Abstract Background and aims The I148M PNPLA3, the rs641738 in MBOAT7/TMC4 locus and the E167K TM6SF2 polymorphisms represent the main predisposing factors to non-alcoholic fatty liver disease (NAFLD) development and progression. We previously generated a full knockout of MBOAT7 in HepG2 cells (MBOAT7 -/- ), homozygous for the I148M PNPLA3. Therefore, we aimed to:1) investigate the synergic impact of the 3 at-risk variants on liver injury and hepatocellular carcinoma (HCC) in a large cohort of NAFLD patients;2) create in vitro models of genetic NAFLD by silencing TM6SF2 in both HepG2 and MBOAT7 -/- cells. Methods NAFLD patients (n=1380) of whom 121 had HCC were stratified with a semi-quantitative score ranging from 0 to 3 according to the number of PNPLA3 , TM6SF2 and MBOAT7 at-risk variants. TM6SF2 was silenced in HepG2 (TM6SF2 -/- ) and MBOAT7 -/- (MBOAT7 -/- TM6SF2 -/- ) through CRISPR/Cas9. Results In NAFLD patients, the additive weight of these mutations was associated with liver disease severity and increased risk to develop HCC. In HepG2 cells, TM6SF2 silencing altered lipid composition and induced the accumulation of micro-vesicular LDs, whereas the MBOAT7 -/- TM6SF2 -/- cells showed a mixed micro/macro pattern of LDs. TM6SF2 deletion strongly affected endoplasmic reticulum (ER) and mitochondria ultrastructures thus increasing ER/oxidative stress. Mitochondrial number raised in both TM6SF2 -/- and MBOAT7 -/- TM6SF2 -/- models, suggesting an unbalancing in mitochondrial dynamics and the silencing of both MBOAT7 and TM6SF2 impaired mitochondrial activity with a shift towards anaerobic glycolysis. MBOAT7 -/- TM6SF2 -/- cells also showed the highest proliferation rate. Conclusions The co-presence of the 3 at-risk variants impacts on NAFLD course, in both patients and experimental models affecting LDs accumulation, mitochondrial functionality and metabolic reprogramming towards HCC.

TM6SF2/PNPLA3/MBOAT7 loss-of-function genetic variants impact on NAFLD development and progression both in patients and in in vitro models