Liver mitochondrial DNA damage and genetic variability of Cytochrome b - a key component of the respirasome - drive the severity of fatty liver disease.

BACKGROUND AND AIMS: The progression of nonalcoholic fatty liver disease (NAFLD) into severe histological forms (steatohepatitis - NASH) is paralleled by the occurrence of complex molecular processes. Mitochondrial dysfunction is a hallmark feature of advanced disease. Mitochondrially encoded cytochrome B (cytochrome b, MT-CYB), a member of the oxidative phosphorylation system, is a key component of the respirasome supercomplex. Here, we hypothesized that NAFLD severity is associated with liver tissue cytochrome b mutations and damaged mitochondrial DNA (mtDNA). METHODS: We included 252 liver specimens of NAFLD patients - in whom histological disease ranged from mild to severe - which were linked to clinical and biochemical information. Tissue molecular explorations included MT-CYB sequencing and analysis of differential mtDNA damage. Profiling of circulating Krebs cycle metabolites and global liver transcriptome was performed in a subsample of patients. Tissue levels of 4-hydroxynonenal - a product of lipid peroxidation and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative damage - were measured. RESULTS: Compared to simple steatosis, NASH is associated with a higher level of MT-CYB variance, 12.1 vs. 15.6 substitutions per 103  bp (P = 5.5e-10). The burden of variants was associated with increased levels of 2-hydroxyglutarate, branched-chain amino acids, and glutamate, and changes in the global liver transcriptome. Liver mtDNA damage was associated with advanced disease and inflammation. NAFLD severity was associated with increased tissue levels of DNA oxidative adducts and lipid peroxyl radicals. CONCLUSION: NASH is associated with genetic alterations of the liver cellular respirasome, including high cytochrome b variation and mtDNA damage, which may result in broad cellular effects.

Systematic review with meta-analysis: the significance of histological disease severity in lean patients with nonalcoholic fatty liver disease.

BACKGROUND: Current evidence suggests that lean and obese patients with nonalcoholic fatty liver disease (NAFLD) share an altered metabolic and cardiovascular profile. However, there is an incomplete understanding of the natural history of "lean-NAFLD." Indeed, an unanswered question is whether lean (BMI ≤ 25 Kg/m2 ) NAFLD-patients are protected from severe histological outcomes. AIM: To perform a meta-analysis with the goal of providing a quantitative estimation of the magnitude of fibrosis, as well as histological features associated with the disease severity, in lean versus overweight/obese-NAFLD patients. METHODS: Through a systematic search up to July 2017, we identified eight studies that compared histological outcomes in lean (n = 493) versus overweight/obese (n = 2209) patients. RESULTS: Relative to lean-NAFLD, overweight/obese-NAFLD patients showed significantly (P = .032) higher fibrosis scores; the observed difference in means between the two groups, which is the absolute difference between the mean value of fibrosis score [0-4] ± standard error, was 0.28 ± 0.13. The risk of having nonalcoholic steatohepatitis-NASH (OR 0.58 95% CI 0.34-0.97) was significantly lower in lean-NAFLD (n = 322) than in overweight/obese-NAFLD (n = 1357), P = .04. Relative to lean-NAFLD, overweight/obese-NAFLD patients also have significantly greater NAFLD activity (difference in means ± SE: 0.58 ± 0.16, P = .0004) and steatosis (difference in means ± SE: 0.23 ± 0.07, P = .002) scores. CONCLUSIONS: Lean-NAFLD patients tend to show less severe histological features as compared to overweight/obese-NAFLD patients. Subsequent longitudinal assessment is needed to understand the clinical impact of these findings; however, the significant ~ 25% increment of mean fibrosis score in overweight/obese patients suggests that obesity could predict a worse long-term prognosis.

Systematic review with meta-analysis: risk factors for non-alcoholic fatty liver disease suggest a shared altered metabolic and cardiovascular profile between lean and obese patients.

BACKGROUND: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is closely associated with the co-occurrence of multiple pathological conditions characterising the metabolic syndrome (MetS), obesity in particular. However, NAFLD also develops in lean subjects, whose risk factors remain poorly defined. METHODS: We performed a meta-analysis of 15 studies, along with the data pertaining to our own population (n=336 patients). Data from lean (n=1966) and obese (n=5938) patients with NAFLD were analysed; lean (n=9946) and obese (n=6027) subjects without NAFLD served as controls. RESULTS: Relative to the lean non-NAFLD controls, lean patients with NAFLD were older (3.79±0.72 years, P=1.36×10-6 ) and exhibited the entire spectrum of the MetS risk factors. Specifically, they had a significant (P=10-10 ) increase in plasma glucose levels (6.44±1.12 mg/dL) and HOMA-IR (0.52±0.094-unit increment), blood lipids (triglycerides: 48.37±3.6, P=10-10 and total cholesterol: 7.04±3.8, mg/dL, P=4.2×10-7 ), systolic (5.64±0.7) and diastolic (3.37±0.9) blood pressure (mm Hg), P=10-10 , and waist circumference (5.88±0.4 cm, P=10-10 ); values denote difference in means±SE. Nevertheless, the overall alterations in the obese group were much more severe when compared to lean subjects, regardless of the presence of NAFLD. Meta-regression suggested that NAFLD is a modifier of the level of blood lipids. CONCLUSION: Lean and obese patients with NAFLD share a common altered metabolic and cardiovascular profile. The former, while having normal body weight, showed excess of abdominal adipose tissue as well as other MetS features.

Mendelian randomisation suggests no beneficial effect of moderate alcohol consumption on the severity of nonalcoholic fatty liver disease.

BACKGROUND: Previous epidemiological studies suggest that patients diagnosed with nonalcoholic fatty liver disease (NAFLD) who drink light to moderate amounts of alcohol (up to ~30 g per day) have less severe histological lesions compared with nondrinkers. However, while the cross-sectional nature of current evidence precludes assessment of causality, cumulative lifetime-exposure of moderate alcohol consumption on histological outcomes has never been evaluated. AIM: To overcome these limitations, a Mendelian randomisation study was performed using a validated genetic variant (rs1229984 A;G) in the alcohol dehydrogenase (ADH1B) gene as a proxy of long-term alcohol exposure. METHODS: We first assessed whether the instrumental variant (rs1229984) was associated with the amount of alcohol consumption in our cohort. We further explored the association between the variant and histological outcomes; a sample of 466 individuals, including 266 patients with NAFLD confirmed by liver biopsy, was studied. RESULTS: We found that carriers of the A-allele consumed significantly lower amounts of alcohol compared with noncarriers (2.3 ± 5.3 vs. 8.18 ± 21 g per day, mean ± s.d., P = 0.03). The analysis of association with the disease severity showed that carriers of the A-allele had lower degree of histological steatosis (1.76 ± 0.83 vs. 2.19 ± 0.78, P = 0.03) and lower scores of lobular inflammation (0.54 ± 0.65 vs. 0.95 ± 0.92, P = 0.02) and NAFLD-Activity Score (2.9 ± 1.4 vs. 3.7 ± 1.4, P = 0.015) compared with noncarriers. CONCLUSION: Mendelian randomisation analysis suggests no beneficial effect of moderate alcohol consumption on NAFLD disease severity.

Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease.

OBJECTIVE & DESIGN: Nonalcoholic fatty liver disease (NAFLD) is a clinical condition that refers to progressive histological changes ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH). We evaluated the status of cytosine methylation (5mC) of liver mitochondrial DNA (mtDNA) in selected regions of the mtDNA genome, such as D-loop control region, and mitochondrially encoded NADH dehydrogenase 6 (MT-ND6) and cytochrome C oxidase I (MT-CO1), to contrast the hypothesis that epigenetic modifications play a role in the phenotypic switching from SS to NASH. METHODS: We studied liver biopsies obtained from patients with NAFLD in a case-control design; 45 patients and 18 near-normal liver-histology subjects. RESULTS: MT-ND6 methylation was higher in the liver of NASH than SS patients (p < 0.04) and MT-ND6 methylated DNA/unmethylated DNA ratio was significantly associated with NAFLD activity score (p < 0.02). Liver MT-ND6 mRNA expression was significantly decreased in NASH patients (0.26 ± 0.30) versus SS (0.74 ± 0.48), p < 0.003, and the protein level was also diminished. The status of liver MT-ND6 methylation in NASH group was inversely correlated with the level of regular physical activity (R = -0.54, p < 0.02). Hepatic methylation levels of D-Loop and MT-CO1 were not associated with the disease severity. DNA (cytosine-5) methyltransferase 1 was significantly upregulated in NASH patients (p < 0.002). Ultrastructural evaluation showed that NASH is associated with mitochondrial defects and peroxisome proliferation. CONCLUSION: Hepatic methylation and transcriptional activity of the MT-ND6 are associated with the histological severity of NAFLD. Epigenetic changes of mtDNA are potentially reversible by interventional programs, as physical activity could modulate the methylation status of MT-ND6.

The influence of common gene variants of the xenobiotic receptor (PXR) in genetic susceptibility to intrahepatic cholestasis of pregnancy.

BACKGROUND: The xenobiotic nuclear pregnane X receptor is implicated in many physiological pathways and diseases, including bile acid detoxification and cholestasis. Aim To estimate the contribution of common gene variants of the xenobiotic receptor (pregnane X receptor, PXR) to genetic susceptibility to intrahepatic cholestasis of pregnancy. METHODS: A total of 101 intrahepatic cholestasis of pregnancy patients and 171 healthy pregnant women in the third trimester of their pregnancies were included. Four tag single nucleotide polymorphisms (SNPs) (rs12488820 C/T, rs2472671 C/T, rs2461823 A/G, and rs1054191 A/G) encompassing 36 kb in chromosome 3, with a minor allele frequency > or =0.10 and representing 33 polymorphic sites were genotyped. Besides these, three additional SNPs (rs3814057, rs6785049, and rs7643645) were included because they showed previous evidence of functionality. RESULTS: Genotypic test for single SNPs showed that rs2461823 genotypes were significantly associated with intrahepatic cholestasis of pregnancy (P < 0.0069), OR per G allele: 1.44, 95% CI: 1.01-2.05, P < 0.042. The Cochran-Armitage test for trend and the allelic test showed a significant association with disease status (P < 0.04 and 0.03 respectively), G being the risk allele. A positive association between rs2461823 and ALT, AST, and bilirubin concentrations was observed. Neonate birth weight adjusted by the Capurro index was significantly associated with rs2461823 (P < 0.05); the proportion of the total variation attributed to rs2461823 genotypes was 7.8%. CONCLUSION: Common PXR polymorphisms may contribute to the genetic susceptibility to intrahepatic cholestasis of pregnancy.

Role of the C-344T aldosterone synthase gene variant in left ventricular mass and left ventricular structure-related phenotypes.

AIM: We performed a systematic review of the literature by means of a meta-analysis to evaluate the influence of the aldosterone synthase gene (CYP11B2) C-344T polymorphism on left ventricular mass (LVM) and related phenotypes. DESIGN: From 485 reports, we included 14 studies about the association between the C-344T variant and left ventricular mass and left ventricular structure-related phenotypes, from which information about number of subjects in each category, outcomes data and genotyping performed with a validated molecular method could be extracted. Fixed and random effect models were used to pool data from individual studies, and the results in the abstract show the extreme genotype comparison, homozygous TT vs homozygous CC. RESULTS: From a total of 2157 subjects, we found no significant association between LVM and the C-344T variant (D: 0.049, 95% CI: 0.091 to 0.179, p = 0.462). Similarly, no significant association was found for interventricular septal-wall thickness (D: 0.027, 95% CI: -0.090 to 0.143, p = 0.654, n: 2105). However, homozygous TT hypertensive subjects had increased LVM (D: 0.251, 95% CI: 0.020 to 0.481, p = 0.04, n: 332). Lastly, in 10 homogeneous studies posterior wall thickness (PWT) was lower in homozygous CC individuals (D: 0.142, 95% CI: 0.016 to 0.268, p = 0.028, n = 1994). CONCLUSION: Independently of hypertension, homozygous individuals for the -344T allele may have 2.4% higher PWT compared to homozygous subjects for the C-344 allele. Besides, homozygous hypertensive TT subjects show a 6.9% increase in LVM compared to CC homozygous subjects.

Effects of rotating shift work on biomarkers of metabolic syndrome and inflammation.

OBJECTIVE: The major function of the circadian system is the internal cycling of physiological and metabolic events. The present study sought to explore the effect of rotating shift work schedule on leucocyte count and its relationship with risk factors of metabolic syndrome (MS). DESIGN AND PARTICIPANTS: From a population-based design, 1351 men of self-reported European ancestry were included in a cross-sectional study: 877 day workers were compared with 474 rotating shift workers. Medical history, health examination including anthropometric and arterial blood pressure measurements, a questionnaire on health-related behaviours and biochemical determinations was given to all participants. RESULTS: In comparison with day workers, rotating shift workers had elevated (mean +/- SE) body mass index (27.1 +/- 0.3 vs. 26.3 +/- 0.2, P < 0.0154), waist-hip ratio (0.95 +/- 0.01 vs. 0.93 +/- 0.01, P < 0.00024), diastolic arterial blood pressure (78 +/- 1 vs. 76 +/- 1, P < 0.033), fasting insulin (65.5 +/- 2.9 vs. 55.9 +/- 1.9 pmol L(-1), P < 0.017), Homeostasis Model Assessment index (2.12 +/- 0.11 vs. 1.77 +/- 0.07, P < 0.0027), triglycerides (1.71 +/- 0.1 vs. 1.5 +/- 0.1 mmol L(-1), P < 0.002), uric acid (292.7 +/- 2.8 vs. 282 +/- 3.4 micromol L(-1), P < 0.01) and leucocyte count (7030 +/- 84 vs. 6730 +/- 58, P < 0.0094). In multiple regression analysis, leucocyte count was correlated with rotating shift work independently of age, smoking, education and components of MS. CONCLUSION: The odds ratio for MS in rotating shift workers compared with day workers was 1.51 (95% CI 1.01-2.25), independently of age and physical activity. Increased leucocyte count, a biological marker of systemic inflammation, was associated with rotating shift work.

Peroxisome proliferator-activated receptor gamma and its coactivator-1 alpha may be associated with features of the metabolic syndrome in adolescents.

Our objective was to search for differences in genotypes of peroxisome proliferator-activated receptor gamma (PPARgamma) (Pro12 Ala) and its coactivator PGC-1alpha (Gly482 Ser) in adolescents harboring features of metabolic syndrome. In a population-based study, we determined medical history, anthropometric variables, biochemical measurements and arterial blood pressures of 934 high-school students of Caucasian origin. We selected 220 adolescents who had systolic or diastolic blood pressures more than the 80th or less than the 20th percentiles based on the previous single set of measurements. One hundred and seventy-five adolescents completed the study and underwent two additional blood pressure measurements on different days, as well as biochemical analysis and genotyping. We found no association between insulin resistance, body mass index (BMI) and leptin levels and PPARgamma and PGC-1alpha genotypes. The 12 Ala PPARgamma allele was associated with increased waist-to-hip ratio (WHR) and carriers seemed to have higher diastolic blood pressure and lower pulse pressure than non-carriers, particularly in the hypertensive and overweight group. Although Ser482 Ser PGC-1alpha homozygotes had lower WHRs than other PGC-1alpha genotypes, they were more frequent in the hypertensive group than in the normotensive (44.4 vs 24.5%, P<0.03), so the 482 Ser PGC-1 allele was in our population a risk factor for hypertension independently of WHR, homeostasis model assessment of insulin resistance, BMI and Pro12 Ala PPARgamma variant (odds ratio=4.0, 95% confidence interval 1.5-10.6, P<0.01). Multiple regression analysis showed that age- and sex-adjusted systolic blood pressure correlated with the 482 Ser PGC-1 allele regardless of those covariates. In conclusion, the Gly482 Ser variant of the PGC-1alpha gene may be an independent genetic risk factor for young-onset hypertension.

Combined therapy with interferon and ribavirin in chronic hepatitis C does not affect serum quasispecies diversity.

Our aim was to investigate if interferon plus ribavirin has any effect on serum HCV quasispecies distribution and the relationship between diversity of HCV quasispecies and treatment response. In all, 21 patients were treated with interferon plus ribavirin for 48 weeks. The presence of HCV quasispecies was determined in serum samples at baseline and at the fourth week of treatment by SSCP analysis of the hypervariable region. SSCP pattern was defined as single or multiple band. A single band was found in six patients and multiple bands in nine. No significant difference was found between SSCP pattern in pretreatment samples and response to the therapy. In none of the patients were observed changes in number of SSCP bands between samples taken at baseline and in the fourth week of the therapy. In conclusion, the complexity of HCV quasispecies before the therapy was not related to treatment response; combined therapy did not affect serum HCV quasispecies.

Phylogenetic characterization of genotype 4 hepatitis C virus isolates from Argentina.

Among 114 patients infected with hepatitis C virus, three genotype 4 isolates, unusual in Argentina, were detected by phylogenetic analysis over different genomic regions. The patients were not related. One sample was associated with Egyptian sequences, and the others were associated with a Zairean isolate, a fact which reinforces the idea that they are from independent sources.

Development of cutaneous sarcoidosis in a patient with chronic hepatitis C treated with interferon alpha 2b.

BACKGROUND: Sarcoidosis is a multisystemic granulomatous disorder of unknown etiology that most commonly affects young adults. A probable induction of sarcoidosis by interferons (IFN) has been published. To this date, few cases of cutaneous sarcoidosis inpatients with chronic hepatitis C under interferon treatment have been reported. OBJECTIVE: We describe a 50-year-old woman with chronic hepatitis C who developed lesions of cutaneous sarcoidosis three months after IFN treatment. CONCLUSIONS: The possible role of INF therapy in the development of cutaneous sarcoidosis in a patient with chronic hepatitis C should be considered.

Identification of 2 novel isolates of hepatitis E virus in Argentina.

Hepatitis E virus (HEV) has been identified in 2 Argentine patients with acute hepatitis who reported no history of travel to regions in which HEV is considered endemic. These isolates are the first to be identified in South America. By use of degenerate primers from open reading frames 1 and 2, HEV sequences were obtained from these patients' serum and compared with published HEV sequences. The Argentine isolates are different from all previously identified HEV isolates and are most closely related to each other. The Argentine isolates are distinct from the most geographically related isolate from Mexico as well as isolates from other endemic (China, Southeast Asia, and India) and nonendemic (the United States and Europe) regions. Phylogenetic analysis indicate that the Argentine isolates represent a new genotype of HEV, genotype 8, distinct from the Burmese-like genotype 1, Mexican genotype 2, US genotype 3, Chinese/Taiwan genotype 4, and European genotypes 5-7.

Relationship between diversity of hepatitis C quasispecies and histological severity of liver disease.

The aim of this work was to assess if the diversity of hepatitis C virus (HCV) quasispecies is related to histological severity and duration of infection in a cohort of untreated patients with an estimated onset of the disease. A total of 27 patients with diagnosis of chronic liver disease and history of blood transfusion (n = 16) or intravenous drug use (IDU) (n = 11) were included. All were anti-HCV positive and had detectable serum HCV-RNA. The onset and the duration of the disease were estimated from the time of the transfusion or the first drug injection. Patients who consumed drugs for more than 2 years, or were coinfected with HBV or HIV were excluded. History of alcohol intake (> 80 g/day), ALT level and age at infection were recorded. Histological assessment of grading and staging was performed according to Knodell score. The quasispecies diversity was investigated by single strand conformation polymorphism (SSCP) targeted to HVR-E2 region and SSCP pattern was evaluated as a single or multiple bands. The number of quasispecies did not correlate with the estimated duration of the disease. Patients who acquired hepatitis C by blood transfusion did not differ in number of bands from patients who were IDU. There was no correlation between the heterogeneity of HCV quasispecies and age, serum ALT, Knodell score, HAI and fibrosis. In conclusion the quasispecies diversity of E2 had no correlation with grade and stage of chronic HCV infection and the presence of quasispecies was independent of the duration of the disease.

[Propranolol and 5-isosorbide mononitrate in patients with cirrhosis: systemic and portal hemodynamic events]. / Propranolol y 5-mononitrato de isosorbide en pacientes con cirrosis: episodios hemodinámicos sistémicos y portales.

AIM: To study the acute variations in portal and systemic hemodynamics after propranolol and 5-isosorbide mononitrate (IMN) administration in cirrhotic patients. PATIENTS AND METHODS: Seventeen cirrhotic patients with portal hypertension were studied with catheterization and Doppler duplex Ultrasound Systemic hemodynamics. Hepatic venous pressure gradient (HVPG), portal blood flow and resistance were evaluated in baseline, after intravenous propranolol (0.15 mg/kg), and after 20 mg p.o. of IMN. Patients who showed a decrease > or = 20% and/or < 12 mm/hg in HVPG were considered responders. RESULTS: There were no significant differences in clinical or portal hemodynamic baseline data between responders and non-responders to the drugs. After propranolol administration cardiac index decreased (p < 0.05) and pulmonary capillary pressure increased (p < 0.0001). Six patients (35%) were responders; lack of response was associated with an insufficient decrease in portal blood flow or with an increase in portal resistance. After IMN administration cardiac index decreased (p < 0.05) with normalization of pulmonary capillary pressure (p < 0.05). Seven patients were responders to the addition of IMN (5 non-responders to propranolol) and showed a decrease in HVPG associated with a reduction in portal blood flow and resistance; in the remaining 10 patients HVPG did not decrease despite a reduction in portal blood flow, with an increase in portal resistance. CONCLUSIONS: Addition of IMN increased the number of responders and reduced portal blood flow with a variable effect in portal resistance.

In vivo down regulation of HIV replication after hepatitis C superinfection.

There are increasing molecular and clinical evidences that the effects of human immunodeficiency virus (HIV) infection can be modified by coinfection with other viruses. The objective was to investigate the viral interaction between HIV and hepatitis C virus (HCV) after HCV superinfection. A 16 year-old pregnant woman was evaluated because of icteric acute hepatitis. Admission laboratory tests showed the following results: ALT 877 IU/L; AST 1822 IU/L; bilirubin 6.79 mg/dl. Diagnosis of acute HCV was based on detection of serum HCV RNA by PCR and anti-HCV seroconversion. ELISA for anti HIV testing was positive and confirmed by western blot. Serum markers for other viruses were negative. The patient was followed during 19 months; serum samples were taken monthly during this period for detection of plasma HIV and HCV RNA. Levels of plasma HIV-RNA were positive in all samples tested before and after the onset of acute hepatitis C. Six months later and a for two month period, and 13 months later for a period of one month HIV viremia was undetectable; then HIV-RNA in plasma was detectable again. In conclusion, HCV superinfection may have temporarily interfered with HIV replication in our patient. The following observations support our hypothesis: it has been demonstrated that HIV-1 replication is suppressed by HCV core protein which has transcriptional regulation properties of several viral and cellular promoters. Clinical implications of this event are not generally known and the interaction between these two viruses in dual infections is worth considering.