I型钠通道（Nav1.1）基因框内缺失突变在癫痫发病机制中的作用及其与疗效的关系

Nav1.1 sodium channel, encoded by SCN1A gene, plays a critical role in controlling the excitability of brain. Mutations in SCN1A are associated with epilepsy. In-frame deletion mutations, which cause deletion of one or more amino acids, usually lead to mild diseases. However, in-frame deletion mutants of Nav1.1 protein almost manifested severe clinical phenotypes. And the underlying mechanism between in-frame deletion mutation and clinical phenotype is still unknown. We recently identified 5 in-frame deletion of SCN1A in patients with epilepsy, who revealed phenotypes of variant severity and different responses to antiepileptic drugs (AEDs). It is speculated that various position and length of in-frame deletions result in electrophysiological functions and expression changes by affecting folding and trafficking defectiveness of Nav1.1 protein. This project, therefore, plans to study the electrophysiological alteration, cell surface expression, sub-cellular localization, and kinetics of degradation of the in-frame deletion mutants. With an analysis of the relationship between the biophysiological alteration and clinical phenotype, we aim to explore the pathogenic mechanism of in-frame deletion of SCN1A. Additionally, we plan to explore the effect of ERAD mechanism in epilepsy with treatment of Ankyrin G and lactacystin. This study will shed light on the pathogenesis of in-frame deletion mutation and providing a clue for clinical precise management of the patients with epilepsy.

钠通道蛋白Nav1.1由SCN1A基因编码，主要控制大脑兴奋性且与癫痫密切相关。框内缺失突变是指一个或多个密码子缺失，蛋白质一级结构基本完整且临床表现较轻。而目前报道的SCN1A框内缺失突变多致重型癫痫，尚无机制研究。本课题组前期新发现5个框内缺失突变，其癫痫严重程度及药物反应各异，推测框内缺失的位置、长度影响钠通道电生理特性，以及蛋白折叠、运输功能和膜表达变化，继而决定癫痫临床表型。本研究拟体外表达Nav1.1不同框内缺失突变体，检测其电生理功能、蛋白表达量及亚细胞定位变化，探讨Nav1.1框内缺失-临床表型-AEDs药效关系。给予锚蛋白G、乳胞素干预，探讨内质网相关蛋白降解（ERAD）机制在Nav1.1框内缺失突变致痫机制中的作用及补救措施。本研究将阐明Nav1.1框内缺失突变致痫的全新机制，AEDs对框内缺失突变患者的疗效及其药理机制，为临床癫痫患者精细化管理提供有力依据。

研究背景: 框内缺失突变是指一个或多个密码子缺失，蛋白质一级结构基本完整且临床表现较轻。而目前报道的SCN1A框内缺失突变多致重型癫痫，尚无机制研究。通过对SCN1A截短突变和缺失突变电生理功能的研究，并与既往文献报道的突变的功能改变进行对比，探讨突变所产生的钠通道结构改变对电生理功能以及蛋白表达的影响及其在癫痫机制中的作用。.研究内容:详细收集携带SCN1A基因截短突变和缺失突变的EFS+患者的临床资料及突变特点，并纳入文献中报道的表现为LGS的1例缺失突变。构建突变质粒，并异源性的表达于HEK293T细胞，通过膜片钳技术检测其电生理功能改变。.重要结果与关键数据:.1、5例框内缺失突变，其中1例表现为FS+，1例PEFS+，3例DS。.2、电生理检测发现5个SCN1A框内缺失突变的电生理改变基本一致，属于钠通道功能缺失（LOF）。.3、激光共聚焦亚细胞定位与Western blotting发现，不同Nav1.1蛋白缺失突变体中，p.T303-R322del膜蛋白表达与Nav1.1野生型大致相同，p.Thr160_Tyr202del、p.V1335_V1428del 的膜蛋白表达与Nav1.1野生型相比明显减少。.科学意义: SCN1A框内缺失突变的电生理改变基本一致，钠通道功能属于LOF。结合本课题组对于框内缺失突变细胞膜蛋白表达水平的研究结果，我们推测不能，我们推测不能单纯从缺失突变的电生理改变和细胞膜蛋白表达水平来解释临床症状的差异。