Novel Point and Combo-Mutations in the Genome of Hepatitis B Virus-Genotype D: Characterization and Impact on Liver Disease Progression to Hepatocellular Carcinoma

<div><p>Background</p><p>The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC.</p><p>Methods</p><p>The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence.</p><p>Results</p><p>Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively.</p><p>Conclusions</p><p>Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.</p></div

Frequencies of naturally occurring single and combination of mutations accumulated in the HBV genome were found significantly higher in hepatocellular carcinoma (HCC) than non-HCC patients.

<p>p<0.05 was considered as significant. ** indicates p>0.05.</p

List of primers used to generate PCR products and promoter construct.

<p>List of primers used to generate PCR products and promoter construct.</p

Frequencies of mutations showing escalating trend with the progression of the liver diseases from no liver fibrosis (nLF) or liver fibrosis (LF) to liver cirrhosis (LC) and hepatocellular carcinoma (HCC).

<p>Significant mutations were indicated in bold (p≤0.05) and marginal significant values were represented in italics.</p><p>Frequencies of mutations showing escalating trend with the progression of the liver diseases from no liver fibrosis (nLF) or liver fibrosis (LF) to liver cirrhosis (LC) and hepatocellular carcinoma (HCC).</p

Multivariate analysis to identify independent risk factors for HCC development in HBeAg positive and negative patients.

<p>p value <0.05 was considered as significant.</p><p>Multivariate analysis to identify independent risk factors for HCC development in HBeAg positive and negative patients.</p

Clinical, biochemical and demographic profile of sixty-eight patients included in the study.

<p>SD =  Standard deviation; NS =  Not significant; ALT =  Alanine aminotransferase; AST = Aspartate aminotransferase; INR =  International normalized ratio; ALP = Alkaline phosphatase; IU =  International unit; Δ Parameters presented as Mean±SD; β parameters presented as Median [Range]; δ Mutations with statistically significant difference between the groups are presented.</p><p>Clinical, biochemical and demographic profile of sixty-eight patients included in the study.</p

Quantification of ROS generated after HBV/genotype D and HBV/genotype C infection.

<p>Huh7 cells were transfected with control pTriEX vector and pTriEX-HBV/D and pTriEX-HBV/C plasmids, which contain 1.2× HBV genome independently. After 48 hours, cells were treated with DCFDA and DCF positive cells were quantified on (A) FACSCaliber and (B) Confocal microscope. Scale bar corresponds to 20 µm. <i>p</i><0.05 was considered as significant.</p

Distribution pattern of HBV/genotype C and HBV/genotype D in hepatocellular carcinoma (HCC) (A) and in liver cirrhotic (LC) (B) patients.

<p>HBV genotypes were detected from sequence analysis of five clones of amplified HBx or HBs region from chromosomal DNA and serum viral DNA using virus specific primer pairs. This will amplify replicating virus in liver tissue and circulating virus in serum. Genotypes of Integrated viruses were determined from sequence analysis of five clones of Alu-PCR product. T = tumor, NT = adjacent non-tumor and LC = liver cirrhosis.</p

Genetic Association and Gene-Gene Interaction Reveal Genetic Variations in ADH1B, GSTM1 and MnSOD Independently Confer Risk to Alcoholic Liver Diseases in India

<div><p>Genetic susceptibility is an important modifier of clinical outcome and natural history of progression in Alcoholic liver disease (ALD). While the significance of ethnicity in this evolution is very clear, subtle inter-individual genetic variant(s) might be important and thus we investigated those in an Indian population. Fourteen markers were genotyped within two alcohol metabolism genes [Alcohol dehydrogenase (<i>ADH</i>) gene clusters (<i>ADH1B</i> and <i>ADH1C</i>) and Aldehyde dehydrogenase (<i>ALDH2</i>)], one microsomal ethanol oxidizing enzyme cytochrome p450 (<i>CYP2E1</i>) and three oxidative stress response (OSR) genes (<i>MnSOD</i>, <i>GSTT1</i> and <i>GSTM1</i>) among 490 Bengali individuals (322 ALD and 168 control) from Eastern and North-Eastern India and validation was performed in a new cohort of 150 Bengali patients including 100 ALD and 50 advanced non-alcoholic steatohepatitis (NASH). Out of 14 genetic variants, carriage of 5 genotypes (rs2066701CC in <i>ADH1B</i>, rs1693425TT in <i>ADH1C</i>, rs4880TT in <i>MnSOD</i> and <i>GSTT1</i>/<i>GSTM1</i> null, p-value <0.05) were noted significantly higher among ALD patients while inter or intra group gene-gene interaction analysis revealed that addition of risk genotype of any OSR gene enhanced the possibility of ALD synergistically. Multiple logistic regression analysis showed independent association of rs2066701CC, rs4880TT and <i>GSTM1</i> null genotype with ALD while lower frequencies of those genotypes in advanced NASH patients further confirmed their causal relation to ALD. Thus these findings suggest that the three variants of <i>ADH1C</i>, <i>MnSOD</i> and <i>GSTM1</i> can be used to identify individuals who are at high risk to develop ALD and may be helpful in proper management of Indian alcoholics.</p></div

Quantification of DNA double strand breaks generated after HBV infection.

<p>Huh7 cells (4×10⁴) were transfected with control pTriEX vector and pTriEX-HBV/D, pTriEX-HBV/C plasmids, which contain 1.2× HBV genome, separately in 4 wells glass chamber slide. After 48 hours, (A) cells were fixed, immuno-stained with γ-H2AX antibody and observed under confocal microscope. Scale bar corresponds to 20 µm. (B) The number of foci per cell in ten different fields was counted and average of foci number per cell was plotted. (C) Total protein was isolated and western blot analysis was performed with γ-H2AX antibody. The ratio of band intensity between γ-H2AX and tubulin was determined. The experiment was repeated three times. <i>p</i><0.05 was considered as significant variation.</p