Mutation Screening Of SCN5A Gene With Brugada Syndrome And Discussion Of Its Pathogenic Mechanism

BackgroundBrugada syndrome (BrS) have been researched more and more thoroughly since it was reported first by Brugada brothers in1992, Brs is characterized by right precordial ST elevation, frequently associated with conduction delays at different cardiac levels, potentially lethal arrhythmias, and a positive family history for sudden premature death, it typically expresses in males in their third to fourth decade of life and is responsible for a significant subset of sudden death in young individuals BrS typically presents during adulthood. Although most patients are in their30’s to40’s, BrS has been reported in patients as young as2years of age and as old as84years. The reason for this male preponderance is possibly related to underlying differences in hormonal status. It is estimated that BrS is responsible for at least4-12%of all sudden deaths and at least20%of all sudden deaths occurring in patients without structural heart disease. The prevalence of the disease manifesting with clinical symptoms is estimated to be1in5,000to10,000in Western countries and may be more prevalent in Southeast Asia and Japan,0.12-0.14%.Of note,BrS is the second lethal reason for the young in Southeast Asia,while traffic accident is the first. Recently,Cardiologists in the world have spent more time and money in the research of sudden death patients who were "healthy" before,and BrS is a secret no longer.It is reported that BrS is a inherted Arrhythmic Disorders like LQTS which is also a Channelopathy because of gene mutation.Its an autosomal dominant inheritan-ce,there are many factors (fenver, alcohol,gene SNP et al.) effecting its phenotype wi-th incomplete penetrance.There are more than100mutations in SCN5A gene associ-ated with BrS since Chen discovered the first mutation in this gene in1998. Some of them is confirmed by expression in exvivo cells.SCN5A is on the chromosome3in human, encoding a protein of2016aminoacids with a calculated molecular weight of227kDa. The protein each consist of four homologous domains (DI-DIV),and each domain contains six transmembrane subunits (S1-S6).There is a "P-Loop" connecting each domain intra-cellular area, and the fore "P-Loop"contribute a pore through which sodium get into the cell.Channel defect resulted from gene mutation makes Ito potentiation,which results a deep notch in the phase1of action potential(AP),and In general, the epicardium has the shortest AP duration and the highest concentration of transient outward (Ito) current. The M cells(middle myocardium cells) have the longest AP duration and the lowest concentration of Ito current.The endocardium, on the other hand, has intermediate AP duration and Ito concentration. A decrease in the AP duration is observed when the ICa currents are overwhelmed by Ito currents, which leads to a significant shortening of phase2of the AP. This phenomenon occurs in some myocytes in the epicardial tissue; the others maintain normal AP durations. This difference in electrical properties, observed in myocytes within the same epicardial tissue, creates a heterogenous population of cells with different AP durations and, subsequently, different refractory periods,and this is the reason why the ECG of BrS patients showing "sellaeform"like ST-elevation or "cove"like.Cells with shorter refractory periods have the potential to be re-excited by cells in the surrounding tissue that have normal AP duration. This phenomenon, referred to as phase-2re-entry, is believed to be the cause of the ventricular arrhythmias in patients who have Brugada syndrome.Nowaday,the mechanism of BrS is more and more clear because of cardologists’ endeavour,other gene associated with Brugada syndrome was found,such as GPD1-L、SCN1b、SCN3b、CACNA1C、CACNBp2b、KCNE3and KCNH2.2011HRS/EHRA Expert Consensus Statement on the State of Genetic Testing for the Channelopathies and Cardiomyopathies showed that:at least eight genes may be causally involved. SCN5A, the gene encoding the cardiac sodium channel,accounts for the vast majority (>75%) of BrS genotype positive cases (Table2). However, the yield of SCN5A genetic testing for robust clinical cases of BrS is approximately25%.92Thus, the majority(>65%) of BrS cases remain genetically elusive.So it is recommended that Comprehensive or BrS1(SCN5A) targeted BrS genetic testing can be useful for any patient in whom a cardiologist has established a clinical index of suspicion for BrS based on examination of the patient’s clinical history, family history, and expressed electrocardiographic (resting12-lead ECGs and/or provocative drug challenge testing) phenotype.It may help us to find some new related gene mutation. In September of2006,The National Heart, Lung, and Blood Institute (NHLBI) and the Office of Rare Disease held a symposium meeting jointly about Proposing to strengthen the study of hereditary ion channel diseases for promoting scientific research in order to improve the diagnosis and treatment of inherited arrhythmia diseases. At present, the study of the genetics of hereditary arrhythmia has become a hot research field today, it can not only reveal the nature of the arrhythmias behind, but also provide strong evidence of the guidance for making clinical decision. Objective We choose3patients with BrS definitly, who visited our hospital because of unexplained syncope, as research objects, whose whole blood was collected for extracting genomic DNA, and SCN5A gene is the candidate gene. All the28exons of SCN5A were amplified by polymerase chain reaction (PCR) and the PCR products were then sequenced directly. We hope to perform mutation screening of SCN5A gene in chinese patients with BrS, which may clarify the cause of the disease further and investigate the pathogenesis to enrich our understanding of the molecular genetics of Brugada syndrome in the Chinese population according to review related literatures. Subjects3male subjects with Brugada syndrome diagnosed definitly in department of cardiology of Nanfang Hospital from January2010to January2011,whose age ranges from32-70years old, received some examinations such as blood biochemical, myocardial enzymes, troponin, chest X-ray, EEG, CT and echocardiography, and coronary angiography was performed in patients over the age of45. all patients had syncope but not the obvious structure cardiovascular and cerebrovascular disease, their ECG showed type I Brugada ECG (at least one of V1-3leads showing complete right bundle branch block and ST-segment elevation like a cove, accompanied by T wave inversion), their family history were negative, and ventricular fibrillation was induced when electrophysiological study was performed to them. Diagnostic criteria for Brugada syndrome based on the2005ESC expert consensus:if the secondary factors (drugs, hypothermia, acute anterior myocardial infarction and so on) was excluded,a definitive diagnosis of BrS can be made when a type1ST-segment elevation pattern is observed in>1right precordial lead (V1to V3) along with one of the following:1) documented polymorphic VT or VF;2) a family history of sudden cardiac death at<45years of age;3) similar type ECG in family members;4) inducibility of VT/VF during an electrophysiology study;5) unexplained syncope or history of nocturnal agonal respiration.103of healthy cases were selected as a control study group, the criteria were:(1) no history of syncope, seizures and ventricular arrhythmias, and a family history of sudden death;(2) exclusion of atrial fibrillation (AF), dilated cardiomyopathy (DCM), long QT syndrome (LQTS), sick sinus syndrome (SSS), atrioventricular block (AVB) and left ventricular Non-Compaction (LVNC) has been confirmed with the SCN5A gene abnormalities related diseases. The above study were informed consent.MethodsGenomic DNA from all subjects was extracted from peripheral blood leukocytes. All the28exons of SCN5A were amplified by polymerase chain reaction (PCR) and the PCR products were then sequenced directly. The PCR reaction system are as follows:the Platinum(?) Taq reaction system25uL including1uL template DNA and24uL of Supermix as follows:2.5uL10×PCR buffer,2uL2.5mM of dNTP mixture,0.75uL50mM MgC12,0.2uL Platinum(?)Taq DNA polymerase and5uM upstream and downstream primers1uL respectivly,the rest of the volume was made up by adding sterilized distilled water. The amplification conditions of the reaction system as follows:predenaturation at94℃for5min, then denaturation at94℃for30s, annealing at58℃for45s, extension at72℃for90s, there were35cycles from denaturation to extension, extension at72℃again for5min after the cycles, and keep the products at4℃forever. The products were identified using a1.5%agarose electrophoresis. The products were sequenced in Invitrogen Biotechnology Co. by3730XL type DNA sequencing instrument.And if some abnormal gene base which could make amino acid sequence change was detected by comparing in NCBI database, the same exon was detected in103nomal control people. ResultsExcept some reported synonymous SNPs, a nucleotide substitution changing from adenine (A) to guanine (G) was found in the20th exon in one of the three patients, resulting in the1192amino acid codon of sodium channel a subunit protein became CGG for arginine [Arg (R)] instead of CAG for glutamine [Gin (Q)]. The same exon of103control cases was screened,and we found that one of them carrying the same variation (n=1/103cases,0.0097), and the variant have not been announced in the NCBI SNP database of SCN5A,but it had been reported that the same variat was found in Japanese patients with Brugada syndrome in2002.Conclusion1、The abnormality due to a G�'A substitution (G3769A) in exon20of SCN5A gene encoding the cardiac sodium channel protein a subunit also exists in Chinese patients with Brugada syndrome,and it results in a substitution of arginine by glutamine at codon1192(R1192Q);2、There are other genes associated with Brugada syndrome except SCN5A.