title: Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease.
abstract: Liver biopsy is currently the most reliable way of evaluating liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). Its inherent risks limit its widespread use. Differential liver DNA methylation of peroxisome proliferator-activated receptor gamma (PPARγ) gene promoter has recently been shown to stratify patients in terms of fibrosis severity but requires access to liver tissue. The aim of this study was to assess whether DNA methylation of circulating DNA could be detected in human plasma and potentially used to stratify liver fibrosis severity in patients with NAFLD.
  
  
  
  Patients with biopsy-proven NAFLD and age-matched controls were recruited from the liver and gastroenterology clinics at the Newcastle upon Tyne Hospitals NHS Foundation Trust. Plasma cell-free circulating DNA methylation of PPARγ was quantitatively assessed by pyrosequencing. Liver DNA methylation was quantitatively assessed by pyrosequencing NAFLD explant tissue, subjected to laser capture microdissection (LCM). Patients with alcoholic liver disease (ALD) were also subjected to plasma DNA and LCM pyrosequencing.
  
  
  
  26 patients with biopsy-proven NAFLD were included. Quantitative plasma DNA methylation of PPARγ stratified patients into mild (Kleiner 1-2) and severe (Kleiner 3-4) fibrosis (CpG1: 63% vs 86%, p<0.05; CpG2: 51% vs 65% p>0.05). Hypermethylation at the PPARγ promoter of plasma DNA correlated with changes in hepatocellular rather than myofibroblast DNA methylation. Similar results were demonstrated in patients with ALD cirrhosis.
  
  
  
  Differential DNA methylation at the PPARγ promoter can be detected within the pool of cell-free DNA of human plasma. With further validation, plasma DNA methylation of PPARγ could potentially be used to non-invasively stratify liver fibrosis severity in patients with NAFLD. Plasma DNA methylation signatures reflect the molecular pathology associated with fibrotic liver disease.
authors.affiliation: Fibrosis Laboratories, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.|Department of Gastroenterology and Hepatology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Timothy
authors.initials: T
authors.lastname: Hardy
authors.affiliation: School of Medicine, Koç University, Istanbul, Turkey.
authors.forename: Mujdat
authors.initials: M
authors.lastname: Zeybel
authors.affiliation: Fibrosis Laboratories, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Day
authors.affiliation: Department of Gastroenterology and Hepatology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Dipper
authors.affiliation: Department of Gastroenterology and Hepatology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Steven
authors.initials: S
authors.lastname: Masson
authors.affiliation: Department of Gastroenterology and Hepatology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Stuart
authors.initials: S
authors.lastname: McPherson
authors.affiliation: Department of Gastroenterology and Hepatology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Elsbeth
authors.initials: E
authors.lastname: Henderson
authors.affiliation: Fibrosis Laboratories, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.|Department of Cellular Pathology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Dina
authors.initials: D
authors.lastname: Tiniakos
authors.affiliation: Department of Hepatobiliary Surgery, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Steve
authors.initials: S
authors.lastname: White
authors.affiliation: Department of Hepatobiliary Surgery, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Jeremy
authors.initials: J
authors.lastname: French
authors.affiliation: Fibrosis Laboratories, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Derek A
authors.initials: DA
authors.lastname: Mann
authors.affiliation: Fibrosis Laboratories, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.|Department of Gastroenterology and Hepatology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
authors.forename: Quentin M
authors.initials: QM
authors.lastname: Anstee
authors.affiliation: Fibrosis Laboratories, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Jelena
authors.initials: J
authors.lastname: Mann
mesh_terms: D016022:Case-Control Studies; D019175:DNA Methylation; D005260:Female; D005819:Genetic Markers; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008103:Liver Cirrhosis / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D065626:Non-alcoholic Fatty Liver Disease / Q000235:genetics* / Q000473:pathology; D047495:PPAR gamma / Q000235:genetics* / Q000378:metabolism; D012720:Severity of Illness Index
chemical_list: D005819:Genetic Markers; D047495:PPAR gamma
keywords: HEPATIC FIBROSIS; MOLECULAR GENETICS; MOLECULAR PATHOLOGY; NONALCOHOLIC STEATOHEPATITIS; PPAR GAMMA