Studies On Correlation Between Disease Progression And Mutations In X Gene Of Hepatitis B Virus

Background and Objectives:Around 2 billion people worldwide have been infected with hepatitis B virus (HBV), of which 350 million people are chronic infection. About 1 million people die from chronic HBV infection related diseases each year. HBV infection seriously threatens human health.HBV is a small partial double stranded DNA virus, which consists of 3,200 nucleotides. Negative strand has four open reading frames (ORF), including S, P, C and X regions. HBV X gene is the most complicated region in the structure and function of HBV genome, overlapping Enhancer 2, the core promotor and direct repeat sequences (DR1 and DR2). The overlapping regions play important roles in replication, transcription regulation and expression of HBV. The protein coded by HBV X gene (HBx) consists of 154 amino acids with six regions (A-F). HBx is the unique protein in HBV genome, which has various sorts of regulative functions, such as activating transcription of host cells and HBV genes, regulating apoptosis, inhibiting exterior restoration of intracellular injured DNA, enhancing the ability of oncogene transcription, activating reverse transcriptase of human telomerase, activating the path of signal transduction, and promoting liver fibrosis. It also plays an important role in carcinogensis of HBV. HBV X gene and HBx protein have become the hot topics recently.Chronic HBV infection has different clinical stages, including asymptomatic carrier (ASC), chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Nevertheless, why different people infected with HBV have different outcomes remains unsolved. HBV replication is quite special. It needs an intermediate process, i.e. reverse transcription. Since HBV replication is very fast and active, and its reverse transcriptase lacks proofreading function, mutations of HBV are common. Studies on the relationship between HBV X gene and hepatocellular carcinoma are accumulating. However, detailed researches on the correlation between mutations of HBV X gene and progression of the disease are few. Therefore, we sequenced HBV X gene of 122 patients with chronic HBV infection, compared their sequences with the standard ones, and studied the correlation between HBV X gene mutations and the disease progression. Association of hot mutations in HBV X gene with HBV serum markers and HBV DNA quantity were analyzed as well. This study refreshed HBV pathogenicity and supplied new thoughts for the judgment of disease progression and HCC prevention.Methods:We randomly selected 122 chronic HBV infection patients, who were autochthons in Shandong province, including 35 patients of ASC,35 patients of CHB, 22 patients of LC and 30 patients of HCC. Fasting blood of all patients was collected and serum was made. HBV X gene was amplified with nested polymerase chain reaction (nested PCR) to all the 122 patients. The amplified products after purification were bilaterally sequenced with a fluorescence automatic sequencer (ABI-PRISM3730). The results were analyzed and compared with the standard sequences by the software of BioEdit7.0 and Clustal W. Enzyme-linked immunosorbent assay (ELISA) and fluorescence quantitative polymerase chain reaction (FQ-PCR) were used to detect HBV serum marker and HBV DNA quantity respectively. Relationship of hot mutations in HBV X gene with HBV serum markers and HBV DNA quantity were analyzed.Results:1. The mutation accumulation rates of HBV X gene in ASC, CHB, LC and HCC patients were 1.06%,1.20%,1.92% and 2.34% respectively. And the rates of accumulation mutations in HCC and in LC patients were higher than that in ASC and in CHB patients (p<0.05).2. The rates of A1762T/G1764A double mutations, T1753C point mutation and C1653T point mutation were much higher in HCC and in LC patients than in ASC and in CHB patients (p<0.05).3. The rates of T1719G point mutation in HCC and in LC patients were significantly less than that in ASC and in CHB patients (p<0.05).4. No differences were found among the 4 groups on the other two hot point mutations (i.e. C1485T and A1499G) (p>0.05).5. The incidences of A1762T/G1764A double mutations, T1753C point mutation and C1653T point mutation were much higher in 36 HBeAg negative patients (83.33%,44.44% and 30.56% respectively) than in 86 HBeAg positive patients (25.58%,4.65% and 3.49% respectively. p<0.05). No difference was found between the incidence of T1719G point mutation in 36 HBeAg negative patients (41.67%) and that in 86 HBeAg positive patients (45.35%, p=0.71).6. The quantity of HBV DNA in 52 patients with A1762T/G1764A double mutations (6.02±1.17 log copies/mL) was significantly lower than that in 70 patients without the double mutations (7.43±0.96 log copies/mL, p=0.00). The quantity of HBV DNA in 20 patients with T1753C point mutation (4.62±1.96 log copies/mL) was significantly lower than that in 102 patients without the mutation (6.99±1.60 log copies/mL, p=0.00). No difference was found between the quantity of HBV DNA in 14 patients with C1653T point mutation and that in 108 patients without the mutation (p=0.69). No difference was found between the quantity of HBV DNA in 54 patients with T1719G point mutation and that in 68 patients without the mutation (p=0.09).Conclusions:1. The development of HCC and LC are closely related to the accumulation of HBV X gene mutations.2. A1762T/G1764A double mutations, T1753C point mutation and C1653T point mutation may play important roles in the development of HCC and LC. 3. T1719G point mutation might weaken the pathogenicity and carcinogenicity of HBV.4. A1762T/G1764A double mutations, T1753C point mutation and C1653T point mutation might inhibit the expression of HBeAg, while T1719G point mutation had no influence on it.5. A1762T/G1764A double mutations and T1753C point mutation might inhibit the replication capability of HBV, while C1653T point mutation and T1719G point mutation had no influence on it.