Identification And Primary Functional Study Of PCSK9 Gene And Its Mutants In Familial Hypercholesterolemia

BackgroundFamilial Hypercholesterolemia (FH) is a dominant hereditary metabolic disease and is caused by mutations in multiple genes, which are related to lipid metabolism. Genetic analytical research on pedigrees confirmed that dysfunctions in low-density lipoprotein receptor (LDL-R) due to genetic mutations in LDL-R and proprotein convertase subtilisin kexin 9 (PCSK9) could all lead to the same clinical phenotype: early onset of atherosclerosis by great elevation in level of homozygous serum LDL-R, where it could develop into severe coronary disease and even death at childhood. Infants with heterozygous presentation, at birth, would immediately show signs of dysfunction in LDL-R followed by a situation in high level of cholesterol and progress of arteriosclerosis was significantly accelerated. In recent years of genetic research into PCSK9, scholars gained more insights into metabolic mechanism of LDL-cholesterol. The latest research result discovered that PCSK9 gene had important regulatory function on metabolism of LDL-R, where different point mutations of the PCSK9 gene could lead to two completely reciprocal phenomena: acquired functional mutation in PCSK9 was related to hypercholesterolemia and the lost of function in PCSK9 due to deletion mutation was related to hypocholesterolemia. This research project intended to based on a case of familial hypercholesterolemia, screen the pathogenic mutation and conducted an in vitro research on the functions of mutator, in order to investigate the underlying mechanism, where it could provide new insights for clinical diagnosis and lipid metabolism in case of hypercholesterolemia. Part 1 Clinical and gene analysis on patient with familial hypercholesterolemiaObjectiveCollected a clinically diagnosed case from Han's ethnic pedigree with familial hypercholesterolemia (FH) and measured the pathogenic mutations in genes in relation to LDL-R metabolism.MethodsWith a pedigree with FH as the research subject, details of the patient's diet, habit, and familial history were inquired and a complete examination on cardiovascular system was performed. DNA of peripheral white blood cells were extracted and by measurement for nucleotide sequences of genes LDL-R, apoB, and PCSK9 from a patient of Hans ethnicity, mutations were detected. Prediction and simulation on the secondary and tertiary structures of mutator protein.Results1. The clinically diagnosed patient with FH, by physical examination, revealed severe atherosclerotic changes in cardiovascular system, where there were signs of ischemic injury to the heart. 2. There was no mutational change in LDL-R and apo B100 and nucleotide sequential measurement discovered a change in G and T at the 918th position of nucleotides of PCSK9 gene in the patient and his father, which caused replacement of Arginine by Serine at the 306th position of the 6th exon. 3. By simulation on the secondary and tertiary structures of mutator protein, changes such asα-helix,β-angling, and unknown sequential alteration in encoded protein after R306S mutation in PCSK9 gene led to further conformational change.Conclusion1. This investigation discovered a missense mutation R306S in PCSK9 gene of the FH pedigree of Han's ethnicity. 2. There are changes on the secondary and tertiary structures of R306S in PCSK9 gene by prediction and simulation.Part 2 Expression and primary functional study on new mutant of PCSK9 GeneObjectiveIn vitro genetic recombination technique was employed to construct the eukaryotic expression carrier for mutational genetic type of the patient with FH, in order to observe its expression in hepatocytes and its influence on expression of LDL-R, as well as relationships of structure & function and genotype & phenotype. Initial investigation of the effect on LDL metabolism and molecular mechanism for inducing FH due to mutation would also be performed, where it could provide theoretical and laboratorial evidences for prevention & treatment of cardiovascular diseases and expansion of knowledge on LDL metabolism.MethodsThe cDNA of wild type gene PCSK9 of whole length (WT-PCSK9) was acquired from BEL-7402 cell strain of hepatic carcinoma. Recombinant eukaryotic expression plasmid with genetically pathogenic PCSK9 was constructed by using fixed mutation before it was verified of sequence and inserted as a fragment. With an empty carrier as control, recombinant plasmid, by lipofection, was transfected to BEL-7402 cells. RT-PCR was performed to test expression of LDL-R mRNA and Western blot was performed to measure protein expressions of PCSK9 and LDL-R. Hertz experiment would observe the influence on LDL metabolism. Immunofluorescent test was performed to observe localization of PCSK9 gene and LDL-R in cells. Flow cytometry was used to test the changes in binding capability of fluorescently marked LDL in transfected cells of LDL-R. Stable transfected hepatocytic cell strain was then used to establish a Tet on system, where the influence of PCSK9 gene mutation on expression of LDL-R was quantitatively observed.Results1. Nucleotide sequential measurement would verify the size and the correct order of the insertion fragment in constructed expression carrier. 2. The level of LDL-R mRNA in each transfected mutational BEL-7402 cells showed no significant difference from the wildtype (p > 0.05). 3. Western blot yielded that there was no significant variation between proprotein and mature protein of PCSK9 in each transfected mutational BEL-7402 cells (p > 0.05). In comparison to the control group, after transfecting with wildtype PCSK9 plasmid, the expression of mature protein of LDL-R was lowered. After transfection of positive control plasmid, the band of mature LDL-R disappeared. Transfection with R306S mutant, the level of mature LDL-R was even more significantly decreased (p < 0.05). 4. Lipid stress test results showed a decrease in lipid level in hepatocytes transfected with wildtype PCSK9. The reduction level in hepatocytes of the R306S group was even more significantly higher than that of the control group (p < 0.01), but lower than the wildtype PCSK9 transfected BEL cells (p < 0.05). 5. Immunofluorescence tested co-localization of PCSK9 and LDL-R in hepatocytes, which were primarily located on cell membrane and intracellularly before and after transfection. 6. Flow cytometry was used to test the change in binding activity of fluorescently marked LDL in transfected cells with LDL-R. In comparison with blank control, the fluorescent intensity in wildtype PCSK9 transfected group decreased significantly (p < 0.05), and a even more remarkable decrease in fluorescent intensity was found in the positive control F216R group (p < 0.01). The decrease in average fluorescent intensity in the group with the mutatnt tranfection was also more significant than in the wildtype group. 7. The mutator system of Tet on was highly effective and stably expressed in BEL-7402 cells. In the PCSK9 wildtype and the mutator Tet on/off system, following the increased concentration of Doxycycline, the expression of wildtype and mutant PCSK9 were gradually increased, while the level of mature protein of LDL-R was gradually decreased. Unglycosylated LDL-R was not significantly changed. In comparison to the wildtype, the capability of R306S to reduce mature LDL-R was greatly enhanced (p < 0.05).Conclusion1. The eukaryotic expression carriers with the wildtype and mutant PCSK9 gene were successfully constructed .2. In vitro experimentation discovered that there was no significant influence on transcription of LDL-R by R306S mutation.3. PCSK9 R306S mutant could significantly lower the level of mature protein of LDL-R4. PCSK9 R306S mutant could decrease the absorption of LDL, consequently lead to hypercholesterolemia, which indicates that the mutated PCSK9 gene could be the pathogenic gene for FH.