Prognostic significance of vitamin D receptor (VDR) gene polymorphisms in liver cirrhosis

Several polymorphisms in the vitamin D receptor (VDR) are associated with the occurrence of chronic liver disease. Here, we investigated the association between BsmI, ApaI, TaqI and FokI VDR polymorphisms and the severity of liver cirrhosis in relation to serum cytokine and lipopolysaccharide binding protein (LBP) levels and their role on survival in cirrhotic patients. We found that patients harboring the BB genotype had higher MELD score, and they were mainly at CP stage C; patients harboring the AA genotype had increased LBP, IL-1β and IL-8 levels, and they were mostly at CP stage C; TT genotype carriers had higher MELD score and they were mainly at CP stage C and FF genotype carriers had lower IL-1β levels when compared to Bb/bb, Aa/aa, Tt/tt and Ff/ff genotypes respectively. In the multivariate analysis ApaI, BsmI and TaqI polymorphisms were independently associated with liver cirrhosis severity. In the survival analysis, the independent prognostic factors were CP score, MELD and the FF genotype. Our results indicate that the ApaI, TaqI and BsmI polymorphisms are associated with the severity of liver cirrhosis, through the immunoregulatory process. Survival is related to the FF genotype of FokI polymorphism, imparting a possible protective role in liver cirrhosis.

Cytokines are key mediators in the pathophysiology of liver disease as they play an essential role in hepatic regeneration and fibrosis 20 . The hepatic non parenchymal cells which are involved in liver fibrosis development, can rapidly produce profibrogenic cytokines which lead to hepatic inflammation and fibrosis 21 . In contrast, antifibrogenic cytokines downregulate the pro-inflammatory response promoting the hepatic regeneration 20,21 . VDR polymorphisms may influence the immune regulation by affecting cytokine levels and, thus, they might play a role in the progression of liver disease 11,13 .
In this study, we have investigated the potential associations between VDR gene polymorphisms and the severity of liver cirrhosis, in relation to the cytokine and bacterial profiles, vitamin D and vitamin D binding protein (VDBP) levels, and their role on patient survival.

Results
The main demographic and clinical characteristics of the examined patients are presented in Table 1 and the main characteristics of the examined VDR polymorphisms are presented in Table 2.

Distribution of clinical variables and serum cytokine expression according to the VDR genotypes.
As shown in Table 3 the presence of BsmI polymorphism, in particular the BB genotype, was associated with advanced Child-Pugh (CP) stage (p = 0.044) and higher model for the end-stage liver disease (MELD) score (p = 0.045). The AA genotype of the ApaI polymorphism was associated with advanced CP stage (p = 0.001) and increased LBP levels (p = 0.014). The presence of TaqI polymorphism (TT genotype) was associated with advanced CP stage (p = 0.027) and MELD score (p = 0.025). As regards to the FokI polymorphism, the FF genotype was associated with lower levels of the pro-inflammatory cytokine IL-1β (p = 0.045).

Comparisons of clinical parameters and serum cytokine expression between VDR polymorphisms.
As shown in Table 4, BsmI patients harboring the BB genotype had higher MELD score (p = 0.026) and were mainly at CP stage C (p = 0.020) compared to Bb/bb genotypes. ApaI patients harboring the AA genotype had increased levels of LBP (p = 0.004), IL-1β (p = 0.036) and IL-8 (p = 0.03) and were mostly at CP stage C (p = 0.001) compared to patients with the Aa/aa genotypes. The TT genotype carriers of the TaqI polymorphism had higher MELD score (p = 0.026) and were mainly at CP stage C (p = 0.02). Finally, FokI patients who had the FF genotype showed lower levels of IL-1β (p = 0.013) compared to patients with the Ff/ff genotypes. In the multivariate analysis, in the presence of other significant covariates, as well as cirrhosis '   Association between vitamin D and VDBP levels with VDR polymorphisms. We found no statistically significant differences between serum 25(OH) vitamin D levels and VDBP levels in relation to VDR polymorphisms. Haplotype analysis of VDR polymorphisms in relation to disease severity. Haplotype association with cirrhosis severity was evaluated by the distribution of VDR haplotypes in the different CP stages. Estimated VDR haplotype frequencies of FokI, BsmI, ApaI and TaqI polymorphisms are reported in Table 6. The results showed that in patients with CP stage C, BAT haplotype was more frequent suggesting a potential increased risk for advanced cirrhosis, whereas the complementary haplotype bat was more common in patients with CP stage A; however, this difference was no significant (LR test p = 0. 581).

Discussion
This is the first report of an association between polymorphisms of the VDR gene and cytokine levels, severity of liver disease and survival in patients with liver cirrhosis. In particular, an independent association between BsmI, ApaI, and TaqI VDR polymorphisms and the severity of liver cirrhosis is clearly shown. Moreover, the presence of FF genotype of FokI polymorphism is associated with a better prognosis regarding survival in this cohort. These features appear to be independent of the etiology of liver cirrhosis, as they observed in patients of any cause. Vitamin D promotes the stimulation of innate immunity, the differentiation of monocytes, the inhibition of lymphocyte proliferation and cytokine secretion by T and B cells 22,23 . VDR acts as a ligand-stimulated  transcription factor and activates 1,25(OH) 2 D 3 at the transcriptional level. The activation of VDR contributes to the regulation of immune response by inhibiting T helper 1 (Th1) cell proliferation and pro-inflammatory cytokine production and inducing Th2 cell proliferation and anti-inflammatory cytokine production 7,22-26 . The presence of VDR polymorphisms possibly leads to a dysfunctional receptor, affecting VDR activity and the subsequent vitamin D-mediated effects 26 . The association between VDR polymorphisms and the occurrence of chronic liver disease from different etiologies such as autoimmune hepatitis, PBC, HCC or HBV infection has been investigated with conflicting results 8,[10][11][12][13][14][15][16]27 . Previous reports have identified gene polymorphisms which affect the progression of liver fibrosis [28][29][30][31] . The relationship between liver fibrosis progression and the presence of VDR polymorphisms (ApaI, TaqI and BsmI) has been investigated, indicating that in PBC patients, BsmI and TaqI were associated with progressive cirrhosis 10 and in NAFLD patients, VDR mRNA expression and profibrogenic genes were significantly affected by BsmI polymorphism 18 . The effect of bAt haplotype in fibrosis progression has been investigated in HCV patients as well, giving conflicting results 18,19,32 . Our results indicate that the presence of ApaI polymorphism (AA genotype) is related to significant higher levels of IL-1β and IL-8. The increased levels of these pro-inflammatory cytokines suggest that the ApaI VDR polymorphism leads to a less active VDR protein which may contribute to a disturbance of Th1/Th2 balance, a transition to Th1 polarization and a decreased activity of vitamin D-related signaling pathways.
Several studies have demonstrated a positive correlation between higher pro-inflammatory cytokine levels and the severity of liver disease [33][34][35][36][37] . In this study, we have shown that the AA genotype of the ApaI polymorphism is related to decreased levels of platelets and increased levels of LBP, which are consistent with the progression of cirrhosis and portal hypertension development 38,39 . The presence of ApaI, TaqI and BsmI VDR polymorphisms could impede the interaction between vitamin D and VDR, resulting in ineffective vitamin D-VDR complex, impaired VDR-mediated transcription, decreased activity of vitamin D related signaling pathways, transition to a Th1 polarization, and consequently, to a more progressive form of liver cirrhosis (Fig. 2).
A second novel finding of this study, is the inverse association between the FokI polymorphism, particularly the FF genotype, with mortality in liver cirrhotic patients, imparting a protective role of this genotype in cirrhosis. The FokI polymorphism is located in the coding region of the VDR gene and results in a VDR protein with a different structure, creating a new start codon and consequently a VDR protein shortened by three amino acids 38,40 . This protein is more functional and has higher transcriptional activity compared to the long-length VDR protein 38,40,41 . FokI is the only polymorphism that was not associated with severity of liver cirrhosis in our study. The length of the VDR protein influences the regulation of gene transcription through occupation of recognition sites of other transcription factors and interference with their signaling pathways 7 . Therefore, a longer VDR protein may lead to a decreased transcriptional activity and an increased risk of susceptibility to disease 40 . These observations are in line with our study as we have shown that the presence of FokI polymorphism (FF genotype) is associated with significantly lower levels of IL-1β. Patients with the FF genotype produce a shorter form of VDR,   leading to higher transcriptional activity, formation of more active complexes of VDR-vitamin D, inhibition of the Th1 response and induction of the Th2 cell response. Hence, patients with FokI FF genotype may have a better response to vitamin D resulting in a lower progression rate of cirrhosis. However, due to the fact that this hypothesis is of high interest, we suggest that it should be further explored in larger and more specific cohorts with more patients harboring the FokI polymorphism in order to be confirmed. We have also shown the existence of strong linkage disequilibrium between the BsmI, ApaI and TaqI polymorphic sites in our cirrhotic population. These results are in agreement with previous reports suggesting an extensive LD between these genetic markers 10,14 . As these polymorphisms are in strong LD, it can be assumed that these single nucleotide polymorphisms (SNPs) contribute to the severity of cirrhosis in a dependent manner. Nevertheless, as these polymorphisms do not cause a functional change in the VDR gene, it is possible that BsmI, TaqI and ApaI are possibly genetic markers of other functional variations of the VDR gene or in other closely linked genes that are in linkage disequilibrium with the identified polymorphisms.
Some limitations of the current study should be acknowledged. The first limitation concerns the relatively small sample size, however our results are consistent with the reports on the association between VDR polymorphisms and the susceptibility to liver fibrosis 10,18,19 . Secondly, our study was performed on Caucasians patients and it would be interesting to perform the same analysis in different ethnic groups. Lastly, the single measurement of 25(OH)D at baseline may not be representative of the respective concentrations over time. However, there are reports supporting that although 25(OH)D levels present seasonal fluctuation, its levels remain stable over time 42,43 .
In conclusion, our results indicate that VDR polymorphisms are independently associated with the severity of liver cirrhosis and the survival of patients with liver disease, regardless of disease etiology, suggesting a potential influence of them in disease progression. Based on these results future studies will delineate causation between specific VDR polymorphisms and outcome/severity of liver cirrhosis, and the importance of VDR polymorphism analysis in clinical practice to identify patients at greater risk of disease progression and to modify patients' surveillance and treatment accordingly.

Methods
Study design and participants. This study was a prospective cohort study, on 89 consecutive Caucasian patients with liver cirrhosis. During the recruitment, all cirrhotic patients were in stable clinical condition, without any severe complication of liver disease including gastro-intestinal bleeding, hepatorenal syndrome, moderate to severe hepatic encephalopathy, spontaneous bacterial peritonitis, malignancy, or organ failure. Patients with indications or history of bacterial infection at last 4 weeks prior to recruitment in the study, human immunodeficiency virus (HIV) infection and severe cardiopulmonary disease or renal failure were excluded. Severity of cirrhosis was assessed by the CP stage and the MELD score 44 . Diagnosis of cirrhosis was based on histological or compatible clinical, laboratory and imaging data [45][46][47] . After baseline examination, patients were followed in the   VDR is an intracellular ligand-activated transcription factor that specifically binds 1,25(OH)2D 3 and regulates the expression of several target genes. Upon the activation of vitamin D, the ligated VDR heterodimerizes with retinoid X receptor (RXR) which is necessary for DNA binding, translocates to the nucleus, binds to vitamin D response elements (VDRE) and recruits other nuclear proteins to the transcriptional pre-initiation complex. This process results in the transcriptional activation or suppression of the target genes through the interaction with nuclear receptor co-activators or co-repressors. The binding of VDR with vitamin D may modulate cytokine responses by T cells, inhibiting Th1 cell proliferation and pro-inflammatory cytokine secretion and activating Th2 cell proliferation and anti-inflammatory cytokine secretion. (b) Presence of AA genotype of ApaI VDR polymorphism. The presence of polymorphisms may impair the activity of the VDR resulting in a dysfunctional receptor. The dimerization of the 1,25(OH)2D 3 -VDR with RXR may be hindered by the existence of genetic variations thus affecting VDR activity and subsequent downstream vitamin D-mediated effects. This impaired process may lead to disturbance of the Th1/Th2 balance, resulting in a transition to Th1 cell response and pro-inflammatory cytokine secretion that is closely related the progression of liver cirrhosis.
hepatology clinic at regular intervals according to current guidelines 48  DNA extraction. Genomic DNA was extracted using the NucleoSpin® Blood QuickPure kit (Macherey-Nagel, Germany). The DNA concentration of the samples was determined using a Nanodrop spectrophotometer (UV spectrophotometer Q3000, Quawell Technology, Inc., USA).
VDR Genotyping. Genotyping was carried out using TaqMan SNP Genotyping Assays (Applied Biosystems; Foster City, USA). The PCR reactions were carried out in MicroAmp® Fast Optical 96-Well Reaction Plates (Applied Biosystems) on the Step One Plus real-time PCR system (Applied Biosystems, CA, USA). The rs731236 (TaqI), rs1544410 (BsmI), rs7975232 (ApaI) and rs2228570 (FokI) probes were designed using TaqMan pre-designed SNP genotyping assays (Applied Biosystems). Two non-template-control wells were included on each plate. DNA amplification was performed as follows: 95 °C for 10 min, followed by 40 cycles of 92 °C for 15 sec and 60 °C for 1 min.

Statistical analysis. Continuous variables were summarized as medians and interquartile ranges (IQRs)
while counts and corresponding percentages were calculated for categorical variables. All comparisons were performed using non-parametric tests: Fisher's exact tests in case of frequencies' comparisons, Mann-Whitney and Kruskal-Wallis tests for the comparison of median values between two groups and more than two groups, respectively. Correlations between vitamin D and VDBP levels with VDR polymorphisms were assessed by the Spearman's coefficient. Multivariable ordinal logistic regression models were fitted, to test the hypothesis that the VDR polymorphisms are associated with the CP stage. Further analysis was conducted to explore whether these polymorphisms' effect interacts with the etiology of cirrhosis, i.e. whether the effect of the polymorphisms is different in the subgroups of viral, alcoholic or other etiology's cirrhosis. The VDR gene polymorphisms' Hardy-Weinberg equilibrium was examined by means of chi square test goodness of fit test, i.e by comparing observed and expected count in each of the polymorphisms groups (wt/wt, mt/wt, mt/mt). Pairwise linkage disequilibrium (LD) analysis between the VDR gene polymorphisms was performed using the genetics package of R software. Allelic frequencies were estimated by the hapipf stata command, based on the expectation-maximization (EM) algorithm. The hypothesis of allelic association with the CP stage was tested using the likelihood-ratio (LR) test. Time to death was analyzed using the Cox survival model. Before fitting the models, the proportional hazards assumption was assessed for all variables based on Schoenfeld residuals. Individuals' baseline clinical and laboratory variables, including the VDR polymorphisms, were considered as potential risk factors. For all models selection, the Collett's approach was followed 52 . More specifically, all variables with a p-value < 0.200 were initially included and then eliminated using backwards selection. When a model that included only significant covariates was reached, variables initially excluded entered the final model one by one and tested for significance in the presence of already included significant variables. Analysis was performed using Stata 13.1 (StataCorp LP, College Station, Texas, USA). Level of significance α was set at 0.05.