The HBV web: An insight into molecular interactomes between the hepatitis B virus and its host en route to hepatocellular carcinoma.

Hepatitis B virus (HBV) is a major aetiology associated with the development and progression of hepatocellular carcinoma (HCC), the most common primary liver malignancy. Over the past few decades, direct and indirect mechanisms have been identified in the pathogenesis of HBV-associated HCC which include altered signaling pathways, genome integration, mutation-induced genomic instability, chromosomal deletions and rearrangements. Intertwining of the HBV counterparts with the host cellular factors, though well established, needs to be systemized to understand the dynamics of host-HBV crosstalk and its consequences on HCC progression. Existence of a vast array of protein-protein and protein-nucleic acid interaction databases has led to the uncoiling of the compendia of genes/gene products associated with these interactions. This review covers the existing knowledge about the HBV-host interplay and brings it down under one canopy emphasizing on the HBV-host interactomics; and thereby highlights new strategies for therapeutic advancements against HBV-induced HCC.

In Silico Integration of Transcriptome and Interactome Predicts an ETP-ALL-Specific Transcriptional Footprint that Decodes its Developmental Propensity.

Early T precursor acute lymphoblastic leukemia (ETP-ALL) exhibits poor clinical outcomes and high relapse rates following conventional chemotherapeutic protocols. Extensive developmental flexibility of the multipotent ETP-ALL blasts with considerable intra-population heterogeneity in terms of immunophenotype and prognostic parameters might be a target for novel therapeutic interventions. Using a public gene expression dataset (GSE28703) from NCBI GEO DataSets with 12 ETP-ALL and 40 non-ETP-ALL samples, such heterogeneity was found to be reflected in their transcriptome as well. Hub genes were identified from the STRING-derived functional interaction network of genes showing differential expression between ETP-ALL and non-ETP-ALL as well as variable expression across ETP-ALL. Nine genes (KIT, HGF, NT5E, PROM1, CD33, ANPEP, CDH2, IL1B, and CXCL2) among the hubs were further validated as possible diagnostic ETP-ALL markers using another gene expression dataset (GSE78132) with 17 ETP-ALL and 27 non-ETP-ALL samples. Linear dimensionality reduction analysis with the expression levels of the hub genes in ETP-ALL revealed their divergent inclinations towards different hematopoietic lineages, proposing them as novel indicators of lineage specification in the incompletely differentiated ETP-ALL blasts. This further led to the formulation of a personalized lineage score calculation algorithm, which uncovered a considerable B-lineage-bias in a substantial fraction of ETP-ALL subjects from the GSE28703 and GSE78132 cohorts. In addition, STRING-derived physical interactome of the potential biomarkers displayed complete segregation of the B-lineage-skewed markers from other lineage-associated factors, highlighting their distinct functionality and possible druggability in ETP-ALL. A panel of these biomarkers might be useful in pinpointing the dominant lineage specification programmes in the ETP-ALL blasts on a personalized level, urging the development of novel lineage-directed precision therapies as well as repurposing of existing therapies against leukemia of different hematopoietic lineages; which might overcome the drawbacks of conventional chemotherapy.

Familiarity Breeds Strategy: In Silico Untangling of the Molecular Complexity on Course of Autoimmune Liver Disease-to-Hepatocellular Carcinoma Transition Predicts Novel Transcriptional Signatures.

Autoimmune liver diseases (AILD) often lead to transformation of the liver tissues into hepatocellular carcinoma (HCC). Considering the drawbacks of surgical procedures in such cases, need of successful non-invasive therapeutic strategies and treatment modalities for AILD-associated-HCC still exists. Due to the lack of clear, sufficient knowledge about factors mediating AILD-to-HCC transition, an in silico approach was adopted to delineate the underlying molecular deterministic factors. Parallel enrichment analyses on two different public microarray datasets (GSE159676 and GSE62232) pinpointed the core transcriptional regulators as key players. Correlation between the expression kinetics of these transcriptional modules in AILD and HCC was found to be positive primarily with the advancement of hepatic fibrosis. Most of the regulatory interactions were operative during early (F0-F1) and intermediate fibrotic stages (F2-F3), while the extent of activity in the regulatory network considerably diminished at late stage of fibrosis/cirrhosis (F4). Additionally, most of the transcriptional targets with higher degrees of connectivity in the regulatory network (namely DCAF11, PKM2, DGAT2 and BCAT1) may be considered as potential candidates for biomarkers or clinical targets compared to their low-connectivity counterparts. In summary, this study uncovers new possibilities in the designing of novel prognostic and therapeutic regimen for autoimmunity-associated malignancy of liver in a disease progression-dependent fashion.

Virological Characteristics of Acute Hepatitis B in Eastern India: Critical Differences with Chronic Infection.

Hepatitis B Virus (HBV) manifests high genetic variability and is classifiable into ten genotypes (A-J). HBV infection can lead to variable clinical outcomes, ranging from self-limiting acute hepatitis to active chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study characterizes HBV strains circulating among patients with acute (AHB) and chronic HBV infection (CHB). Among a total of 653 HBsAg positive cases, 40 manifested acute infection. After sequencing the surface(S), basal core promoter/pre-core(BCP/PC) and the X gene regions, phylogenetic tree was constructed using MEGA4 by neighbor-joining method. Statistical robustness was established with bootstrap analysis. Nucleotide diversity was determined by Shannon entropy per site using the Entropy program of the Los Alamos National Laboratories. Analyses of acute patients revealed that HBV/D2 is the major circulating sub-genotype and commonly associated with sexual promiscuity and the age group between15-30 years. Comparison of AHB and CHB patients revealed that HBeAg positivity, ALT levels and genotype D were significantly high in AHB, whereas CHB patients were predominantly male, had a high viral load, and were commonly associated with genotype C. The frequencies of mutations in the S, BCP/PC, and X gene were low in AHB as compared to CHB. Drug resistant mutations were not detectable in the polymerase gene of AHB. Average nucleotide diversity in AHB was considerably low as compared to CHB. Further, the highest average ΔH (average difference in entropy between chronic and acute infection) was observed in the BCP/PC region implying that this region was most vulnerable to mutations upon HBV persistence, especially in case of genotype C. Additionally, among all substitutions, the A1762T and G1764A BCP mutations were the strongest indicators of chronicity. In conclusion, the study exhibits a general portrait of HBV strains circulating among acute hepatitis B patients in Eastern India and their intricate differences with chronic patients which should be useful from the clinical point of view.

Tumor suppressor micro RNA miR-145 and onco micro RNAs miR-21 and miR-222 expressions are differentially modulated by hepatitis B virus X protein in malignant hepatocytes.

BACKGROUND: Hepatitis B Virus (HBV) X protein (HBx) is known to be involved in the initiation and progression of hepatocellular carcinoma (HCC) through modulation of host gene response. Alterations in miRNA expressions are frequently noted in HCC. This study is aimed to examine the role of HBx protein in the modulation of oncogenic miRNA-21, miRNA-222 and tumor suppressor miRNA-145 in malignant hepatocytes. METHODS: Expressions of miRNA-21, miRNA-222 and miRNA-145 were measured in HepG2 cells transfected with HBx-plasmid (genotype D) and with full length HBV genome (genotype D) and also in stably HBV producing HepG2.2.15 cells using real time PCR. Their target mRNAs and proteins - PTEN, p27 and MAP3K - were analyzed by real time PCR and western blot respectively. miRNA expressions were measured after HBx/D mRNA specific siRNA treatment. The expressions of these miRNAs were analyzed in liver cirrhosis and HCC patients also. RESULTS: The study revealed a down-regulation of miRNA-21 and miRNA-222 expressions in HBx transfected HepG2 cells, pUC-HBV 1.3 plasmid transfected HepG2 cells as well as in HepG2.2.15 cells. Down regulation of miRNA-21 and miRNA-222 expression was observed in patient serum samples. Down regulation of miRNA-145 expression was observed in HepG2 cells transiently transfected with HBx and pUC-HBV1.3 plasmid as well as in patient samples but the expression of miRNA-145 was increased in HepG2.2.15 cells. Target mRNA and protein expressions were modulated in HepG2 cells and in HepG2.2.15 cell line consistent with the modulation of miRNA expressions. CONCLUSION: Thus, HBx protein differentially modulated the expression of miRNAs. The study throws light into possible way by which HBx protein acts through microRNA and thereby regulates host functioning. It might suggest new therapeutic strategies against hepatic cancer.

Molecular characterization of HBV strains circulating among the treatment-naive HIV/HBV co-infected patients of eastern India.

Previously we reported that the exposure to hepatitis B virus (HBV) infection serves as a major threat among the treatment naive HIV infected population of eastern India. Hence, molecular characterization of these strains is of utmost importance in order to identify clinically significant HBV mutations. A total of 85 treatment naive HIV/HBV co-infected participants were included of whom the complete basal core promoter/precore region, the core and the whole envelope gene could be successfully sequenced for 59, 57 and 39 isolates respectively. Following phylogenetic analysis, it was found that HBV/D was the predominant genotype with HBV/D2 (38.5%) being the most prevalent subgenotype followed by HBV/A1. The major mutations affecting HBeAg expression includes the A1762T/G1764A (13.6%), G1896A (22%) and G1862T mutation (33.9%) which was predominantly associated with HBV/A1. Moreover, the prevalence of G1896A was considerably high among the HBeAg negative HIV/HBV co-infected subjects compared to HBV mono-infection. The main amino acid substitutions within the MHC class II restricted T-cell epitope of HBcAg includes the T12S (15.8%) and T67N (12.3%) mutation and the V27I (10.5%) mutation in the MHC class I restricted T-cell epitope. PreS1/S2 deletion was detected in 3 isolates with all harboring the BCP double mutation. Furthermore, the frequently occurring mutations in the major hydrophilic loop of the S gene include the T125M, A128V and M133I/L. Therefore, this study is the first from India to report useful information on the molecular heterogeneity of the HBV strains circulating among the treatment naive HIV/HBV co-infected population and is thus clinically relevant.

Association of TNF-α Promoter Polymorphism with HBV Associated Disease Outcome Among HBV Infected Patients from Orissa, Southern Part of East India.

BACKGROUND: TNF-α promoter polymorphism has been known to be a potential predictive factor in patients with HBV infection. We therefore tried to investigate whether the TNF-α promoter polymorphism at position -238, -857 and -863 was associated with the outcome of HBV infection in a population from Orissa, southern part of East India. METHODS: A total of 195 patients recruited for the study were classified into 85 controls and 110 HBV infected cases, which included 34 IC, 30 CLD, 32 LC and 14 HCC patients. The polymorphisms at the respective sites were detected by a PCR-RFLP followed by statistical analysis. RESULTS: The frequency of the genotype -238 GG and the allele -238G in the cases (89.0% and 92.7% respectively) was significantly higher than that in the controls (68.2% and 82.2% respectively) (P < 0.001, OR = 3.8 and P = 0.001, OR = 2.73). Whereas the -238 GA genotype was significantly high in the control group (28.2%) when compared to the cases (7.2%) (P < 0.001, OR = 0.2). Similarly, the frequency of -863CC and the allele -863C was significantly higher among the cases (24.5% and 49.5%) compared to controls (1.17% and 34.7%), (P < 0.001, OR = 27.32 and P = 0.003, OR = 1.85), whereas the -863CA genotype was significantly high in the controls (67.0%) when compared to the cases (50.0%) (P = 0.01, OR = 0.49). Haplotype -863C/-857C/-238G in cases was significantly higher than controls (P = 0.002). Multivariate logistic regression analysis indicates that the genotype -863CC bears a negative association with liver disease progression. CONCLUSION: The present study established an association of polymorphisms at site -238 and -863 of the TNF-α promoter with the outcome HBV infection and disease progression.

Influence of HIV-associated degree of immune suppression on molecular heterogeneity of hepatitis B virus among HIV co-infected patients.

We have investigated the molecular diversity of Hepatitis B virus (HBV) among the HIV co-infected patients from eastern-India. HBsAg/HBV-DNA positive subjects (n=73) from 874 HIV-infected patients were analyzed by sequencing followed by genetic diversity quantification. HBV/genotype-D and HBV/sugenotype-D2 were predominant. HBV/D2 isolates from patients with low CD4 count manifested significantly lower non-synonymous substitutions (p<0.0001) and Shannon entropy (p=0.0006) in their surface and polymerase gene in comparison to those from moderately increased CD4 count. ART-induced immune-reconstitution therefore might raise non-synonymous immune/therapy escape substitutions among these HBV/D2 isolates. Decreased genetic diversity and increased viral load in the HBV/D2 isolates might facilitate the maintenance of their wild type characteristics in the low CD4 count, leading to its increased prevalence in this group. Interestingly, genetic diversity in HBV/A1, the next common subgenotype, was modified in the opposite manner. Together our results underscore the need for proper HBV molecular monitoring in HIV co-infection.

Association of Interleukin-1ß and Gene Polymorphisms with Liver Pathogenesis in Hepatitis B Virus Infection among Eastern Indian Population.

BACKGROUND: Interleukin-1ß (IL-1ß) is an important member of the family of the proinflammatory cytokines that modulate outcome of hepatitis B virus (HBV) infection. OBJECTIVES: This study was designed to investigate the relationship between the polymorphic genotypes of the interleukin-1ß (IL-1ß) promoter region and the interleukin-1 receptor antagonist gene (IL-1RN) and disease outcome in HBV-infected individuals. METHODS: DNA was extracted from 395 study subjects including HBV carriers with varying clinical presentations, as well as healthy controls and spontaneously recovered cases (SRC). Polymorphisms in IL-1ß (at position -511) and IL-1RN (variable nucleotide tandem repeats, VNTR) were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR based assay respectively. RESULTS: Among the study subjects, different IL-1ß (at position -511) (CC, CT and TT) and IL-1RN (1/1, 1/2, 2/2 and 1/3) polymorphic genotypes were found at variable proportions. Logistic regression analysis revealed, no notable difference at the level of IL-1ß promoter (P = 0.244; OR = 0.78; 95% CI = 0.52-1.18) or IL-1RN genotype polymorphism (P = 0.840; OR = 1.03; 95% CI = 0.78-1.36) among the HBV carriers and controls or SRC cases. Pairwise proportion testing showed, IL-1ß -511 genotype CC was significantly higher among asymptomatic carriers (ASC) in comparison with liver cirrhosis (LC) patients (P value = 0.028) and healthy control group (P-value = 0.036). IL-1RN genotype 2/2 was considerably higher in LC group than SRC as well as control group. Combinations of IL-1ß (-511) and IL-1RN polymorphisms were associated with disease progression, such as CC-1/2 with ASC and TT-2/2 with LC. CONCLUSION: IL-1ß polymorphisms are found to be associated with disease severity. Different polymorphic combinations are associated with degree of disease severity. Overall this is the first report from Eastern India, which shows association of IL-1ß polymorphisms with HBV-related hepatic complications.

Subgenotype D5, BCP and MHR mutations in hepatic complications among hepatitis B virus infected patients from Orissa, India.

The study was undertaken to investigate the clinical implications of hepatitis B virus (HBV) genotypes, basal core promoter (BCP), precore (PC) and surface gene mutations in HBV infected patients from Orissa, southeastern India. HBV infections were identified by serology testing and HBV DNA amplification by polymerase chain reaction among the 152 patients. After sequencing, surface gene mutation were studied by sequence analysis as well as by using BLOSUM scores and BCP mutations were studied only by sequence analysis. A high proportion of HBV/D5 (66.0%) was found among the study samples having significant relation with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) patients (p<0.05). The BCP mutation, TA (81.4%) and C1753/TA (75.0%) was found in significant proportion (p<0.05) among HCC cases and in fact a gradual increase in these mutations were noted between inactive carriers (IC) to HCC group and also showed higher viral load. An increasing trend of major hydrophilic region (MHR) mutations in S gene was also observed from IC (56.0%) to chronic liver disease (CLD) (60.4%) to LC (72.4%) to HCC (95.0%) patients. In conclusion, our study suggests that the predominant HBV subgenotype HBV/D5 with high viral load and BCP mutations (double and triple) and high mutations in MHR region was significantly associated with advanced liver disease (LC and HCC) and might act as predictor of severe hepatic complications.

Anti-hepatitis B core antigen testing with detection and characterization of occult hepatitis B virus by an in-house nucleic acid testing among blood donors in Behrampur, Ganjam, Orissa in southeastern India: implications for transfusion.

BACKGROUND: Occult hepatitis B virus (HBV) infection might transmit viremic units into the public blood supply if only hepatitis B surface antigen (HBsAg) testing is used for donor screening. Our aim was to evaluate the prevalence of occult HBV infection among the HBsAg negative/antiHBc positive donations from a highly HIV prevalent region of India. METHODS: A total of 729 HBsAg negative donor units were included in this study. Surface gene and precore region were amplified by in house nucleic acid test (NAT) for detection of occult HBV infection and surface gene was analyzed after direct sequencing. RESULTS: A total of 220 (30.1%) HBsAg negative donors were antiHBc positive, of them 66 (30%) were HBV DNA positive by NAT. HBV DNA positivity among 164 antiHBc only group, was 27.1% and among 40 antiHBs positive group was 30.0%. HBV/D (93.3%) was predominant and prevalence of both HBV/C and HBV/A was 3.3%. Single or multiple amino acids substitutions were found in 95% samples. CONCLUSION: Thus, a considerable number of HBV infected donors remain undiagnosed, if only HBsAg is used for screening. Addition of antiHBc testing for donor screening, although will lead to rejection of a large number of donor units, will definitely eliminate HBV infected donations and help in reducing HBV transmission with its potential consequences, especially among the immunocompromised population. The HBV genetic diversity found in this donor population are in accordance with other parts of India.