RESUMEN
Hydroxysteroid 17-ß dehydrogenase 13 (HSD17B13) is a lipid droplet-associated protein; its gene-encoding variants affect the chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD). To estimate the effect of rs72613567, a splice variant with an adenine insertion (A-INS), on NAFLD susceptibility and severity, we performed a case-control study with 609 individuals. We investigated the effect of carrying the A-INS allele in 356 patients with biopsy-proven disease and explored the relationship between rs72613567 genotypes and the hepatic transcriptome. The A-INS allele protected against NAFLD [odds ratio (OR) per adenine allele = 0.667; 95% CI, 0.486-0.916; P = 0.012]; this effect was nonsignificant when logistic regression analysis included BMI. The A-INS allele protected against nonalcoholic steatohepatitis (NASH) (OR = 0.612; 95% CI, 0.388-0.964; P = 0.033), ballooning degeneration (OR = 0.474; 95% CI, 0.267-0.842; P = 0.01), lobular inflammation (OR = 0.475; 95% CI, 0.275-0.821; P = 0.007), and fibrosis (OR = 0.590; 95% CI, 0.361-0.965; P = 0.035). In patients carrying A-INS, HSD17B13 levels decreased proportionally to allele dosage. Whole-transcriptome genotype profiling showed overrepresented immune response-related pathways. Thus, the rs72613567 A-INS allele reduces the risk of NASH and progressive liver damage and may become a therapeutic target.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Polimorfismo de Nucleótido Simple , Anciano , Alelos , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Genotipo , Humanos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/inmunologíaRESUMEN
We report on the presence of a rare nonsense mutation (rs149847328, p.Arg227Ter) in the glucokinase regulator (GCKR) gene in an adult patient with nonalcoholic fatty liver disease (NAFLD), morbid obesity, and type 2 diabetes; this patient developed a progressive histological form of the disease. Analysis of paired (5 years apart) liver biopsies (at baseline and follow-up) showed progression of simple steatosis to severe nonalcoholic steatohepatitis and cirrhosis. Study design involved an initial exploration that consisted of deep sequencing of 14 chromosomal regions in 96 individuals (64 of whom were patients with NAFLD who were diagnosed by liver biopsy that showed the full spectrum of histological severity). We further performed a replication study to explore the presence of rs149847328 that included a sample of 517 unrelated individuals in a case-control study (n = 390), including patients who were morbidly obese (n = 127). Exploration of sequence variation by next-generation sequencing of exons, exon-intron boundaries, and 5' and 3' untranslated regions of 14 genomic loci that encode metabolic enzymes of the tricarboxylic acid cycle revealed the presence of heterozygosity for the p.Arg227Ter mutation, the frequency of which is 0.0003963 (4:10,000; Exome Aggregation Consortium database). GCKR protein expression was markedly decreased in the liver of the affected patient compared with patients with NAFLD who carry the wild-type allele. Sequencing of the same 14 genomic loci in 95 individuals failed to reveal the rare mutation. The rarity of p.Arg227Ter was confirmed in a more extensive screening. Conclusion: While rare variants/mutations are difficult to detect in even reasonably large samples (frequency of the mutant allele of p.Arg227Ter was ~1:1,000 in our data set), the presence of this mutation should be suspected as potentially associated with NAFLD, particularly in young adults at the extreme of histological phenotypes. Hepatology Communications 2018;0:0-0).
RESUMEN
Long noncoding RNAs (lncRNAs) are functional molecules that orchestrate gene expression. To identify lncRNAs involved in nonalcoholic fatty liver disease (NAFLD) severity, we performed a multiscale study that included: (a) systems biology modeling that indicated metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a candidate lncRNA for exploring disease-related associations, (b) translational exploration in the clinical setting, and (c) mechanistic modeling. MALAT1 liver profiling was performed in three consecutive phases, including an exploratory stage (liver samples from patients with NAFLD who were morbidly obese [n = 47] and from 13 individuals with normal liver histology); a replication stage (patients with NAFLD and metabolic syndrome [n =49]); and a hypothesis-driven stage (patients with chronic hepatitis C and autoimmune liver diseases, [n = 65]). Liver abundance of MALAT1 was associated with NAFLD severity (P = 1 × 10-6); MALAT1 expression levels were up-regulated 1.75-fold (P = 0.029) and 3.6-fold (P = 0.012) in patients with nonalcoholic steatohepatitis compared to those diagnosed with simple steatosis (discovery and replication set, respectively; analysis of covariance adjusted by age, homeostasis model assessment, and body mass index). Quantification of liver vascular endothelial growth factor A messenger RNA, a target of MALAT1, revealed a significant correlation between the two RNAs (R, 0.58; P = 5 × 10-8). Increased levels of MALAT1 were also associated with autoimmune liver diseases. Interactome assessment uncovered significant biological pathways, including Janus kinase-signal transducers and activators of transcription and response to interferon-γ. Conclusion: Deregulated expression of MALAT1 stratifies patients into the histologic phenotypes associated with NAFLD severity. MALAT1 up-regulation seems to be a common molecular mechanism in immune-mediated chronic inflammatory liver damage. This suggests that convergent pathophenotypes (inflammation and fibrosis) share similar molecular mediators. (Hepatology Communications 2018;2:654-665).
RESUMEN
Current knowledge on the genetic basis of nonalcoholic fatty liver disease (NAFLD) suggests that variants contributing not only to the disease predisposition but histological severity as well are located in genes that regulate lipid metabolism. We explored the role of rs641738 C/T located in TMC4 (transmembrane channel-like 4) exon 1 (p.Gly17Glu) and 500 bases- downstream of MBOAT7 gene (TMC4/MBOAT7), in the genetic risk for developing NAFLD in a case-control study. Our sample included 634 individuals (372 patients with NAFLD diagnosed by liver biopsy and 262 control subjects); genotyping was performed by a Taqman assay. Genotype frequencies in controls (CC: 84, CT: 137, TT: 41) and patients (CC: 134, CT: 178, TT: 60) were in Hardy-Weinberg equilibrium; minor allele frequency 40.8%. Our sample had 84-99% power if an additive genetic model is assumed for estimated odds ratios of 1.3-1.5, respectively. We found no evidence of association between rs641738 and either NAFLD (Cochran-Armitage test for trend, p = 0.529) or the disease severity (p = 0.61). Low levels of MBOAT7 protein expression were found in the liver of patients with NAFLD, which were unrelated to the rs641738 genotypes. In conclusion, the role of rs641738 in the pathogenesis of NAFLD is inconclusive.
Asunto(s)
Aciltransferasas/genética , Proteínas de la Membrana/genética , Mutación Missense , Enfermedad del Hígado Graso no Alcohólico/genética , Polimorfismo de Nucleótido Simple , Adulto , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The human transcriptome comprises a myriad of non protein-coding RNA species, including long noncoding RNAs (lncRNAs), which have a remarkable role in transcriptional and epigenetic regulation. We hypothesized that variants in lncRNAs influence the susceptibility to nonalcoholic fatty liver disease (NAFLD). Using next generation sequencing, we performed a survey of genetic variation associated with randomly selected lncRNA-genomic regions located within both experimentally validated and computationally predicted regulatory elements. We used a two-stage (exploratory, n = 96 and replication, n = 390) case-control approach that included well-characterized patients with NAFLD diagnosed by liver biopsy. We sequenced > 263 megabase pairs at quality score > Q17, in a total of 2,027,565 reads, including 170 lncRNA-genomic regions. In the sequencing analysis and the validated dataset, we found that the rs2829145 A/G located in a lncRNA (lnc-JAM2-6) was associated with NAFLD and the disease severity. Prediction of regulatory elements in lnc-JAM2-6 showed potential sequence-specific binding motifs of oncogenes MAFK and JUND, and the transcription factor CEBPB that is involved in inflammatory response. The A-allele was significantly associated with NAFLD as disease trait (p = 0.0081) and the disease severity (NASH-nonalcoholic steatohepatitis vs controls: OR 2.36 [95% CI: 1.54-3.62], p = 0.000078). The A-allele carriers also have significantly higher body mass index and glucose-related traits compared with homozygous GG. Hence, our results suggest that variation in lncRNAs contributes to NAFLD severity, while pointing toward the complexity of the genetic component of NAFLD, which involves still unexplored regulatory regions of the genome.
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Enfermedad del Hígado Graso no Alcohólico/genética , ARN Largo no Codificante/genética , Epigénesis Genética , Femenino , Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) develops from a complex process, which includes changes in the liver methylome. Betaine plays a pivotal role in the regulation of methylogenesis. We performed a two-stage case-control study, which included patients with biopsy-proven NAFLD to explore circulating levels of betaine and its association with the histological spectrum. We also explored the association between a missense rs1805074, p.Ser646Pro variant in DMGDH (dimethylglycine dehydrogenase mitochondrial) and NAFLD severity (n=390). RESULTS: In the discovery phase (n=48), betaine levels were associated with the disease severity (P=.0030), including liver inflammation (Spearman R:-0.51, P=.001), ballooning degeneration (R: -0.50, P=.01) and fibrosis (R: -0.54, P=.0008). Betaine levels were significantly decreased in nonalcoholic steatohepatitis (NASH) in comparison with nonalcoholic fatty liver (NAFL). Further replication (n=51) showed that betaine levels were associated with advanced NAFLD (P=.0085), and patients with NASH had a 1.26-fold decrease in betaine levels compared with those with NAFL. The rs1805074 was significantly associated with the disease severity (P=.011). CONCLUSION: NAFLD severity is associated with a state of betaine-insufficiency.
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Betaína/sangre , Dimetilglicina-Deshidrogenasa/genética , Progresión de la Enfermedad , Proteínas Mitocondriales/genética , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/genética , Adulto , Argentina , Biomarcadores , Estudios de Casos y Controles , Hígado Graso/patología , Femenino , Fibrosis , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Mutación Missense , Enfermedad del Hígado Graso no Alcohólico/patología , Análisis de Regresión , Índice de Severidad de la EnfermedadRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction, a decreased liver mitochondrial DNA (mtDNA) content, and impaired energy metabolism. To understand the clinical implications of mtDNA diversity in the biology of NAFLD, we applied deep-coverage whole sequencing of the liver mitochondrial genomes. We used a multistage study design, including a discovery phase, a phenotype-oriented study to assess the mutational burden in patients with steatohepatitis at different stages of liver fibrosis, and a replication study to validate findings in loci of interest. We also assessed the potential protein-level impact of the observed mutations. To determine whether the observed changes are tissue-specific, we compared the liver and the corresponding peripheral blood entire mitochondrial genomes. The nuclear genes POLG and POLG2 (mitochondrial DNA polymerase-γ) were also sequenced. We observed that the liver mtDNA of patients with NAFLD harbours complex genomes with a significantly higher mutational (1.28-fold) rate and degree of heteroplasmy than in controls. The analysis of liver mitochondrial genomes of patients with different degrees of fibrosis revealed that the disease severity is associated with an overall 1.4-fold increase in mutation rate, including mutations in genes of the oxidative phosphorylation (OXPHOS) chain. Significant differences in gene and protein expression patterns were observed in association with the cumulative number of OXPHOS polymorphic sites. We observed a high degree of homology (â¼98%) between the blood and liver mitochondrial genomes. A missense POLG p.Gln1236His variant was associated with liver mtDNA copy number. In conclusion, we have demonstrated that OXPHOS genes contain the highest number of hotspot positions associated with a more severe phenotype. The variability of the mitochondrial genomes probably originates from a common germline source; hence, it may explain a fraction of the 'missing heritability' of NAFLD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Genoma Mitocondrial , Enfermedad del Hígado Graso no Alcohólico/genética , Adulto , Estudios de Casos y Controles , ADN Mitocondrial/genética , Femenino , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Haplotipos , Humanos , Cirrosis Hepática/genética , Masculino , Persona de Mediana Edad , Mitocondrias Hepáticas/genética , Mutación , Mutación Missense/genética , Fosforilación Oxidativa , Polimorfismo Genético , Índice de Severidad de la EnfermedadRESUMEN
Ballooning degeneration (BD) of hepatocytes is a distinguishing histological feature associated with the progression of nonalcoholic fatty liver disease (NAFLD). Under the assumption that NAFLD severity is associated with metabolic-stress we explored the hypothesis that heat shock 27 kDa protein 1 (HSP27), a protein chaperone involved in stress resistance and cytoskeletal-remodeling, might be deregulated in ballooned hepatocytes. We observed that fasting plasma glucose (fpG) (p = 0.00002), total cholesterol (p = 0.02) and triglycerides (p = 0.01) levels, and female sex (p = 0.01) were significantly associated with the presence of BD. A logistic regression model showed that BD was independently associated with fpG (p = 0.002); OR per unit of glucose concentration 1.05, 95% confidence interval 1.02-1.09. Furthermore, BD was associated with a significant 2.24-fold decrease in the expression level of HSP27-mRNA in comparison with absence of ballooning, p = 0.002. Ballooned hepatocytes showed very low HSP27 immunoreactivity compared with hepatocyes without ballooning (p = 0.009); HSP27 immunoreactivity was inversely correlated with fpG levels (R: -0.49, p = 0.01). In conclusion, BD is associated with down-regulation of liver HSP27 gene and protein expression, suggesting that ballooned hepatocytes fail to ensure a robust physiological response to metabolic-induced stress.
Asunto(s)
Regulación hacia Abajo , Proteínas de Choque Térmico HSP27/biosíntesis , Hepatocitos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Adulto , Femenino , Proteínas de Choque Térmico , Hepatocitos/patología , Humanos , Masculino , Persona de Mediana Edad , Chaperonas Moleculares , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/terapiaRESUMEN
BACKGROUND: Extensive epidemiologic studies have shown that cardiovascular disease and the metabolic syndrome (MetS) are associated with serum concentrations of liver enzymes; however, fundamental characteristics of this relation are currently unknown. OBJECTIVE: We aimed to explore the role of liver aminotransferases in nonalcoholic fatty liver disease (NAFLD) and MetS. DESIGN: Liver gene- and protein-expression changes of aminotransferases, including their corresponding isoforms, were evaluated in a case-control study of patients with NAFLD (n = 42), which was proven through a biopsy (control subjects: n = 10). We also carried out a serum targeted metabolite profiling to the glycolysis, gluconeogenesis, and Krebs cycle (n = 48) and an exploration by the next-generation sequencing of aminotransferase genes (n = 96). An in vitro study to provide a biological explanation of changes in the transcriptional level and enzymatic activity of aminotransferases was included. RESULTS: Fatty liver was associated with a deregulated liver expression of aminotransferases, which was unrelated to the disease severity. Metabolite profiling showed that serum aminotransferase concentrations are a signature of liver metabolic perturbations, particularly at the amino acid metabolism and Krebs cycle level. A significant and positive association between systolic hypertension and liver expression levels of glutamic-oxaloacetic transaminase 2 (GOT2) messenger RNA (Spearman R = 0.42, P = 0.03) was observed. The rs6993 located in the 3' untranslated region of the GOT2 locus was significantly associated with features of the MetS, including arterial hypertension [P = 0.028; OR: 2.285 (95% CI: 1.024, 5.09); adjusted by NAFLD severity] and plasma lipid concentrations. CONCLUSIONS: In the context of an abnormal hepatic triglyceride accumulation, circulating aminotransferases rise as a consequence of the need for increased reactions of transamination to cope with the liver metabolic derangement that is associated with greater gluconeogenesis and insulin resistance. Hence, to maintain homeostasis, the liver upregulates these enzymes, leading to changes in the amounts of amino acids released into the circulation.
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Alanina Transaminasa/metabolismo , Aspartato Aminotransferasa Citoplasmática/metabolismo , Aspartato Aminotransferasa Mitocondrial/metabolismo , Inducción Enzimática , Gluconeogénesis , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Alanina Transaminasa/sangre , Alanina Transaminasa/genética , Aminoácidos/metabolismo , Aspartato Aminotransferasa Citoplasmática/sangre , Aspartato Aminotransferasa Citoplasmática/genética , Aspartato Aminotransferasa Mitocondrial/sangre , Aspartato Aminotransferasa Mitocondrial/genética , Biomarcadores/sangre , Estudios de Casos y Controles , Línea Celular Tumoral , Ciclo del Ácido Cítrico , Estudios de Cohortes , Estudios Transversales , Hígado Graso/etiología , Femenino , Humanos , Resistencia a la Insulina , Isoenzimas/sangre , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/patología , Hígado/fisiopatología , Masculino , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Síndrome Metabólico/patología , Síndrome Metabólico/fisiopatología , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Polimorfismo de Nucleótido SimpleRESUMEN
The 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification whose role in the pathogenesis of metabolic-related complex diseases remains unexplored; 5-hmC appears to be prevalent in the mitochondrial genome. The Ten-Eleven-Translocation (TET) family of proteins is responsible for catalyzing the conversion of 5-methylcytosine to 5-hmC. We hypothesized that epigenetic editing by 5-hmC might be a novel mechanism through which nonalcoholic fatty liver disease (NAFLD)-associated molecular traits could be explained.Hence, we performed an observational study to explore global levels of 5-hmC in fresh liver samples of patients with NAFLD and controls (nâ=â90) using an enzyme-linked-immunosorbent serologic assay and immunohistochemistry. We also screened for genetic variation in TET 1-3 loci by next generation sequencing to explore its contribution to the disease biology. The study was conducted in 2 stages (discovery and replication) and included 476 participants.We observed that the amount of 5-hmC in the liver of both NAFLD patients and controls was relatively low (up to 0.1%); a significant association was found with liver mitochondrial DNA copy number (Râ=â0.50, Pâ=â0.000382) and PPARGC1A-mRNA levels (Râ=â-0.57, Pâ=â0.04).We did not observe any significant difference in the 5-hmC nuclear immunostaining score between NAFLD patients and controls; nevertheless, we found that patients with NAFLD (0.4â±â0.5) had significantly lower nonnuclear-5-hmC staining compared with controls (1.8â±â0.8), meansâ±âstandard deviation, Pâ=â0.028. The missense p.Ile1123Met variant (TET1-rs3998860) was significantly associated with serum levels of caspase-generated CK-18 fragment-cell death biomarker in the discovery and replication stage, and the disease severity (odds ratio: 1.47, 95% confidence interval: 1.10-1.97; Pâ=â0.005). The p.Ile1762Val substitution (TET2-rs2454206) was associated with liver PPARGC1A-methylation and transcriptional levels, and Type 2 diabetes.Our results suggest that 5-hmC might be involved in the pathogenesis of NAFLD by regulating liver mitochondrial biogenesis and PPARGC1A expression. Genetic diversity at TET loci suggests an "epigenetic" regulation of programmed liver-cell death and a TET-mediated fine-tuning of the liver PPARGC1A-transcriptional program.
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Citosina/análogos & derivados , Proteínas de Unión al ADN/genética , Hígado , Mitocondrias Hepáticas/metabolismo , Enfermedad del Hígado Graso no Alcohólico , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , 5-Metilcitosina/análogos & derivados , Adulto , Citosina/metabolismo , Metilación de ADN/genética , Epigénesis Genética , Femenino , Predisposición Genética a la Enfermedad , Proteínas de Choque Térmico/genética , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Persona de Mediana Edad , Oxigenasas de Función Mixta , Enfermedad del Hígado Graso no Alcohólico/epidemiología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Biogénesis de Organelos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gammaRESUMEN
UNLABELLED: We explored the role of transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 C/T nonsynonymous (p.Glu167Lys) variant in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and disease severity. A total of 361 individuals (135 control subjects and 226 patients with histologically proven NAFLD) were included in a sample with 97% power for the additive genetic model. A discrete trait analysis of NAFLD showed that rs58542926 was associated with a modest risk of fatty liver (P = 0.038; odds ratio [OR]: 1.37; 95% confidence interval [CI]: 1.02-1.84); nevertheless, conditioning on patatin-like phospholipase domain-containing 3 (PNPLA3)-rs738409 abolished this effect. We did not observe an interaction between rs738409 and rs58542926 variants on the risk of NAFLD. We observed a significant association of rs58542926 and disease severity (P = 0.027), but not lobular inflammation or fibrosis; rs58542926 was not associated with levels of liver enzymes. An allelic test showed that the T (Lys167) allele was significantly associated with disease progression (P = 0.021; OR, 1.66; 95% CI: 1.08-2.55). A significant association was found with the histological degree of liver steatosis (ß, 0.15; standard error: 0.06; P = 0.0299) that was independent of rs738409. Homozygous carriers of the C (Glu167) allele showed increased risk for cardiovascular disease. TM6SF2 protein expression was decreased markedly in liver of NAFLD patients, compared to controls. In addition, TM6SF2 immunoreactivity was reduced in subjects carrying at least one copy of the T allele, consistent with a difference in liver allele-specific transcript abundance. CONCLUSION: rs58542926 is a low-frequency variant with a modest effect on NAFLD, suggesting that carriers of the T allele are slightly more likely to accumulate fat in the liver and develop nonalcoholic steatohepatitis than those without. TM6SF2 appears to play a significant role in disease biology.
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Hígado/patología , Proteínas de la Membrana/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Adulto , Anciano , Alelos , Enfermedades Cardiovasculares/genética , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Fibrosis , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/patología , Polimorfismo de Nucleótido SimpleAsunto(s)
Hígado Graso/etiología , Lipasa/genética , Proteínas de la Membrana/genética , Animales , Femenino , Humanos , MasculinoRESUMEN
OBJECTIVES: We used a screening strategy of global serum microRNA (miRNA) profiling, followed by a second stage of independent replication and exploration of liver expression of selected miRNAs to study: (1) the circulating miRNA signature associated with non-alcoholic fatty liver disease (NAFLD) progression and predictive power, (2) the role of miRNAs in disease biology and (3) the association between circulating miRNAs and features of the metabolic syndrome. METHODS: The study used a case-control design and included patients with NAFLD proven through biopsy and healthy controls. RESULTS: Among 84 circulating miRNAs analysed, miR-122, miR-192, miR-19a and miR-19b, miR-125b, and miR-375 were upregulated >2-fold (p<0.05) either in simple steatosis (SS) or non-alcoholic steatohepatitis (NASH). The most dramatic and significant fold changes were observed in the serum levels of miR-122 (7.2-fold change in NASH vs controls and 3.1-fold change in NASH vs SS) and miR-192 (4.4-fold change in NASH vs controls); these results were replicated in the validation set. The majority of serum miR-122 circulate in argonaute2-free forms. Circulating miR-19a/b and miR-125b were correlated with biomarkers of atherosclerosis. Liver miR-122 expression was 10-fold (p<0.03) downregulated in NASH compared with SS and was preferentially expressed at the edge of lipid-laden hepatocytes. In vitro exploration showed that overexpression of miR-122 enhances alanine aminotransferase activity. CONCLUSIONS: miR-122 plays a role of physiological significance in the biology of NAFLD; circulating miRNAs mirror the histological and molecular events occurring in the liver. NAFLD has a distinguishing circulating miRNA profile associated with a global dysmetabolic disease state and cardiovascular risk.
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Hígado/patología , MicroARNs/sangre , Enfermedad del Hígado Graso no Alcohólico/genética , ARN no Traducido/sangre , Adulto , Antropometría/métodos , Proteínas Argonautas/sangre , Proteínas Argonautas/genética , Biomarcadores/sangre , Biopsia , Estudios de Casos y Controles , Células Cultivadas , Simulación por Computador , Femenino , Perfilación de la Expresión Génica/métodos , Estudios de Asociación Genética/métodos , Predisposición Genética a la Enfermedad , Humanos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Enfermedad del Hígado Graso no Alcohólico/patología , Transaminasas/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
The intrahepatic cholestasis of pregnancy (ICP) is a multifactorial liver disorder which pathogenesis involves the interplay among abnormal bile acid (BA) levels, sex hormones, environmental factors, and genetic susceptibility. The dynamic nature of ICP that usually resolves soon after delivery suggests the possibility that its pathobiology is under epigenetic modulation. We explored the status of white blood peripheral cells-DNA methylation of CpG-enriched sites at the promoter of targeted genes (FXR/NR1H4, PXR/NR1I2, NR1I3, ESR1, and ABCC2) in a sample of 88 ICP patients and 173 healthy pregnant women in the third trimester of their pregnancies. CpG dinucleotides at the gene promoter of nuclear receptors subfamily 1 members and ABCC2 transporter were highly methylated during healthy pregnancy. We observed significant differences at the distal (-1890) and proximal promoter (-358) CpG sites of the FXR/NR1H4 and at the distal PXR/NR1I2 (-1224) promoter, which were consistently less methylated in ICP cases when compared with controls. In addition, we observed that methylation at FXR/NR1H4-1890 and PXR/NR1I2-1224 promoter sites was highly and positively correlated with BA profiling, particularly, conjugated BAs. Conversely, methylation level at the proximal FXR/NR1H4-358 CpG site was significantly and negatively correlated with the primary cholic and secondary deoxycholic acid. In vitro exploration showed that epiallopregnanolone sulfate, a reported FXR inhibitor, regulates the transcriptional activity of FXR/NR1H4 but seems to be not involved in the methylation changes. In conclusion, the identification of epigenetic marks in target genes provides a basis for the understanding of adverse liver-related pregnancy outcomes, including ICP.
Asunto(s)
Colestasis Intrahepática/metabolismo , Metilación de ADN , Fenotipo , Complicaciones del Embarazo/metabolismo , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/metabolismo , Adulto , Línea Celular Tumoral , Colestasis Intrahepática/genética , Colestasis Intrahepática/patología , Receptor de Androstano Constitutivo , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/genética , Femenino , Humanos , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Embarazo , Complicaciones del Embarazo/genética , Complicaciones del Embarazo/patología , Pregnanolona/análogos & derivados , Pregnanolona/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
OBJECTIVES AND DESIGN: Epidemiological studies have suggested a role of nonalcoholic fatty liver disease (NAFLD) in the development of cardiovascular disease. We evaluated liver mRNA expression of 84 genes encoding proteins involved in the atherosclerosis pathway in patients with NAFLD proven through biopsy in a case-control design, and examined the putative role of the histological disease severity in the molecular events associated with the atherogenic profile. RESULTS: Nonalcoholic steatohepatitis (NASH), when compared with simple steatosis (SS), significantly increases the expression of TGFB1 (6.8, p<0.005), angiotensin I-converting enzyme (ACE) (2.1, p<0.007), LAMA1 (2.1, p<0.007), SERPINB2 (2.1, p<0.007), CSF2 (2.5, p<0.002), IL1A (2.5, p<0.005), IL3 (2.1, p<0.007), IL4 (2.1, p<0.007), LIF (2.1, p<0.007), and MMP1 (2.1, p<0.007), and decreases the transcript levels of genes involved in the negative regulation of cell-death pathways. A post hoc analysis of liver biopsies of NASH patients who were treated with enalapril monotherapy because of arterial hypertension showed a significant association with lower fibrosis scores in comparison with untreated patients. BIRC3, a severe hypoxia-activated gene, was significantly increased in SS (8.2, p<0.004), when compared with the controls. NASH, but not SS, was also associated with a significant increase in platelet abundance of TGFB1 mRNA. Systems biology analysis revealed highly scored pathways involved in the regulation of programmed cell death, angiogenesis, and immune system, in which TGFB1 was mostly involved. CONCLUSION: NASH, but not SS, may increase atherosclerotic and cardiovascular risk by local overexpression of mediators of atherogenesis, endothelial damage, and regulators of blood pressure; this observation may have therapeutic implications, because ACE inhibitors may improve both cardiovascular outcomes and liver fibrosis. Hepatocyte hypoxia seems to have an important role in the molecular events activated by liver steatosis.
Asunto(s)
Aterosclerosis/patología , Hígado Graso/metabolismo , Hígado/metabolismo , Adulto , Biopsia , Presión Sanguínea , Enfermedades Cardiovasculares/metabolismo , Estudios de Casos y Controles , Femenino , Fibrosis , Regulación de la Expresión Génica , Hepatocitos/citología , Humanos , Hipoxia/patología , Hígado/patología , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico , ARN Mensajero/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
UNLABELLED: Insulin resistance (IR) and mitochondrial dysfunction play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). We hypothesized that genetic factors and epigenetic modifications occurring in the liver contribute to the IR phenotype. We specifically examined whether fatty liver and IR are modified by hepatic DNA methylation of the peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A) and mitochondrial transcription factor A (TFAM) promoters, and also evaluated whether liver mitochondrial DNA (mtDNA) content is associated with NAFLD and IR. We studied liver biopsies obtained from NAFLD patients in a case-control design. After bisulfite treatment of DNA, we used methylation-specific polymerase chain reaction (PCR) to assess the putative methylation of three CpG in the PPARGC1A and TFAM promoters. Liver mtDNA quantification using nuclear DNA (nDNA) as a reference was evaluated by way of real-time PCR. Liver PPARGC1A methylated DNA/unmethylated DNA ratio correlated with plasma fasting insulin levels and homeostasis model assessment of insulin resistance (HOMA-IR); TFAM methylated DNA/unmethylated DNA ratio was inversely correlated with insulin levels. PPARGC1A promoter methylation was inversely correlated with the abundance of liver PPARGC1A messenger RNA. The liver mtDNA/nDNA ratio was significantly higher in control livers compared with NAFLD livers. mtDNA/nDNA ratio was inversely correlated with HOMA-IR, fasting glucose, and insulin and was inversely correlated with PPARGC1A promoter methylation. CONCLUSION: Our data suggest that the IR phenotype and the liver transcriptional activity of PPARGC1A show a tight interaction, probably through epigenetic modifications. Decreased liver mtDNA content concomitantly contributes to peripheral IR.
Asunto(s)
Epigénesis Genética/fisiología , Hígado Graso/genética , Proteínas de Choque Térmico/genética , Resistencia a la Insulina/genética , PPAR gamma/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Adulto , Estudios de Casos y Controles , Islas de CpG/genética , Metilación de ADN , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias Hepáticas/fisiología , Proteínas Mitocondriales/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Mensajero/metabolismoRESUMEN
OBJECTIVES: We evaluated circulating levels of biomarkers of atherosclerosis (soluble intercellular adhesion molecule: sICAM-1, plasminogen activator inhibitor: PAI-1 and soluble CD40 ligand: sCD40L) in patients with NAFLD proven through biopsy and control subjects, and correlated them with the histological disease severity. We further explored liver protein expression of ICAM-1, CD40 and PAI-1 in patients with different histological forms of NAFLD and control liver biopsies. PATIENTS AND METHODS: We included 215 individuals: 113 patients with NAFLD (simple steatosis n=45 and NASH n=68) and 102 control subjects. Circulating levels of the biomarkers were measured by ELISA. Liver expression of ICAM-1, CD40 and PAI-1 was assessed by immunohistochemistry using monoclonal antihuman antibodies. RESULTS: Patients with NAFLD, in comparison with control subjects, showed significantly higher circulating levels of sICAM-1 (605.3+/-34.6ng/ml vs. 356.5+/-24.6ng/ml, p=5.9 x 10(-6)), PAI-1 (22.8+/-1.7ng/ml vs. 19.0+/-2.1ng/ml, p=0.0149) and sCD40L (1347.5+/-513.7pg/ml vs. 804.5+/-396.1pg/ml, p=0.0229), results expressed as mean+/-SE. sICAM-1 was a strong predictor of histological severity of NAFLD, after adjusting for potential confounders. In addition, patients with NAFLD showed significantly higher liver staining scores for ICAM-1 and PAI-1 than control liver biopsies. ICAM-1 immunoreactivity in lobular inflammatory infiltrate showed high scores in NASH patients; a significant correlation was found between both the degree of liver steatosis and the severity of necroinflammatory activity and liver ICAM-1 expression. CONCLUSIONS: Our study shows that NAFLD is associated with elevated circulating levels and abnormal liver expression of molecular mediators of atherosclerosis. Additionally, ICAM-1 may be involved in liver damage and inflammation.