Functional Expression Of A Novel SCN5A Mutation G1712C Identified In Brugada Syndrome

Background:Brugada syndrome(BrS)is a rare heritable arrhythmia syndrome characterized by an electrocardiographic(ECG)pattern consisting of coved-type ST-segment elevation in the right precordial leads V1 through V3(often referred to as a type-1 Brugada ECG pattern)with a high risk for sudden cardiac death(SCD)in young and otherwise healthy adults,Often accompanied by varying degrees of heart block.Its typical electrocardiogram shows that the right chest leads’(V1 and V3)ST segments elevation 2 mm or higher,secondary T wave inversion,and QT interphase is normal.When attacking,electrical diagrams for a very short phase matching between contracts before sexual period,polymorphic ventricular tachycardia and ventricular fibrillation,and physical examination and auxiliary examination(chest radiograph,echocardiography,cardiac magnetic resonance imaging(MRI),etc.)are no organic disease.The prevalence of the disease is difficult to estimate because the pattern is not always recognized or because it may transiently normalize.Nevertheless,it is considered endemic in Southeast Asian countries,where the prevalence is higher,with prevalence varying from 5 to 10 in every 10,000.In some parts of Asia,Brugada syndrome is of the most common cause for the deaths of young men under the age of 50 years old.Clinical cardiology study is paying more and more attention to use the method of molecular genetics to explain some normal cardiac structure patients’ mechanism of sudden cardiac death.The past decade,researches show that the gene SCN5A coding of human cardiac sodium channel alpha subunit is closely related to the pathogenesis of Brugada syndrome.The reason may be that SCN5A gene mutations cause the dysfunction of cardiac sodium channel protein.Objective:To further elucidate the molecular and electrophysiological mechanism of Brugada syndrome through a primary in-vitro assay and analysis of the functional expression of the novel SCN5A gene mutation,G1712C.Methods:The first,through the in vitro PCR-based site-directed mutagenesis technique,a recombinant plasmid vector pcDNA3.1 containing the mutant human cardiac sodium channel a subunit cDNA was constructed.The purified PCR product and plasmid pcDNA3.1 were digested with HindⅢ and XbaⅠ respectively,then ligated into a full length pcDNA3.1.DNA sequencing to identify site-directed mutagenesis was successful.The second,Lipofectamine 3000 was used to transfect HEK293 cell line with plasmid DNA establishing stable expression of Na+ channel β1-subunit.Then,the standard liposome method was used to transiently transfect HEK293 cells with either the wild type or mutant Na+ channel subunits pcDNA3.1 and pcDNA3.1-SCN5A respectively.The transfection cells were incubated in the 37℃ incubator for 48 hours.Then macroscopic Na+ currents were recorded by using whole cell patch-clamp technique mode.Therebefore,A recombinant plasmid vector pcDNA3.1 containing the mutant SCN5A green fluorescent protein,GFP was constructed.Lipofectamine 3000 was used to transfect HEK293 cell line logarithmic phase with pcDNA3.1 green fluorescent protein and pcDNA3.1-SCN5A green fluorescent protein,respectly.After transfecting 36 hours observed the fluorescence transfection efficiency with microscope.Then observed the distribution of green fluorescent protein with the laser confocal microscope(leica TCS SP8).Results:We successfully constructed carry mutations G1712C pcDNA3.1(mpcDNA3.1)expressing recombinant.Observed under laser confocal microscope,GFP-SCN5A mutant(G1712C)mainly distributed in the cytoplasm of peripheral(membrane),mutations successful positioning on the cell membrane.In the transient transfection of wild-type pcDNA3.1 cell lines,when instruction potential from 60 mV gradually rises,sodium current was larger gradually,also fully activated when instruction potential was-20 mV.The activating voltage was in 60 mV to-50 mV,and inversion of potential around 50 mV.However,after transient transfection the G1712C subunit,a Na+ current was not recorded.Conclusion:Compared with normal Na+ channel,the wild-type channel exhibited a similar sodium current.The characteristic kinetics of sodium channel of WT was the same as normal cardiac muscle cell.G1712C mutation of SCN5A gene can lead to Nav1.5 channel lose function,may be the cause of the family’ s Brugada syndrome.