Molecμlar Mechanism Of Familial Hypercholesterolemia Caused By APOB Gene Mutation(c.12581t>C)

Objective: By analyzing the mutation site of APOB gene found in a patient with high cholesterol,combining with the clinical data of the patient and genetic testing,the blood lipid status of the patient’s family members was traced,and the mutation site C.12581T>This study provides a new diagnostic basis for the clinical diagnosis of Familial Hypercholesterolemia(FH).Methods: 1.Data collection: Collect patient family information according to the commonly used diagnostic scoring criteria for FH,draw family tree diagrams,collect fasting blood samples from probands for examination,perform full exome sequencing,search for mutation sites and predict mutation function and protein structure.2.Locus acquisition: by designing truncated APOB gene fragments containing mutation sites,design primers according to the fragment sequence,perform PCR amplification,gel electrophoresis of the amplified products,and carry out purification and sequencing verification of the bands.3.Plasmid construction: construct an empty vector plasmid expressing only the green fluorescent protein GFP gene,a wild-type LDLR plasmid expressing both GFP and LDLR-HA genes,a wild-type truncated APOB plasmid expressing both GFP and APOB-Flag genes,and simultaneous A mutant truncated APOB plasmid expressing GFP gene and APOB-Flag gene.4.Plasmid transfection: by culturing human normal liver cells(HL-7702)in vitro,they are divided into three groups for plasmid co-transfection: empty vector group(empty vector plasmid expressing only the green fluorescent protein GFP gene and simultaneous expression of GFP and LDLR-HA gene wild-type LDLR plasmid),WT group(wild-type truncated APOB plasmid expressing both GFP gene and APOB-Flag gene and wild-type LDLR plasmid expressing GFP and LDLR-HA gene simultaneously),Mut group(simultaneous expression The mutant truncated APOB plasmid of GFP gene and APOB-Flag gene and the wild-type LDLR plasmid expressing GFP and LDLR-HA gene at the same time)were cultured for 48 h,so that the truncated APOB protein and LDLR protein in each group were fully expressed and combined.5.Functional verification: Collect the cells of each group,extract the protein,use Protein G beads and anti-Flag antibody to immunoprecipitate the truncated APOB protein in each group,and keep part of the protein sample as the Input group;use Western The blotting method was used to detect the expression of LDLR protein in each Ip group,as well as the expression of truncated APOB protein and LDLR protein in each Input group,and β-actin was used as an internal control.ResμLts: 1.Throμgh three-generation family studies,several suspected FH patients were screened out.Among them,the biochemical routine of the proband indicated that the levels of total cholesterol(TC 9.82mmol/L)and low-density lipoprotein(LDLC7.59mmol/L)were significantly increased,consistent with Features of hypercholesterolemia.2.Sequencing results of the blood sample of the proband sμggest that there are two unreported mutations in the APOB gene: c.12581T>C(nucleotide 12581 in the coding region is mutated from thymine to cytosine),resulting in amino acid changes p.I4194T(Amino acid No.4194 is mutated from isoleucine to threonine),which is located on exon 29 and is a missense mutation;c.4538G>A(nucleotide No.4538 in the coding region is mutated from guanine to adenine Purine),resulting in an amino acid change p.R1513Q(amino acid 1513 is changed from arginine to glutamine),which is located on exon 26 and is a missense mutation.The protein prediction software predicts that c.12581T>C may be a pathogenic mutation.After the mutation,a hydrogen bond is added between I4194 and the 4190 th amino acid,which may affect the spatial structure of the protein.3.Design primers for the target gene fragment containing the c.12581T>C mutation site,and amplification and sequencing indicate that the target fragment is successfully extracted.4.The mutant truncated APOB overexpression plasmid containing the c.12581T>C mutation site and the wild-type truncated APOB overexpression plasmid containing the target sequence were constructed.Sequencing indicated that the mutant plasmid was constructed successfully compared with the target gene m RNA sequence..5.Measured by Image J after plasmid transfection,statistics of the three groups of green fluorescence values indicate that the gene expression levels of the three groups of cells after plasmid transfection are equivalent.6.Western blotting results indicate that the input group of wild-type and mutant truncated APOB plasmids can normally express truncated APOB protein and LDLR protein;the molecular weight of the mutant truncated APOB protein in the Ip group and the molecular weight of wild-type truncated APOB protein It was equivalent,the protein binding level was only slightly reduced(P>0.05),and the difference was not statistically significant after testing.Conclusion: 1.Throμgh the collection of clinical history data of an FH family,as well as the blood lipid index and gene sequencing analysis of the proband,two mutations in the coding region of the APOB gene that are highly correlated with the clinical phenotype of FH were found: c.12581T>C And c.4538G>A.Both can cause amino acid changes,and the mutation c.12581T>C may be an influencing factor causing FH.2.The overexpression truncated plasmid was successfully constructed throμgh in vitro cell experiments,and the mutant truncated APOB protein and wild-type truncated APOB protein on the receptor binding domain were simulated by immunoprecipitation technology to bind to LDLR respectively,and the mutation position was verified.Whether the point causes disease by affecting the ability of truncated APOB protein to bind to LDLR.3.From the function of truncated APOB protein,it is verified that the c.12581T>C mutation site does not cause abnormal expression of APOB gene,and has no significant effect on the binding of truncated APOB protein to LDLR.It is preliminarily confirmed that this site does not affect truncation.The secretion and binding function of APOB protein.