This study is designed as a case-control study involved 298 patients [chronic hepatitis B (CHB) (n = 104), liver cirrhosis (n = 89), hepatocellular carcinoma (HCC) (n = 105)] and 238 healthy individuals. VDR genotyping was performed by ARMS-PCR.
Journal of military pharmaco-medicine no5-2020 ASSOCIATION OF VDR ApaI POLYMORPHISM WITH CLINICAL OUTCOMES AND LIVER DISEASE PROGRESSION IN PATIENTS WITH CHRONIC HEPATITIS B VIRUS INFECTION Nguyen Khuyen1,2, Nghiem Xuan Hoan3 Nguyen Binh An3, Dao Phuong Giang3 Do Tuan Anh1, Le Huu Song3, Le Van Nam1 SUMMARY Objectives: To investigate the association of vitamin D receptor (VDR) variants [TaqI (rs731236), FokI (rs10735810), ApaI (rs7975232) and BsmI (rs1544410)] with hepatitis B virus (HBV) susceptibility and liver disease progression in chronic HBV infection and also to evaluate the relationship between these VDR variants and the serum vitamin D levels in HBV-infected patients Methods: This study is designed as a case-control study involved 298 patients [chronic hepatitis B (CHB) (n = 104), liver cirrhosis (n = 89), hepatocellular carcinoma (HCC) (n = 105)] and 238 healthy individuals VDR genotyping was performed by ARMS-PCR Results: The frequency of genotype ApaI rs7975232 GT and allele T was significantly lower in HBV infected patients compared to healthy control [OR = 0.3 (0.1 - 0.6), p = 0.006 and OR = 0.7 (0.5 - 0.98), p = 0.048] VDR-AapI rs7975232 TT was significantly correlated with HBV-related liver disease [HCC vs CHB: OR = 3.7 (1.1 - 13.5), p = 0.041] Both VDR variants ApaI and FokI were associated with vitamin D levels in the serum in HBV infected patients Conclusions: There was a significant association of VDR variant ApaI (rs7975232) with the clinical outcomes of HBV infected patients Additionally, ApaI and FokI variant may be a genetic factor supporting the valuation of vitamin D levels in HBV infected patients * Keywords: Vitamin D receptor; Vitamin D deficiency; Hepatitis B virus; Chronic liver disease INTRODUCTION Hepatitis B virus infection is a leading cause of life-threatening complications, including acute hepatitis, cirrhosis and liver cancer HBV is a virus that does not directly damage liver cells The study suggests that the outcome and clinical progression of HBV-infected patients are determined to be due to an interaction between the host immune response and the virus [1] The host genetic factor plays an important role in the progression of chronic hepatitis, including genetic variants of the VDR coding gene [1, 2] Military Hospital 103, Vietnam Military Medical University My Duc General Hospital 108 Military Central Hospital Corresponding author: Nguyen Khuyen (nguyenkhuyenbvdg@gmail.com) Date received: 9/6/2020 Date accepted: 18/6/2020 138 Journal of military pharmaco-medicine no5-2020 Vitamin D plays an important role in coordinating the skeletal metabolic system, and it is of great importance in modulating both the innate and adaptive immune responses through the VDR VDR belongs to the receptor family of transcription factors This receptor manifests itself in a variety of cell types including immune cells (mononuclear cells, macrophages, astrocytes, T and B cells) and more than 36 different types of parenchyma [3] Recent studies suggest that VDR is one of the risk factors for developing cancer, cardiovascular disease, autoimmune disease and infectious pathology including chronic hepatitis B [4] and its function Through the engagement with VDR receptor, vitamin D is involved in many pathogenetic processes including cell proliferation, differentiation of the cell, and therefore involved in the process of oncogenes including hepatocellular carcinoma (HCC) Domestic studies on VDR variants in patients infected with HBV are, however inconsistent and no studies have been published on chronic HBV patients in Vietnam where high rates of HBV infection range from 10 - 20% The chronic hepatitis and the complications of HBV infection are expected to remain a public health burden even for decades to come Therefore, we conducted this study for two objectives: - To investigate the relationship between the polymorphism of the gene encoding VDR and clinical manifestations in patients infected with chronic HBV - To assess the association of VDR polymorphism and VDR in patients with HBV infection including CHB, liver cirrhosis and HCC SUBJECTS AND METHODS Subjects 298 HBV-infected patients were classified into groups: CHB (n = 104), liver cirrhosis (n = 89), HCC (n = 105) In addition, 238 healthy individuals were included in the study as controls The study was conducted at 108 Military Central Hospital from 10/2013 - 10/2018 Methods * Design of study: Case-control study The ARMS-PCR technique is used to determine the genotypes of VDR variants The results of the ARMS method are checked by Sanger sequencing The total serum vitamin D concentration was quantified by ELISA method In this study, we classified vitamin D deficiency as follows: Normal vitamin D concentration (≥ 30 ng/mL), moderate vitamin D deficiency (20 - 29.9 ng/mL), severe vitamin D deficiency (10 - 19.9 mg/mL), severe vitamin D deficiency (< 10 ng/mL) Statistical analysis The data were compared with the R program Chi-square test to compare the frequencies between groups The Kruskal-Wallis test and the MannWhitney-Wilcoxon test were used to compare the groups for continuous variables Multivariate regression analysis and logistic regression analysis, adjusted for age and sex, were used to compare genotypic frequencies between study groups All comparisons were statistically significant (p < 0.05) 139 Journal of military pharmaco-medicine no5-2020 RESULTS Charateristics of gender and age in the healthy group and hepatitis B virus group Table 1: Charateristics of gender between the groups Gender Group Male (n, %) Female (n, %) Total Healthty group 160 (67.2) 78 (32.8) 238 (100.0) HBV patients 272 (91.3) 26 (8.7) 298 (100.0) 423 (80.6) 104 (19.4) 536 (100.0) Total Males were predominant with a high incidence of 91.2% in HBV group and 67.2% in the healthy group Table 2: Age group distribution in the controls and patients Age group The controls n (%) < 30 HBV patients n (%) 186 (78.1) 27 (9.1) 30 - 40 (3.8) 47 (15.8) 40 - 50 24 (10.1) 68 (22.8) 50 - 60 15 (6.3) 85 (28.5) > 60 (1.7) 71 (23.8) 238 (100) 298 (100) Total Patients’ age with hepatitis B virus were between 19 - 85 years with more than a half (51.4%) aged 40 - 60 years The age group less than 30 years in the controls made up the majority (78.1%) due to the withdrawal of voluntary blood donors Laboratory characteristics of the controls and patients Table 3: Paraclinical features between the two groups Subclinical characteristics Controls HBV patients p 6.99 (3.9 - 13.3) 6.42 (1.81 - 20.5) 0.0022 4.8 (3.9 - 6.7) 4.47 (1.9 - 7.4) 0.0001 260 (2.9 - 422) 144,5 (6.7 - 641) 0.0001 AST (IU/L) 21 (10 - 59) 98 (17 - 7,700) 0.0001 ALT (IU/L) 18 (4 - 113) 64 (4 - 4,908) 0.0001 Total bilirubin (µmol/L) 22 (9 - 230) 22.7 (6 - 733) 0.0001 Direct bilirubin (µmol/L) 2.2 (1.8 - 21) 8.8 (0 - 449.1) 0.0475 43.9 (11.8 - 47) 36 (16 - 54) 0.0001 White blood count (x 10 /L) Red blood count (x 10 /L) Platelet (x 10 /L) Albumin (g/L) Prothobin (%) 75.85 (14.1 - 140) HBV DNA (copies/mL) AFP (UI/mL) 140 4.5e5 (212 - 1.1e10) 1.6 (0.6 - 8.9) 18.4 (0.9 - 400) 0.0001 Journal of military pharmaco-medicine no5-2020 HBV-infected patients had a lower number of red blood cells, white blood cells and platelets than healthy people, the difference was statistically significant (p < 0.05) HBV-infected patients had higher biochemical parameters than the healthy ones, the test was statistically significant (p < 0.05) * Laboratory characteristics of HBV patient subgroups: A B Figure 2: Characteristics of biochemical parameters (A) and haematological parameters (B) in patient groups 141 Journal of military pharmaco-medicine no5-2020 (A) The level of AST and ALT in the CHS group were significantly higher than in the liver cirrhosis and HCC groups (p < 0.0001) Serum albumin levels were significantly lower in the liver cirrhosis group than in the CHB and HCC groups (p < 0.0001), whereas bilirubin concentration was significantly lower in the HCC group than in the other two groups (p < 0.0001) (B) In patients with liver cirrhosis, the red blood cells were lower than in groups of liver cirrhosis and HCC, and this difference was statistically significant (p < 0.0001) Platelet count in the liver cirrhosis patient group was lower than in the CHB and HCC groups (p < 0.0001) For leukocytes, there was no difference between the subtypes of HBV patients (p > 0.05) Red cells and platelets were significantly lower in the liver cirrhosis group than in the other two groups (p < 0.0001) The association between ApaI and HBV-related chronic liver diseases Table 4: Comparison of genotypic and allelic frequency between healthy group and HCC group HCC Healthy Groups OR (95%CI) p n % n % GG 39 47.7 114 48.7 TG 36 41.4 106 45.3 1.0 (0.6 - 1.7) 0.978* 2.5 (1.1 - 5.9) 0.035* TT 12 13.9 14 6.0 3.17 (1.05 - 9.94) 0.042 Co-dominant # Allele G 114 65.5 334 71.4 T 60 34.5 134 28.6 1.3 (0.9 - 1.9) GG+TG 75 86.2 220 94.0 TT 12 13.8 14 6.0 0.152 Recessive model 2.76 (1.2 - 6.1) 0.013* 3.5 (1.14 - 10.9) 0.024 Dominant model GG 39 44.8 114 48.7 TG+TT 48 55.2 120 51.3 1.2 (0.7 - 1.9) (Note: * χ2: test; #: logistic regression model adjusted for age and gender) 142 0.44 # Journal of military pharmaco-medicine no5-2020 Table 5: Comparison of genotype and allen distribution between CHB and HCC HCC Groups CHB OR (95%CI) p n % n % GG 39 44.8 45 44.1 TG 36 41.4 52 51.0 0.8 (0.4 - 1.5) 0.466* TT 12 13.8 4.9 2.8 (0.9 - 8.6) 0.077* G 114 65.5 142 69.6 T 60 34.5 62 30.4 1.2 (0.8 - 1.9) Co-dominant Allele 0.397 Ressesive model GG+TG 75 86.2 97 95.1 TT 12 13.8 4.9 GG 39 44.8 45 44.1 TG+TT 48 55.2 57 55.9 (0.5 - 1.7) 3.1 (1.1 - 9.2) 0.033* 4.3 (1.2 - 15) 0.017 # 0.922 $ Dominant model Table 6: Genotype and allele distribution in patients with CHB and advanced liver disease (HCC+LC) Groups CHB (n = 87) LC + HCC (n = 183) OR (95%CI) p* n % n % GG 45 44.1 80 47.6 TG 52 51.0 68 40.5 0.7 (0.4 - 1.2) 0.241 TT 4.9 20 11.9 2.3 (0.8 - 6.4) 0.129 G 142 69.6 228 67.9 T 62 30.4 108 32.1 1.1 (0.7 - 1.6) GG+TG 97 95.1 148 88.1 TT 4.9 20 11.9 GG 45 44.12 79 47.02 TG+TT 57 55.88 89 52.98 0.89 (0.54 - 1.46) Co-dominant Allele 0.67 Ressesive model 2.62 (0.95 - 7.22) 0.054* 3.19 (1.03 - 9.88) 0.032 # Dominant model 0.642 (Note: * χ2: test; #: logistic regression model adjusted for age and gender) 143 Journal of military pharmaco-medicine no5-2020 Correlation between VDR variants and vitamin D levels in HBV patients and healthy group A B C D Figure 2: The association between vitamin D levels and genotypes of VDR SNPs in hepatitis B patients and healthy individuals Vitamin D receptor ApaI variant (rs7975232) (figure A): Vitamin D levels in genotype TT carriers were significantly lower than those with GG or TG genotypes in all research groups including healthy control, CHB, liver cirrhosis and HCC VDR FokI variant (rs10735810) (figure C): Vitamin D levels in genotype TT carriers were significantly lower than those of genotypes CC and TC in HCC groups There was no difference in other research groups There was no difference in vitamin D concentration between genotypes in patient groups or healthy control in the remaining two variants BsmI (rs1544410) and TaqI (rs731236) (figures B and D) Liver enzyme levels Correlation between VDR ApaI and subclinical parameters Figure 3: Correlation between VDR ApaI variant and subclinical parameters For VDR-ApaI (rs7975232): Liver enzyme levels significantly elevated in patients carrying TG genotype compared to GG and TT genotypes 144 Journal of military pharmaco-medicine no5-2020 DISCUSSION Vitamin D is considered to be a hormone that not only plays an important role in calcium metabolism and in the mechanism of regulation of the metabolic balance of the skeletal system but also has many effects in the regulation of the immune system in the body [3] The physiological role of vitamin D in the body is expressed by VDR which plays a very important role in many infectious diseases The gene encoding the VDR receptor is located on chromosome 12 and consists of 11 exons Exons 1A, 1B, and 1C form a region "encoding region" exons (exons - 9) encode the structural components of the VDR receptor The TaqI variant is located in exon 9, while the ApaI and BsmI variants are located in the position of intron The polymorphism at these positions has no influence on the protein synthesis and expression of the VDR gene It is assumed that only the FokI variant located at the 2-terminal exon "influences the structural changes and the transcriptional activity of the VDR gene The transcriptional activity of VDR in the T allele carrier (variant: FokI rs10735810) is lower than that of the nonmutated allele (allele C) [5] Our research also showed that the vitamin D concentration in patients with the genotype FokI rs10735810 TT was significantly lower than in patients with the genotype CC and CT Studies on the correlation between the above-mentioned VDR variants and the risk of HBV infection are controversial [6, 7] A recent meta-analysis on 15 studies, investigating the correlation between VDR gene variants for HBV susceptibility and clinical outcomes in HBV-infected patients in different populations [7] In this study, 4.218 HBV-infected patients and 2.298 healthy subjects were analyzed The analysis showed that genotype FokI rs10735810 CC and allele C were considered risk factors for HBV infection [OR = 1.54 (1.2 - 2.0), p < 0.01 and OR = 1.23 (1.04 - 1.45), p = 0.02] However, in our study, the ApaI variant (rs7975232) was the only variant significantly associated with HBV sensitivity and clinical course in chronic hepatitis B patients For the ApaI variant, our study showed that carriers of allen T were less likely to be infected with HBV than carriers of allele G Carriers of genotype TT were significantly associated with the risk of developing liver cancer In patients with CHB, Li et al’s report showed [8] a significant association of this variant with the progression of cirrhosis in patients with chronic HBV infection Suneetha et al [9] also reported the role of ApaI variant in HBV-infected patients In this study, VDR-ApaI rs7975232 TT was considered a significant factor associated with a significantly higher HBV DNA load compared to other ApaI genotypes Our previous study revealed an inverse correlation of vitamin D concentration with HBV DNA load in patients with chronic HBV infection [4], which is consistent with our finding, as carriers of VDR-ApaI rs7975232TT had lower serum vitamin D concentrations in all patient groups, including CHB, liver cirrhosis and HCC, than those with liver types VDR-ApaI rs7975232GT and GG 145 Journal of military pharmaco-medicine no5-2020 With regard to the progression of CHB disease, many studies suggest that vitamin D is involved in the inhibition of inflammation and progression of liver cirrhosis This evidence has been demonstrated in mice in which the VDR receptors were switched off Other studies also clearly show that vitamin D deficiency is the cause of the progression of cirrhosis in patients with non-alcoholic steatohepatitis and in patients with HBV infection [4], although the mechanism explaining why VDR variants play a role in HBV sensitivity and disease progression remains unclear CONCLUSIONS We reported for the first time in Vietnam on the role of VDR variants in HBV susceptibility and disease progression in patients with CHB VDR-ApaI is a variant that is significantly associated with HBV sensitivity, disease progression and its significant association with serum vitamin D levels in patients with chronic HBV REFERENCES Zeng Z Human genes involved in hepatitis B virus infection World J Gastroenterol 2014; 20: 7696-7706 Karatayli SC, Ulger ZE, Ergul AA, et al Tumour necrosis factor-alpha, interleukin-10, 146 interferon-gamma and vitamin D receptor gene polymorphisms in patients with chronic hepatitis delta J Viral Hepat 2014; 21:297-304 Deluca HF, Cantorna MT Vitamin D: Its role and uses in immunology FASEB J 2001; 15:2579-2585 Hoan NX, Khuyen N, Binh MT, et al Association of vitamin D deficiency with hepatitis B virus - related liver diseases BMC Infect Dis 2016; 16:507 Whitfield GK, Remus LS, Jurutka PW, et al Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene Mol Cell Endocrinol 2001; 177:145-159 Zhu Q, Li N, Han Q, et al Singlenucleotide polymorphism at CYP27B1-1260, but not VDR Taq I, is possibly associated with persistent hepatitis B virus infection Genet Test Mol Biomarkers 2012; 16:1115-1121 He Q, Huang Y, Zhang L, Yan Y, Liu J, Song X, Chen W Association between vitamin D receptor polymorphisms and hepatitis B virus infection susceptibility: A meta-analysis study Gene 2018; 645:105-112 Li J Dong, PH Jin, YH Lu, MQ Pan, FF Wang, BS Chen, YP The relationship between vitamin D receptor gene polymorphism and liver fibrosis Zhejiang Med J 2006; 28:426-434 Suneetha PV, Sarin SK, Goyal A, Kumar GT, Shukla DK, Hissar S Association between vitamin D receptor, CCR5, TNF-alpha and TNF-beta gene polymorphisms and HBV infection and severity of liver disease J Hepatol 2006; 44:856-863 ... clinical manifestations in patients infected with chronic HBV - To assess the association of VDR polymorphism and VDR in patients with HBV infection including CHB, liver cirrhosis and HCC SUBJECTS... variant with the progression of cirrhosis in patients with chronic HBV infection Suneetha et al [9] also reported the role of ApaI variant in HBV-infected patients In this study, VDR- ApaI rs7975232... inconsistent and no studies have been published on chronic HBV patients in Vietnam where high rates of HBV infection range from 10 - 20% The chronic hepatitis and the complications of HBV infection