| Qidong, Jiangsu province, is an area with a high mortality for hepatocellular carcinoma (HCC). The high incidence of HCC is the consequence of high prevalence of hepatitis B virus (HBV) infection and exposure to aflatoxin B1. Recently, the impact of HBV heterogeneity such as genotypes, viral load and viral mutations has been recognized to contribute to HCC development. However, there are only a few reports about the relationship between HBV heterogeneity and HCC in Qidong, China. To screen the new risk variants for HCC, we carried out a study comparing the complete sequences of HBV isolated from patients in Qidong. In addition, further studies were carried out to verify the association between the newly identified mutations and HCC. Our study provides additional data on HBV variations in relation to HCC in Qidong.PART 1. Comparison study of complete sequences of hepatitis B virus identifies new mutations associated with hepatocellular carcinoma in Qidong, ChinaAIM: To investigate the prevalence of HBV genotypes and identify new risk variations for HCC in Qidong high-risk area.METHODS:HBV DNA was extracted from serum samples of 20 HCC and 35 non-HCC patients by phenol-chloroform method. HBV full-length sequences were amplified by polymerase chain reaction (PCR). PCR product was cloned into T-vector and sequenced directly. In addition, a consecutive series of serum samples from four HCC cases were employed to compare the HBV sequences before and after the occurrence of HCC. Genotype was classified by Phylogenetic tree using software MEGA 4.1. Sequences of the complete genome were compared using software Bioedit. Statistical analysis was made by SPSS version 12.0.RESULTS:Genotype C2 was predominant in Qidong area. It was prevalent in 47 out of the 55 cases (85.5%), whereas genotype B2 only existed in 8 cases (14.5%). There was no significant difference in the distribution of genotype between HCC and non-HCC groups (P>0.05). The average rate of nucleotide substitutions within the whole genome of HBV was 0.0150±.0.0037 per site for HCC patients and 0.0110±0.0047 per site for non-HCC patients (P＜0.01). HCC group had significantly more nucleotide substitutions in pre-S2 (P=0.017), X (P＜0.001), precore/core (pre-C/C) (P=0.001) and P (P=0.013) regions. Pre-S deletion and twelve point mutations, i.e., pre-S2 start codon mutation, T53C in pre-S2 gene, T766A in S gene, G1613A, C1653T, A1762T, G1764A in X gene, and G1899A, C2002T, A2159G, A2189C and G2203W (A or T) in pre-C/C gene, showed close association with HCC. The longitudinal study demonstrated that the HCC-related mutations were gradually accumulated during the development of HCC.CONCLUSION: There was no association between genotype C and HCC in Qidong. The complete genome analysis of HBV provided pilot data for identification of other novel mutations related to HCC in Qidong high-risk area.PART 2. Mutations in precore/core gene of hepatitis B virus is associated with hepatocellular in Qidong, ChinaAIM:To investigate the mutations in pre-C/C gene of HBV that might be related to HCC.METHODS:HBV DNA was extracted from serum samples of 103 HCC and 103 age-, sex-matched chronic hepatitis (CH) patients by phenol-chloroform method. DNA sequences of HBV pre-C/C gene were amplified by PCR and sequenced directly. In addition, a consecutive series of serum samples from 13 HCC cases were employed to compare the core sequences before and after the occurrence of HCC. Genotype was classified by Phylogenetic tree using software MEGA 4.1. Sequences of the pre-C/C gene were compared using software Bioedit. Hepatitis B e antigen (HBeAg) was tested by commercially available assay. Statistical analysis was made by SPSS version 12.0.RESULTS:G1899A, A2159G, A2189C, G2203W showed consistent association with HCC by univariate analysis. Multivariate analysis demonstrated G1899A, A2189C and G2203W were independent risk factors for HCC. The odds ratio (95%CI) were 6.313 (2.067-19.283),4.251 (1.621-11.151) and 8.837 (1.009-77.428), respectively, for G1899A, A2189C and G2203W. The longitudinal study demonstrated that G1899A mutation was accumulated during the development of HCC. Reverse mutation was observed for G1896A. Although reverse mutations were also observed for A2159G and A2189C, these two variants have been keeping their properties from the 2-4 years before occurrence of HCC.CONCLUSION:G1899A, A2189C and G2203W were the new viral markers for HCC in Qidong high-risk area.PART 3. Hepatitis B virus core protein variations differ in tumor and surrounding liver tissues from patients with hepatocellular carcinomaAIM:To investigate the properties of HBV core protein in tumor and surrounding liver tissues from patients with HCC.METHODS:DNA was extracted from 98 tumor tissue from patients with HCC by phenol-chloroform method. DNA from 33 corresponding non-tumor liver tissues from the same group of the patients was also extracted. DNA sequences of HBV pre-C/C gene nt.1901-2365 were amplified by PCR and sequenced directly. Genotype was classified by Phylogenetic tree using software MEGA 4.1. Sequences of the core protein were compared using software Bioedit. Statistical analysis was made by SPSS version 12.0.RESULTS:In tumor tissues, most of the changes in HBV core protein clustered in small segments. The six mutation cluster regions (MCRs) were codon 21-38 (MCR1), codon 59-63 (MCR2), codon 83-87 (MCR3), codon 95-104 (MCR4), codon 130-135 (MCR5), and codon 151-155 (MCR6). Among these regions, codon 130 (38.8%),97 (37.8%),87 (23.5%),135 (14.3%),27 (14.3%) 100 (11.2%) and 5 (10.2%) had higher mutation rates than other codons. Codon 84 to 97 was the common region affected by the all nine truncations. Comparision study of the core sequences from the 33 HCC tissues and the corresponding non-tumor liver tissues showed that the mutation rate of HBV core in non-tumor tissues was significantly higher than that in tumor tissues (0.0217±0.0163 vs.0.0141±0.0112, P=0.03). Especially, the missense mutation rate in codon 39 to 58 and codon 64 to 82 in non-tumor tissues was significantly higher than that in tumor tissues, respectively (P=0.028 and P=0.006). CONCLUSION:The distribution of mutations in HBV core protein was not random in tumor tissues. The missense mutations clustered in codon 39 to 58 and codon 64 to 82 may be negatively correlated with the development of HCC. |
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