Identification And Mechanism Investigation Of Pathogenic Gene Mutation In VHL Syndrome

Von Hippel-Lindau syndrome is a rare autosomal dominant genetic disease,and the main cause of the disease is the mutation of the VHL gene.VHL syndrome manifests in multiple locations,such as tumors in kidney cells,chromaffin cells,and angioblast.We found a case of a proband with VHL syndrome from Anhui,China,and collected whole blood samples from 33 other members of the proband’s family.According to all the medical records,clinical manifestations and Sanger sequencing method verification provided by the doctor,it was found that 5patients in this family contained a missense mutation of the VHL gene（c.239C>T,p.S80I）,and the clinical data showed they all have tumors.Although the mutation site has been reported by researchers before,the pathogenic function of the mutation site has not been studied.Therefore,a cell model was established to study the pathogenic function of the mutation point.1.The Sanger sequencing method was used to perform first-generation sequencing to verify the three exons of the VHL gene in the proband and other members of the family.The results showed that the patient had a missense mutation VHL c.239C>T in the VHL gene exon1,and normal people did not have this mutation site.2.The qRT-PCR technology was used to analyze the m RNA expression levels extracted from the blood of 2 normal people and 2 patients.The results showed that the m RNA expression level of the patients was significantly lower than the normal expression level,and the site may be pathogenic Sex.3.Construct eukaryotic expression vectors of VHL^WT and VHL^MT,carry out cell transfection,perform cell transfection to explore the location of mutations in cells and their effects on cell phenotypes.Using immunofluorescence technology to observe whether the VHL gene mutation will affect the location in the cell,the results show that VHL^MT will not affect it.The proliferation,senescence and migration of VHL^WT and VHL^MT cells were detected by CCK8experiment,β-galactosidase experiment and cell scratch experiment,the results showed that when VHL^MT was transfected into cells,the proliferation ability of cells was strengthened,cell senescence was weakened,and the cell senescence was weakened.The rate of migration has accelerated.4.Construct stable overexpression vectors of VHL^WT and VHL^MT,and use Westeron blot,ELISA and q PCR experiments to detect the expression and concentration of E3 ubiquitinase,the protein level of the downstream hypoxia inducible factor（HIF-1α）of p VHL and the level of vascular endothelial growth factor VEGF.The expression level of m RNA,the results show that VHL^MT will cause the expression and concentration of E3 ubiquitin ligase to decrease,affect the degradation of downstream substrate HIF-1α,cause HIF-1αaccumulation and transfer to the nucleus,and then promote vascular VEGF generate.5.Use the pyruvate content and pyruvate kinase to determine the biochemical kit,Westeron blot and q PCR experiments to detect the expression of enzymes and products in the glycolysis process.The results show that VHL^MTcompared with VHL^WT,it is found that VHL^MTcan cause the increase of pyruvate content and the expression of pyruvate kinase.It is further explained that the mutation of the VHL gene will promote the occurrence of glycolysis to regulate the consumption of oxygen.In summary,this article is based on a relatively comprehensive clinical data and blood samples,using Sanger sequencing method to verify all three exons of the VHL gene,and find the missense mutations carried by patients in this family Site.Through biochemical experiments and cell experiments,the pathogenic function research of the family and the mutation site was initially revealed.It provides an important reference for the treatment and clinical diagnosis of the mutation site in the later period.