Studies On Association Of Nafld With Gene Polymorphisms And Influences Of UGT Gene Polymorphisms On MMF Metabolism

In recent years,the incidence of non-alcoholic fatty liver disease(NAFLD)is increasing year by year.There is a significant correlation between the occurrence of NAFLD and heredity.The purpose of this research is to study the relationship between SAMM50 gene and LEPR gene single nucleotide polymorphisms(SNPs)and the occurrence of NAFLD.Based on the previous work,a reasonable combination of SNP loci was selected,and a gene chip was made to detect the genotypes of NAFLD-related sites for the purpose of early warning.Peripheral blood and the clinical data were collected from 380normal subjects and 380patients with NAFLD.DNA was extracted from peripheral blood and Taqman probe was used to detect the genotypes of rs738491,rs2073082 and rs6700896 loci in each sample by real-time pcr.The best combination of NAFLD related SNPs was screened and the gene chip was prepared based on the principle of single base extension reaction.The susceptibility to NAFLD was significantly increased in rs738491 T allele carriers and rs2073082 G allele carriers(OR=1.39;95%CI=1.141.71,p=0.001;OR=1.31;95%CI=1.05-1.62,p=0.016,respectively).Compared with the results of taqman probe,the coincidence rate of microarray genotype was 90%,and the sensitivity of DNA detection was 10 ng.SAMM50 rs738491 and rs2073082 polymorphisms are associated with susceptibility to NAFLD.The gene chip based on single base extension reaction can effectively detect the genotype of NAFLD-associated SNP loci.Mycophenolate mofetil is a first-line drug after organ transplant,but there are differences in metabolism of mycophenolate mofetil among individuals.The UDP glucuronosyltransferase enzyme is the key metabolic enzyme for mycophenolate mofetil,and UGT1A8 gene polymorphisms may affect the elimination of mycophenolate mofetil in patients.Here,we conducted an in vitro study to explore the relation between UGT1A8 gene polymorphisms and mycophenolate mofetil metabolism.Five mutant loci overexpression vectors(UGT1A8 128C>T,157C>A,431C>T,518C>G,and 830G>A)were constructed by genetic recombination and site-directed mutagenesis.We used Lipo2000(Invitrogen,Carlsbad,CA,USA)to transfect the vectors into HEK293 cells.Mycophenolic acid,the active ingredient of mycophenolate mofetil,was added to different groups of cells.We then used the liquid chromatography tandem-mass spectrometry technique to detect production of the metabolite 7-O-mycophenolic acid glucuronide and to evaluate activity of the UDP glucuronosyltransferase enzyme in cells with different overexpression vectors.Mutations of UGT1A8 157C>A and 518C>G vectors can lead to increased activity of UDP glucuronosyltransferase enzymes and increased production of the 7-O-mycophenolic acid glucuronide metabolite,which showed 116%(P<0.001)and 107%(P=0.0191)production changes of 157C>A and 518C>G mutations,respectively,relative to wild-type UGT1A8.However,mutations of UGT1A8 431C>T and 830G>A loci resulted in decreased activity of UDP glucuronosyltransferase enzymes and decreased production of the metabolite,respectively showing 62.9%(P<0.001)and 9.05%(P<0.001)activity relative to wild-type UGT1A8.UGT1A8 128C>T had little effect on enzyme activity.Our results showed that UGT1A8 gene polymorphisms can affect the activity of UDP glucuronosyltransferase enzyme,which may influence the elimination of mycophenolate mofetil in different patients.