SCN1A基因突变导致钠通道不同功能表型和临床表型的机理及其与药物反应的关系

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 mutations vary widely, for which the underlying mechanisms remain elusive. The functional defects of Nav1.1 channel caused by SCN1A mutations are assumed to be one of the key determinants for the heterogeneous phenotype, supported by our recent findings in partial epilepsy with febrile seizures (PEFS+). PEFS+ is an intermediate type of SCN1A-related epilepsy syndromes in terms of both phenotype and molecular defect, characterized by seizure aggravation by AEDs and loss of function of Nav1.1. 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 mutation types, 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, we will test in vitro the electro-property changes of Nav1.1 with different AEDs. Finally, we will study the functional changes and the altered responses to AEDs in iPS-derived cell lines obtained from patients with 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，亦是多种抗癫痫药物作用的靶点。SCN1A基因与癫痫关系最为密切，其突变致病的特点为量的依赖性和表型的多样性。各种突变导致不同表型的机理不清。我们新报道了一种中间类型的癫痫-部分性癫痫伴热性惊厥附加症，此类患者的癫痫可被抗癫痫药物加重，功能学研究证实其钠通道功能丧失。本项目前期研究已发现42例SCN1A基因突变，我们将进一步扩大筛查范围，并根据突变类型体外定点诱变构建异源细胞表达体系，研究不同类型、不同位点突变、同一位点不同氨基酸置换导致的钠通道功能差异，分析其与临床表型、抗癫痫药物反应的关系。并利用病人体细胞构建诱导多能干细胞，建立自源体外细胞研究模型，研究突变体的电生理差异及对药物的反应等。综合病人临床资料、药物反应及体外研究结果将建立个体化的癫痫基因诊疗体系，对揭示钠通道突变的致病机制、通道结构与功能、抗癫痫药物作用机制均有重要意义和实用价值。

I 型电压依赖型钠通道是神经电活动的基础，亦是多种抗癫痫药物作用的靶点。SCN1A基因与癫痫关系最为密切，其突变致病的特点为表型的多样性。各种突变导致不同表型的机理不清，特别是在部分性癫痫伴热性惊厥附加症中尤为明显。本研究拟根据突变类型体外定点诱变构建异源细胞表达体系，研究不同类型、不同位点突变、同一位点不同氨基酸置换导致的钠通道功能差异，分析其与临床表型、抗癫痫药物反应的关系。将利用病人体细胞构建诱导多能干细胞，建立自源体外细胞研究模型，研究突变体的电生理差异及对药物的反应等。本课题组前期研究已发现42例SCN1A基因突变，我们对本课题组发现以及文献报道的1257个SCN1A基因突变进行总结归纳，建立突变数据库http://www.gzneurosci.com/scn1adatabase/。研究发现SCN1A基因错义突变的频率与临床表型的严重性呈负相关，而且基因型，通道功能改变与临床表型紧密相关。本课题组构建NaV1.1与NaV1.3同源突变的研究模式。首次提出NaV1.1与NaV1.3孔区突变在电生理上存在的显著差异： NaV1.1与NaV1.3孔区突变与野生型电生理特征比较说明NaV1.1-N301S孔区突变电生理功能基本丧失，而NaV1.3-N302S孔区突变存在部分电生理功能。在国际上首次系统阐述了NaV1.1与NaV1.3孔区突变在电生理上的显著差异，并对可能的作用机制进行了探讨和分析。此外，我们通过CRISPR/Cas9技术在iPS细胞水平上敲入荧光蛋白基因来标记神经元网络中的GABA能神经元亚型，首次在病人来源的神经元网络中对表达Nav1.1的神经元亚型做了电生理检测。同时，对网络中自发抑制性及兴奋性突触后电活动进行了分析。研究发现，患者来源的神经元网络突触后活动从抑制为主的状态转化为兴奋性为主的激发态，这表明，仅仅sIPSCs的变化就足以显著逆转神经元网络的兴奋性水平。建立了高效iPSCs产生及诱导分化为神经元技术，为个性化药物的筛选及在神经元模型上开展调控元件功能研究奠定了良好的基础。目前已发表标明资助的SCI论文3篇。