Functional Analysis Of The Mutation About The Sodium Channel Gene SCN1A 3' Untranslated Region (c.*20A>G) Related To The Dravet Syndrome

Background and purposeDravet syndrome is,one of the most intractable epileptic encephalopathy.It begins in the first year of life in an otherwise healthy infant.The patients usually suffer from prolonged febrile and non-febrile seizures within the first year,and it progresses to other seizure types,including myoclonic,atypical absence,and complex focal seizure.The disease normally accompanied with cognitive impairment,motor deficit and behavior disorder.Current standard antiepileptic medication to Dravet syndrome is limited,and prognosis is poor.Previous studies indicate that 70-80% of the Dravet syndrome patients are caused by mutations in the SCN1 A gene.In mouse and i PSCs?Induced pluripotent stem cells,i PSc?models of DS,the SCN1 A gene mutations have been observed to result in a impairment in sodium currents.However,there are still around 15% of the patients remain unknown etiology.Researches indicate that,the 3'UTR plays an important role in the translation,localization and stability of the m RNA.The 3'UTR produces an effect on gene expression on the posttranscriptional level,by affecting the binding ability of RNA binding proteins,microRNAs and non-coding RNAs.The aberrant regulation of 3'UTR will result in abnormal level of expressed protein and lead to disease.Our research group previous study on DS patients had found a novo variant?NM₀06920c.*20 A>G?in SCN1 A 3'UTR.The purpose of this study is to analyze the function of the novo variant in SCN1 A 3'UTR,and investigate mechanism of SCN1 A in DS.Methods Functional analysis of the 3'UTR mutation in SCN1 A gene.1.Bioinformatics analysis: using the Vector NTI to detect the conservation of the mutation site,and then using the RNA-structure 5.3 to predict the RNA second structure of the mutation in SCN1 A 3'UTR.2.To construct the plasmid of the Dual-Luciferase Reporter System,then transfect into NT2 cells,and detect the Luciferase activity.3.Using the RNA Electrophoretic Mobility Shift Assay?RNA-EMSA?to analyze the effects of the mutation on the sequence of UTR binding ability with the cytoplasmic proteins.4.Using q-PCR to detect the effects of the mutation on the m RNA stability of the reporter gene.5.RNA-Protein Pull-Down and RNA Electrophoretic Mobility Shift Assay were carried out to separate the cytoplasm proteins of the NT2 cells and the RNA probe.Then the LCMS/MS was used to identify the specific RNA binding protein.Finally confirm by the analyses of RNA-EMSA.GAPDH lentivirus interference experiments 1.To construct the lentivirus for the interference of RNA binding protein.2.To transfect the SH-SY5 Y cells with lentivirus,and then the stable cell lines were selected.3.Using Western Blot to detect the expression of the Nav1.1 and GAPDH protein in the SH-SY5 Y cells.Results 1.The analysis of conservation among the analyzed species showed that the site is highly conservative.And the RNA secondary structure prediction found that the mutation changed RNA secondary structure significantly.2.To construct the plasmid of the Dual-Luciferase Reporter System,psi CHECK2-SCN1 A 3u?20A?of wild type and psi CHECK2-SCN1 A 3u?20G?of mutation type,then they were transfect into NT2 cells.The results of the relative Luciferase activity showed that,compared with wild type,the activity of mutation type was reduced 30%?t=8.5,P < 0.01?.This indicated that the mutation negatively regulated the expression of reporter gene.The q-PCR results of the renal luciferase expression showed that,compared with wild type,the half-life of mutation type of reporter gene m RNA were shorten,which means that the stability report gene were decreased because of the mutation.3.The RNA probes of wild type?20A?and mutation type?20G?labeled by Biotin were Synthesize.Then RNA-EMSA with cytoplasm proteins of the NT2 cells were carried out.The results indicated that,compared with wild type of RNA probes,specificity cytoplasm proteins of the NT2 cells were combined with the mutation type.The results imply that,the binding ability of mutation type RNA probes with cytoplasm proteins were increased.4.A protein band was obtained from RNA-Protein Pull-Down and SDS-PAGE analyses.According to the Nano LC/MS/MS analyses,the protein band may be glyceraldehyde 3-phosphate dehydrogenase?GAPDH?.RNA-EMSA experiment were carried out by the purificatory GAPDH,and a specific block belt was found form mutation probes,which confirm the binding protein is GAPDH.5.To construct the stable SH-SY5 Y cell lines infected by the lentivirus of GAPDH interference,than detect the expression of the Nav1.1 and GAPDH protein.The results of Western Blot showed that,compared with the blank group and the control group,the protein of GAPDH in the positive group were decreased significantly?P < 0.01,n=3?,but Nav1.1 protein in the positive group were increased significantly?P < 0.01,n=3?.Conclusion 1.The novo variant?c.*20A>G?in SCN1 A 3'UTR in DS is a functional mutation.The mutation enhances the binding ability of RNA protein GAPDH with the 3'UTR,thus decrease the stability of the report gene m RNA,which negatively regulate the expression of SCN1 A gene in the post-transcriptional level.2.The pathogenic of the novo variant may be by enhancing the binding ability of RNA protein GAPDH with the 3'UTR of the SCN1 A,thus decrease the stability of the Nav1.1 m RNA,and reduce the expression of Nav1.1,which lead to haploinsufficiency.And this need to be confirm by the in vivo experiments.