1298C are not associated with the risk of developing lung cancer, with and without dietary folate intake [19]. Our findings support that the MTHFR A1298C polymorphism may be as relevant in predicting GBC but further studies are needed.

In conclusion, our study suggests that increased frequency of the A1298C MTHFR gene polymorphism observed may be associated with risk of developing GBC. Further research is needed to establish the relationship proposed by the present results. However, we did not find any association with C677T and gallbladder cancer.