The Associated Ion between the VDR Gene Polymorphisms and Susceptibility to Hepatocellular Carcinoma and the Clinicopathological Features in Subjects Infected with HBV

Aim. To evaluate the possible association between the vitamin D receptor (VDR), single-nucleotide polymorphisms (SNPs), and hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV) infection. Method. 968 chronic HBV infection patients were enrolled, of which 436 patients were diagnosed HCC patients, and 532 were non-HCC patients. The clinicopathological characteristics of HCC were evaluated. The genotypes of VDR gene at FokI, BsmI, ApaI, and TaqI were determined. Results. The genotype frequencies of VDR FokI C>T polymorphism were significantly different between HCC and non-HCC groups. HCC patients had a higher prevalence of FokI TT genotype than non-HCC subjects. With FokI CC as reference, the TT carriage had a significantly higher risk for development of HCC after adjustments with age, sex, HBV infection time, α-fetoprotein, smoking status, and alcohol intake. In addition, we also found that the TT genotype carriage of FokI polymorphisms were associated with advanced tumor stage, presence of cirrhosis, and lymph node metastasis. The SNP at BsmI, ApaI, and TaqI did not show positive association with the risk and clinicopathological features of HCC. Conclusion. The FokI C>T polymorphisms may be used as a molecular marker to predict the risk and to evaluate the disease severity of HCC in those infected with HBV.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the second leading cause of cancer-related death in China [1,2]. The carcinogenesis of HCC is a multifactor, multistep, and complex process. It is known that multiple risk factors, including chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, cirrhosis, carcinogen exposure, and excessive alcohol consumption, contribute to hepatocarcinogenesis [3][4][5]. Epidemiological studies also showed that a variety of genetic factors mediate an individual's susceptibility to cancer [6][7][8]. The identification of genetic factors related to HCC susceptibility may help to elucidate the complex process of hepatocarcinogenesis and improve the scientific basis for preventative intervention.
The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily of ligand-inducible transcription factors, which are involved in many physiological processes, including cell growth and differentiation, embryonic development, and metabolic homeostasis. The role of VDR in cancer has recently attracted much attention [9][10][11].
Several single nucleotide polymorphisms (SNP) have been described in the VDR gene, and some SNPs are associated with tumor development. For instance, VDR polymorphisms have been related to the risk and prognosis of breast, prostate, skin, colon rectum, bladder and renal cell carcinoma, and malignant melanoma [12][13][14][15][16]. VDR polymorphisms have been investigated in the context of some chronic liver diseases, such as primary biliary cirrhosis and autoimmune hepatitis [17][18][19]. In a very recent published study, VDR  genetic polymorphisms are significantly associated with the occurrence of HCC in Caucasian patients with alcoholic liver cirrhosis [20]. In China, hepatitis virus infection is a highly endemic factor for HCC [21][22][23][24]. However, the association between the VDR gene polymorphisms and the risk and pathological development in Chinese subjects with chronic hepatitis virus infection remains unknown. This case-control study, therefore, aimed to evaluate the role of VDR gene SNPs in the susceptibility and clinicopathological status of HCC in Chinese subjects with chronic HBV infection.

Subjects and Specimen
Collection. This is a hospital-based case-control study. A total of 968 chronic HBV infection patients were enrolled at our hospital between Jan 2006 and Mar 2010. Of 968 patients, 436 patients were recruited as a HCC group, and 532 were enrolled as non-HCC group according to the presence or absence of HCC. We also enrolled 132 patients with determined HCC, but without HBV infection as control. The patients were diagnosed with HCC based on the characteristic criteria of the national guidelines for HCC, such as liver injury diagnosed by either histology or cytology irrespective of -fetoprotein (AFP) titer where imaging data showed any one of following three items: (1) one or more liver masses ≥2 cm in diameter via both computed tomography and magnetic resonance imaging; (2) imaging data with early enhancement and a high level of AFP ≥400 ng/mL; (3) imaging data with early arterial phase contrast enhancement plus early venous phase contrast washout regardless of AFP level. Liver cirrhosis was determined by histology, imaging, or clinical indications, such as esophageal varices or ascites. Age, gender, smoke status, and alcohol use were recorded. Relevant medical information including stage of HCC, liver cirrhosis history, lymph node metastasis, portal invasion, AFP, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) was also collected from patients by medical chart review. Heavy alcohol intake was defined as ethanol intake ≥80 g/day for >10 years. Patients with other cancers and chronic diseases, especially kidney, bone metabolism diseases were strictly excluded. This study was approved by the Institutional Review Board of our Hospital, and written informed consent was obtained from all study subjects.

Results
The characteristics of the study population are presented in Table 2. There were no significant differences of age, gender, HBV infection time, smoker status, and family cancer history between HCC and non-HCC. However, HCC group had a significantly higher rate of heavy alcohol intake compared with controls ( = 0.034). We observed a significant difference in BMI between HCC and non-HCC subjects. The mean serum AFP levels were significantly higher in HCC subjects than non-HCC subjects (all < 0.001). HCC patients had a significantly higher rate of liver cirrhosis compared with non-HCC patients ( < 0.001).
The genotype frequencies of VDR gene in HCC and non-HCC subjects are presented in Table 3. The genotype frequencies of all SNPs in control patients were in Hardy-Weinberg equilibrium (all > 0.05). The genotype frequencies of VDR FokI C>T polymorphism were To identify the independent risk factor for the development of HCC, we performed the multivariate regression analyses. With FokI CC as reference, our data showed that the TT carriage had a significantly higher risk for development of HCC after adjustments with age, gender, HBV infection time, smoker status, family cancer history, alcohol intake, BMI, and serum AFP level (OR = 2.269, = 0.006). With C allele as reference, the OR of T allele carriage for HCC was 1.553 ( < 0.001). For SNPs of BsmI, ApaI, and TaqI, their genotype and allele frequencies did not significantly differ between HCC and non-HCC group (all > 0.05). Multivariate regression analyses showed no association between the above-mentioned SNPs of VDR gene and susceptibility of HCC in this study. Table 4 showed the genotype frequencies of VDR gene in HCC patients with chronic HBV infection and HCC patient without HBV infection. We found that the VDR genotype frequencies were similar between HCC with HBV infection and HCC without HBV infection. None of the VDR SNPs showed significant difference between HCC with HBV infection and HCC without HBV infection (All > 0.05).
We further analyzed the association between VDR polymorphisms and the clinicopathological features in HCC subjects (Table 5). We found that only the SNPs at FokI locus were significantly different when all HCC patients were stratified by tumor stage, presence of liver cirrhosis history, and lymph node metastasis (all < 0.05), but not tumor size and portal invasion. We further performed the multivariate regression analyses to explore the role of SNPs at FokI locus in determining the clinicopathological features in HCC subjects. Taking the CC genotype as reference, we found that the TT genotype carriage of FokI polymorphisms was associated with advanced tumor stage (OR for III + IV stage = 2.335, = 0.001), presence of cirrhosis (OR for presence = 2.714, < 0.001), and lymph node metastasis (OR for presence of lymph node metastasis = 2.122, = 0.004).
No association between the FokI polymorphisms, tumor size, and portal invasion was observed (both adjusted > 0.05). The SNP at other loci including BsmI, ApaI, and TaqI did not show positive association with the the clinicopathological features of HCC (data not shown).
AFP is the common clinical pathological markers of HCC. We compared the serum AFP levels among different VDR genotype carriers in HCC patients. We found HCC patients with FokI TT genotype had a much higher AFP level than CT and CC carriers (Figure 1(a), = 0.011 versus CC; = 0.015 versus CT, resp.). The AFP levels were similar among BsmI, ApaI, and TaqI genotype carriers (data not shown). We also compared the liver function marker, namely, serum ALT and AST level according to the VDR genotypes. We found that only the FokI TT carriers had higher serum AST and ALT levels compared with TC and CC (all < 0.05, Figures 1(b) and 1(c)) in HBV infection patient with HCC, but not in the HBV infection patients without HCC (data not shown).

Discussion
In the study, we investigated the possible association between the VDR gene polymorphisms and HCC in a Chinese population with HBV infection. We found that the FokI C>T polymorphisms was significantly associated with the susceptibility and clinical features of HCC, including advanced tumor stage, presence of liver cirrhosis history, and lymph node metastasis. Since the association between the VDR genetic polymorphism and HCC had not been previously reported, our data, for the first time, provide new information in this aspect. The results of this study suggest the FokI C>T polymorphisms may be used as a molecular marker to predict the risk and to evaluate the disease severity of HCC in those infected with HBV. Hepatitis virus infection is associated with the increase of oxidative stress in liver cells and results in DNA changes and instability, thus increasing the risk of developing cirrhosis and/or HCC [21,25]. Some previous studies provided evidence that genetic polymorphisms of certain genes may predict the HCC occurrence in hepatitis virus infection [26][27][28][29].
The VDR is an intracellular hormone receptor that specifically binds the biologically active form of vitamin D, 1,25-dihydroxyvitamin D or calcitriol and interacts with specific nucleotide sequences (response elements) of target genes to produce a variety of biologic effects [30]. The VDR gene is located on chromosome 12q12-q14, and several single-nucleotide polymorphisms have been identified that may influence cancer risk [31].
The FokI restriction fragment length polymorphism, located in the coding region of the VDR gene, results in the production of a VDR protein that is three amino acids longer and functionally less effective [32]. Arai et al. demonstrated that compared with the FokI T/T genotype, FokI C/C had 1.7fold greater function of vitamin D-dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells [33]. It has been hypothesized that a less active VDR could be associated with either an increased susceptibility to cancer risk or to a more aggressive disease [31].
Vogel et al. detected a significant association between the F allele of the FokI C>T (F/f) polymorphism and autoimmune hepatitis patients, indicating a genetic link of VDR polymorphisms to autoimmune liver diseases such as primary biliary cirrhosis in German patients [19]. In Chinese population, Fan et al. reported a significant difference in FokI polymorphism between autoimmune hepatitis patients and controls, and a significant association in BsmI polymorphisms between primary biliary cirrhosis patients and controls [34]. In Italian population who underwent liver transplantation with the etiologies of liver disease as hepatitis C, hepatitis B, and alcoholic liver disease, carriage of the GG genotype of BsmI and the TT genotype of TaqI was significantly associated with occurrence of HCC. The authors concluded that VDR genetic polymorphisms are significantly associated with the occurrence of HCC in patients with liver cirrhosis, and this relationship is more specific for patients with an alcoholic etiology [20]. Our study showed that only the FokI C>T polymorphisms were associated with the HCC susceptibility in Chinese patient with chronic HBV infection; the other SNPs, including BsmI, were not related with the development of HCC. The HCC patients had a significantly higher cirrhosis rate than non-HCC patients ( < 0001), suggesting that the VDR polymorphisms at FokI locus may relate to HCC risk by enhancing the occurrence of liver cirrhosis. Although many factors may be account for the discrepancies among these studies, the ethnic difference should be predominately considered since Fan et al. found the distribution of FokI, BsmI, ApaI, and TaqI gene types significantly differed between Chinese healthy controls and Caucasian healthy controls [34].
The prognostic role of FokI C>T (F/f) polymorphism in cancer patients has been reported. Hama et al. reported the VDR FokI TT genotype was associated with a poor progression-free survival rate in patients with head and neck squamous cell carcinoma. In contrast, the other polymorphisms (BsmI, ApaI, and TaqI) showed no significant associations with progression-free survival [35]. In this study, we did not perform prognostic analyses; however, we found that the TT genotype carriage of TaqI polymorphisms was associated with advanced tumor stage, worse tumor differentiation, presence of cirrhosis, and lymph node metastasis. In addition, the TT carriers had higher serum AFP level than CC and CT carriers. The SNP at other loci including BsmI, ApaI, and TaqI did not show positive association with the clinicopathological features of HCC. Since tumor stage, tumor differentiation, presence of cirrhosis, and lymph node metastasis are conventional factors associated with HCC prognosis, our data imply that the TT carriers had poorer prognosis than CC and CT carriers.
Some limitation in this study should be addressed. Firstly, this is a hospital-based case-control study, thus the enrollment bias was inevitable. Secondly, no information was available regarding vitamin D intake (dietary or supplemental) and circulating vitamin D levels of patients. Thirdly, we only enrolled subjects with HBV infection, and all the subjects were Chinese. As HCV infection is another major cause for HCC in China, future study with larger sample size including HCV subjects is warranted.