Association of TNF-Alpha gene polymorphisms and susceptibility to hepatitis B virus infection in Egyptians

Abstract

Tumor necrosis factor alpha (TNF-α) is one of the important cytokine in generating an immune response against hepatitis B virus (HBV). Genetic polymorphisms might influence gene transcription, leading to disturbance in cytokine production. We hypothesized that single nucleotide polymorphism (SNPs) in TNF-α gene could affect the pathogenesis of HBV. To test this hypothesis, we investigated the role of TNF-α polymorphism [−863C/A (rs1800630), −308G/A (rs1800629), −376G/A (rs1800750), −857C/T (rs1799724) and +489G/A (rs1800610)] in the susceptibility to chronic hepatitis B (CHB) infection. Polymorphisms of the TNF-α (−863C/A (rs1800630), −308G/A) were analyzed by Polymerase chain reaction sequence specific primer (PCR-SSP) while TNF-α (−376G/A, −857C/T and +489G/A) by PCR-restriction fragment length polymorphism (PCR-RFLP) in 104 patients with CHB and 104 healthy controls. The plasma level of TNF-α was measured using Enzyme-linked immunosorbent assay (ELISA). The study showed a significant increase in the frequency of −863CC, −376GA, −857CC, −857TT and +489GA genotypes and −863C, −376A, −857C, and +489A alleles in CHB patients compared to controls. In addition, CAGCG haplotype had a highest frequency in CHB patients. A strong Linkage Disequilibrium (LD) between TNF-α −863C/A (rs1800630) and −376G/A (D′ = 0.7888, r² = 0.0200); −308G/A and −857C/T (D′ = 0.9213, r² = 0.1770); −308G/A and +489G/A (D′ = 0.9088, r² = 0.1576) was demonstrated. CHB patients had significantly lower levels of TNF-α compared to controls. In conclusion, our preliminary results suggest that −863C/A (rs1800630), −308G/A, −376G/A, and +489G/A of the TNF-α gene may play a role in HBV susceptibility in Egyptians. The significant reduction in TNF-α in CHB patient was independent of any particular genotype/haplotype in TNF-α.

Introduction

Hepatitis B virus (HBV) infection is a main cause of chronic hepatitis, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) [1]. Globally, 240 million people have been chronically infected with HBV and it leads to the death of more than 686,000 people annually [2]. In Egypt there is an intermediate level of endemicity where the surface antigen (HBsAg) prevalence ranges from 2 to 8% of the population, so according to some studies there are more than 3 million chronic carriers of HBV in Egypt [3]. Additionally, there are some areas such as Upper Egypt and the countryside have the highest prevalence of 78% [4]. HBV persistence risk is related to two major factors: the age at the time of infection and the immune status of the host [5]; which has been reported to be major factor affecting the natural history of liver diseases [6]. Chronic HBV infection is a dynamic and non-linear process, with different stages characterized by different combinations of viral replication levels, humoral (antibody) and cellular (T cells) response features, and changes in liver disease activity [7], [24]. To what extent the immune response is responsible for chronic infection’s these distinctive features is critical for development of more successful immunotherapeutic interventions.

From multiple immune mediators implicated in the pathogenesis of CHB, Tumor necrosis factor alpha (TNF-α) is one of the most important cytokines involved in the immune response to HBV infection [1]. It is a potent proinflammatory cytokine secreted by macrophages, neutrophils, monocytes, T-cells and NK-cells [8]. It suppresses expression and replication of HBV in the liver, thus inducing the clearance of HBV [9]. It considered as a key player in HBV infection; it can facilitate immune-mediated virological control. On the contrary; it can cause collateral hepatocyte damage, cirrhosis and possibly promote liver cancer formation [10].

Human TNF-α gene is 850 kb telomeric to the class II HLA-DR locus on the short arm of chromosome 6 (6p21.3) composed of 4 exons with several polymorphisms [11], [12]. Its expression is tightly controlled at the transcriptional and post-transcriptional level. TNF-α production in patients with HBV infection has been shown to be affected by the polymorphisms in promoter region [13] and thus may affect the disease progression [14], [15], [16]. Polymorphisms in TNF-α is considered to be correlated with the severity of liver disease in HBV patients. It can also influence the susceptibility to chronicity and outcome of HBV infection such as cirrhosis or HCC [17]. Several investigations [18], [19], [20] have stressed on the importance of TNF-α polymorphisms not only in increasing the risk of persistence but also on dictate clinical outcomes including the risks of cancer formation or liver cirrhosis.

Single nucleotide polymorphisms (SNPs) have been proposed to be the next generation marker for the identification of loci associated with diseases and its progression [21]. In TNF-α gene, most of these SNPs occur in the promoter region of the gene and of particular interest are polymorphisms at nucleotide 308. In continuation with our previous studies on the relationship between cytokine gene polymorphim and HBV infection [22], [23], the aim of the current study was to investigate the association of TNF-α polymorphism [−863C/A (rs1800630), −308G/A (rs1800629), −376G/A (rs1800750), −857C/T (rs1799724) and +489G/A (rs1800610)] with the susceptibility to CHB infection. We also measured the plasma level of TNF-α in the same HBV patients and control groups to determine whether the change in the level of TNF-α level is associated or independent of the genetic polymorphism. Although some studies have been performed elsewhere, no such study has been carried out in Egypt.