Assessment of miR-212 and Other Biomarkers in the Diagnosis and Treatment of HBV-infection-related Liver Diseases.

OBJECTIVES: Our study aims to detect the sensitivity of the new biomarker miR-212 existing in serum exosomes along with other hepatocellular carcinoma biomarkers such as AFP (alpha-fetoprotein), CA125 (carbohydrate antigen-ca125), and Hbx protein in the diagnosis of HBV-related liver diseases. We also aim to study the roles of these biomarkers in the progression of chronic hepatitis B and provide scientific data to show the clinical value of these biomarkers. METHODS: We selected 200 patients with HBV-infection (58 cases of chronic hepatitis B, 47 cases of hepatocellular carcinoma, 30 cases of compensatory phase cirrhosis, and 65 cases of decompensatory phase cirrhosis), 31 patients with primary liver cancer without HBV infection, and 70 healthy individuals as the control group. The expression level of serum AFP and CA125 was detected with electrochemiluminescence immunoassay. The expression level of the Hbx protein was detected with ELISA. Meanwhile, the expression level of miR-212 in serum was analyzed with RT-qPCR. We collected patients' clinical information following the Child-Pugh classification and MELD score criterion, and statistical analysis was made between the expression level of miR-212 and the collected clinical indexes. Lastly, we predicted the target genes of the miR-212 and its functions using bioinformatics methods such as cluster analysis and survival prediction. RESULTS: Compared to the control group, the expression level of miR-212 in HBV infected patients was remarkably increased (P<0.05), especially between the HBV-infection Hepatocellular carcinoma group and the non-HBVinfection liver cancer group (P<0.05). The expression of miR-212 was increased in patients' Child-Pugh classification, MELD score, and TNM staging. Moreover, the sensitivity and specificity of miR-212 were superior to AFP, CA125, and HBx protein. CONCLUSION: There is a linear relationship between disease progression and expression level of miR-212 in the serum of HBV infected patients. This demonstrates that miR-212 plays a significant role in liver diseases. miR-212 is expected to be a new biomarker used for the diagnosis and assessment of patients with HBV-infection-related liver diseases.

Assessment of HLADP gene rs3128917 and rs9380343 polymorphisms in chronic HBV infection.

BACKGROUND/AIMS: About 400 million people worldwide have been exposed to Hepatitis B (HBV) infection. A range of 10%-15% of chronic HBV carriers may present with various liver diseases including cirrhosis and hepatic cancer. The chronicity or clearance of HBV infection is dependent on viral and genetic variables. Genome-wide association studies (GWAS) have reported that the variants of human leukocyte antigen (HLA), rs3128917 and rs9380343, are significantly related to persistent HBV infection. HLA molecules are responsible for introducing various antigens into the immune system. These variants might affect antigen presentation by influencing HLA mRNA expression, therefore, antigen presentation may not be performed properly. This study aims to assess the relationship of HLA gene variants to chronic HBV infection. MATERIALS AND METHODS: HLA variants were explored in 238 chronic HBV patients and in 238 individuals with spontaneous clearance of HBV using PCR-RFLP assay. RESULTS: The allele and genotype of rs9380343 polymorphism were associated with persistent HBV infection risk (allele: p=0.038, genotype: p=0.029), but rs3128917 polymorphism was not significant. Additionally, rs9380343 polymorphism was also related to increased risk of HBV infection in males (p<0.05). CONCLUSION: The current study is the first report demonstrating the HLA rs9380343 polymorphism as a genetic risk factor for chronicity of HBV infection. Further independent studies are required to confirm the current findings using a larger sample size in different populations.

Serum leptin and homeostasis model assessment-IR as novel predictors of early liver fibrosis in chronic hepatitis B virus infection.

BACKGROUND: The relationship between hepatitis B virus (HBV) infection, leptin and insulin resistance remains unclear. We hypothesised links between serum leptin and insulin resistance in non-diabetic patients with chronic viral hepatitis B infection and their relation to liver fibrosis. METHODS: We recruited 190 untreated patients with chronic HBV infection and 72 healthy controls. Serum leptin, fasting glucose, insulin, liver function tests (LFTs), C-peptide and Homeostasis model assessment-IR (HOMA-IR) were measured/calculated by ELISA and standard techniques. RESULTS: Serum leptin, C-peptide (both P < 0.001), HOMA-IR (P = 0.021) and several LFTs were increased in patients with chronic HBV-infection. In multivariate regression analysis, both HOMA-IR (P = 0.003) and leptin (P = 0.002) were significant independent predictors of HBV infection. There were significant positive correlations (P < 0.01) between leptin and HOMA-IR (r = 0.81), between serum leptin and METAVIR activity (r = 0.95), and between HOMA-IR and BMI (r = 0.75), fasting glucose (r = 0.005), and fasting insulin (r = 0.81). Several LFTs, glucose and insulin correlated modestly (r = 0.61-0.69, P < 0.05) with leptin. CONCLUSION: Serum leptin may be related to the rate of fibrosis progression in nondiabetic patients with chronic HBV infection. Follow-up by serial measurement of serum leptin and HOMA-IR in non diabetic HBV-infected patients may be used as a non-invasive marker of early liver fibrosis.

Comprehensive assessment showed no associations of variants at the SLC10A1 locus with susceptibility to persistent HBV infection among Southern Chinese.

The sodium taurocholate cotransporting polypeptide (NTCP) encoded by SLC10A1 was recently demonstrated to be a functional receptor for hepatitis B virus (HBV). The role of SLC10A1 polymorphisms, particularly the Ser267Phe variant (rs2296651) in exon 4, has been frequently investigated in regard to risk of persistent HBV infection. However, these investigations have generated conflicting results. To examine whether common genetic variation at the SLC10A1 locus is associated with risk of persistent HBV infection, haplotype-tagging and imputed single nucleotide polymorphisms (SNPs) were assessed in two case-control sample sets, totally including 2,550 cases (persistently HBV infected subjects, PIs) and 2,124 controls (spontaneously recovered subjects, SRs) of Southern Chinese ancestry. To test whether rare or subpolymorphic SLC10A1 variants are associated with disease risk, the gene's exons in 244 cases were sequenced. Overall, we found neither SNPs nor haplotypes of SLC10A1 showed significant association in the two sample sets. Furthermore, no significant associations of rare variants or copy number variation covering SLC10A1 were observed. Finally, expression quantitative trait locus analyses revealed that SNPs potentially affecting SLC10A1 expression also showed no significant associations. We conclude that genetic variation at the SLC10A1 locus is not likely a major risk factor of persistent HBV infection among Southern Chinese.

THE NEED FOR HBV GENOTYPING: A COST-EFFICIENT APPROACH.

The outcome of chronic HBV infection is variable; approximately one half of individuals transition to an inactive carrier state, 30% progress to cirrhosis, and the remainder to chronic hepatitis. Ten different HBV genotypes and many subtypes have been identified with distinct geographical distributions. Over the years, a lot of studies presented the efficiency of different genotyping methods; for this reason we aimed to present a cost efficient genotyping diagnosis algorithm of CHB infected patients, especially useful to identify those at risk of disease progression and determine optimal anti-viral therapy as useful instrument for physicians.

Assessment of chronic hepatitis B: the importance of hepatitis B virus DNA testing.

BACKGROUND: Chronic hepatitis B (CHB) has an estimated prevalence of 90 000 to 160 000 in Australia. Cirrhosis and hepatocellular carcinoma are important complications of CHB and appropriate evaluation of hepatitis B surface antigen (HBsAg)-positive individuals is vital to identify treatment candidates. METHODS: A review of the database of a tertiary hospital was performed and 348 HBsAg-positive individuals with baseline demographic, virological, serological and biochemical variables were identified and evaluated cross-sectionally. A small subgroup of hepatitis B e antigen (HBeAg)-negative patients with normal alanine aminotransferase (ALT) at baseline were identified and followed longitudinally. RESULTS: 175/348 (50%) of patients were in the HBeAg-negative, chronic hepatitis phase of disease, 22% in the HBeAg-positive immune clearance and 6% in the immune tolerant phases. HBeAg-negative patients were older and more likely to be male than HBeAg-positive patients. The correlation between hepatitis B virus (HBV) DNA and ALT levels was examined. ALT and HBV DNA levels showed no correlation in HBeAg-positive CHB and only a weak correlation in HBeAg-negative patients. Furthermore, 35% of HBeAg-negative patients with detectable HBV DNA had a normal ALT. Conversely 38% of HBeAg-negative patients with no detectable HBV DNA had an elevated ALT. A persistently normal ALT over 24 months was seen in five of nine HBeAg-negative patients with normal initial ALT and detectable HBV DNA. CONCLUSION: Appropriate evaluation of HBeAg-negative CHB must include HBV DNA because the ALT is not a reliable guide to underlying viral replication.

Tenofovir disoproxil fumarate for the treatment of chronic hepatitis B infection.

This paper presents a summary of the evidence review group (ERG) report into the clinical effectiveness and cost-effectiveness of tenofovir disoproxil fumarate for the treatment of chronic hepatitis B, in accordance with the licensed indication, based upon the evidence submission from Gilead to the National Institute for Health and Clinical Excellence (NICE) as part of the single technology appraisal process. The submitted clinical evidence included two international randomised controlled trials (RCTs) comparing tenofovir with adefovir, and a mixed treatment comparison (MTC) using Bayesian methodology to compare tenofovir with other nucleos(t)ide analogues using direct and indirect RCT evidence. There were no statistically significant differences between tenofovir and adefovir in overall adverse events although, in hepatitis B 'e' antigen (HBeAg)-positive patients, there was a higher incidence of mild nausea in the tenofovir treatment group. The primary outcome, 'complete response', was a composite end point defined as histology response and hepatitis B virus DNA below 400 copies/ml. For both HBeAg-positive and HBeAg-negative patients, a significantly greater proportion had a complete response after 48 weeks with tenofovir than with adefovir. There was no statistically significant difference in histological response in either group of patients compared with adefovir. The MTC could only generate results for HBeAg positive nucleos(t)ide naive patients as there was insufficient evidence for other subgroups. The probability of achieving undetectable HBV DNA with tenofovir was found to be significantly higher than that for all other treatments considered in the analysis at the 0.05 level. The analysis demonstrated that there is a 98% probability that tenofovir is the most potent nucleos(t)ide in terms of this outcome. The manufacturer's submission concluded that tenofovir is a cost-effective option as first-line treatment. For HBeAg-positive patients, tenofovir followed by lamivudine has an incremental cost-effective ratio (ICER) of 9940 pounds per quality-adjusted life-year (QALY) gained, compared with lamivudine followed by tenofovir. A more appropriate treatment strategy of tenofovir followed by tenofovir plus lamivudine has an ICER of 13,619 pounds per QALY gained, compared with lamivudine followed by tenofovir. For HBeAg-negative patients, tenofovir followed by lamivudine has an ICER of 9811 pounds per QALY gained, compared with best supportive care. A more clinically appropriate treatment strategy of tenofovir followed by tenofovir plus lamivudine has an ICER of 13,854 pounds per QALY gained, compared with tenofovir followed by lamivudine. The ERG uncovered a number of errors in the submission and these ICERs approximately doubled when the analysis was corrected and reran. The guidance issued by NICE on 22 July 2009 states that tenofovir disoproxil, within its marketing authorization is recommended as an option for the treatment of people with chronic HBe-Ag-positive or HBe-Ag-negative hepatitis B in whom antiviral treatment is indicated.

Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.

AIM: To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS: Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS: Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 (33%), 41 (33%) and 45 (36%) samples, and genotype C in 76 (60%), 76 (60%) and 81 (64%) samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real-time PCR was the rapidest and cheapest among the three assays. CONCLUSION: Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.

Semiquantitative and qualitative assessment of hepatic iron in patients with chronic viral hepatitis: relation with grading, staging and haemochromatosis mutations.

BACKGROUND: Hepatic iron overload is a common but still poorly characterized finding in patients with chronic viral hepatitis. AIM: To evaluate lobular and cellular distribution of iron in patients with chronic viral hepatitis, the relation between hepatic iron distribution, grading and staging, and the frequency of haemochromatosis gene mutations. PATIENTS: Thirty-four patients with chronic viral hepatitis and iron overload; 34 matched chronic viral hepatitis controls without iron overload; 139 healthy controls. METHODS: Hepatic iron was assessed by hepatic iron concentration and Deugnier's score, histological grading and staging by Ishak's score, and frequency of haemochromatosis gene mutations by polymerase chain reaction-restriction assays. RESULTS AND CONCLUSIONS: Iron deposits were found in hepatocytes (94% of the patients), sinusoidal tracts (88%) and portal cells (59%). In 41%, iron deposits were homogeneously distributed in the hepatic specimen. Hepatocytic iron showed a decreasing gradient from Rappaport's zone 1 to 3. Heavy alcohol intake influenced the distribution rather than the amount of hepatic iron by increasing sinusoidal iron. Haemochromatosis gene mutations were more frequent in chronic viral hepatitis patients with iron overload than in those without iron overload and in healthy controls suggesting they contribute to pathogenesis of hepatic iron accumulation. The correlation between hepatic fibrosis and portal iron supports the fibrogenetic role of iron in chronic viral hepatitis.