The Association And Mechanism Of Patatin-like Phospholipase Domain-containing3Gene And Nonalcoholic Fatty Liver Disease

Hereditary factors play an important role in the development of non-alcoholic fattyliver disease (NAFLD). A single nucleotide polymorphism (SNP) in the PNPLA3gene(rs738409, representing a substitution from cytosine to guanine, resulting in a switchfrom isoleucine to methionine at148residue, I148M) was reported to be significantlyassociated with NAFLD. It is unknown if the variant can induce NAFLDindependently, it is also unclear by which mechanism the variant affecting the lipidmetabolism of liver. In this study, we researched the physiological functions ofPNPLA3I148M variant at the individual, cellular and molecular levels. We hope toverify the correlation between this variant and NAFLD in Chinese Han population, toreveal the mechanism by which this variant affecting hepatocytes’ metabolism, and toelucidate the molecular mechanism by which this variant affecting the activity of theenzyme.Firstly, we explored the relationship between PNPLA3I148M variant andhereditariness to NAFLD and chronic hepatitis B (CHB) in Chinese Han population ofQingdao. The study included315NAFLD patients with336healthy controls and185CHB patients with164healthy controls. Genotypes of blood samples were examinedby polymerase chain reaction (PCR) and genotyping method. Allele frequencydistribution of rs738409G were65.40%,71.87%and56.47%in patients with NAFLD,NASH and SS and33.18%in control,31.9%in CHB patients and21.9%in controlgroup（P﹤0.05）, respectively, which had statistical signification(P﹤0.05). Non-conditional Logistic regression showed that, comparing with CC gene carriers, oddsratio of occurrence of NAFLD was3.81(95%CI:3.03to4.79, P <0.05), and the oddsratio of occurrence of NASH OR was1.97(95%CI=1.41~2.75, P <0.05) in GGgene carriers. A case-control analysis revealed a1.67-fold (95%CI=1.18—2.34, P=0.003) excess risk of developing CHB for rs738409G allele compared with C allele.Unconditional logistic regression model adjusted for age, sex, OR is1.63-fold (95%CI=1.17—2.68P=0.04). PNPLA3I148M variant was associated with the level ofserum ALT、γ-GT. Through stratified analysis of NASH group, the BMI, ALT, andFINS of GG genotype were all higher than that of the CC genotype (P <0.05), whilethe serum HDL level of GG genotype was lower than that of both CC genotype andGC genotype (P <0.05). The results suggested that, PNPLA3I148M variant wasassociated with NAFLD and CHB hereditary susceptibilities; the variant was asignificant factor which could determine NAFLD and CHB hereditary susceptibilitiesin Chinese Han population.Secondly, we explored the mechanism of PNPLA3I148M variant in the culturedhepatocytes. We have improved the classic two-step perfusion technique andconstructed a simple method to isolate and culture primary mouse hepatocytes withhigh purity and high viability. Adequate hepatocytes were isolated by this method; themotility rate of the hepatocytes was higher than90%, the viability of the primaryhepatocyts lasted stably for7days. We constructed the recombinant lentivirusescontaining PNPLA3gene or PNPLA3gene I148M variant successfully. Huh-7cellswere infected by the recombinant lentiviruses containing PNPLA3or PNPLA3I148M.Western blot showed that PNPLA3protein was overexpressed in Huh-7cells. PNPLA3gene improved the level of triglyceride, total cholesterol, lipoproteins, as well as theaminotransferance in the cells; the I148M variant possessed a more enormousenhancing effect. Simvastatin depressed the triglyceride level in the cells, and theeffect showed a time and dose dependent tendency, the cells expressing I148M variantwere more sensitive to the simvastatin. The results suggested that, PNPLA3geneI148M variant can induce the liver steatosis independently, and the I148M variant wasmore sensitive to the simvastatin. In the future work, we can make individualtherapeutics to NAFLD according to the patients’ genotypes.Thirdly, transgenic mouse expressing definite gene in definite tissue has become apowerful tool to study gene function. DNA pronucleus microinjection is the mostlyused technique to construct transgenic mouse, and this need us to construct liverspecific expression plasmid of PNPLA3I148M variant. We got the complete codingsequences of PNPLA3I148M variant and the liver specific promoter ALB by PCR. The PNPLA3I148M and the ALB were connected by PCR. GeneHogs weretransformed by the PCR products and the plasmids were harvested and then verified byelectrophoresis and sequencing. SMMC-7721cells were infected by the ALB-PNPLA3I148M plasmids, western blot showed that ALB-PNPLA3I148M wasoverexpressed in SMMC-7721cells. The liver specific expression plasmid of PNPLA3I148M will lay a foundation for the construction of transgenic mouse.Lastly, to glean insights into the variant’s effect on enzymatic activity, we performedmolecular dynamics simulation and flexible docking studies. Our data showed that thesize of the substrate-access entry site was significantly reduced in mutants, whichlimited the access of palmitic acid to the catalytic dyad. Besides, the binding freeenergy calculations suggested low affinity for substrate to mutant enzyme. Thesubstrate-bound system simulations revealed that the spatial arrangement of palmiticacid was distinct in wild-type from that in mutant. The substrate recognition specificitywas lost due to the loop where the I148M mutation was located. Our results providedstrong evidence for the mechanism by which I148M affected the enzyme activity andsuggested that mediating the dynamics might offer a potential avenue for NAFLD.