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Hepatitis B virus pre-S2 start codon mutations in Indonesian liver disease patients.

ABSTRACT: To identify the prevalence of pre-S2 start codon mutations and to assess their association with liver disease progression.The mutations were identified by direct sequencing from 73 asymptomatic carriers, 66 chronic hepatitis (CH), 66 liver cirrhosis (LC) and 63 hepatocellular carcinoma (HCC) patients. Statistical significances were determined using Fisher's exact test, ?² test, and t-test analyses whenever appropriate. Pre-S mutation as a risk factor for advanced liver disease was estimated by unconditional logistic regression model adjusted with age, sex, and hepatitis B e antigen (HBeAg). P < 0.05 was considered significant.Mutation of the hepatitis B virus (HBV) pre-S2 start codon was found in 59 samples from 268 subjects (22.0%), with higher prevalence in patients with cirrhosis 27/66 (40.9%) followed by HCC 18/63 (28.6%), chronic hepatitis 12/66 (18.2%) and asymptomatic carriers 2/73 (2.7%) (P < 0.001). Logistic regression analysis showed that pre-S2 start codon mutation was an independent factor for progressive liver disease. Other mutations, at T130, Q132, and A138, were also associated with LC and HCC, although this was not statistically significant when adjusted for age, sex, and HBeAg. The prevalence of pre-S2 start codon mutation was higher in HBV/B than in HBV/C (23.0% vs 19.1%), whilst the prevalence of T130, Q132, and A138 mutation was higher in HBV/C than in HBV/B. The prevalence of pre-S2 start codon mutation was higher in LC (38.9%) and HCC (40.0%) than CH (5.6%) in HBeAg? group, but it was similar between CH, LC and HCC in HBeAg? group.Pre-S2 start codon mutation was higher in Indonesian patients compared to other Asian countries, and its prevalence was associated with advanced liver disease, particularly in HBeAg? patients.

SUBMITTER: Utama A

PROVIDER: S-EPMC3471111 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Hepatitis B virus pre-S2 start codon mutations in Indonesian liver disease patients.

Utama Andi A Siburian Marlinang Diarta MD Fanany Ismail I Intan Mariana Destila Bayu MD Dhenni Rama R Kurniasih Tri Shinta TS Lelosutan Syafruddin A R SA Achwan Wenny Astuti WA Zubir Nasrul N Arnelis Lukito Benyamin B Yusuf Irawan I Lesmana Laurentius Adrianus LA Sulaiman Ali A

World journal of gastroenterology 20121001 38

<h4>Aim</h4>To identify the prevalence of pre-S2 start codon mutations and to assess their association with liver disease progression.<h4>Methods</h4>The mutations were identified by direct sequencing from 73 asymptomatic carriers, 66 chronic hepatitis (CH), 66 liver cirrhosis (LC) and 63 hepatocellular carcinoma (HCC) patients. Statistical significances were determined using Fisher's exact test, χ² test, and t-test analyses whenever appropriate. Pre-S mutation as a risk factor for advanced live ...[more]

PMID: 23082059

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Project description:AimTo investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH) technique, and to pave the way for elucidating the pathogenesis of HBV infection.MethodspcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1(-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-galactosidase (beta-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.ResultsThe pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of beta-gal in HepG2 cells transfected with pcDNA3.1(-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein.ConclusionThe pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transduction and cell apoptosis. This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.

Project description:Although hepatitis B virus (HBV) has been established to cause hepatocellular carcinoma (HCC), the exact mechanism remains to be clarified. Type II ground glass hepatocytes (GGH) harboring the HBV pre-S2 mutant large surface protein have been recognized as morphologically distinct hallmark of HCC in advanced stages of chronic HBV infection. Considering its pre-neoplastic nature, we hypothesized that type II GGH may exhibit high genomic instability, which is important for the carcinogenic process in chronic HBV carriers. In this study we found that pre-S2 mutant LHBS directly interacted with importin 1, the key factor that recognizes cargos undergoing nuclear transportation mediated by the importin 1 associated nuclear pore complex (NPC). By interacting with importin 1, which inhibits its function as an NPC factor, pre-S2 mutant LHBS blocked nuclear transportation of an essential DNA repair and recombination factor Nijmegen breakage syndrome 1 (NBS1) upon DNA damage, thereby delaying formation of nuclear foci at the sites of DNA double strand breaks. Pre-S2 mutant LHBS was also found to block NBS1-mediated homologous recombination repair and induce multi-nucleation of cells. In addition, pre-S2 mutant LHBS transgenic mice showed genomic instability indicated by increased global gene copy number variations (CNV), which were significantly higher than those in hepatitis B virus X mice, indicating that pre-S2 mutant LHBS is the major viral oncoprotein inducing genomic instability in HBV-infected hepatocytes. Consistently, the human type II GGHs in HCC patients exhibited increased DNA double-strand breaks representing significant genomic instability. In conclusion, type II GGH harboring HBV pre-S2 mutant oncoprotein represents a high-risk marker for the loss of genome integrity in chronic HBV carriers and explains the complex chromosome changes in HCCs. Non-tumorous liver sections from four each the HBx and pre-S2 mutant LHBS transgenic mice and 3 double transgenic mice between 9 and 22 months old were examined for gene gain and loss.

Dataset Information

Hepatitis B virus pre-S2 start codon mutations in Indonesian liver disease patients.

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Hepatitis B virus pre-S2 start codon mutations in Indonesian liver disease patients.

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