Abstract
Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a complex metabolic disorder with a diverse spectrum. This study aimed to classify patients with MASLD into molecular subtypes based on the underlying pathophysiology. Methods: We performed high-throughput RNA sequencing on 164 liver tissue samples from healthy controls and patients with MASLD. The clustering was based on individual genes or pathways that showed high variation across the samples. Second, the clustering was based on single-sample gene set enrichment analysis. Results: Optimal homogeneity was achieved by dividing the samples into four clusters (one healthy control and three MASLD clusters I-III) based on the top genes or pathways with differences across the samples. No significant differences were observed in waist circumference, blood pressure, glucose, alanine transaminase (ALT), or aspartate transferase (AST) levels between cluster I patients with MASLD and the healthy controls. Cluster I showed the clinical features of lean MASLD. Cluster III of MASLD demonstrated hypertension and a T2DM prevalence of 57.1% and 50.0%, respectively, with a significantly higher fibrosis burden (stage of fibrosis, liver stiffness, and FIB-4 value) than clusters I and II. Cluster III was associated with severe fibrosis and abnormal glucose homeostasis. In MASLD cluster I, the sphingolipid and GPCR pathways were upregulated, whereas the complement and phagocytosis pathways were downregulated. In MASLD cluster II, the cell cycle and NOTCH3 pathways increased, whereas the PI3K and insulin-related pathways decreased. In MASLD cluster III, increased activity occurred in the interleukin-2 and -4 and extracellular matrix pathways, coupled with decreased activity in the serotonin 2A and B pathways. Conclusions: MASLD can be divided into three distinct molecular phenotypes, wherein each is characterized by a specific molecular pathway.