title: Epigenetic Modifications in the Biology of Nonalcoholic Fatty Liver Disease: The Role of DNA Hydroxymethylation and TET Proteins.
abstract: 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 (n = 90) 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 (R = 0.50, P = 0.000382) and PPARGC1A-mRNA levels (R = -0.57, P = 0.04).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 (0.4 ± 0.5) had significantly lower nonnuclear-5-hmC staining compared with controls (1.8 ± 0.8), means ± standard deviation, P = 0.028. 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; P = 0.005). 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.
authors.affiliation: From the Department of Molecular Genetics and Biology of Complex Diseases, Institute of Medical Research A Lanari-IDIM, University of Buenos Aires-National Scientific and Technical Research Council (CONICET), Ciudad Autónoma de Buenos Aires, Argentina (CJP, RS, TFG); Biomedical Genomics and Evolution Laboratory, Ecology, Genetics and Evolution Department, Faculty of Science, IEGEBA, University of Buenos Aires-National Scientific and Technical Research Council (CONICET), Ciudad Autónoma de Buenos Aires, Argentina (HD, CR); Department of Pathology, Hospital Diego Thompson, San Martin, Buenos Aires, Argentina (JSM); Liver Unit, Medicine and Surgery Department, Hospital Abel Zubizarreta, Ciudad Autónoma de Buenos Aires, Argentina (GOC); and Department of Clinical and Molecular Hepatology, Institute of Medical Research A Lanari-IDIM, University of Buenos Aires-National Scientific and Technical Research Council (CONICET), Ciudad Autónoma de Buenos Aires, Argentina (RS, SS).
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Pirola
authors.forename: Romina
authors.initials: R
authors.lastname: Scian
authors.forename: Tomas Fernández
authors.initials: TF
authors.lastname: Gianotti
authors.forename: Hernán
authors.initials: H
authors.lastname: Dopazo
authors.forename: Cristian
authors.initials: C
authors.lastname: Rohr
authors.forename: Julio San
authors.initials: JS
authors.lastname: Martino
authors.forename: Gustavo O
authors.initials: GO
authors.lastname: Castaño
authors.forename: Silvia
authors.initials: S
authors.lastname: Sookoian
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000328:Adult; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019175:DNA Methylation / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D044127:Epigenesis, Genetic; D005260:Female; D020022:Genetic Predisposition to Disease; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008930:Mitochondria, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases; D065626:Non-alcoholic Fatty Liver Disease / Q000453:epidemiology / Q000235:genetics / Q000378:metabolism; D001678:Organelle Biogenesis; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; C486497:PPARGC1A protein, human; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human