Lennox-Gastaut综合征(LGS)是儿童难治性癫痫，目前发病机制未明。我们前期发现两个特发性LGS患者中分别携带GABRB3基因新生错义突变E180G和N328D。GABRB3基因编码抑制性γ-氨基丁酸A型受体（GABAAR）的主要亚基β3，基因缺失的动物中有癫痫表型，但其错义突变如何导致LGS，有待阐明。通过电生理初步试验及生物信息学分析，突变可能导致GABAAR电流及功能受体送达改变。本项目将利用全细胞膜片钳技术、糖基化检测、粗面内质网相关降解（ERAD）、溶酶体降解和內吞分析，了解不同错义突变导致GABAAR功能改变的途径；并进一步利用患者来源的iPSCs，诱导分化为神经元，进行功能研究与抗癫痫药物的疗效分析，从而了解GABAB3在LGS的发生发展中的作用机制，为难治性癫痫的发病机制、个性化诊治、遗传咨询及新药的开发提供理论依据。

Lennox-Gastaut syndrome (LGS) is one of the most severe epileptic encephalopathies with childhood onset. We recently identified two de novo missense mutations, E180G and N328D, for GABRB3 encoding the β3 subunit, a major constituent of the GABAAR, in two unrelated families with idiopathic LGS by whole exome sequencing and resequencing. In silico analysis and whole cell patch clamp record of missense substitutions indicates potential functional alterations in the GABAAR. The fact that mice lacking GABRB3 present with epilepsy phenotypes also suggests that GABRB3 mutations could lead to human LGS. The project will investigate the functional alterations caused by these GABRB3 missense mutations, and the underlying mechanisms leading to LGS as well. By expressing mutations in HEK 293 cells, we will study possible alterations in receptor properties, assemblies of subunits with the use of electrophysiological recording. We will also investigate surface expression functional GABAAR through N-glycosylation detection, ERAD, lysosome degradation and endocytosis analysis in vitro. Furthermore, based on a mature technology in which the induced pluripotent stem cells (iPSCs) from patients could be differentiated into neurons, we will analysis the functional altertions of mutant neurons and the effects of different anti-epilepsy drugs (AEDs). These studies will reveal the molecular mechanisms underlying the refractory epileptic encephalopathies resulted from the GABRB3 mutation, assist with the novel target GABRB3 for the LGS treatment and genetic counseling in family members and future children.