Hepatocellular carcinoma (HCC) is a major worldwide malignancy. Hepatocarcinogenesis is a complex process of variable etiologies mainly secondary to viral hepatitis infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) [1].

Egypt suffers the highest prevalence of HCV infection, overwhelming their local health services. Chronic HCV infection is characterized by a high rate of progression to fibrosis, chronic hepatitis, and eventually cirrhosis and ultimately to HCC [2]. Some of the host- and viral-related pathogenetic mechanisms of hepatocarcinogenesis are still not well known. There is a continued need for the identification of hepatocarcinogenesis genetic risk factors for early identification and preventative interventions [3].

Vitamin D is a systemic hormone that plays an important role in the metabolism of the skeleton and helps mainly the regulation of host immune responses and fibrogenesis. It has been established that mutations in genes involved in response to hormones, their metabolism or actions may affect the outcome of the disease and thus act as modifiers [4]. This could explain its role in carcinogenesis through vitamin D receptor gene (VDR) polymorphism that made it unable to bind to 1, 25-OH-D [5, 6].

Vitamin D insufficiency has been identified in different populations worldwide as well as its correlation with the presence of various malignancies, including HCC [6]. The VDR gene is a highly polymorphic gene located on chromosome 12q12–q14 [7]. Various single-nucleotide polymorphisms (SNPs) were identified. These genetic variations in the VDR gene could explain the inter-individual variations in rates of vitamin D synthesis and activation in the skin, liver and kidney, transport, metabolism and degradation. These phenotypic variations could influence the anti-tumor effect of vitamin D through the change in cell behaviors such as proliferation, differentiation and apoptosis. Moreover, cumulative data shows the relation between the VDR gene variants and increased risk of cancers including breast [8], prostate [9], and colorectal [10]. VDR polymorphisms were investigated in various chronic liver diseases, including primary biliary cirrhosis and autoimmune hepatitis [11]. So, the aim of this work was to investigate the relation of VDR FokI (rs2228570), BsmI (rs3782905) and ApaI (rs7975232) gene polymorphism and the risk of HCC development in chronic HCV Egyptian patients.

This cross-sectional multi-center study was carried out between May 2018 and June 2019. A total of 468 were recruited for participation from University Hospitals in Kafrelsheikh, Banha, Minia, Cairo and Alexandria Medical research Institute Tanta outpatient hepatology clinics and inwards, 258 were excluded and 210 Egyptian participants were enrolled. They were divided into two groups; chronic HCV patients group I included 107 patients with cirrhosis and 103 patients with HCC group II. A third control group was included, 101 normal healthy subjects with matched age and sex from blood bank donors were enrolled. The study was approved by the Ethics Committee of each Faculty of Medicine in accordance with the Helsinki Declaration of 1975. In addition, written informed consent was obtained from each participant prior to their inclusion in the study.

Total 311 participants of Egyptian origin were enrolled in this study. They were divided into 3 groups: 103 patients with HCV liver Cirrhosis, 107 patients with HCV related HCC and 101 normal healthy subjects as a control group.

3.2. Genotypic and Allelic Distribution of VDR SNPs (FokI, Bsm1and ApaI) among the Three Studied Groups.

Patients carrying the corresponding ApaI CC genotype had a higher prevalence (48.3%) of HCC than those with CA (35.5%) or AA type (19.7%) (x²=18.5 and P <0.001). Patients with HCC carried a higher ratio of ApaI CC genotype compared to those with liver cirrhosis (x²=5.4 and P = 0.03) or controls (x²=6.8 and P = 0.01). CI (0.031-0.038). For the FokI and BsmI polymorphisms, no significant associations were found (Table 2) (Fig. 1).

Table 1. Demographical and laboratory features of the studied population.

Parameter	Liver Cirrhosis (n = 103) Mean ± SD	HCC (n = 107) Mean ± SD	Control (n = 101) Mean ± SD	P-value
Age(years)	54.5±9.74	59.19±7.69	55.96±8.68	< 0.001*
Male gender, n (%)	59 (57.3)	60 (56.1)	59 (58.4)	0.477
Smoking, n (%)	63(61.1)	72(67.3)	61(60.3)	0.023*
BMI (kg/m2)	26.63±2.9	26.36±2.1	25.57±3.9	0.07
AST (U/L)	60.6±30.2	102.1±53.9	32.5±6.6	< 0.001*
ALT (U/L)	44.2±25.4	59.3±28.1	30±5.5	< 0.001*
T-Bil (mg/dL)	3.9±1.3	6.27±2.5	0.8±0.3	< 0.001*
ALB(g/L)	2.5±0.8	2.6±0.5	3.9±0.2	< 0.001*
Creatinine(mg/dL)	1.54±1.14	2.26±1.79	0.96±0.16	< 0.001*
Platelet (103/μL)	140.5±72	114±41.6	286.2±90.1	< 0.001*
INR	1.4±0.3	1.6±0.7	1±0.1	< 0.001*
AFP (ng/ml)	135.2±20	1567±202.5	5.72±1.7	< 0.001*

Table 2. Genotypic and allelic distribution of the VDR SNPs (FokI, Bsm1and ApaI) among the three studied groups.

Gene SNP/rs No.	Allele	Genotype Distribution			P-value	Allelic OR* (95% CI)
		Liver Cirrhosis N=103 (%)	HCC N=107(%)	Control N=101(%)	-	-
-	GG	34 (33.7)	44 (41.1)	31 (30.1)	-	-
FokI (rs2228570)	GA	57 (56.4)	55 (51.4)	56 (54.4)	0.22	1.45 (0.8-2.6)
-	AA	10 (9.9)	8 (7.5)	16 (15.5)	0.03	2.8 (1.08-7.45)
MAF	G vs. A allele	125: 77	143: 71	118: 88	-	-
P value of HWE	-	-	-	-	0.26	-
	CC	24 (23.8)	19 (17.8)	26 (25.2)	-	-
Bsm1 (rs3782905)	CG	68 (67.3)	76 (71)	69 (67.0)	0.3	1.5 (0.77-2.96)
	GG	9 (8.9)	12 (11.2)	8 (7.8)	0.3	2 (0.7-5.9)
MAF	G vs. C allele	86:116	100:114	89: 121	-	-
P value of HWE	-	-	-	-	0.39	-
	AA	35 (34.7)	15 (14)	34 (33)	-	-
ApaI (rs7975232)	AC	44 (43.6)	49 (45.8)	45 (43.7)	0.2	2.5(1.19-5.12)
	CC	22 (21.8)	43 (40.2)	24 (23.3)	<0.001	4.1(1.86-8.92)
MAF	C vs. A allele	88:114	135:79	93:113	-	-
P value of HWE	-	-	-	-	0.8	-

Fig (1). The association of VDR gene polymorphisms with the three studied groups.

HCV-related HCC is a major health problem, with significant mortality and morbidity rates being the fifth most common cancer and the second leading cause of cancer-related death worldwide [15-35]. Nevertheless, only a fraction of infected patients develop HCC during their lifetime, suggesting that genetic factors might modulate HCC development [16]. Consequently, the identification of additional genetic factors affecting transcription of specific regulatory genes could help to select high-risk populations. So, the aim of the present work was to investigate the possible association between the VDR gene polymorphisms (FOKI rs 2228570, BsmI rs3782905 and ApaI rs7975232) and HCC in the Egyptian population with chronic HCV infection.

The present work demonstrated that smoking among HCC patients was significantly higher than those with liver cirrhosis and controls. In agreement with our result, a recent meta-analysis has shown a significant increase in relative risk of HCC in smokers with HCV compared with nonsmokers with HCV, with relative risks of 23 and 7.9 respectively [17] this probably could be explained by the fact that HCC risk is linked to lifestyle factors, such as smoking which accelerate progression to HCC in HCV patients likely via increased oxidative stress.

In the present study, we found a significantly higher level of serum transaminases (ALT and AST) in the patients’ groups than in control. AST and ALT are excellent markers of hepatocellular injury, especially in the setting of chronic HCV infection [18]. Also, we demonstrated impairment of both secretory (as evident by increased bilirubin level) and synthetic (as evident by decreased albumin level and prolongation of prothrombin time) functions of the liver, which were significantly obvious in the patients’ groups than in the control group. Also, the platelets count was significantly lower in the patients’ groups than in the control group. These findings were due to the necro-inflammatory effect of chronic HCV infection. Furthermore, due to the tumor mass cholestatic and necrotic effects of HCC on the liver tissue, AST and serum bilirubin level were significantly higher in the HCC group than liver cirrhosis group [19].

AFP can be mildly elevated in those with regenerating nodules in viral cirrhosis, giving more false-positive results. However, a rising AFP over time is more accurate in the diagnosis of HCC, even if the level does not reach 400 ng/ml [19]. In agreement with the previously mentioned data, the present work demonstrated a significantly higher level of AFP in the patients’ group than the control group. Furthermore, the level of AFP was significantly higher in HCC group than in liver cirrhosis group. Also, in 2018 the American association of study of liver diseases guidelines stated that surveillance should be accomplished by ultrasound with or without AFP, as it is “not possible to determine which type of surveillance test, US [ultrasound] alone or the combination of US plus AFP, leads to greater improvement in survival” [20].

In the present work, we demonstrated the prevalence of VDR gene polymorphism (FokI rs 2228570, BsmI rs3782905 & ApaI rs7975232) in HCC and liver cirrhosis, and we found that patients with HCC had a higher frequency of ApaI CC genotype (40.2%) than in those with liver cirrhosis (23.35%) and the control group (21.8%). Moreover, logistic regression analysis showed that ApaI CC genotype was an independent risk factor for HCC making ApaI CC genotype a potential risk factor for HCC in patients with HCV liver cirrhosis. On the other hand, the other ApaI variant AA and AC genotypes were found to be associated with a significantly increased risk of renal cell carcinoma compared with the CC genotype [21].

In agreement with our results, Hung et al. [22] suggested a significant association of VDR ApaI rs 7975232 polymorphism with the development of HCC in chronic HCV infection. In his hospital-based study, patients with HCC also had a higher frequency of ApaI CC genotype. Other two studies have reported the significant relationship between VDR gene polymorphisms and HCC development in patients with chronic HCV infection [23, 24].

In another Egyptian study [25] on chronic HCV patients, in accordance with our result, Rawhia et al. found that VDR Apa-I gene polymorphism could be associated with an increased risk of HCC development in chronic hepatitis C virus-infected patients. Also, Galal et al. [26] had found that the carriage of the ApaI CC genotype was an independent predictor for HCC in HCV-related liver cirrhosis.

Although, Falleti et al. [27], in their study of VDR genetic polymorphisms in transplantation patients of variable etiologies, found that A strong association was observed between carriage of the BAT A-T-C and G-T-T haplotypes and HCC only in alcoholic liver disease rather than to those with viral cirrhosis. Triantos et al. [28] showed that the carriage of VDR ApaI AA genotype was associated with more severe liver disease compared to ApaI CC/CA genotypes. This contradiction could be justified by the difference in the inclusion criteria as the authors investigated patients with chronic liver disease of variable causes (viral, autoimmune, cryptogenic, etc.) and not complicated by HCC on top, while in our cohort, we selected only the HCV-related cirrhotic patients.

Moreover, in an Italian study [29] on chronic HCV patients treated with direct acting antivirals (DAAs), the authors elucidated vitamin D pathway gene SNPs and HCC relationship in the Italian population. They showed that HCC risk factors were age, ribavirin administration, IL28Brs12979860CC and previous treatments; VDR FokI CC, sex and insulin resistance were protective factors. Although the studied SNPs were different from our work.

Our study has the advantage of being multicentric, covering most of the geographical areas of Egypt, but the study has its own limitations. The relatively small number of patients included but this can be explained by the pilot nature of the study and trial to cover more geographical areas. Our study is still a cross-sectional study, and we are now following our cirrhotic patients that have VDR ApaI CC polymorphism to confirm the causal relationship.

ApaI CC VDR gene mutation is an independent risk factor for HCC development in Egyptian Cirrhotic HCV patients.