Establish Of The Standard Sequence Of Full-length Hepatitis B Virus Genome And Associtions Between Mutations And Hepatitis B Virus Related Disease

Hepatitis B virus (HBV) chronic infection is a major risk factor for hepatocellular carcinoma(HCC). Epidemiological studies provide a large number of data to prove a causal association between HBV infection and HCC. Since the advent of PCR amplification of viral DNA technology, the heterogeneity of HBV genome and its expression in the pathogenesis and the role of viral protein research, has become the focus of the study of hepatotropic virus. Mutation selection frequently happened in HBV genes in the process of development in chronic HBV infection or liver disease. The most important immune escape mutations include "a" epitope mutation in the S region, CTL recognition site mutation in the C region, and the stop codon mutation in the preC region. Mutations in other regions are rarely studied. China is an endemic aera for HBV infection with a high prevalence rate in the population. The epidemic strains of HBV in Europe, America, Japan, and other low-prevalence rate areas are different from ours, which is predominantly genotype B and genotype C. As yet, the standard sequence of the whole HBV genome is built with HBV strains prevalent abroad. Therefor, establishing the gene database and the standard sequence of HBV strains prevalent in China is a fundamental work for HBV variation studies. By analyzing the associations between HBV mutations and some HBV-related diseases, we can know if these mutations are playing a role in the progression of some diseases.ObjectiveFirst, we sequence the whole HBV genome of asymptomatic HBsAg carriers(ASCs) who were hepatitis B e antigen(HBeAg) seropositive and infected with HBV genotype B2 or genotype C2. Then we establish the standard full-length HBV sequences of genotype B2 and C2 which are prevalent in China. Second, we sequence some parts of the HBV genome of patients with chronic hepatitis B(CHB), liver cirrhosis(LC) and hepatocellular carcinoma who were infected with HBV genotype B2 and C2. At last, we explore the associations between HBV genotypes, mutations and different HBV-related liver disease states.MethodsUsing routine and nested polymerase chain reaction (PCR) method, amplify the whole genome of 486 cases of ASCs with HBeAg seropositive, and partial genome of 93 cases of CHB, 101 cases of LC, and 140 cases of HBV-related HCC. All of the cases were infected with HBV genotype B2 or C2. After amplifying, all the samples were sent to sequencing right away. MEGA5.0 clustal w software was used in sequence comparison and concatenation. We searched the NCBI Genebank and downloaded 10 HBV B2 and 10 HBV C2 sequences as references, and the bases with the highest frequent in all of the ASCs cases were considered as the wildtype. Using this method, we acquired the standard full-length sequences for HBV genotype B2 and C2. The bases with point mutation rate higher than 10% in all the samples of CHB, LC and HCC were assigned as"hotspots". The SPSS 16.0 and simple statistics software were used to analyze the data. Univariateχ2 test and unconditional logistic regression model were used to measure the relationship between genotypes, mutations and their interactions with the CHB, LC and HCC. The image processing software ORIGIN6.0 was used to analyze the mutation rate alterations in patients at different age group with different disease states.Results1.The standard full-length sequences of HBV subgenotype B2 and C2 from HBeAg seropositive ASCs were generated in this study, the homology between the two genotypes were 91.8%. In all the cases, CHB, LC and HCC patients were older than ASCs, and there were more males and more genotype C infected patients than females and genotype B.2.According to the"wildtype"of HBV genotype B2 and C2, we analyzed 66 hotspots in certain regions, most of the mutation rate was higher in genotype C than genotype B. In genotype C, when ASCs were considered as control group, A1206C mutation was a risk factor for LC; C873T,G1229A,A1314G,T1344C,T1353C,A1359G,C1362T,T1497C,C1504T,T1508A,G1512A,A1727T,A2574G mutations were risk factors for HCC; C1338T,G1386A,T1464C,T1500C,T1544A,G1613A,C1653T,T1674C,G1719T,A1727G,G2699C mutations were risk factors for both LC and HCC; G915A,A993G,A1053G,C1218T,A1221T,G1230C,T2441C,G2699A mutations were risk factors for CHB,LC and HCC. While T929A was a protect factor for CHB; A1329C was a protect factor for LC; T1078G,A1329C,C1505T,A1574T were protect factors for HCC; T796G,A934C,T2465A were protect factors for CHB and LC; T1488C,C1491C were protect factors for LC and HCC; G834A,G912A,C2456T,A2558G were protect factors for CHB,LC and HCC. In addition, A1206C was a risk factor for LC compared with CHB; T796G,G1229A,T1344C,A1359G,T2441C, A2574G were risk factors for HCC compared with LC; G1230C was a protect factor for HCC compared with LC.3.In HBeAg seropositive patients, T796G and G1229A were risk factors for HCC, while G1230C was a protect factor for HCC compared with LC, which illustrate that the HBeAg seropositive LC patients with T796G and G1229A mutations had a higher risk to develop HCC, while patients with G1230C had a lower risk to develop HCC compared with HBeAg seronegative LC patients. Compared with LC patients, A2574G was a risk factor for HCC in HBeAg seronegative patients,which inferred that the HBeAg seronegative LC patients with A2574G mutation had a higher risk to develop HCC compared with HBeAg seropositive LC. Compared with CHB patients, A1206C was a risk factor for LC in HBeAg seropositive patients, which inferred that the HBeAg seropositive CHB patients with A1206C mutation had a higher risk to develop HCC compared with HBeAg seronegative CHB patients.4.Univariant and multivariant regression analysis showed that T796G,C1218T,A1221T,G1229A,T1344C,A2574G were independent risk factors for HCC,G912A,G1230C,C2456T were independent protective factors for HCC. In HCC, LC and ASCs patients, the mutation rates did not increase with increasing age. The average mutation frequencies of T796G, G912A and C2456T were much higher in ASCs than HCC, LC and CHB. However, the average mutation frequencies of C1218T,A1221T and G1230C were much lower in ASCs than HCC, LC and CHB. The average mutation frequencies of G1229A and A2574G were much higher in HCC(49.6%,38.5%) than LC , CHB and ASCs (G1229A were 32.9%,28.1%,38.6%, respectly; A2574G were 15.2%,15.6%,21.7%, respectly).5. Analysis the YMDD (M204V/I) variation of HCC patients did not receive antiviral treatment. We examined the nt.739(YVDD) and nt.742(YIDD) of P gene found A739G mutation is only 5.81%, G741T variation of only 4.65% and lamivudine therapy 5-year resistance rate of nearly 70%, indicating that the natural mutation rate of YMDD is not high in the patients did not receive lamivudine treatment .Conclusions1.The heterogeneity between standard full-length sequences of HBV subgenotype B2 and C2 is 8.2%. 2.The HBeAg seropositive LC patients with T796G and G1229A mutations had a higher risk to develop HCC, while patients with G1230C had a lower risk to develop HCC compared with HBeAg seronegative patients. The HBeAg seronegative LC patients with A2574G mutation had a higher risk to develop HCC compared with HBeAg seropositive LC. The HBeAg seropositive CHB patients with A1206C mutation had a higher risk to develop HCC compared with HBeAg seronegative CHB patients.3. C1218T,A1221T,G1229A,A2574G mutations were independent risk factors for HCC development.