Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease

Abstract

Objective 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 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. Design 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 ALD were also subjected to plasma DNA and LCM pyrosequencing. Results 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. Conclusions 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 noninvasively stratify liver fibrosis severity in patients with NAFLD. Plasma DNA methylation signatures reflect the molecular pathology associated with fibrotic liver disease.

DNA methylation signatures reflect aging in patients with nonalcoholic steatohepatitis

“…Using liver biopsy tissue from NAFLD patients, Sookoian et al identified methylation of promoter sites on PPARγ coactivator 1α (PPARGC1A) with markers of insulin resistance such as HOMA-IR and serum insulin (19). Other investigators have demonstrated that specific methylation sites on promoters of genes are linked to more severe liver fibrosis (17,18). Although these reports establish a potential relationship between fibrosis and specific epigenetic modifications, targeted individual CpG sites may not accurately reflect the complex interaction between causal and compensatory measures in chronic diseases such as NASH.…”

“…Previous studies of DNAm in NAFLD have examined gene-specific epigenetic modifications that may be involved in disease pathogenesis by targeting genes known to regulate fibrogenesis or metabolic function (16)(17)(18)(19)(20). For instance, Hardy et al examined two specific CpG loci on the PPARγ promoter in circulating DNA and found differences in methylation between NAFLD patients with severe fibrosis and those with mild fibrosis (17).…”

“…For instance, Hardy et al examined two specific CpG loci on the PPARγ promoter in circulating DNA and found differences in methylation between NAFLD patients with severe fibrosis and those with mild fibrosis (17). Comprehensive integration of genome-wide methylation and gene expression studies has also been performed on liver tissue and been used to identify differential targets between mild and severe NAFLD (18,20).…”

Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention

“…Remodelling of DNA methylation at key fibrosis modifier genes underpinned protection against carbon tetrachloride (CCl 4 ) induced liver fibrosis in male offspring of male mice that were themselves subjected to CCl 4 mediated liver injury 104 . Remarkably, DNA methyl ation remodelling occurred in the same genes in patients with NASH with mild fibrosis and intriguing data sug gests that epigenetic signatures present on circulating cell free DNA could be a potential biomarker of this effect and, therefore, disease severity 104,105 .…”

Current status and future perspectives of circulating cell-free DNA methylation in clinical diagnostics

“…Finally, exocrine pancreas-derived DNA was identified in patients with pancreatitis. Other researchers suggest ccfDNA methylation as a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease [24]. Cardiac diseases might be accompanied by elevated levels of ccfDNA and is found in patients with acute coronary syndrome [25].…”