The Screening For Key Gene Variants Of Statin Resistant And Mechanism Of LDLR In Different Tissues On Lipid Profile

Objective:(1)Statins are the preferred drugs for lowering blood lipids,and the lipid-lowering efficiency of statins varies greatly among different individuals.Genetic variations of statins efficiency were identified among different ethnic groups.(2)To explore the correlation between Trip12 polymorphisms and dyslipidemia and coronary heart disease in different ethnic population.(3)To investigate the effect of LDLR expression in different tissues on blood lipid in mice.Methods:(1)A total of 536coronary heart disease(CAD)patients of Han and Uygur population were collected,and enrolled them randomly into the resistance group(55 cases)and the sensitive group(55cases)according to the decreasing of low-density lipoprotein-cholesterol level(LDL-C)decline after oral statin administration.The total exome sequencing and bioinformatics analysis was used to screen out genes that might affect the lipid-lowering efficiency of statin.(2)A case-control study including 3108 CAD patients and 2129 gender,age,and ethnic-matched controls was conducted to genotype and haplotype construction of Trip12gene polymorphism and analyze the relationship between Trip12 polymorphism and coronary heart disease.(3)Mice with LDLR specific knockout in different tissues were constructed by cre-loxp technology to monitor the changes in blood lipid and analyze the effect of macrophages LDLR knockout on atherosclerosis under the background of Apo E knockout.Results:(1)Total exon sequencing results showed no significant difference in the mean mutation number between the resistance group and the sensitive group.In the resistance group,there were 96,995 mutations in each patient,including22041 exons(22.7%).The total variation of each patient in the sensitive group was 96815,including 22997 exons(23.8%).(2)The bioinformatics analysis showed that there were23,963 functional high-frequency mutation sites in Han population and 21,608 in Uygur population.Rare functional mutation sites:5genes were screened strictly and 54genes were not screened strictly.(3)The main mutations affecting statin resistance were:high frequency variation(CCLX,PPP1R14A),low frequency functional variation(TRIP12,DOCK8,SKIDA1,CEP162),candidate gene variation(INSIG1,ABCC1,ABCG8,LDLR,HMGCS1).The main variants that affect statins sensitivity are:high frequency variants(TNK2,SLC6A1,SYNE1),low frequency functional variants(KIF14,MAN2B1,KIAA1586,TMEM132C,SVEP1,DNHD1),and candidate gene variants(INSIG2,ABCA1).(4)By exon sequencing,rs200837058 of Trip12,a rare functional variant gene,were significantly correlated with coronary heart disease in Uygur population.(5)To verify the effect of LDLR on serum lipids at the animal level,we compared serum levels of TC,LDL-C,VLDL-C and HDL in LDLR^F/FCMV Cre group and WT group mice after 8weeks of normal diet.Further comparison of the TC levels in the liver and small intestine showed that TC levels in the liver higher than that in the WT group,but there was no significant difference in the TC levels in the small intestine.(6)To further clear the influence of different tissue organ LDLR on blood lipid,we respectively constructed LDLR^F/FAlb Cre,LDLR^F/FVillin Cre and LDLR^F/Flyz2 Cre mice,respectively to compare the influence of liver,small intestine and macrophages LDLR on blood lipid.We found the liver,small intestine LDLR knockout mice serum TC level were significantly higher than that of WT group,macrophages LDLR knockout mice serum TC there was no significant difference in the WT group.(7)To further reveal the macrophages LDLR influence on atherosclerosis,we built the APOE KO LDLR^F/Flyz2 Cre and APOE KO mice,fed by high cholesterol feed,results show that APOE KO LDLR^F/Flyz2 Cre mice were less prone to atherosclerosis.Conclusion:(1)The main variants identified for statin resistance were:CCLX,PPP1R14A,TRIP12,DOCK8,SKIDA1,CEP162,INSIG1,ABCC1,ABCG8,LDLR,HMGCS1.(2)The rs962906182 and rs200837058 mutations of Trip12gene were significantly correlated with the incidence of coronary heart disease.(3)Serum lipid level of LDLR knockout mice was significantly higher than that of WT mice.(4)Liver specific LDLR knockout mice showed more significant lipid changes than small intestine specific LDLR knockout mice(5)In APOE knockout background,macrophage specific LDLR knockout had a protective effect on atherosclerosis in mice.