ABSTRACT: Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide, but its pathophysiological mechanisms remain elusive. It is a progressive disease, encompassing hepatic steatosis, steatohepatitis with(out) fibrosis and ultimately cirrhosis and hepatocellular carcinoma. DNA methylation (DNAm) is dysregulated in MASLD and may underpin key pathogenic mechanisms. Additionally, aging is associated with MASLD and shares common processes of chronic inflammation and oxidative stress. Therefore, this study focuses on DNAm changes in relation to MASLD progression and epigenetic age acceleration (EAA). Results: Liver biopsies from 22 MASLD patients were analyzed using Infinium MethylationEPIC BeadChip arrays. Strikingly, progression of MASLD was characterized by gradual DNAm changes, revealing associated KEGG pathways. Additionally, Horvath’s EAA significantly correlated with MASLD stage and individual histological MASLD parameters while LiverClock’s EAA correlated only with MASLD stage. In contrast, both Horvath’s intrinsic EAA and HepClock’s EAA showed no significant correlations. Integrative analyses, leveraging both gradual MASLD and Horvath’s EAA DNAm signatures, gene expression (n = 118), and a MASLD-specific transcriptional regulatory network, identified (regulon-specific) transcription factors implicated in MASLD and EAA progression, representing a transcription factor-network of redox (ferroptosis), immune, and metabolic/endocrine related epigenetic processes. Conclusion: Gradual DNAm changes were found to align with progression of MASLD and EAA, with EAA a potential non-biased quantitative biomarker for MASLD. Integrative analysis highlighted potential new therapeutic transcription factor targets, with special emphasis on AEBP1 and emerging nuclear receptors including CAR(NR1I3), MR(NR3C2), GR(NR3C1), and ESRRG, underscoring the potential of epigenetic redox-metabolic therapies for MASLD
