EMX2基因罕见突变致神经元迁移发育障碍在癫痫发生中的作用机制研究

Genetic factors play an important role in epilepsy, and the genetic heritability of epilepsy is up to 40%. In previous study, whole exome sequencing was carried out in more than 600 epilepsy families, and a rare mutation of EMX2 gene was found in the DNA samples of several epileptic patients from fatal epileptic family. Then, we transfected the mutant plasmid in primary cultured neuron by high efficient nuclear power transfer instrument, the neurite out-growth was impaired in mutant group; in addition, with the help of in utero electroporation technology, we transfered mutant plasmid into embryonic cortical neurons, the process of neuronal migration was blocked, and neurons were trapped in the area of neurogenesis; After injection with pentylenetetrazol, the seizure frequency and grade of the mice transfected with mutant plasmid were significantly higher than those in the control group. However, it is not clear the development and migration disorder mechanisms by which involved in epileptogenesis. Based on the conditional point mutation knock in transgenic mouse model, we plan to systemically explore the function of rare mutation of EMX2 gene as well as the molecular mechanisms by which involved in epileptogenesis through behavioral screening and with help of molecular biology, chromatin immunoprecipitation sequencing (CHIP-seq), neuron culture, in utero electroporation, electrophysiology and multi-channel in vivo field potential recording techniques , so as to provide new treatment strategies for epilepsy.

遗传因素在癫痫发生中起非常重要的作用，癫痫的基因遗传度达到了40%以上。课题组前期通过全外显子测序技术对已有的600多个癫痫家系进行了高通量筛查，在致死性癫痫家系多名患者DNA样本中筛查到EMX2基因发生罕见突变；将突变型质粒通过高效核电转仪导入原代培养神经元中，神经元神经突生长严重障碍；此外，借助胚胎电转技术，将突变型质粒转入皮层迁移神经元，阻碍了神经元迁移过程；利用戊四氮进行癫痫造模后，电转突变质粒组小鼠发作频率和等级都显著高于对照组；提示EMX2罕见突变介导的神经元迁移发育障碍可能参与癫痫发生发展。基于己构建的条件点突变变敲入小鼠模型，故本研究计划利用染色质免疫共沉淀-测序技术找出异常信号通路，并结合分子生物学、神经元培养、胚胎电转、行为学筛查以及多通道在体场电位记录等技术，多维度去探究EMX2基因罕见突变通过何种分子机制参与调控神经元迁移发育及癫痫的发生发展，探索新的癫痫治疗策略。

遗传因素在癫痫发生中起非常重要的作用，癫痫的基因遗传度达到了40%以上。课题组前期通过全外显子测序技术对已有的600多个癫痫家系进行了高通量筛查，在致死性癫痫家系多名患者DNA样本中筛查到EMX2基因发生罕见突变；并构建了EMX2点突变条件性敲入小鼠模型，基于该模型我们利用染色质免疫共沉淀-测序技术找出异常信号通路，并结合分子生物学、神经元培养、胚胎电转、行为学筛查以及多通道在体场电位记录等技术，多维度去探究EMX2基因罕见突变通过何种分子机制参与癫痫的发生发展。本研究发现EMX2突变以后对SrGAP2基因调控出现异常，导致SrGAP2的表达量降低，进而使得神经元的神经突发育异常，同时也造成了神经元的迁移异常，使得突变组小鼠的脑部组织出现异常，最终使得突变小鼠在戊四氮癫痫造模以后，癫痫发作频率和发作等级都显著高于对照组。研究提示EMX2-SrGAP2这条神经发育和迁移相关的分子通路可能是治疗癫痫的新靶点。