Impact of Hepatitis B Virus (HBV) X Gene Mutations on Hepatocellular Carcinoma Development in Chronic HBV Infection

The hepatitis B virus (HBV) PreS mutations C1653T, T1753V, and A1762T/G1764A were reported as a strong risk factor of hepatocellular carcinoma (HCC) in a meta-analysis. HBV core promoter overlaps partially with HBx coding sequence, so the nucleotide 1762 and 1764 mutations induce HBV X protein (HBx) 130 and 131 substitutions. We sought to elucidate the impact of HBx mutations on HCC development. Chronically HBV-infected patients were enrolled in this study: 42 chronic hepatitis B (CHB) patients, 23 liver cirrhosis (LC) patients, and 31 HCC patients. Direct sequencing showed HBx131, HBx130, HBx5, HBx94, and HBx38 amino acid mutations were common in HCC patients. Of various mutations, HBx130 + HBx131 (double) mutations and HBx5 + HBx130 + HBx131 (triple) mutations were significantly high in HCC patients. Double and triple mutations increased the risk for HCC by 3.75-fold (95% confidence interval [CI] = 1.101 to 12.768, P = 0.033) and 5.34-fold (95% CI = 1.65 to 17.309, P = 0.005), respectively, when HCC patients were compared to CHB patients. Functionally, there were significantly higher levels of NF-kappa B activity in cells with the HBx5 mutant and with the double mutants than that of wild-type cells and the triple-mutant cells. The triple mutation did not increase NF-kappa B activity. Other regulatory pathways seem to exist for NF-kappa B activation. In conclusion, a specific HBx mutation may contribute to HCC development by activating NF-kappa B activity. The HBx5 mutation in genotype C2 HBV appears to be a risk factor for the development of HCC and may be used to predict the clinical outcomes of patients with chronic HBV infection.