Genetic insights into hepatocellular carcinoma: the role of VDR FokI (rs2228570) and TaqI (rs731236) polymorphisms in Egyptian patients.

Introduction
Hepatocellular carcinoma (HCC) still represents a global health issue [1]. HCC poses a significant public health challenge in Egypt, primarily attributable to the widespread incidence of chronic HCV infection. In Egypt, liver cancer represents a major public health burden. According to GLOBOCAN 2022 estimates, liver cancer accounted for 27,946 new cases in 2022 (ASR 32.0 per 100,000) and a 5-year prevalence of 39,028 cases (36.8 per 100,000) [2, 3]. Several reports indicate that vitamin D deficiency is widespread among Egyptian HCC patients and may be linked to poorer disease outcomes [4]. Vitamin D, a lipid-soluble precursor of steroid hormones, is essential in sustaining bone structure and maintaining calcium–phosphate homeostasis. Its active form, calcitriol (1,25-dihydroxyvitamin D3), mediates its physiological effects mainly via vitamin D receptors (VDRs), which are broadly distributed across many organs such as the intestine, bone, and lungs [5]. The VDR functions as a nuclear transcription factor by interacting with activated vitamin D [6]. Once engaged, the VDR makes a complex with the retinoid X receptor (RXR), which translocates into the nucleus and binds vitamin D response elements (VDREs) within target genes that regulate calcium homeostasis, inhibit uncontrolled cell proliferation, promote cell differentiation, suppress angiogenesis, and influence insulin secretion [7–9]. Vitamin D insufficiency is associated with increased risk of various pathological conditions, including cancers, autoimmune disorders, hypertension, and infectious diseases [10].
The VDR gene (OMIM#: 601769) has been widely investigated across different populations and linked to a variety of diseases. It is located on chromosome 12q13.11, spans approximately 101,500 base pairs, and consists of 14 exons. More than 900 allelic variants have been identified within this locus [8]. Genetic variations in VDR can influence the body’s responsiveness to vitamin D, thereby altering susceptibility to several disorders, including HCC [11]. Evidence suggests that specific VDR polymorphisms may elevate the risk of HCC, particularly in HCV patients [12]. Understanding how VDR variants contribute to liver carcinogenesis may therefore aid in developing preventive and therapeutic strategies for populations such as Egyptians, where HCC prevalence is high.
Among the greatest extensively examined VDR single nucleotide polymorphisms (SNPs) are ApaI (rs7975232, A > C (A > a)), BsmI (rs1544410, C > T (B > b)), TaqI (rs731236, T > C (T > t)), and FokI (rs2228570, T > G (F > f)) [13]. These SNPs can affect VDR protein function and alter its capacity to regulate vitamin D–D-mediated pathways [14]. Several studies have reported associations between these variants and heightened risk of HCC, especially chronic viral hepatitis cases [15–18]. However, few investigations have explored this association among Egyptian patients with HCV-induced HCC. Therefore, this study intended to evaluate the role of VDR FokI (c.2T > G; p.Met1Arg; rs2228570) and TaqI (c.1056T > C; p.Ile352; rs731236) variants in predisposing Egyptians to HCC, and their relationship with clinical and laboratory outcomes.

Materials and methods

Study participants
This case–control study included 100 individuals with HCC and 100 healthy controls. Healthy control subjects were age- and sex- matched recruited from individuals attending routine health check-ups at the same hospital during the study period. All participants were of the same ethnic background and matched for age and sex. Patient recruitment took place at the Oncology Center, Mansoura University, Egypt, between April 2022 and February 2023. Diagnosis of HCC was confirmed by histopathological assessment and radiological examinations, including MRI and CT. Individuals with renal failure, HIV infection, autoimmune disorders, or other cancers were excluded. Ascites was classified following the International Ascites Club guidelines into 3 grades [19]. Ethical acceptance for the study was granted by the Institutional Review Board of the Faculty of Medicine, Mansoura University (Approval code: R.23.05.2158).

Collection of samples and analysis
Peripheral blood samples were obtained using sterile, disposable syringes. The samples were divided into two portions: the 1 st was placed in an EDTA-containing tube for molecular and hematological analyses. At the same time, the remaining blood was collected in plain tubes without anticoagulant, then centrifuged, and the serum was collected for subsequent biochemical assays.

Biochemical measurements
Biochemical analyses included measurement of serum transaminases, albumin, bilirubin, and creatinine were determined using colorimetric assay kits on a Jenway 6051 bench colorimeter (UK). Serum alpha-fetoprotein (AFP) levels were quantified using an ELISA kit (ab108631, Abcam, USA). Screening for HCV Abs and HbsAg was performed with qualitative ELISA kits (MyBioSource, USA). A complete blood count was performed on an automated hematology analyzer (Abbott Diagnostics, USA).
The DNA was isolated using the DNA Purification Kit (Thermo Fisher Scientific, UK) according to the manufacturer’s guidelines. DNA yield was evaluated using a NanoDrop spectrophotometer.

Genotyping of the VDR (FokI T > G rs2228570)
Genotyping of the VDR FokI variant (T > G, rs2228570) located in exon 3 was performed using the multiplex amplification refractory mutation system–polymerase chain reaction (ARMS-PCR) method, as previously reported by Tangoh et al. [20].
Allele-specific amplification was conducted in two separate reaction tubes: one for the wild-type allele (F) and the other for the mutant allele (f). The primers, initially designed by Jafari et al. [21] and their sequences were: 5′-TGGCCGCCATTGCCTCCG-3′ for the F allele, 5′-TGGCCGCCATTGCCTCCA-3′ for the f allele, and 5′-AGCTGGCCCTGGCACTGA-3′ for the FokI/C.
PCR was performed using Master Mix (Promega, USA), comprising Taq polymerase, buffer (pH 8.5), dNTPs (400 µM each), 3 mM MgCl₂, and nuclease-free water. Reaction optimization included an additional 2 mM MgCl₂. The final 25 µL reaction mixture, which contained 12.5 µL Master Mix, 1 µL MgCl₂, 10 ng/µL DNA, and 0.17 µL (0.07 µM) of each primer (FokI/C and F/f). All reagents were mixed on ice to minimize primer-dimer formation.
Thermocycling conditions began with an initial denaturation at 95 °C for 2 min, followed by 29 cycles comprising denaturation at 95 °C for 25 s, primer annealing at 58 °C for 30 s, and extension at 72 °C for 1 min. A final elongation step was conducted at 72 °C for 5 min. The amplified DNA fragments were resolved on a 2% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. Genotypes were interpreted as follows: a band at 77 bp only in the wild-type tube indicated FF, a band at 77 bp only in the mutant tube indicated ff, and bands in both tubes indicated Ff. Images of the PCR products were captured using a digital imaging system (Fig. 1A). For validation, > 10% of the samples were randomly regenotyped, yielding identical results.

Genotyping of the VDR (TaqI T˃C rs731236)
Genotyping of the VDR TaqI variant (T > C rs731236) in exon 10 was performed using the tetra ARMS-PCR assay as outlined by Hoan et al. [17]. In this approach, both the wild-type and mutant alleles were amplified within the same reaction tube. The primers included the following sequences: forward outer 5′-GCTGCCGTTGAGTGTCTGTGTGGGTG-3′, reverse outer 5′-ACAAGGGGCGTTAGCTTCATGCTGCACTC-3′, inner forward (T allele) 5′-CAGGACGCCGCGCTGCTT-3′, and inner reverse (t allele) 5′-CGGT CCTGGATGGCCGCG-3′. The PCR reaction (25 µL) contained 1X PCR buffer, 0.2 mM dNTPs, 1 mM MgCl₂, 0.15 mM of each primer, 1 unit of Taq polymerase, and 50 ng of template DNA. The applied thermal profile was an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 35 s), and extension (72 °C, 45 s), with a terminal elongation at 72 °C for 7 min. PCR products were separated on 2% agarose gels, stained with ethidium bromide, and visualized using UV light. Expected fragment sizes included a 415 bp product, a 300 bp fragment, and in heterozygotes (Tt), two fragments at 300 bp and 150 bp. The amplified products were documented by digital photography (Fig. 1B). To ensure reliability, genotyping quality control included negative controls in each run and randomly repeated genotyping of > 10% of the samples to assess reproducibility.

Statistical analysis
Data was analyzed using SPSS Statistics, version 26. Statistical power was assessed using G*Power (Heinrich-Heine-Universität Düsseldorf, Germany) at α = 0.05. In addition, a post hoc power analysis based on the observed allele distributions/effect sizes indicated adequate power to detect the reported associations (approximately 86% for rs2228570 and 78% for rs731236). Qualitative variables were expressed as numbers and percentages and compared using Fisher’s exact test. At the same time, continuous non-parametric data were summarized as medians (interquartile ranges, IQR) and evaluated by the Mann–Whitney U test. The normal distribution was defined via the Kolmogorov–Smirnov test. Genotype and allele frequencies of VDR FokI and TaqI polymorphisms were determined using the SNPstats online analysis tool (https://www.snpstats.net/), and Hardy–Weinberg equilibrium (HWE) was tested in both the HCC and control groups using the chi-square test. The genetic inheritance models were evaluated to compute odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression. The R software (version 4.1.3) was applied for the multivariate analysis. A probability value < 0.05 was deemed significant.

Results

The basic characteristics of the studied groups
The demographic, clinical, and lab profiles of the study participants are presented in Table 1. No significant differences were recorded between the two regarding age (p = 0.36) or sex distribution (p = 0.60). In contrast, smoking was more frequent among HCC patients, showing a significant difference compared with controls (p = 0.002).

Among the patients, ascites was detected in 68 cases: 21 (30.9%) had mild ascites, 22 (32.3%) had moderate ascites, and 25 (36.8%) had severe ascites. Splenomegaly was present in 64 patients, of whom 40 (62.5%) had mild enlargement, 18 (28.1%) had moderate enlargement, and 6 (9.4%) had marked splenomegaly.
Biochemical and hematological analyses showed that HCC patients had considerably higher levels of ALT, AST, total bilirubin, creatinine, AFP, and WBC counts, while albumin, RBC counts, hemoglobin, and platelet levels were markedly reduced compared with healthy controls. Additionally, serological testing revealed that most patients (85%) were positive for anti-HCV antibodies, whereas only 10% tested positive for HBsAg.

The genotypic and allelic frequencies of the VDR (FokI T˃G rs2228570) variant
The distribution of the VDR FokI (T > G, rs2228570) SNP conformed to HWE in both groups (p > 0.05). Compared with controls, HCC patients had significantly higher frequencies of the Ff (59% vs. 41%, p = 0.001) and ff (18% vs. 12%, p = 0.01) genotypes (Table 2).

At the allelic level, the minor f allele was considerably prevalent in patients (47.5%) compared to controls (32.5%), conferring nearly a twofold increase in HCC risk (OR = 1.94, p < 0.002) (Fig. 2A). Under the dominant model (Ff + ff vs. FF), carriers of the f allele among HCC patients were markedly more frequent among controls group (77% vs. 53%, OR = 3.00, p < 0.001). Likewise, in the overdominant model (Ff vs. FF + ff), heterozygous carriers were significantly overrepresented in patients (59% vs. 41%, OR = 2.18, p = 0.007).

These outcomes indicate a clear relationship between the f allele and increased risk of HCC in the Egyptian population. However, under the recessive model (ff vs. FF + Ff), although the ff genotype was more frequent among patients (18% vs. 12%), without meaning difference (OR = 1.51, p = 0.31) (Table 3). The frequency of the f allele in this cohort was also higher than other populations as displayed in (Fig. 2B).

The genotypic and allelic frequencies of the VDR (TaqI T˃C rs731236) variant in the studied groups
The distribution of the VDR TaqI variant (c.1056T > C; p.Ile352=; rs731236) followed HWE in both groups (p > 0.05). Compared to the control group, HCC patients had a significantly higher frequency of the Tt genotype (52% vs. 36%, p = 0.01). In contrast, although the tt genotype was more common among patients (7% vs. 3%), the difference was non-significant (p = 0.07) (Table 2). Allelic analysis showed that the minor t allele was significantly more frequent in HCC patients (33%) than in controls (21%), nearly doubling disease risk (OR = 1.98, p < 0.006) (Fig. 2C).
Under the dominant model (Tt + tt vs. TT), the combined genotypes were more prevalent in patients (59%) than in controls (39%), with an associated OR of 2.19 (p = 0.007). Similarly, in the overdominant model (Tt vs. TT + tt), heterozygous carriers (Tt) were significantly overrepresented in HCC patients (52% vs. 36%, OR = 1.90, p = 0.03).
These findings suggest that the t allele contributes to increased susceptibility to HCC in this Egyptian cohort. However, when applying the recessive model (tt vs. TT + Tt), no significant difference was observed between patients and controls (7% vs. 3%, OR = 2.23, p = 0.24) (Table 3). The observed allele frequencies in this study were comparable to other populations as displayed in (Fig. 2D).

The associations of the VDR (FokI T˃G rs2228570) and VDR (TaqI T˃C, rs731236) variant with the main demographic, clinical and lab data of the HCC patients
The associations of VDR FokI (T > G rs2228570) with demographic, clinical, biochemical, and hematological parameters in HCC patients are presented in Table 4. No significant correlations were found with any of the assessed variables (p > 0.05).
Similarly, analysis of the VDR TaqI (cT > C rs731236) variant revealed no significant associations with most clinical and laboratory parameters, except for gender, where a significant difference was observed (p = 0.04) (Table 4).

Multivariate analysis
Multivariate analysis using PCA differentiated the study population into two distinct clusters, clearly separating patients from healthy controls. Demographic, clinical, biochemical, and hematological parameters contributed to this separation, with longer vector arrows indicating variables exerting a greater influence. The analysis highlighted that both the VDR FokI (c.2T > G; p.Met1Arg; rs2228570) and TaqI (c.1056T > C; p.Ile352=; rs731236) variants were linked to an elevated risk of HCC (Fig. 2E). In addition, a correlation matrix constructed using Pearson’s correlation test was applied to explore relationships among clinical features and laboratory findings in HCC patients (Fig. 2F).

In Silico data analysis
The bioinformatic characterization of the VDR gene is illustrated in Fig. 3. The VDR gene [ENSG00000111424], also known by synonyms such as NR1I1 and PPP1R163, is located on chromosome 12p13.11 and spans approximately 101,512 base pairs on the reverse strand (chr12:47,841,537–47,943,048). It produces eight transcript isoforms: VDR−201 through VDR−208. The principal transcript (LTA-206) contains 10 exons. The VDR FokI polymorphism (c.2T > G; p.Met1Arg; rs2228570) lies in exon 3 at position chr12:47,879,112, with a maximum minor allele frequency (MAF) of 0.48, according to Ensembl (GRCh38.p14). The VDR TaqI polymorphism (c.1056T > C; p.Ile352=; rs731236) is located in exon 10 at position chr12:47,844,974, with a maximum MAF of 0.47 (GRCh38.p14). The VDR gene encodes the Vitamin D3 receptor, a 427-amino acid protein (~ 48.3 kDa) from the nuclear hormone receptor superfamily of ligand-activated transcription factors [Uniprot ID: P11473]. Gene interaction network analysis (GeneMANIA) indicated that VDR is strongly involved in transcriptional regulation, including RNA polymerase II DNA-binding activity, transcription initiation, intracellular receptor signaling, and ligand-activated transcription factor functions. Subcellular localization data (Cellular Compartment database) showed VDR presence in the cytoplasm, nucleus, cytoskeleton, extracellular matrix, lysosomes, endoplasmic reticulum, peroxisomes, mitochondria, and plasma membrane. Protein–protein interaction predictions (STRING) suggested that VDR interacts in processes such as vitamin D3 binding, retinoic acid receptor binding, RXR binding, and transcriptional regulatory complexes. Finally, Kaplan–Meier survival analysis demonstrated that VDR expression was significantly associated with outcomes in liver cancer patients (p = 0.031) [Kaplan–Meier Plotter database].

Discussion
Understanding the underlying mechanisms of HCC, which ranks among the leading causes of mortality worldwide, is of critical importance [22]. Identifying genetic determinants of HCC susceptibility may not only deepen insights into the biological processes of hepatocarcinogenesis but also provide a stronger foundation for preventive strategies [23].
While vitamin D is conventionally recognized for its role in bone metabolism, it also exhibits immunomodulatory, anti-inflammatory, and antifibrotic properties. Through its receptor (VDR), vitamin D further regulates pathways linked to cell growth, differentiation, and tumorigenesis [24]. Given these functions, numerous investigations have assessed the correlation between VDR gene polymorphisms and the risk or progression of HCC across different populations [11, 12, 25, 26]. Among the VDR variants, FokI and TaqI have received particular attention [7]. However, evidence regarding their association with HCC remains inconsistent [27].
Some reports suggest that individuals carrying the FokI ff or TaqI tt genotypes face an increased risk of developing HCC, whereas other studies have failed to confirm such associations [26]. Moreover, some studies suggest that combinations of VDR polymorphisms may collectively increase HCC risk [28]. The discordant results can arise from several factors, including: population-specific genetic background and linkage disequilibrium patterns; differences in underlying etiological exposures; variation in case definition and control selection; differences in analytical models and adjustment for confounders; and limited sample sizes that may lead to underpowered estimates and variability in effect direction.
In this study, the combined (Ff + ff) genotypes, the heterozygous Ff genotype, and the f allele of the VDR FokI (c.2T > G; p.Met1Arg; rs2228570) variant were found at significantly higher frequencies among Egyptian HCC patients compared to healthy controls (p < 0.001; p = 0.007; p = 0.002, respectively). The prominence of the FokI variant vector in the PCA plot toward the patient group further supports its association with HCC susceptibility (Fig. 2E).
Comparable findings were reported in a Chinese cohort, in which the VDR FokI (rs2228570) SNP was associated with a higher risk of HBV-associated HCC [29]. By contrast, an earlier Egyptian study reported no significant relationship between the FokI variant and HCC susceptibility [26]. Hepatocarcinogenesis is a multifactorial process involving the malignant transformation of normal hepatocytes through both genetic and epigenetic changes [30]. The FokI SNP lies within exon 3 in the 5′ coding region of the VDR gene. This polymorphism results from a thymine-to-guanine substitution, producing a missense mutation that alters the amino acid sequence. Specifically, the F allele encodes methionine (ATG), while the f allele encodes arginine (AGG) at the translation start site [31]. This T > G substitution (Met→Arg) yields a protein isoform with reduced functional activity compared to the wild-type receptor [32].
In addition, our results demonstrated that the combined (Tt + tt) genotypes, the heterozygous Tt genotype, and the t allele of the VDR TaqI (c.1056T > C; p.Ile352=; rs731236) variant were significantly more frequent in Egyptian patients than controls (p = 0.007; p = 0.03; p = 0.006, respectively). The PCA analysis also highlighted the TaqI variant by its extended vector toward the patient group, further supporting its association with increased HCC susceptibility (Fig. 2E). These findings align with the work of Neamatallah et al., who likewise identified an essential link between the TaqI polymorphism and HCC risk in Egyptian patients [16]. Mechanistically, the TaqI SNP alters a restriction enzyme recognition site near the VDR gene, potentially influencing VDR expression levels [33].
Regarding VDR TaqI, the gender-based analysis showed that the homozygous variant genotype (tt) was rare overall and further enriched in one sex after stratification. This resulted in an apparent increased risk under the recessive model in that subgroup. Biologically, sex-specific differences in vitamin D metabolism, immune regulation, and hepatic inflammatory responses may contribute to differential effects of VDR variants between males and females. Vitamin D–VDR signaling has documented interactions with sex hormones, which can influence hepatic fibrosis, cirrhosis progression, and downstream carcinogenic pathways. Statistically, the small number of individuals carrying the tt genotype—particularly after stratification by sex—may also contribute to unstable risk estimates with wide confidence intervals. Therefore, this finding should be interpreted as exploratory and hypothesis-generating rather than conclusive.
Taken together, the relationship between VDR polymorphisms and HCC is multifactorial and complex. Genetic predispositions and environmental exposures interact to shape individual risk, and the influence of VDR variants may vary accordingly.
This study has certain limitations, including its single-center design, the relatively modest sample size, and the absence of data on VDR gene expression in patient samples. To confirm and encompass these findings, larger, multi-center studies with expanded datasets are recommended. It is also recommended to validate VDR variants using Sanger sequencing or high-throughput genotyping platforms.

Conclusions
In summary, this study indicates that the VDR FokI (c.2T > G; p.Met1Arg; rs2228570) and TaqI (c.1056T > C; p.Ile352=; rs731236) polymorphisms are linked to an increased susceptibility to HCC among Egyptian patients. However, these genetic variants do not appear to influence the clinical or biochemical characteristics of the disease.

Supplementary Information