Association of VDR ApaI polymorphism with clinical outcomes and liver disease progression in patients with chronic hepatitis B virus infection

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.

ASSOCIATION OF VDR ApaI POLYMORPHISM WITH CLINICAL

OUTCOMES AND LIVER DISEASE PROGRESSION IN PATIENTS

WITH CHRONIC HEPATITIS B VIRUS INFECTION

Nguyen Khuyen 1,2 , Nghiem Xuan Hoan 3 Nguyen Binh An 3 , Dao Phuong Giang 3

Do Tuan Anh 1 , Le Huu Song 3 , Le Van Nam 1

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]

1

Military Hospital 103, Vietnam Military Medical University

2

My Duc General Hospital

3

108 Military Central Hospital

Corresponding author: Nguyen Khuyen (nguyenkhuyenbvdg@gmail.com)

Date received: 9/6/2020 Date accepted: 18/6/2020

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

1 Subjects

298 HBV-infected patients were classified into 3 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

2 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)

3 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 Mann-Whitney-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)

1 Charateristics of gender and age in the healthy group and hepatitis

B virus group

Table 1: Charateristics of gender between the groups

Gender Group

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

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

2 Laboratory characteristics of the controls and patients

Table 3: Paraclinical features between the two groups

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

(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 2 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)

3 The association between ApaI and HBV-related chronic liver diseases

Table 4: Comparison of genotypic and allelic frequency between healthy group and

HCC group

Groups

Co-dominant

2.5 (1.1 - 5.9) 0.035*

3.17 (1.05 - 9.94) 0.042# Allele

0.152

Recessive model

2.76 (1.2 - 6.1) 0.013*

3.5 (1.14 - 10.9) 0.024# Dominant model

(Note: * χ2 : test; #: logistic regression model adjusted for age and gender)

Table 5: Comparison of genotype and allen distribution between CHB and HCC

Groups

Co-dominant

Allele

Ressesive model

3.1 (1.1 - 9.2) 0.033*

4.3 (1.2 - 15) 0.017#

Dominant model

Table 6: Genotype and allele distribution in patients with CHB and advanced liver

disease (HCC+LC)

Groups

Co-dominant

Allele

Ressesive model

2.62 (0.95 - 7.22) 0.054*

3.19 (1.03 - 9.88) 0.032#

Dominant model

4 Correlation between VDR variants and vitamin D levels in HBV patients and healthy group

Figure 2: The association between vitamin D levels and genotypes of 4 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)

5 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

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 5 "encoding region" 8

exons (exons 2 - 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 8 The polymorphism at

these 3 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

5 "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

non-mutated 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

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

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