A Clinical and Molecular Analysis of the Brugada Syndrome

描述（由申请人提供）：心律失常仍然是发病率和死亡率的主要原因。Brugada综合征是一种罕见的、常染色体显性遗传、男性为主的特发性室颤，其特征是右束分支传导阻滞模式和体表心电图右胸导联ST段抬高。唯一有效的治疗方法是植入式心律转复除颤器。心脏Na+通道SCN 5A的突变通过减少内向Na+电流导致约20%的Brugada综合征病例，Na+通道阻滞剂如普鲁卡因胺加重EKG结果。大多数Brugada综合征患者的遗传基础尚不清楚。在该项目的初期，我们发现了一个Brugada综合征的大家族，其特征是进行性传导疾病，年龄和性别依赖性的心律失常，对Na+通道阻滞剂普鲁卡因胺的反应最小。连锁被鉴定为染色体3 p24上的~ IcM区域（最大LOD得分> 4.0），并且排除SCN 5A（LOD得分< -2）。对该区域中的候选物进行直接测序，鉴定了甘油-3-磷酸脱氢酶1样基因（GPD 1-L，KIAA 0089）外显子6中保守氨基酸（A280 V）的丙氨酸至缬氨酸取代。该突变存在于所有受影响的个体中，并且在>200个混合种族背景的未受影响的对照中不存在。北方和Western印迹分析证实了在心脏中的表达。表达SCN 5A的稳定转染的HEK细胞系的全细胞膜片钳研究显示，与野生型GPD 1-L相比，用A280 V突变体转染的细胞中的峰值Na电流降低约60%（p=0.01）。共聚焦显微镜显示野生型而非突变型GPD 1-L蛋白在质膜上的表达，沿着表达A280 V突变体的细胞膜中的SCN 5A减少。在表达A280 V GPD 1-L的细胞中，NADH增加，并且在过表达A280 V GPD 1-L的转基因小鼠中，表面EKG上的QRS持续时间延长。在这种竞争性的更新，我们将测试的假设，GPD 1-L是一种新的离子通道调节剂，GPD 1-L的突变通过改变细胞内NAD+/NADH水平降低Na+电流。我们将1）使用细胞系和新生大鼠心室肌细胞来确定突变型GPD 1-L在体外改变离子通道运输和电流的机制; 2）确定GPD 1-L影响离子通道运输的下游机制; 3）研究转基因和基因靶向小鼠模型以确认其生理作用。美国每年有超过25万人突然死亡。大多数猝死发生在心脏病发作期间或因先前损伤而心脏虚弱的患者中，但我们没有好的方法来预测谁是死亡风险最高的人，也没有药物治疗来预防猝死。在这项提案中，我们将研究一个具有遗传性猝死遗传形式的家庭，希望我们能够为这种毁灭性和不可预测的疾病的更常见原因开发新的治疗方法。

DESCRIPTION (provided by applicant): Arrhythmias remain a major cause of morbidity and mortality. Brugada syndrome is a rare, autosomal dominant, male predominant form of idiopathic ventricular fibrillation characterized by a right bundle branch block pattern and ST elevation in the right precordial leads of the surface EKG. The only effective therapy is an implantable cardioverter-defibrillator. Mutations of the cardiac Na+ channel SCN5A cause ~20% of cases of Brugada syndrome by decreasing inward Na+ current, and Na+ channel blockers such as procainamide exacerbate the EKG findings. The genetic basis for most remaining Brugada syndrome patients is unknown. During the initial periods of this project, we identified a large family with Brugada syndrome characterized by progressive conduction disease, age- and sex-dependent penetrance, and minimal response to the Na+ channel blocker procainamide. Linkage was identified to a ~1 cM region on chromosome 3p24 (max LOD score > 4.0) and SCN5A was excluded (LOD score < -2). Direct sequencing of candidates in the region identified an alanine to valine substitution in a conserved amino acid (A280V) in exon 6 of the glycerol-3- phosphate dehydrogenase 1-like gene (GPD1-L, KIAA0089). The mutation was present in all affected individuals and was absent in >200 unaffected controls of mixed racial background. Northern and Western blot analysis confirmed expression in the heart. Whole cell patch clamp studies of a stably transfected HEK cell line expressing SCN5A showed an ~60% reduction in peak Na current in cells transfected with the A280V mutant compared to the wild type GPD1-L (p=0.01). Confocal microscopy showed expression of the wild type but not the mutant GPD1-L protein on the plasma membrane, along with reduced SCN5A in the membrane of cells expressing the A280V mutant. NADH is increased in cells expressing A280V GPD1-L, and QRS duration on the surface EKG is prolonged in transgenic mice overexpressing A280V GPD1-L. In this competing renewal, we will test the hypothesis that GPD1-L is a novel ion channel modulator and that mutations of GPD1-L decrease Na+ current by altering intracellular NAD+/NADH levels. We will 1) define the mechanisms by which mutant GPD1-L alters ion channel trafficking and current in-vitro using cell lines and neonatal rat ventricular myocytes; 2) identify downstream mechanisms by which GPD1-L affects ion channel trafficking, and 3) study transgenic and gene-targeted mouse models to confirm its physiological role. Over 250,000 people in the United States die suddenly each year. Most sudden death occurs during heart attacks or in patients with weak hearts from prior damage, but we have no good ways to predict who is at highest risk of deaths and no drug therapies to prevent sudden death. In this proposal, we will study a family with an inherited genetic form of sudden death with the hope that we can develop new treatments for the more common causes of this devastating and unpredictable condition.