SCN1A基因剪切突变导致不同功能表型和癫痫临床表型差异的机理研究

Voltage-gated sodium channels (VGSCs) play an essential role in neuronal excitability, which are also the targets of many antiepileptic drugs (AEDs). SCN1A, the gene encoding sodium channel Nav1.1, is the most clinically relevant epilepsy gene. The phenotypes of SCN1A splicing mutations vary widely, for which the underlying mechanisms remain elusive. To test the hypothesis that the functional defects of Nav1.1 contribute to clinically variability, we will examine the functional defects (funotype) of SCN1A mutations identified in patients with different epilepsy phenotype. Based on the mutations identified, we will develop in vitro heterologous expression systems with SCN1A mutations produced by site-directed mutagenesis, and then analyze the electrophysiological property of the mutant Nav1.1. The correlations between the channel dysfunction and the location of the mutations and the nature of amino acid substitutions will be studied. Further to explore the mechanism of seizure aggravation by AEDs in patients with SCN1A mutations.Finally, we will study the the mechanism of U1snRNA and Morpholino correcting the aberrant splicing of SCN1A mutations. This study is expected to disclose the relationships among genotype, funotype and phenotype of sodium channelopathies, which may be helpful in understanding the function of sodium channel as well as the pathogenesis of epilepsy. The understanding on the mechanism of seizure aggravation by AEDs will benefit the epilepsy patients in improving their outcome by avoiding development of refractory epilepsy.

I型电压依赖型钠通道是神经电活动的基础。癫痫与SCN1A基因关系非常密切，SCN1A基因突变致病的特点为量依赖和多样性表型。剪切位点突变导致不同表型的机理不清楚。我们前期研究已发现6例SCN1A基因剪切位点突变，本项目中我们将进一步扩大筛查范围，并根据突变位置在体外定点诱变构建minigene，研究不同剪切位置突变、同一剪切位置不同碱基置换导致钠通道的功能差异，结合临床表型分析其关系，并进一步建立致病量-临床表型严重程度的关系模型。同时研究SCN1A 基因异常剪接的修复和剪接异常Nav1.1 蛋白在亚细胞的定位和表达。最后综合患者临床资料、体外剪切结果、致病量及电生理改变，建立个体化的癫痫基因诊疗体系，对揭示钠通道剪切位点突变的致病机制、钠通道结构和功能、量依赖作用机制及癫痫的临床治疗和个体化基因治疗均有重要意义和实用价值。

编码I型钠通道α亚基(Nav1.1)的SCN1A基因突变与热性惊厥相关性癫痫关系最为密切。本研究通过对热性惊厥相关性癫痫患者SCN1A基因不同剪切位点突变剪切效应的分析以及对mRNA转录本水平的影响，探讨剪切突变的位置、剪切模式与表型严重程度的关系。筛查所得的1个新的剪切位点突变，结合前期我们筛查到的6个剪切位点，加上文献报道的6个确切剪切位点突变并纳入此项研究。使用体外剪切报告“minigene”分析剪切位点突变的剪切效应，对那些影响剪切的突变，进一步使用实时荧光定量聚合酶链式反应(Q-PCR)分析SCN1A基因突变后mRNA转录本的量。5个位于不变剪切点区域的剪切位点存在一种或者2种异常剪切方式：既可以引起完全的相关外显子的删除，也可以是外显子删除和的方式，或者引起部分相关外显子删除与异常内含子插入相结合的方式；不变剪切点区域的突变，可以存在或者不存在正常转录本，即使存在正常转录本，其正常剪切转录本的量也远远低于异常剪切转录本的量，所对应的临床表型是最严重的癫痫类型Dravet 综合征 和 Lennox-Gastaut 综合征。正常剪切转录本和异常剪切转录本的量的比例也可能随着其位置和剪切模式的变化而改变。这些区域的突变所对应的临床表型以及疾病的严重程度也存在一定差异。我们的研究首次在SCN1A基因中证实了同一个基因上剪切突变位点的位置与剪切效应有关，并且与表型严重程度相关。首次发现了SCN1A基因突变所致的热性惊厥相关性癫痫的临床表型的严重程度与正常剪切转录本的量呈负相关。目前已发表标明资助的SCI论文3篇。