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Mutations at Nucleotide 1762, 1764 and 1766 of Hepatitis B Virus X Gene in Patients with Chronic Hepatitis B and Hepatitis B-Related Cirrhosis

Background and objective : Hepatitis B virus (HBV) is a DNA virus with high tendency toward hepatic tissue. There are currently about 3 million HBV-infected people and 350 to 400 million chronic carriers of this virus in the world. X protein plays a role in the over-expression of oncogenes, carcinogenicity of liver cells and overlaps with the basal core promoter of the virus. Mutations at specific nucleotides of this region increase viral replication and liver disease progression. The aim of this study was to investigate the frequency of mutations at nucleotides 1762, 1764 and 1766 of HBV X gene in patients with chronic hepatitis B and hepatitis B-related cirrhosis. Methods : In this study, 102 patients including 68 chronic hepatitis patients and 34 patients with hepatitis B-related cirrhosis were enrolled. After DNA extraction, HBV X gene was amplified and sequenced using Semi Nested-PCR. Obtained gene sequences were compared with the standard sequence of HBV virus X gene available in the gene bank (Okamoto AB033559). Then, the mutations in the gene X of HBV were identified. Results : Comparison of the standard sequence with sequences obtained from patients showed the presence of A1762T / G1764A mutation in 12 chronic (17.64%) and 13 cirrhotic (38.23%) patients. Also, C1766G / G1764T mutations were found in 8.23% of chronic patients and 17.64% of cirrhotic patients. Conclusion : A1762T / G1764A mutations in the overlapping region of the basal core promoter with gene X C-terminal may lead to liver disease progression from chronic hepatitis to cirrhosis, by changing the amino acid sequence of the X protein. The results were analyzed by SPSS-16 using T-test and Chi-square analytical tests. P-value of less than 0.05 was considered as statistically significant. Data were reported as means ± standard Deviations (SD) and as percentages, if necessary. Confidence interval (CI) of 95% was also calculated.

with chronic hepatitis B and 34 patients with cirrhosis, with confirmed diagnosis by a doctor using FibroScan. Approximately 5 ml of blood were collected and plasma was separated by centrifugation at 3000 rpm for 5 minutes. Then, 200 μl of plasma was used for viral DNA extraction using High Pure Viral Nucleic Acid Kit (Roche Co.) according to the manufactures' instructions. To perform Semi Nested-PCR of the X gene at nucleotide positions 1374 to 1836 of the HBV genome, a pair of external primers and a pair of inner primers were used for the first and second rounds, respectively. First, 35 cycles were considered for the first round of the PCR program, including the initial step at temperature of 94 o C for denaturation of the double-stranded template for 1 minute, annealing temperature of 55 o C for 1 min, extension at 72 o C for 1 minute and final elongation at 72 o C for 5 minutes. Then, the PCR products of the first round (713 bp fragment) were used for the second round. For the second round of the PCR, 35 cycles were considered, including the initial step at temperature of 94 o C for 5 minutes, denaturation at 94 o C for 30 seconds, annealing at 57 o C for 30 seconds, extension at 72 o C for 30 seconds and final elongation at 72 o C for 5 minutes. Then, the PCR products of the second round (516 bp fragment) was electrophoresed on 1.5% agarose gel and after staining with ethidium bromide was finally examined under UV light ( Figure 1). The products were later sent for sequencing to the MacroGene Company, South Korea. In order to analyze and evaluate the mutations at the nucleotide level, Blast and Chromas softwares were used. The obtained sequences from the patients were compared with the standard Iranian HBV strains and standard reference X gene of HBV, available in the GenBank (Okamoto AB033559). The results were analyzed by SPSS-16 using T-test and Chi-square analytical tests. P-value of less than 0.05 was considered as statistically significant. Data were reported as means ± standard Deviations (SD) and as percentages, if necessary. Confidence interval (CI) of 95% was also calculated.

INTRODUCTION
There are currently about 3 billion people infected with hepatitis B virus (HBV) and 350 to 400 million chronic carriers of this virus in the world (1,2). Hepatitis is defined as the inflammation of the liver and HBV is one of the main causes of hepatitis (3). Nearly 3% of Iran's population are chronic carriers of HBV (4). HBV infection has a wide clinical spectrum ranging from chronic to acute hepatitis and cirrhosis to hepatic cancer (4). These mutations also create an appropriate site with tendency to bind to transcription factors of the hepatocyte nuclear factor 1 (HNF1) and thus, increase viral replication (9). It seems that these mutations can be used as prognostic factor of the disease progression. Based on occurrence of these mutations, patients with high risk of liver disease can be screened. The mentioned mutations can be also beneficial to improve the diagnostic methods and treatment strategies. The aim of this study was to evaluate the frequency of mutations at nucleotides 1762, 1764 and 1766 regions of X gene of HBV, in two groups of patients with chronic hepatitis B and hepatitis B-related cirrhosis.

MATERIAL AND METHODS
This study (Ethics Committee code 9003170116) was performed on 102 patients infected with HBV consisted of 68 patients Given that the P21 protein inhibits the function of G1/S cyclins, the cell stops at the G1 phase. Thus, in infected liver cells, P53 protein is deactivated by the X protein and activates the E/Cdk2 cyclin by reducing the P21 protein expression, which ultimately leads to passage of cell from phase G1 to S (10). This process leads to uncontrolled proliferation of HBVinfected hepatocytes.

RESULTS
This study was performed on 68 patients with chronic hepatitis B including 17 females (25%) and 51 males (75%) and 34 patients with hepatitis B-related cirrhosis including seven females (20.5%) and 27 males (79.41%). The mean age of the subjects was 38.32 ± 11.45 years and 53.88 ± 8.78 years for the first and second group, respectively. There was a significant relationship between the parameter of age and disease stage between the two test groups (P=0.021). There was no significant relationship between gender and disease stage between the two groups of patients (P=0.620). A1762T and G1764A mutations were observed simultaneously in 13 cirrhotic patients (38.23%) and 12 patients (17.64%) with chronic hepatitis B. The simultaneous presence of G1764A/A1762T mutations in both groups showed a significant difference (P=0.023). G1764T mutation was observed in 20.50% of cirrhotic patients and 13.23% of patients with chronic hepatitis B. However, this mutation was not significantly different between the two groups (P=0.063). C1766G/G1764T mutations were observed simultaneously in six cirrhotic patients (17.64%) and six patients with chronic hepatitis B (8.82%). The simultaneous presence of this mutation showed no significant difference between the two groups.

DISCUSSION
Overall, the average age of cirrhotic patients was more than the average age of chronic patients, thus the index of age has a  bind to HNF3 and subsequently leads to increased viral replication. Frequency of G1764T/C1766G in the cirrhotic group was more than the group of chronic patients, which indicates the possible role of this mutation in the development of disease from the chronic stage to liver cirrhosis. In agreement with the present study, Poustchi et al. study in Iran on HBV genotype D, showed that the frequency of G1764T / C1766G double mutations in the patients with cirrhosis and patients with chronic hepatitis B are 75% and 42%, respectively. These results indicate the high frequency of this mutation (14). CONCLUSION A1762T / G1764A mutations in the overlapping region of the X gene C-terminal with BCP, leads to disease progression from the chronic stage to cirrhosis, by changing the amino acid sequence of the X protein. It is recommended to study the effect of mutations at overlapping region of the X gene with BCP on other stages of HBV infection such as HCC.

ACKNOWLEDGMENT
The present study was derived from a research project for completion of MSc course in Virology. The authors would like to thank the Deputy of Research and Technology of Golestan University of Medical Sciences for their financial support.

CONFLICT OF INTEREST
Therer are no conflicts of interest.  (12,13). In addition to adenine to guanine nucleotide change at position 1764 of X gene, guanine to thymine nucleotide changes was also observed. The result of G1764T mutation at position 131 of the X protein is Valine to Leucine amino acid change. Concurrency of this mutation with mutation C1766G at codon 131, leads to Valine to Isoleucine change at this position. This change in the X protein creates an appropriate binding site with more tendency to