Background/Aim: Hepatitis B Virus (HBV) mutations play a role in the development of hepatocellular carcinoma (HCC). However, the association between HBV polymerase gene mutations and HCC has not been reported. In this study, we conducted a multi-stage study to identify HCC-related mutations in the reverse transcriptase (RT) domain of the HBV polymerase gene.Methods: A total of 231 HCCs and 237 non-HCC controls from Qidong, China, were included in this study. The entire sequence of HBV RT was first compared between 29 HCC and 35 non-HCC cases, and candidate mutations were then evaluated in two independent validation sets.Results: There were 15 candidate mutations identified from the discovery set, with A799G and T1055A being consistently associated with HCC across all studies. A pooled analysis of samples revealed that A799G, A987G, and T1055A were independent risk factors for HCC, with adjusted odds ratios of 5.53 [95% confidence interval (CI), 1.69-18.10], 4.20 (95% CI, 1.15-15.35), and 3.78 (95% CI, 1.45-9.86), respectively. A longitudinal study showed that these mutations were detectable 45 years prior to HCC diagnosis.Conclusions: Our study provides evidence the first that HBV RT contains naturally occurring mutations that can be used as predictive markers for HCC.

Objective: To explore the relationship between the mutations in the reverse transcriptase(RT) domain of hepatitis B virus polymerase(HBV-P) gene and the occurrence and development of hepatocellular carcinoma(HCC). Methods: In this case-control study, the serum samples from 202 HCC patients and 202 chronic hepatitis B virus(CHB) carriers matching for age and gender were collected. The sequence of the RT domain of HBV-P gene was determined by direct sequencing following PCR amplifi cation. The relationship between the mutations in the RT domain of HBV-P gene and the occurrence and development of HCC was analyzed. Results: The T895A, A904T and C955T mutations in the RT domain of HBV-P gene were all signif icantly associated with HCC compared to non-HCC controls(P = 0.034, 47.5% vs 37.1%; P = 0.011, 5.4% vs 1.0%; P = 0.030, 5.4% vs 1.5%). Furthermore, the Logistic multivariate analysis showed that T895A was an independent risk factor for HCC [odds ratio(OR) = 2.230, 95% confi dence interval(CI): 1.230-4.044, P = 0.008]. A904T or C955T increased the risk of HCC occurrence either(OR = 6.523, 95% CI: 0.838-50.733; OR = 2.904,95% CI: 0.599-14.093), but it did not reach statistical signifi cance. The mutation rate of combined mutation occurrence either in A904 or C955T was 9.4% in HCC group and 2.5% in non-HCC controls(P = 0.003). The adjusted OR was 4.145(95% CI: 1.170-14.681), demonstrating its signif icant association with HCC(P = 0.028). Conclusion: The mutations in the RT domain of HBV-P gene are associated with the tumorigenesis of HCC.

Background and purpose: It was reported that G1896A and G1899A mutation in the hepatitis B virus(HBV) precore region were all signifi cantly associated with hepatocellular carcinoma(HCC). Simple, sensitive and reliable methods to detect precore mutations are needed in order to prevent the occurrence of HCC in clinical detection. The aim of this study was to develop a simple and sensitive reverse hybridization method for the detection of HBV precore mutation in HBV carriers. This method was applied for exploring the relationship between the precore mutations and HCC in patients of Qidong, Jiangsu Province. Methods: The specifi c probes of nt.1896 and nt.1899 were designed and synthesized. In order to improve the sensitivity and specificity, reaction conditions of reverse hybridization were optimized. We used reverse hybridization to detect 50 HCC serum samples and compared the results with direct sequencing. Then we used this method to assess the association between HBV precore mutations and HCC in serum samples from 50 HCC patients and 50 non-HCC controls. Results: The detection limit of reverse hybridization for HBV DNA level was 103 copy/mL and the sensitivity was 10% within a mixed virus population. The coincidence rate of reverse hybridization analysis was 98% compared with the direct sequencing results. It was showed that G1899A mutation was signifi cantly associated with HCC compared to non-HCC controls(P=0.000, OR=4.846, 95%CI: 2.240-10.485), while G1896A mutation was not associated with HCC. Conclusion: Reverse hybridization is a simple andaccurate approach for the detection of low amounts of HBV precore mutants among a mixed viral population. It has potential usage in the large-scale screening of precore mutations in chronic hepatitis B carriers.

AIM:To develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers. METHODS:The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction (PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis (CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 μm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of 114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma (HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis. RESULTS:CGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls (47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC (95%CI: 1.723-5.122, P < 0.001), large deletion (> 99 bp) did not show any association with HCC (P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9% (8/9) had deletions 2-5 years prior to HCC, while only 44.4%4 (4/9) contained such deletions 6-9 years prior to HCC. CONCLUSION:CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.