Impact of hepatitis B virus (HBV) x gene mutations on hepatocellular carcinoma development in chronic HBV infection

Abstract

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-κB 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-κB activity. Other regulatory pathways seem to exist for NF-κB activation. In conclusion, a specific HBx mutation may contribute to HCC development by activating NF-κB 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.

Fig. 1.

Percentage of HBx mutations according to clinical diagnosis of HBV infection. Abbreviations: CHB, chronic hepatitis B; LC, liver cirrhosis; HCC, hepatocellular carcinoma.

Fig. 2.

Phylogenetic tree of enrolled patients developed using the neighbor-joining method. Phylogenetic and molecular evolutionary analyses were performed using MEGA version 4.0.2 (The Biodesign Institute). The genetic distances were estimated by Kimura's two-parameter method. The reliability of the phylogenetic tree analysis was assessed by using the interior branch test with1,000 replicates. Abbreviations: CHB, chronic hepatitis B disease patient; LC, liver cirrhosis patient; HCC, hepatocellular carcinoma patient.

Fig. 3.

Protein expression of cells transiently transfected with wild-type and HBx mutant constructs. (A) HBsAg expression; (B) HBeAg expression.

Fig. 4.

Relative luciferase assays showing the activity of NF-κB in wild-type and HBx mutant Huh7 cells.