Downregulation of Methionine Cycle Genes MAT1A and GNMT Enriches Protein-Associated Translation Process and Worsens Hepatocellular Carcinoma Prognosis.

The major biological methyl donor, S-adenosylmethionine (adoMet) synthesis occurs mainly in the liver. Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are two key enzymes involved in the functional implications of that variation. We collected 42 RNA-seq data from paired hepatocellular carcinoma (HCC) and its adjacent normal liver tissue from the Cancer Genome Atlas (TCGA). There was no mutation found in MAT1A or GNMT RNA in the 42 HCC patients. The 11,799 genes were annotated in the RNA-Seq data, and their expression levels were used to investigate the phenotypes of low MAT1A and low GNMT by Gene Set Enrichment Analysis (GSEA). The REACTOME_TRANSLATION gene set was enriched and visualized in a heatmap along with corresponding differences in gene expression between low MAT1A versus high MAT1A and low GNMT versus high GNMT. We identified 43 genes of the REACTOME_TRANSLATION gene set that are powerful prognosis factors in HCC. The significantly predicted genes were referred into eukaryotic translation initiation (EIF3B, EIF3K), eukaryotic translation elongation (EEF1D), and ribosomal proteins (RPs). Cell models expressing various MAT1A and GNMT proved that simultaneous restoring the expression of MAT1A and GNMT decreased cell proliferation, invasion, as well as the REACTOME_TRANSLATION gene EEF1D, consistent with a better prognosis in human HCC. We demonstrated new findings that downregulation or defect in MAT1A and GNMT genes can enrich the protein-associated translation process that may account for poor HCC prognosis. This is the first study demonstrated that MAT1A and GNMT, the 2 key enzymes involved in methionine cycle, could attenuate the function of ribosome translation. We propose a potential novel mechanism by which the diminished GNMT and MAT1A expression may confer poor prognosis for HCC.

HEV superinfection accelerates disease progression in patients with chronic HBV infection and increases mortality in those with cirrhosis.

BACKGROUND & AIMS: Acute HEV infection causes varying degrees of liver damage. Although liver-related death due to HEV infection alone is rare in healthy individuals, it is unclear whether HEV superinfection is associated with worse outcomes in patients with chronic HBV infection. Thus, we explored whether HEV superinfection was associated with increased incidence of liver-related death, cirrhosis, and hepatocellular carcinoma (HCC). METHODS: Serum and data were collected from 2 independent retrospective cohorts of patients with chronic HBV infection, comprising 2,123 patients without cirrhosis and 414 with cirrhosis at baseline, respectively. All the patients were negative for HEV-IgG at enrolment and HEV superinfection was defined by the presence of HEV-IgG seroconversion. RESULTS: In the non-cirrhotic cohort, 46 of 2,123 patients developed HEV superinfection. Though HEV superinfection was only associated with increased incidence of liver-related death in the overall cohort, it was a risk factor for all 3 endpoints (liver-related death, cirrhosis, and HCC) in a subgroup of 723 HBeAg-negative patients with chronic HBV infection. In addition, the 1-year mortality rate after HEV superinfection was higher in 4 patients who developed cirrhosis during the follow-up than in those who did not (50% vs. 2.4%, p = 0.001). To elucidate the perceived relationship between HEV superinfection and risk of mortality, an independent cohort of cirrhotic patients (n = 414) was further analyzed to control for the inherent increase in mortality risk due to cirrhosis. The 10 cirrhotic patients with HEV superinfection had a higher 1-year mortality rate than those without (30% vs. 0%, p <0.001). CONCLUSIONS: In both cohorts of patients with chronic HBV infection, acute HEV superinfection increases the risk of liver-related death, especially in those with cirrhosis. LAY SUMMARY: The mortality caused by acute hepatitis E virus infection is usually low in the healthy population, but it is unclear how it affects patients with chronic hepatitis B virus infection, as they already have compromised liver function. Our data show that the 1-year mortality rate is 35.7% in patients with hepatitis B-related cirrhosis who contract hepatitis E virus. Hepatitis E may accelerate disease progression in patients with chronic hepatitis B.

Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients.

BACKGROUND: Mongolia has the highest prevalence of hepatitis C virus (HCV) infection worldwide. Ledipasvir/sofosbuvir (LDV/SOF) was introduced to Mongolia since 2016 for HCV eradication. It has been reported that HCV resistance-associated substitutions (RASs) would affect the effectiveness of LDV/SOF in western chronic hepatitis C (CHC) patients. We thus investigated the effectiveness of LDV/SOF and the impact of RAS on the treatment outcome in Mongolian CHC patients. METHODS: Patients with genotype (GT) 1b HCV infection were prospectively enrolled in Mongolia and treated with LDV/SOF for 12 weeks. The proportion of pre-treatment NS5A Y93H RAS in viral quasispecies was measured with next-generation sequencing. The endpoint of LDV/SOF effectiveness was sustained virological response at post-treatment week 12 (SVR12). RESULTS: A total of 94 CHC patients were evaluated. The baseline Y93H proportion was <1% in 74 patients, 1-15% in 7, 15-50% in 2, and ≥50% in 11. All patients completed 12-week LDV/SOF treatment and the SVR rate was 90.4%. The rate of failure to achieve SVR12 for patients with Y93H < 1%, 1-15%, and ≥15% were 0%, 14.3%, and 61.5%, respectively (p for trend = 0.001). In univariable analysis, older age, baseline alanine transaminase level <40 U/mL, and a higher proportion of Y93H were associated with treatment failure. In multivariable analysis, only a higher proportion of Y93H was associated with treatment failure (p = 0.022). CONCLUSION: LDV/SOF therapy achieves a high SVR rate in Mongolian CHC GT1b patients without baseline Y93H RAS. A higher proportion of Y93H may severely undermine the effectiveness of LDV/SOF.

Serum hepatitis B core-related antigen level stratifies risk of disease progression in chronic hepatitis B patients with intermediate viral load.

BACKGROUND: Patients with chronic hepatitis B virus (HBV) infection are at risk of developing liver disease. Serum hepatitis B core-related antigen (HBcrAg) is a new biomarker for intrahepatic templates for HBV replication. AIM: To explore whether a high HBcrAg level is associated with increased risk of cirrhosis, especially in patients with intermediate viral load (HBV DNA 2000-19 999 IU/mL) due to their moderate risk of disease progression. METHODS: A total of 1673 treatment-naïve, non-cirrhotic patients with negative hepatitis B e antigen (HBeAg) and alanine aminotransferase (ALT) level <40 U/L at baseline were enrolled. We explored the relationship between baseline levels of HBcrAg and cirrhosis development in all patients, and whether a higher HBcrAg level (<10 vs ≥10 KU/mL) was associated with an increased risk of disease progression in those with intermediate viral load. RESULTS: Of the 1673 patients, 104 developed cirrhosis after a mean follow-up of 15.9 years. Higher HBcrAg levels were associated with increased incidence of cirrhosis, cirrhosis-related complications, and liver-related death. In 445 patients with intermediate viral load, the cirrhosis risk stratified by HBcrAg level of 10 KU/mL yielded a hazard ratio of 3.22 (95% CI: 1.61-6.47). The risk stratification remained significant when exploring other pre-cirrhosis endpoints, including HBeAg-negative hepatitis, hepatitis flare, and HBV DNA >20 000 IU/mL after 3 years of follow-up. CONCLUSIONS: In HBeAg-negative patients with normal ALT levels, higher HBcrAg levels are associated with increased risk of cirrhosis. Among those with intermediate viral load, HBcrAg <10 KU/mL defines a low-risk group for disease progression.

Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing.

Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated "base editors" (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome.