| IntroductionDilated cardiomyopathy (DCM) is the most common cardiovascular disease and a third leading cause of congestive heart failure with a performance of enlargement of the left or the right ventricular and systolic dysfunction of myocardial, ultimately died of irreversible heart failure[1]. However, the leading factors remain poorly understood and it is an end result of long-tern effects of various factors, including infected/non-infected myocarditis, alcohol poisoning, metabolic and lots of others [2]. Moreover, the role of gene mutations in the pathogenesis of DCM have been increasingly recognized around the worldwide and approximate30%-50%of affected individuals have a familiar form of DCM[3].Cardiac sodium channel gene (SCN5A) encoding the human voltage-gated sodium channel gene mainly express in human myocardial cells which responsible for the rapid depolarization of myocardial action potential[4]. Previous studies have demonstrated SCN5A mutations can lead directly to abnormal myocardial sodium channels, thereby inducing a variety of arrhythmias[6], including Brugada syndrome, sick sinus syndrome (sick sinus syndrome, SSS), long QT syndrome (long QT syndrome, LQTS)[7-9]. Meanwhile, recent studies have found a number of SCN5A mutation were closely associated with the development of DCM[10,11].We have first reported finding All80V of SCN5A in a family with DCM by sequencing all the exons of SCN5A of all the surviving members. Among them, all the patients were found carrying A1180V mutation, on the contrary, all the noncarriers were found with any abnormalities. The examination of ECG after exercise of the carriers who were without symptoms of DCM and arrhythmia were significant with the noncarriers. The mutation, however, was not found in200healthy Han Chinese controls. In addition, vitro data showed that A1180V channels exhibited remarkable dysfunction of intracellular sodium and calcium homeostasis[12]. These results strongly suggest that A1180V mutation is a functional mutation that can contribute to the development of DCM. However, data deposited from the dbSNP database revealed that the frequency of A1180V among Asian was up to6%. Accordingly, we plan to further follow up the family members and expand the control size to screen for A1180V with the purpose of clarifying the pathogenic role of A1180V mutation in familial DCM (Part I).Based on the former pedigree discovers, we further extended our study to the sporadic DCM patients to screen the SCN5A mutations. We have found5new mutations:R225Q, A226V, I1448N,194and Y1434(the latter two are synonymous mutations), in which, R225Q were appeared in3patients. All of the new mutations were not detected in199healthy control cases. In all, the carrier rate of SCN5A mutations among the90patients was about7.8%(7/90). Further clinical studies have found that QTc time of the patients carrying the R225Q or A226V mutation was much longer than the noncarriers DCM patients, which was similar with A1180V carriers. In addition, the R225Q transfected cells also showed a visible result of electrophysiological abnormalities in vitro. Therefore, we intends to build R225Q mutant heterogeneous mouse model by genetic engineering techniques and to observe the changes in cardiac structure and function in the living period to explore the role and mechanisms of R225Q (Part II). Part I:The Pathogenic Role of A1180V Mutation of Cardiac Sodium Channel Gene in Familial Dilated CardiomyopathyObjective:To make sure the pathogenic role of A1180V, a mutation of cardiac sodium channel gene (SCN5A) in a family displaying dilated cardiomyopathy (DCM) and explore the clinical characteristics and progression of the disease resulting from A1180V mutation in the affected family by several years of follow-up and analysis of the carrier frequency of Al180V healthy Han Chinese.Methods:Based on the former data, seven All80V carriers and eight A1180V noncarriers of the affected family were followed up again by inquiring medical history, physical examination,12-lead electrocardiogram (ECG) examination and echocardiography. Clinical data of the ten A1180V carriers (including three dead patients), eight noncarriers and a dead man with unknown genotype were analyzed retrospectively. Next,460normal individuals were recruited voluntarily in Zhongshan Hospital Fudan University and were rolled in the control group. DNA were extracted from the venous blood and the sequence of exon20were examined to analyze whether the one has carried A1180V mutation. Besides, we searched and analyzed the A1180V carrier frequency among Chinese (or Asian) in dbSNP and1000Genomes database as well. The statistical analysis were performed with SPSS16.0and SAS statistical software.Results:During this follow-up period, three carriers were first diagnosed with first-degree atrioventricular block (AVB) or type I second-degree AVB; one carrier’s condition have progressed to cardiac dilation and third-degree AVB. Considering all of the clinical characteristics of all the A1180V carriers, we found that the first-degree AVB were mostly the primary performance among them with a mean diagnosed age of34±3.0. And their disease would progress to DCM with preceding AVB in about5years. Moreover, anyone were found carrying the A1180V mutation among the460healthy Han Chinese (0%,0/460). Data retrieved from the dbSNP represented the total A1180V frequency of Asian in two studies was6.3%(4/63), which was significantly different with us (p=5.3×10-5). In addition, the one was about0.51%(1/197) from the1000Genomics, which was also significantly different with the dbSNP(p=0.013).Conclusion:Our finding suggests that A1180V is a potential risk factor for DCM and it is extremely rare among Healthy Han Chinese. Regular follow-up of the family may provide more opportunity to detect early abnormalities of A1180V carriers. Part II:Cardiac Function Analysis of the Heterozygous Mice Carried R225Q Mutation of Cardiac Sodium Channel Gene AbstractObjective:By constructing the conditional knockout of R225Q mutant heterozygous mice model, we proposed to observe the cardiac structure and function changes of those mice under natural growth or stress intervention condition, and further verify the possible mechanisms.Methods:We have built heterozygote mice model carried conditional knockout of SCN5A gene R225Q mutation by genetic engineering techniques. To identify genotype in mice by building the corresponding primers,we group the mice after them being successfully identified. The ventricular cavity diameter and cardiac function in transgenic and wide type mice of different ages were monitored by small animal cardiac ultrasound, including left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular diastolic anterior wall (LVAW;d), left ventricular diastolic posterior wall (LVPW;d), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS). All the mice were killed when they were60weeks old and the heart were removed and made to paraffin sections, and finally analyzed the pathological form features by hematoxylin-esosin (HE) staining.Results:After the identification of qualified heterozygous mice,we can find the differences between the different groups,compared with the wide type (WT) mice, the mutant mice were of poor station. However, the weight of them were obviously higher than the WT mice (29.67±4.70vs27.36±1.81, p=0.018). At the same time, echocardiographic results indicated that cardiac function of the mutant mice were more than that of WT mice.(EF:69.62±2.25vs72.38±9.72,p=0.035; FS:38.70±1.71vs41.12±8.17,p=0.029) with no significant difference in LVEDD, LVESD, LVPW;d and LVAW;d. In view of the long experimental period and the mismatched age between the two groups, we found that the cardiac function of60weeks was significantly decreased than that of40weeks by the echocardiographic results of mutant mice (EF:62.79±7.50vs69.62±2.25,p=0.000; FS:33.70±5.63vs38.70±1.71, p=0.000; LVAW;d:0.63±0.03vs0.69±0.02,p=0.002), no obvious variation was found in LVEDD, LVESD, LVPW;d. In addition, HE staining did not revealed remarkable abnormalities and differences between the two groups.Conclusion:In absence of any intervention, our results indicated that the heterozygous mice carried conditional knockout R225Q mutation may presented worse heart function than the WT mice without the phenomenon of dilated cardiomyopathy. The result showed that the R225Q mutations might lead to spontaneous decline of cardiac function. But because of the long experimental period and the less mice number of control group, the experiment results and the related mechanism still needs further validation. |
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