Mutation And Significance Analyses Of HBV Precore/Basal Core Promoter In Patients With Chronic Severe Hepatitis B

Chronic severe hepatitis B (CSHB) occurs on the basis of chronic hepatitis B (CHB) and presents extensive hepatocyte inflammatory necrosis. CSHB is also termed as acute-on-chronic liver failure. The disease has a rapid exacerbation of liver function with various severe complications and is hard to be rescued, leading to its high mortality. The occurrence of CSHB is the result of imbalance between the hepatitis B virus (HBV) and host. However, concrete mechanism remains to be clarified. HBV preCcore (preC) and basal core promoter (BCP) mutations may affect HBeAg expression or secretion and viral replication. A few of investigators reported that prC/BCP mutations were associated with the CSHB occurrence, while some others debated the conclusion. The discrepant results may be due to relatively small amount of sampling (less than 70 cases for CSHB for individual study) and less sensitive method for amplifying the viral gene fragment. This study is to clarify preC/BCP mutational characteristics and to verify the association of the mutations with the severity progress in light of population-based analysis. The viral DNA was extracted from patients'sera and subjected to an in-house nested PCR for amplification of target gene fragment, followed by direct sequencing of PCR products. Point mutations at 10 reported hot mutant sites and insertion/deletion mutations were analyzed. The data were statistically treated and the association of mutational characteristics with the disease status was analyzed. The major results from the study are as follows.1. A sensitive and reliable nested PCR assay was developed with the low limit of detection for 103 copies/ml, which is more than 100-fold higher in sensitivity than conventional PCR assay reported.2. The target gene fragments were successfully amplified and sequenced from 399 samples of general CHB patients, 211 samples of severe CHB patients and 348 samples of CSHB patients.3. Analyses showed that mutation occurrence was significantly increased in CSHB patients than in CHB patients, especially at A1762, G1764, and G1896 mutant sites; Insertion/deletion mutations were also significantly increased in CSHB patients. Negative HBeAg was significantly more frequent in CSHB patients than in CHB patients, suggesting the possibility of enhanced immune damage due to HBeAg loss or decrease.4. Independent comparison of occurrences of A1762T+G1764A and A1762T+G1764A+G1896A mutations with various disease statuses showed that statistical significance existed in HBeAg negative patients. With disease progression towards severity, the incidence of two patterns of combined mutations was obviously increased.5. In the presence of A1762T+G1764A or A1762T+G1764A+G1896A mutation, HBV DNA levels were significantly different among various disease statuses of HBeAg negative patients. Severe CHB patients'HBV DNA level was significantly higher than CSHB than patients'HBV DNA level which was significantly higher than general CHB patients'HBV DNA level. In addition, patients with genotype B patients had a tendency of increasing occurrence of A1762T+G1764A or A1762T+G1764A+G1896A mutation with the disease progress towards exacerbation.Together, the analyses of HBV preC/BCP mutations among general CHB patients, severe CHB patients and CSHB patients showed that the regional mutations were associated with the severity of the disease. The results are helpful for understanding mechanisms of the occurrence of CSHB and have potential value for predictive analysis of CSHB development.