Clinical Features And NMD Mechanisms Of Truncation Mutants In SCN1A Gene

PurposeChoosing different truncation mutations in SCN1A gene of patients with Dravetsyndrome，we analysed the clinical characteristics of patients and constructedminigenes to study the mechanism of nonsense-mediated mRNA decay（NMD）. Wewant to investigate whether the different truncation mutations in SCN1A gene shouldmotivate NMD，moreover to analyse the relationship between the NMD andphenotype.MethodsWe collected clinical features and mutation features of truncation mutations inSCN1A gene of patients with Dravet syndrome and constructed minigenes whichcoexpress green fluorescent protein（GFP） and truncation mutations. We appraisedwhether resistance gene （Kana gene） express in different types of cells.The minigenewere transfected into Hela cell,SH-SY5Y cell and NGF induced PC12cellrespectively. The mRNA expression quantity of SCN1A in three cells was estimatedby q-PCR. Experiments in each group were repeated3times to get the mean value.We used the method of2‐△△Ct to analyse relative quantitative data.Data were shown as mean±standard deviation （X±S）. SPSS l7.0software package was used for the statistical methods. Statistical comparisons were done with the multi-sample ANOVAanalyse， P <0.05was statistically significant difference.Results1.Analysis of Clinical features.The patients who carry c.4526delA， c.4547C>A， c.5280₅281delTG，c.5621₅622delGG were diagnosed as SMEI while the patient who carriesc.5540-5541insCCAG was diagnosed as SMEB.And the patients who carryc.5540-5541insCCAG, c.5621₅622delGG were also diagnosed as Autism. AutismBehavior Checklist of the patient who carry c.5540-5541insCCAG andc.5621₅622delGG is78scores and110scores, and childhood autism rating scaleof them is36scores and41scores.2.Constructing and identificating PEGFP-C2-SCN1A minigene plasmidGene segment coexpressing GFP and exons24-26（including intron25）ofSCN1A gene was successfully ligated to the PEGFP-C2vector. PEGFP-C2-SCN1Aminigene plasmid was sequenced and no mutation was found in its coding region.And we also Construct the mutants of PEGFP-C2-SCN1A minigene plasmid.3.Identificating the endogenous expressing of kana gene in four different types ofcell.There are no endogenous expressing of Kana gene in NGF induced PC12cell,Hela cell,SH-SY5Y cell,while there are endogenous expressing of Kana gene inHEK293T cell.4. Real-time quantitative PCR （qPCR）The mRNA expression quantity of SCN1A in three cells was estimated byq-PCR. There are no significant difference between the mutants and wild type inHela cell and SH-SY5Y cell. There are significant difference between the mutants（c.4547C>A，c.5280₅281delTG，c.5540-5541insCCAG，c.5621₅622delGG）and wild type in NGF induced PC12cell.Conclusions1. The clinical features of the patients who carry different truncation mutations in SCN1A gene are not all the same.2. NMD induced by different truncation mutations in SCN1A gene varies indifferent types of cell.3. RNA degradation is different amount truncation mutants in NGF induced PC12cell.We infer that there are more complicated mechanisms which influence thegene expression.