Metabolic dysfunction-associated Fatty Liver Disease (MAFLD) is one of the leading causes of liver disease, characterized by excess lipid accumulation in the liver. Friend of GATA2 (FOG2) is a transcriptional co-regulator protein that is found to regulate hepatic lipid metabolism and regulate lipid accumulation. It is also associated with Metabolic dysfunction-associated Steatohepatitis (MASH) in humans. Preliminary evidence has uncovered a coding variant of FOG2 (A1969G, S657G) predominantly found in individuals of African ancestry (minor allele frequency ~20%) and that has been linked to decreased fatty acid oxidation and an increased incidence of liver failure/cirrhosis (p=0.0053, Genebass - 395k individuals in UK Biobank). Our study aimed to elucidate how FOG2 S657G modulates hepatic lipid metabolism to then promote the onset of MAFLD. By employing a multi-pronged research approach, we sought to define the functional role of the FOG2 point mutation in the pathogenesis of MAFLD and to determine whether this modulation underlies the mutation’s association with the more severe MASH phenotype. 

By utilizing in vitro, in silico, and in vivo approaches, we performed a comprehensive investigation into the role of the FOG2 S657G in hepatic cellular and metabolic environments. Transient lipid-based transfections were performed on the human hepatoma cell line (Huh7) using FOG2 WT and FOG2 S657G DNA vector constructs. Subsequent analyses, including Western blots, qRT-PCR, and total cell triglyceride (TG) assays, were performed to confirm successful transfection and to elucidate the impact of the point mutation on lipid accumulation and metabolism. Results indicated that the FOG2 S657G mutation in Huh7 cells elevates the expression of de novo lipogenesis genes and increases cellular TG mass, pointing to an enhanced lipid accumulation within hepatic cells due to the mutation.

We also interrogated previously published RNAseq & Genotype datasets of Human Induced pluripotent stem cells (iPSCs) differentiated into hepatocytes (iHeps). A number of computational analyses including Differential Gene Expression, Gene Set Enrichment Analysis using the Hallmark Pathway, and Hierarchical Clustering of Genes within the MTORC1 pathway were performed using 24 iHeps cell lines within the database that showed expression of FOG2 wild-type (AA) and heterozygous expression of the point-mutant (AG). The iHeps carrying the FOG2 S657G variant exhibited differential gene expression consistent with increased mTORC1 signaling, suggesting that the point-mutant affects lipid metabolism by regulating mTORC1 signaling. 

Lastly, a novel mouse model with a mutation analogous to S657G (termed FOG2 MUT) was developed and utilized. qRT-PCR and Western Blot analyses were conducted on collected mouse livers on a standard, chow diet. Additionally, Glucose Tolerance Test (GTT) were administered to a cohort of  9-month old mice on chow-diet. The novel FOG2 MUT mouse model showed increased expression of de-novo lipogenesis genes and trended toward increased liver TG, activation of mTORC1 signaling, and impaired insulin sensitivity. 

Taken together, our multi-pronged approach and associated data was consistent with the model whereby the FOG2 S657G point-mutation may promote MAFLD through increased de novo lipogenesis (DNL) and by driving increased Insulin Resistance. Despite the fact that MAFLD is one of the most prevalence forms of liver disease globally, its pathogenesis remains to be a significant black box in hepatology with the transition into the more severe MASH phenotype still largely undefined. This study and our results provides critical insights into the molecular mechanisms by which the FOG2 S657G point mutant operates such that we can begin to understand the complex molecular landscape underlying MAFLD and MASH.