Construction And Functional Study Of Novel LDLR Mutants In Familial Hypercholesterolemia Patients

Familial hypercholesterolaemia (FH) is an autosomal dominant disease characterized by elevated total and low-density lipoprotein (LDL) cholesterol levels in plasma, resulting in skin and tendon xanthomata, and premature cardiovascular disease. The primary defects in FH patients are mutations in the gene encoding the LDL receptor (LDLR).The mutations of LDLR gene account for about a half in FH patients diagnosed by clinical features and blood lipid tests. Over thirty FH familily were analysed and twenty mutations including sixteen point mutations,two deletion mutation and two splicing mutation were identified in our laboratory. However,it's not clear about whether these mutations are pathogenic and theirs pathogenic mechanism . To make clear the cause resulting in hyperlipoidemia and to further understand molecular mechanism and pathogenesis of FH , we take six mutations on the base of mutation analysis, which are used to construction and primary functional study of mutants in our experiments.Objective To analyze and identify LDLR gene mutations in six FH patients which was diagnosed by clinical features and blood lipid tests. To construct and identify of LDLR and its mutant recombinant plasmid . To investigate the impact of mutants on the expression and function of LDLR in HEK-293 cells comparing with mutants and WT-LDLR. And to further explore its effects on LDL metabolism and possible mechanism resulting in FH, which is expected to provide experimental evidence for early treatment , prevention and cure of cardiovascular disease, antenatal diagnosis and familial study.Methods Blood samples were drawn from six FH patients which was diagnosed by clinical features and blood lipid tests after fasting for 12 hours and before lipid-lowering. 0.5ml anticoagulated blood was used for genomic DNA isolation by phenol-chloroform. We eliminated ApoB100 3500 mutations that cause familial defective apoB100(FDB) by PCR- DNA sequence analysis in six FH patients. In addition these patients have been investigated for mutations of promoter and all eighteen exons of the LDLR gene.Screening was carried out by using Touch-down PCR and direct DNA sequence analysis. Hepatic cells were used for total RNA isolation by Trizol. Purified RNA was used for synthesis of LDLRcDNA by reverse transcription PCR. LDLRcDNA was inserted into PTA2 clone vector. After transformation into chemically competent DH-5αE.Coli,the respective destination clones were selected for by plating on ampicilin , 5-Bromo-4-chloro-3-indolyl and Isopropyl-beta-D-thiogalactopyranoside selective agar plat, and plasmids made from the selected clone were digested by special enzyme and were indetified by DNA sequence analysis. The correct plasmids inserted PTA2 clone vector and demonstrated by DNA sequence analysis were inserted into a EGFP tagging eukaryotic expression vector by guangzhou FulenGen company, and LDLR recombinant plasmid was constructed . The point change in the LDL receptor cDNA was made by using QuickChange XL system (Stratagene) and confirmed by DNA sequencing. HEK-293 cells were transfected with the various LDL receptor cDNA constructs by lipofection procedure. HEK-293 cells were transfected without any constructs as control. Forty eight hours later, total proteins from transfected cells were prepared. The expressed LDL receptor proteins were examined by western blot analysis.In addition ,to measure the LDL receptor binding and internalization function by flow cytometric analysis, HEK-293 cells were transfected with the various LDL receptor cDNA constructs and forty eight hours later cells were incubated with DiI-LDL at 4℃and 37℃,respectively.Results No mutations R3500Q of ApoB100 were observed in six FH patients. Six novel mutations in the LDLR gene were identified, including three missense mutations, two deletion mutations and one nonsense mutations. We obtained 6 correct recombined LDLR mutant plasmids by screening and DNA sequencing.Western analysis shows the LDL receptor strap of three missense mutations(C210R, T383I and A459V) were similar to those of wild type . 357delG,675delA and S565X all show a truncated protein strap .All mutations impair the function, displaying 13%-34% of normal receptor binding activity and 13%-35% of normal receptor internalization activity,compared with wild-type control.Conclusion1. Six mutants of LDLR were successfully constructed.2. All mutations impair the function, displaying 13%-34% normal receptor binding activity and 13%-35% of normal receptor internalization activity.Among these mutations,LDL binding activity and internalization activity for A459V are both the highest,which is 34% and 35%,respectively, while those of 675delA are both the lowest,which is 13% and 13%,respectively.3. Six new mutation may result in elevated level of plasma cholesterol and further develop FH proved by phenotype of patients and functional study of mutants in vitro.