A Novel Polymorphism in Codon 25 of the KRAS Gene Associated with Gallbladder Carcinoma Patients of the Eastern Part of India

Introduction

Gallbladder cancer (GBC) is more prevalent than other cancers of the extrahepatic biliary tract in North India (Kapoor and McMichael, 2003). The comparison of different population-based cancer registries indicated that GBC was one of the commonest causes of cancer-related mortality in women in northern and northeastern states of India (Nandakumar et al., 2005). The asymptomatic nature of the disease always remains a problem in the diagnosis and treatment (Rai et al., 2004). GBC is frequently associated with cholelithiasis and cholecystitis in about 75% of cases, but the incidence of documented GBC developing in the presence of cholelithiasis and cholecystitis is only about 0.5% (Bodzioch and Ogiela, 2009). The association of GBC with gallstone increased the risk from four to seven times than that for without gallstone (Geetha, 2002; Gupta and Shukla, 2004). Aside from gallstones and female gender, a number of associated risk factors appear to favor the development of GBC either as neoplastic initiators, such as unknown endo- and exobiotic mutagens, or as neoplastic promoters, including chronic inflammation and infection. The etiology of cholesterol cholelithiasis, like any other chronic disease, is thought to involve the interaction of genetic and environmental factors (Maurer et al., 2009; Wang et al., 2009). There are some reports of familial tendencies toward GBC, although they are based on a very small number of patients (Hsing et al., 2007). A potential relationship between genetic predisposition and GBC incidence has been suggested by population studies of gallstone prevalence in different latitudes (Fernandez et al., 1994).

Analysis of oncogenes or tumor suppressor genes associated with the multistep process of oncogenesis could be important not only for the diagnosis, prognosis, or the follow-up of the disease but also for the management of therapy. The KRAS proto-oncogene is thought to exert control over some of the mechanisms of cell growth and differentiation. The KRAS gene is located on chromosome 12p12.1 and is about 38 kilobases (kb) in length with four exons (O'Connell et al., 1987). This gene is converted to an active oncogene by point mutations significantly concentrated in codon 12, 13, or 61 (Brink et al., 2003). Having the above information, we presume that the KRAS gene is a candidate gene for GBC; only few studies so far have examined the KRAS gene polymorphisms and their association with GBC. Therefore, we conducted a hospital-based case-control study in the eastern part of India to determine the role of KRAS gene polymorphisms in the progression of GBC. An ancestral tribal population diversity cohort (DNA) available from the Anthropological Survey of India has also been screened for the polymorphisms.

Materials and Methods

A total of 150 individuals from the metropolitan and suburban regions of Kolkata were included in this study, recruited through the Cancer Centre Welfare Home and Research Institute, Kolkata, and the National Medical College, Kolkata, during October 2008 to June 2009. The 60 subjects (13 men and 47 women) with gallbladder carcinoma were histologically proved by either pathological examination of gallbladder mass or fine-needle aspiration cytology of inoperable cases. The control subjects comprised a total of 90 individuals (14 men and 76 women) with no diagnosed cancer. Any participant with cholangiocarcinoma was excluded from the study. A separate set of DNA samples (n = 280) from four ancestral tribal populations of the country from a population diversity cohort available from the Anthropological Survey of India comprising apparently normal healthy volunteers were screened for the polymorphisms. After obtaining informed consent, venipuncture on each study subject was performed and a blood specimen was collected into the ethylenediaminetetraacetic acid-containing vaccutainer. Genomic DNA was extracted from all participants, using a standard procedure described elsewhere (Sambrook et al., 1989). This study was approved by the Institutional Review Board of Anthropological Survey of India, Kolkata. Written informed consent was obtained from each participant.

Results

In this study, there were more women than men diagnosed with GBC. The mean age was not statistically significant between cancer cases (51.92 ± 8.42 years) and controls (45.02 ± 11.13 years). The presence of gallstones is also not statistically significant between cancer cases and controls (p = 0.795). None of the subjects in the case or control groups had point mutations, particularly within the mutational hot-spot codons 12, 13, and 61 of KRAS gene that occur at comparable frequencies in human malignancies. However, in this study, we detected one novel polymorphism at codon 25 in exon 1 of the KRAS gene (NC_000012.11: g.5611G>C; CAG>CAT;Gln25His). The polymorphism was predicted to be “not tolerated” by SIFT (probability score = 0.03) and probably “affect protein function.” PolyPhen-2 predicted it to be “possibly damaging” with a score of 0.803 (sensitivity: 0.72; specificity: 0.81). Some differences between the two mRNA structures of wild type and the variant type were observed (Supplementary Figs. S1 and S2; Supplementary Data are available online at www.liebertonline.com/gtmb). We did not find homozygous polymorphism (His25His) in either the cancer or control groups. The distribution of genotypes for this polymorphism along with its minor allele frequency in the GBC and control subjects is reported in Table 1. Observed genotype frequencies were found to be in Hardy-Weinberg equilibrium in the control group (p = 0.109), but not in patients (p = 0.005). This polymorphism was further analyzed for potential association with GBC. Multivariable logistic regression analysis with adjustment for age and sex revealed that the Gln25His polymorphism of the KRAS gene was significantly associated with the prevalence of GBC (Table 1). The additive model for Gln25His compared the GT genotype (53.33% and 28.89% in the GBC and control groups, respectively) with individuals with the GG genotype (46.67% and 71.11% in the GBC and control groups, respectively). The adjusted OR for recessive model was 0.355 (unadjusted p = 0.003).

The results of the analysis of four Indian populations are presented in Table 2. Analysis of the additional four populations revealed presence of the codon 12 mutation only in the Munda population, which followed Hardy-Weinberg equation (p = 0.280), whereas codon 25 polymorphism was observed in the Munda and Toto (East India) populations. Both the Munda and Toto populations followed Hardy-Weinberg equilibrium for codon 25 polymorphism (Table 2). The Kamar (Central India) and Kinnor (North India) populations did not show any evidence for the codon 25 polymorphism.

Discussion

The present study examined the association between the exons 1 and 2 SNPs, which also includes one novel polymorphism within the KRAS gene, and the occurrence of GBC in an unselected East Indian population. This study demonstrates that the incidence of gallbladder cancer is higher in female subjects than in male subjects. The sex-specific prevalence of GBC observed in the present study is consistent with earlier studies in different populations (Kato et al., 1989; Zatonski et al., 1992; Daly et al., 1993; Randi et al., 2006). The presence of gallstones in GBC and controls also demonstrates that gallstones are a very common condition in this region. Interestingly, we did not find the most frequently reported mutations at codons 12, 13, and 61 of KRAS gene that occur at comparable frequencies in human malignancies (Iwase et al., 1997; Wistuba et al., 1999; Brink et al., 2003; Kamisawa et al., 2009). We detected a G-to-T novel polymorphism in codon 25, which leads to a change in amino acid from glutamine to histidine (Gln25His) of the KRAS gene. We found that the Gln25His polymorphism at the KRAS gene was significantly associated with GBC. In silico analysis has shown that the KRAS p.Q25H variant is a disease-causing change predicted to be intolerant by SIFT and possibly damaging by PolyPhen-2. Some differences between the two mRNA structures of wild type and the variant type were observed. Thus, there might be a possibility that alteration in mRNA secondary structure due to this polymorphism has an effect at translational level in the mutant. In future, functional studies will certainly shed some light on the association between genotype and cancer phenotypes.

Cancer is caused mainly due to the activation of oncogenes and inactivation of tumor suppressor genes that occur in a permissive epigenetic milieu, resulting in various pathologic features. About 30% of human tumors carry ras gene mutations (Bos, 1989). Of the three genes in this family (composed of KRAS, N-ras, and H-ras), KRAS is the most frequently mutated member and is said to be one of the most activated oncogenes, with 17%-25% of all human tumors harboring an activating KRAS mutation (Kranenburg, 2005). Critical regions of the KRAS gene for oncogenic activation include codons 12, 13, 59, 61, and 63 (Grimmond et al., 1992). These activating mutations cause Ras to accumulate in the active GTP-bound state by impairing intrinsic GTPase activity and conferring resistance to GTPase-activating proteins (Zenker et al., 2007).

The incidence of mutations has been variously reported to be 17%-59% of gallbladder carcinomas and 23%-100% of bile duct carcinomas. KRAS mutation is more frequently detected in carcinomatous and dysplastic lesions in gallbladder carcinoma cases with gallstones than in those without stones. There was a large difference in the incidence of KRAS mutations between distal (47%-75%) and middle or proximal (0%-8%) bile duct carcinoma. The chronic inflammatory change caused by stone retention and long-time bile exposure may correlate with the Ras gene change. It is generally accepted that physical stimulation by stones is related to carcinogenesis in the gallbladder (Yamaguchi and Enjoji, 1988). Analysis of other ancestral populations also demonstrates that codon 12 is less frequent than codon 25 polymorphism in East Indian populations. Thus, the results of this study conflict with previous studies reporting mutations at codons 12, 13, 59, 61, and 63, thereby warranting population-specific screening, and has significance in light of the genetic structure of the Indian population and determination of an ancestral northern block based on a recent genome-wide analysis (Reich et al., 2009).