Hoang Van Tong

Hepatitis B virus‐induced hepatocellular carcinoma: functional roles of MICA variants

Hepatitis B virus infection is a high‐risk factor for hepatocellular carcinoma. The human major histocompatibility complex class I chain‐related gene A (MICA) is a ligand of the NKG2D receptor that modulates the NK and T‐cell‐mediated immune responses and is associated with several diseases. This study determined the effects of MICA polymorphisms during HBV infection and HBV‐induced HCC. We conducted a case–controlled study in a Vietnamese cohort and genotyped ten functional MICA polymorphisms including the microsatellite motif in 552 clinically classified hepatitis B virus patients and 418 healthy controls. The serum soluble MICA levels (sMICA) were correlated with MICA variants and liver enzyme levels. We demonstrated a significant contribution of MICA rs2596542G/A promoter variant and nonsynonymous substitutions MICA‐129Met/Val, MICA‐251Gln/Arg, MICA‐175Gly/Ser, triplet repeat polymorphism and respective haplotypes with HBV‐induced HCC and HBV persistence. The circulating sMICA levels in HBV patient groups were elevated significantly compared with healthy controls. A significant contribution of studied MICA variants to sMICA levels was also observed. The liver enzymes alanine amino transferase (ALT), aspartate transaminase (AST), total bilirubin and direct bilirubin were positively correlated with sMICA levels suggesting sMICA as a biomarker for liver injury. We conclude that MICA polymorphisms play a crucial role in modulating innate immune responses, tumour surveillance and regulate disease susceptibility during HBV infection.

LRRK2 and RIPK2 Variants in the NOD 2-Mediated Signaling Pathway Are Associated with Susceptibility to Mycobacterium leprae in Indian Populations

In recent years, genome wide association studies have discovered a large number of gene loci that play a functional role in innate and adaptive immune pathways associated with leprosy susceptibility. The immunological control of intracellular bacteria M. leprae is modulated by NOD2-mediated signaling of Th1 responses. In this study, we investigated 211 clinically classified leprosy patients and 230 ethnically matched controls in Indian population by genotyping four variants in NOD2 (rs9302752A/G), LRRK2 (rs1873613A/G), RIPK2 (rs40457A/G and rs42490G/A). The LRRK2 locus is associated with leprosy outcome. The LRRK2 rs1873613A minor allele and respective rs1873613AA genotypes were significantly associated with an increased risk whereas the LRRK2 rs1873613G major allele and rs1873613GG genotypes confer protection in paucibacillary and leprosy patients. The reconstructed GA haplotypes from RIPK2 rs40457A/G and rs42490G/A variants was observed to contribute towards increased risk whereas haplotypes AA was observed to confer protective role. Our results indicate that a possible shared mechanisms underlying the development of these two clinical forms of the disease as hypothesized. Our findings confirm and validates the role of gene variants involved in NOD2-mediated signalling pathways that play a role in immunological control of intracellular bacteria M. leprae.

Mannose binding lectin and susceptibility to Schistosomiasis

Abstract Background. Human ficolin 2 (encoded by FCN2) and mannose-binding lectin (encoded by MBL2) bind to specific pathogen-associated molecular patterns, activate the complement lectin cascade in a similar manner, and are associated with several infectious diseases. Our recently published study established certain FCN2 promoter variants and ficolin-2 serum levels as protective factors against schistosomiasis. Methods. We used the Nigerian cohort from our recently published study, which included 163 Schistosoma haematobium–infected individuals and 183 matched healthy subjects, and investigated whether MBL deficiency and MBL2 polymorphisms are associated with schistosomiasis. Results. MBL serum levels were significantly higher in controls and were associated with protection (P < .0001). The −550H minor allele was significantly associated with protection (P = .03), and the heterozygous genotypes −550HL were observed to confer protection (P = .03). The MBL2*HYPA haplotype was significantly associated with protection (P = .03), with significantly higher serum MBL levels in controls (P = .00073). The heterozygous 6-bp deletion in the promoter was observed to be a susceptibility factor in schistosomiasis (P = .03). Conclusions. In agreement with findings from our recently published study, the findings reported here support the observation that MBL is also associated with protection in schistosomiasis.

Identification of a natural intergenotypic recombinant hepatitis delta virus genotype 1 and 2 in Vietnamese HBsAg‐positive patients

Hepatitis D virus (HDV) infection is acquired as a co‐ /superinfection of Hepatitis B virus (HBV) and can modulate the pathophysiology of chronic hepatitis B and related liver diseases including hepatocellular carcinoma. Among the eight distinct HDV genotypes reported, relatively few studies have attempted to investigate the prevalence of HDV mixed genotypes and RNA recombination of HDV. With a recorded prevalence of 10–20% HBV infection in Vietnam, this study investigated the HDV variability, HDV genotypes and HDV recombination among twenty‐one HDV isolates in Vietnamese HBsAg‐positive patients. HDV subgenomic and full‐length genome sequences were obtained using newly established HDV‐specific RT‐PCR techniques. The nucleotide homology was observed from 74.6% to 99.4% among the investigated full‐length genome of the HDV isolates. We observed HDV genotype 1 and HDV genotype 2 in the investigated Vietnamese patients. Although no HDV genotype mixtures were observed, we report here a newly identified recombinant of HDV genotypes (HDV 1 and HDV 2). The identified recombinant HDV isolate C03 revealed sequence homology to both HDV genotype 1 (nt1 to nt907) and HDV genotype 2 (nt908 to nt1675; HDAg coding region) with a breakpoint at nt908. Our findings demonstrate the prevalence of intergenotypic recombination between HDV genotypes 1 and 2 in a Vietnamese HBsAg‐positive patient. Extended investigation on the distribution and prevalence of HDV, HDV mixed genotypes and recombinant HDV genotypes in a larger Vietnamese population offers vital insights into understanding of the micro‐epidemiology of HDV and subsequent pathophysiology in chronic HBV‐ /HDV‐related liver diseases.

IL-4 Haplotype-590T,-34T and Intron-3 VNTR R2 Is Associated with Reduced Malaria Risk among Ancestral Indian Tribal Populations

Background Interleukin 4 (IL-4) is an anti-inflammatory cytokine, which regulates balance between TH1 and TH2 immune response, immunoglobulin class switching and humoral immunity. Polymorphisms in this gene have been reported to affect the risk of infectious and autoimmune diseases. Methods We have analyzed three regulatory IL-4 polymorphisms; -590C>T, -34C>T and 70 bp intron-3 VNTR, in 4216 individuals; including: (1) 430 ethnically matched case-control groups (173 severe malaria, 101 mild malaria and 156 asymptomatic); (2) 3452 individuals from 76 linguistically and geographically distinct endogamous populations of India, and (3) 334 individuals with different ancestry from outside India (84 Brazilian, 104 Syrian, and 146 Vietnamese). Results The -590T, -34T and intron-3 VNTR R2 alleles were found to be associated with reduced malaria risk (P<0.001 for -590C>T and -34C>T, and P = 0.003 for VNTR). These three alleles were in strong LD (r2>0.75) and the TTR2 (-590T, -34T and intron-3 VNTR R2) haplotype appeared to be a susceptibility factor for malaria (P = 0.009, OR = 0.552, 95% CI = 0.356 –0.854). Allele and genotype frequencies differ significantly between caste, nomadic, tribe and ancestral tribal populations (ATP). The distribution of protective haplotype TTR2 was found to be significant (χ2 3 = 182.95, p-value <0.001), which is highest in ATP (40.5%); intermediate in tribes (33%); and lowest in caste (17.8%) and nomadic (21.6%). Conclusions Our study suggests that the IL-4 polymorphisms regulate host susceptibility to malaria and disease progression. TTR2 haplotype, which gives protection against malaria, is high among ATPs. Since they inhabited in isolation and mainly practice hunter-gatherer lifestyles and exposed to various parasites, IL-4 TTR2 haplotype might be under positive selection.

A trivial role of STAT4 variant in chronic hepatitis B induced hepatocellular carcinoma

Two polymorphisms in the STAT4 and HLA-DQ loci were more recently reported to associate with chronic hepatitis B (CHB) induced hepatocellular carcinoma (HCC). We utilised an independent Vietnamese cohort of clinically classified HBV patients of chronic hepatitis B carriers (n=206), liver cirrhosis (n=222) and hepatocellular carcinoma (n=239) and assessed the influence of the reported variants. The STAT4 variant (rs7574865) was marginally associated with HCC susceptibility in CHB carriers in allelic and recessive genetic models (OR=0.84, 95%CI=0.7-0.99, P=0.048 and OR=0.7, 95%CI=0.5-0.99, P=0.047). No significant association between the studied variant with several clinical parameters such as liver enzymes (ALT, AST), total and direct bilirubin, AFP, HBV genotype and viral loads were observed. Our study highlights the reported variant to be a trivial factor and possibly other confounding factors may regulate STAT4 expression during HCC development.

Genetic insights on host and hepatitis B virus in liver diseases

Hepatitis B virus (HBV) infection is a major global health problem and many studies have underlined the importance of inter individual variability and somatic mutations during the clinical course of HBV infection. In recent years, high-throughput technologies have provided new possibilities to study the genetic basis of many diseases. We reviewed all literature available on genome-wide association studies (GWASs), whole genome, exome and RNA sequencing studies as well as studies on HBV infection and the pathogenesis of related liver disease. Many GWASs conclude that the genetic variants in the HLA region (HLA-DP, HLA-DQ, HLA-DR and MICA), KIF1B, DEPDC5 and PNPLA3 influence HBV infection, its clinical course and the response to hepatitis B vaccination. The next generation sequencing approach provides important clues on the mutational landscape of genes involved in signaling pathways in particular JAK/STAT, Wnt/β-catenin, p53 pathways and multiple chromatin regulator genes that significantly promote hepatocarcinogenesis. In addition, the hotspots of recurrent integrations of HBV-DNA into host chromosomes such as hTERT, PDGF receptor, MLL are involved in pathogenesis of hepatocellular carcinoma (HCC). Additionally, the transitions T>C/A>G, C>T/G>A, C>A/G>T and T>A/A>T remain specific for HCC induced by viral infection and the DNA methylation in the CpG island is proposed as a biomarker for HCC. We have described common mutations in the HBV genome (G1896A, rtM204V, rtM204I) which modulate the pathogenesis and carcinogenesis of the liver. Further GWASs in different ethnic groups and additional functional studies are required to warrant the significance of such defined genetic factors. Such findings continue to shape our understanding of the genetic architecture of host-virus interactions and provide new clues and directions in determining genetic markers that modulate HBV infection and related liver diseases. The studies using high-throughput technologies help identifying potential genetic threats however the utility of mutational information can be complex in predicting prognostic significance and shall pose challenges to its clinical implementation.

Hepatitis E Virus Mutations: Functional and Clinical Relevance

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis and affects more than 20 million individuals, with three million symptomatic cases and 56,000 recognized HEV-related deaths worldwide. HEV is endemic in developing countries and is gaining importance in developed countries, due to increased number of autochthone cases. Although HEV replication is controlled by the host immune system, viral factors (especially specific viral genotypes and mutants) can modulate HEV replication, infection and pathogenesis. Limited knowledge exists on the contribution of HEV genome variants towards pathogenesis, susceptibility and to therapeutic response. Nonsynonymous substitutions can modulate viral proteins structurally and thus dysregulate virus-host interactions. This review aims to compile knowledge and discuss recent advances on the casual role of HEV heterogeneity and its variants on viral morphogenesis, pathogenesis, clinical outcome and antiviral resistance.

Occult Hepatitis B Virus Infection in Nigerian Blood Donors and Hepatitis B Virus Transmission Risks

Background Occult hepatitis B virus infection (OBI) characterized by the absence of detectable HBsAg remains a potential threat in blood safety. We investigated the actual prevalence, viral factors and genotype of OBI infections in Nigerian blood donors. Methods Serum collected from two blood banks were reconfirmed as HBsAg seronegative by ELISA. Forty HBsAg positive samples were employed as controls. HBV-DNA was amplified from all donors and viral loads were determined using quantitative real-time PCR. Antibodies to the HBV core, surface and HBe antigen (anti-HBc,anti-HBs,HBeAg) were measured. The PreS/S and PreC/C regions of the HBV genome were sequenced. Results Of the 429 blood donors, 72(17%) were confirmed as OBI by DNA detection in different reference labs and excluded the concern of possible contamination. Of the 72 OBI samples, 48(67%) were positive for anti-HBc, 25(35%) positive for anti-HBs, and 2(3%) positive for HBeAg. Of the 72 OBI samples, 31(43%) were seropositive for either anti-HBc, anti-HBs or HBeAg, 21 (30%) positive for both anti-HBc and anti-HBs,one positive for both anti-HBc and HBeAg. None of the OBI samples were positive for all three serological markers. The viral load was <50copies/ml in the OBI samples and genotype E was predominant. The L217R polymorphism in the reverse transcriptase domain of the HBV polymerase gene was observed significantly higher in OBI compared with HBsAg positive individuals (P<0.0001). Conclusion High incidence of OBI is relevant in high endemic areas worldwide and is a general burden in blood safety. This study signifies the high prevalence of OBI and proposes blood donor samples in Nigeria should be pre-tested for OBI by nucleic acid testing (NAT) and/or anti-HBc prior to transfusion to minimize the HBV infection risk.

Hepatitis E Virus Superinfection and Clinical Progression in Hepatitis B Patients

Hepatitis E virus (HEV) infection may cause acute hepatitis and lead to hepatic failure in developing and developed countries. We studied HEV seroprevalences in patients with hepatitis B virus (HBV) infection to understand the consequences of HEV superinfection in a Vietnamese population. This cross-sectional study was conducted from 2012 to 2013 and included 1318 Vietnamese patients with HBV-related liver diseases and 340 healthy controls. The case group included patients with acute (n=26) and chronic hepatitis B (n=744), liver cirrhosis (n=160), hepatocellular carcinoma (n=166) and patients with both liver cirrhosis and hepatocellular carcinoma (n=222). Anti-HEV IgG and IgM antibodies were assessed in patients and controls by ELISA. HEV-RNA was identified by PCR assays and sequencing. Seroprevalences of anti-HEV IgG among hepatitis B patients and controls were 45% and 31%, respectively (adjusted P=0.034). Anti-HEV IgM seroprevalences were 11.6% and 4.7% in patients and controls, respectively (adjusted P=0.005). Seroprevalences were higher among the elder individuals. When stratifying for patient groups, those with liver cirrhosis had the highest anti-HEV IgG (52%) and anti-HEV IgM (19%) seroprevalences. Hepatitis B patients with current HEV infection had abnormal liver function tests compared to patients with past or without HEV infection. One HEV isolate was retrieved from a patient with both liver cirrhosis and hepatocellular carcinoma and identified as HEV genotype 3. This study indicates high prevalences of HEV infection in Vietnamese HBV patients and among healthy individuals and shows that HEV superinfection may influence the outcome and progression of HBV-related liver disease.Hepatitis E virus (HEV) infection may cause acute hepatitis and lead to hepatic failure in developing and developed countries. We studied HEV seroprevalences in patients with hepatitis B virus (HBV) infection to understand the consequences of HEV superinfection in a Vietnamese population. This cross-sectional study was conducted from 2012 to 2013 and included 1318 Vietnamese patients with HBV-related liver diseases and 340 healthy controls. The case group included patients with acute (n = 26) and chronic hepatitis B (n = 744), liver cirrhosis (n = 160), hepatocellular carcinoma (n = 166) and patients with both liver cirrhosis and hepatocellular carcinoma (n = 222). Anti-HEV IgG and IgM antibodies were assessed in patients and controls by ELISA. HEV-RNA was identified by PCR assays and sequencing. Seroprevalences of anti-HEV IgG among hepatitis B patients and controls were 45% and 31%, respectively (adjusted P = 0.034). Anti-HEV IgM seroprevalences were 11.6% and 4.7% in patients and controls, respectively (adjusted P = 0.005). Seroprevalences were higher among the elder individuals. When stratifying for patient groups, those with liver cirrhosis had the highest anti-HEV IgG (52%) and anti-HEV IgM (19%) seroprevalences. Hepatitis B patients with current HEV infection had abnormal liver function tests compared to patients with past or without HEV infection. One HEV isolate was retrieved from a patient with both liver cirrhosis and hepatocellular carcinoma and identified as HEV genotype 3. This study indicates high prevalences of HEV infection in Vietnamese HBV patients and among healthy individuals and shows that HEV superinfection may influence the outcome and progression of HBV-related liver disease.