Vitamin D receptor gene polymorphism and hepatocellular carcinoma in chronic hepatitis C patients

Hepatocellular carcinoma (HCC) is a prevalent malignancy worldwide. Vitamin D receptor (VDR) gene polymorphisms were linked to different cancers. This study was carried out to assess the possible relation between VDR gene polymorphism and the occurrence of HCC in chronic hepatitis C patients. This study included 102 subjects classified into three groups. Group A included 34 healthy subjects as control. Group B included 34 chronic hepatitis C patients with HCC. Group C included 34 chronic hepatitis C patients without HCC. Estimation of Apa-1 VDR gene polymorphism was performed by restriction fragment length polymorphism-Polymerase chain reaction (RFLP-PCR). In HCC group, C allele was more frequent than A allele (80.88% and 19.12%), respectively. In chronic hepatitis group, C allele was more frequent than A allele (64.71% and 35.29%), respectively. In control group, A allele was more frequent than C allele (73.53% and 26.47%), respectively. Genotype CC + CA was dominant in HCC group (91.18%) and chronic hepatitis group (79.41%). In the control group, the dominant genotype was AA (58.82%). Moreover, there was a significant relation between Apa-1 VDR genotype CC and tumor size. There is an association between VDR Apa-1 polymorphism and the occurrence of HCC in chronic hepatitis C patients.


Background
Hepatitis C virus (HCV) infection is a common health problem worldwide. Chronic HCV commonly proceeds to cirrhosis which may predispose to hepatocellular carcinoma (HCC). Hepatocarcinogenesis is affected by environmental and genetic features [1].
Risk factors for HCC include alcohol use, viral hepatitis, and metabolic diseases. Host genetics may play an important role. Improving our recognition of molecular factors may help us in early detection, stratification, individual treatment, and predicting the prognosis [2].
The importance of vitamin D has widened, from being only involved in bone metabolism to a broad scope of other physiological and pathological processes. It has an effect on immunity, intellectual and fetal development, insulin secretion, cancer, cell proliferation, and differentiation, and the cardiovascular system via the vitamin D receptor (VDR) which is a nuclear receptor. Vitamin D binds to its receptor to form a complex that enters the nucleus and binds to DNA regulating gene expression [3].
The VDR gene is located in chromosome 12q and has multiple allelic variants. Some of these variants may cause changes in the VDR function and may lead to immune-mediated disorders and cancer development. The most common of these variants are single nucleotide polymorphisms (SNPs). Four polymorphisms, Fok1, Bsm1, APa1, and Taq1, are being most investigated [4].
Several studies were performed and proved the association between VDR gene polymorphism and different types of cancer as cancer breast, prostate, bladder, and kidney. In our study, we clarified the effect of VDR gene polymorphism Apa1 in the occurrence of HCC among chronic hepatitis C patients.

Methods
This is a case-control study, and it was conducted in the Medical Biochemistry& Molecular Biology and Internal medicine Departments, Faculty of Medicine, Zagazig University, Egypt. Our study included 102 subjects classified into three groups. Group A comprised of 34 healthy subjects as control. Group B comprised of 34 chronic hepatitis C patients with HCC. Group C comprised of 34 chronic hepatitis C patients without HCC. Written informed consents from the participants were obtained before being involved in the study.
The patients participated in the study were chronic hepatitis C infection diagnosed by HCV-Ab and quantitative real-time polymerase chain reaction (PCR) and HCC was diagnosed by a triphasic CT scan. Patients excluded from the study were those with chronic hepatitis B infection, patients with other chronic liver diseases, patients having any other malignancy and patients who had diabetes mellitus, chronic renal failure or bone disorders.
All the participants were completely evaluated by complete history, examination, and full investigations in the form of laboratory studies as complete blood count (CBC) and complete renal and liver functions. DNA extraction was performed from whole blood from all cases. Serum levels of alpha-fetoprotein (AFP) were assayed using enzyme linked immunosorbent assay (ELISA).
Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was done to recognize the distribution of allele and genotype frequencies of the Apa-1 C/A polymorphism in the VDR gene. Genomic DNA extraction was performed from whole blood using GeneJET whole Blood Genomic DNA Extraction Mini Kit (Molecular Biology, Thermo Fisher Scientific, USA). The following primers: F: 5′-CAGAGCATGGACAGGG AGCAA-3′ and R: 5′-GCAACTCCTCATGGCTGA GGTCTC-3′ [3] were used.
Cycling conditions were carried out using the Thermal Cycler. An initial denaturation at 95°C for 2 min was performed, followed by 40 cycles; each cycle consisted of denaturation at 94°C for 45 s then annealing at 67°C for 45 s, elongation at 72°C for 60 s. The final elongation at 72°C for 2 min was carried out after the last PCR cycle. The PCR products were digested with Apa-I restriction enzyme. The following reaction components were mixed gently at room temperature: 7 μl nuclease-free water, 2 μl 10 × fast digest green buffer, 1 μl Apa-I restriction enzyme (Thermo Fisher Scientific), and 10 μl of DNA amplification product. The mixture was incubated at 65°C for 5 min. The reaction mixture was loaded directly and electrophoresed on 2% agarose gel containing ethidium bromide and visualized under ultraviolet illumination. PCR products with C at the polymorphic site were digested into two fragments, 531 and 214 bp, where those with A were not because of the absence of Apa-I enzyme restriction site. Samples yielding 531 bp and 214 bp fragments were scored as C/C, those with single 745 bp fragment as A/A, and 745-bp, 531-bp, and 214-bp fragments as A/C. The amount of size marker to load on the gel is 10 μl per lane and the result was photographed (Fig. 1).

Statistical analysis
Data were checked, entered, and analyzed by using (SPSS version 17 for windows, SPSS Inc., Chicago, IL, USA) and Medical 13 for windows (MedCalc Software bvba, Ostend, Belgium). Data were expressed as mean ± SD for quantitative variables or number and percentage for qualitative variables. Chi-squared (χ 2 ), t test, and analysis of variance (ANOVA) tests were performed. Unpaired Student's t test was used to compare between two groups in quantitative data.

Results
In our study, there were significant differences between the three groups regarding direct bilirubin, total bilirubin, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum albumin, serum alkaline phosphatase (ALP), serum AFP, serum creatinine, serum urea, platelet count, INR, and PT. But, there were no significant differences between the three studied groups regarding hemoglobin and body mass index (BMI) ( Table 1).
According to allele frequency, the control group shows that A allele more frequent than the C allele (73.53%, 26.47%), respectively. In the HCC group, the C allele was more frequent than A allele (80.88%, 19.12%), respectively. In the chronic hepatitis C group, the C allele was more frequent than A allele (64.71%, 35.29%) respectively. There was a significant difference between the groups regarding allele frequency (Table 3).
Our study showed a significant relationship between VDR genotypes in HCC and both AFP (P = 0.02) and tumor size (P = 0.008). There was a significant relationship between genotype CC and tumor size (Table 4).

Discussion
HCC is resistant to radiotherapy and chemotherapy, and long-term survival of patients occurred only with small asymptomatic HCC [5]. Early detection of HCC is helpful as it improves the prognosis [6]. And, it allows using several modalities as resection, radiofrequency ablation, and transplantation. These modalities improve the outcome in these patients [7].
It is important to recognize HCV-infected patients who are at a higher risk of developing HCC [8]. The difference in the incidences of HCC among different populations may be explained by genetic background [9]. Besides, multiple susceptible genetic loci of HCC were also recognized and validated [10]. Thus, the study of genetics can be added to the tools of risk prediction, allowing better stratification and personalized assessment of optimal long-term management, thereby increasing the efficacy of surveillance programs [2]. The mechanism of development of HCC among chronic hepatitis C patients, including host-and viralrelated factors, is still unknown. The differences in the prevalence and the strong gender distribution in HCC are due to differences in the exposure to the causative agents as well as genetic factors, particularly gene polymorphisms of inflammatory cytokines and growth factor ligands and receptors [11].
Vitamin D is not only involved in bone metabolism as a hormone but it also has immunomodulatory, antiinflammatory, and antifibrotic properties. It also plays an important role in the regulation of cell proliferation, differentiation, and carcinogenesis via VDR [12]. VDR is a member of the nuclear receptor superfamily of ligandinducible transcription factors, which are involved in many physiological processes, including, cell growth and differentiation, embryonic development, and metabolic homeostasis [13]. VDR performs heterodimerization with auxiliary proteins for effective DNA interaction as the retinoid-X receptors (RXRs). Vitamin D response elements have been recognized in many genes responsible for cellular growth, differentiation, apoptosis, invasion, and metastasis of tumor cells as cell cycle regulators. Therefore, it can be assumed that VDR-mediated signaling pathways and VDR gene polymorphisms are related to carcinogenesis. Furthermore, VDR gene variants can modulate vitamin D effect without affecting serum vitamin D levels [14].
VDR polymorphisms have been studied in chronic liver diseases [15]. Yao et al. (2013) and Hoan et al. (2019) found that VDR polymorphism was a risk predictor and a prognostic molecular marker of HCC in patients with chronic hepatitis B [3,16].
Polymorphisms are defined as variations in DNA sequence which occur in at least 1% of a certain population and have real biological effects. They have been studied with the aim of explaining association with the risk for common diseases [17]. According to Shastry (2002) and Li et al. (2001), humans have a huge number of polymorphisms that lead to different cellular effects due to different mechanisms such as transcription level modification, posttranscriptional, or posttranslational activity or changes in the tertiary structure of the gene product [17,18]. Bai et al. (2012) stated that several SNPs have been described in the VDR gene, and some polymorphisms are associated with tumor occurrence in the breast, prostate, skin, colon-rectum, bladder, and kidney [19]. The most common allelic variants studied included a start codon polymorphism Fok1 (T/C) in exon II, Bsm1 (A/G), and APa1 (C/A) polymorphisms in the intron between exon VII and IX and a Taq1 (T/C) variant in exon IX [20]. The Apa-I is a silent SNP. No replacement of the amino acid occurs in the protein. However, it affects mRNA stability [21].
In this study, there was no statistically significant difference between the three studied groups regarding  sex distribution. There was a significant difference between HCC and control regarding age but not significantly higher than chronic hepatitis patients. These results were similar to those found by Barooah et al. (2019) [22]. In contrary to our results, Yang and Roberts (2010) reported that the risk of HCC is 2-7 times higher in men than in women, although this ratio varies across the world. Their explanation for this might be due to higher rates of environmental exposure to liver carcinogens (such as smoking or alcohol) and hepatitis virus infections. Also, estrogen might suppress interleukin IL-6 mediated inflammation in women, reducing both liver injury and compensatory proliferation. Moreover, testosterone effects could increase androgen receptor signaling in men, promoting liver cell proliferation [23]. Hammad et al. (2013) reported that HCC is significantly higher in men than women (77.7 and 22.3%, respectively) [24]. In our study, there were significant differences between the three groups regarding direct bilirubin, total bilirubin, serum ALT, serum AST, serum albumin,  Rowida et al. (2020). They reported that serum levels of ALT, AST, and bilirubin were higher among patients of HCC than those of chronic liver disease [22,25]. Also, Bruix and Sherman (2011) showed that the serum level of ALT and AST were elevated in HCC especially the advanced cases, and the difference becomes greater as the disease progresses [26]. On the other hand, AFP levels may be normal in up to 40% of patients with HCC, particularly during the early stages (low sensitivity). Elevated AFP levels may be seen in patients with cirrhosis or exacerbations of chronic hepatitis (low specificity) [27].
Our study revealed that patients with HCC had a higher frequency of Apa-I CC genotype compared to those with chronic hepatitis C or control. These results are similar to those found by Barooah [22,25,28]. Hung et al. (2014) revealed that patients carrying the corresponding APa-1 CC genotype had a higher prevalence of HCC than those with CA or AA type. They revealed that age, male gender, lower platelet count (< 15 × 104/μL), and Apa-1 CC genotype were independent predictors for developing HCC [28]. Barooah et al. (2019) found that the frequency of the Apa-I CC genotype and ApaI C allele of the VDR gene was significantly higher in HCC and cirrhotic patients than controls. After adjusting for other covariates (age, gender, platelet count, AST, ALT, serum albumin, and viral load), logistic regression analysis showed that the Apa-I CC genotype was independent predictor of HCC development [22]. Baur et al. showed that Apa1 CC genotype was associated with a rapid fibrosis progression in cirrhotic HCV patients [29].
Our study showed a significant relation between Apa-1 VDR genotypes and AFP levels. This result was similar to that found by Raafat Rowida et al. (2020). Similar to our findings, they found that AFP was highest in those with the CC variant and platelet count was low in both CC and CA groups. However, in our study, platelet count did not reach a significant level as detected by them. They also found that Child-Pugh class C was more frequent in the CC group [25].

Conclusions
Apa-1 polymorphism is a possible predictor and prognostic marker in HCV patients that develop HCC.