LRP6 Intron SNP Rs12823243 Affects The Individual Susceptibility To NAFLD By SPI1-PPAR Pathway

Background and objective:The phenomenon of individual risk difference in non-alcoholic liver disease（NAFLD）is very common.Previously known influencing factors can only explain part of individual risk difference.Discovering unknown influencing factors and revealing their influencing mechanism is a necessary challenge to achieve precise prevention and treatment of NAFLD.Single nucleotide polymorphism（SNP）is the main cause of genetic factors contributing to the risk difference of individuals with NAFLD.Although there have been many previous studies on the effect of SNPs on the individual susceptibility of NAFLD,most of them focused on functional SNPs in gene exons,while few reports on the effect of intron SNPs on the risk differences and mechanisms of individuals with NAFLD.The rs12823243 intron SNP of low-density lipoprotein receptor-6（LRP6）is a new factor that significantly affects the risk difference of individuals with NAFLD.This study will further verify this phenomenon and explore its influencing mechanism,providing a scientific basis for the precise prevention and treatment of NAFLD.Methods:（1）Expand the scale of clinical study,recruit NAFLD and non-NAFLD patients,collect diagnosis and treatment information and collect blood for DNA extraction,use Massarray high-throughput technology to detect candidate SNPs including LRP6 rs12823243,and analyze the relationship between the frequency of each SNP and the risk of NALFD disease.（2）According to the prediction of bioinformation,the influence of intron SNPs on the expression of LRP6 was explored from two possible mechanisms of intron SNPs influencing pseudo-exon formation and enhancer activity,and verified by Sanger sequencing and double luciferase reporter gene assay,respectively,to determine the influence of intron SNPs on LRP6 expression.（3）LRP6^（+/-）knockdown mouse model was constructed by loxp technique to determine the risk between LRP6 knockdown expression and the severity of NALFD in mice,and to bridge the relationship between LRP6 rs12823243 intron SNP and NAFLD risk.Transcriptomics and western blot were further used to explore the molecular mechanism.Results:（1）A total of 292 NAFLD and 387 non-NAFLD patients were recruited,and 46 candidate SNPs were analyzed by high-throughput assay and found that LRP6 rs12823243 T carriers significantly increased the risk of NAFLD（OR=1.826;95%CI,1.053-3.166;P=0.032）,confirming the earlier findings of our research group.In addition,association analysis revealed that the other four intron SNPs of LRP6（rs2417085,rs10492120,rs7956548,rs10845495）were associated with NAFLD risk.Further analysis showed that compared with ATTCC haplotype carriers,TTTTA haplotype carriers constituted by SNPs of the five risk introns of LRP6significantly increased the risk of NAFLD（OR=5.349;95%CI,1.178-24.292;P=0.030）;In addition,functional SNP of LRP6（rs2302685）and functional SNPs of other genes including rs2241883（FABP1）,rs28365927（SIRT3）,rs8113704（NFKBID）were also found to be associated with NAFLD risk.（2）Bioinformation prediction analysis indicated that SNPs of these introns might activate the formation of pseudo-exons,and the possibility of activation of pseudo-exons was found to be small by 39samples,suggesting that this mechanism should not be common.The combined analysis of multiple databases suggested that rs12823243 was located in Enh74957 of LRP6 intron 7,and the polymorphism of this site affected the function of enhancer.Double luciferase reporter gene assay showed that the main allele A had enhancer activity,while the secondary allele T silent LRP6 promoter activity.Western blot assay indicated that the secondary allele T may inhibit the LRP6 promoter activity through SPI1,thereby decreasing the expression of LRP6 protein and increasing the risk of NAFLD in individuals.（3）In NAFLD model induced by high-fat diet,LRP6 protein expression was decreased in LRP6^（+/-）knockout mice,accompanied by more severe liver steatosis,inflammation and fibrosis.KEGG enrichment analysis of transcriptome sequencing revealed that changes in PPAR signaling pathway were closely related to the severity of NAFLD disease,and LRP6^（+/-）knockdown expression significantly reduced the expression of key proteins PPARαand PPARγin this pathway.Conclusion:The intron SNP of LRP6 rs12823243 was associated with the risk difference of NAFLD individuals.The secondary allele T may inhibit the LRP6 promoter activity and reduce the expression of LRP6 by SPI1,thereby increasing the risk of NAFLD disease.PPAR signaling pathway may be the molecular mechanism that mediates the reduction of LRP6 expression influences the difference in NAFLD disease severity,but further experimental verification is needed.This study found and elaborated the relationship between LRP6 intron SNP rs12823243 and individual risk difference of NAFLD and its possible mechanism,providing a scientific basis for precise prevention and treatment of NAFLD.