Lennox-Gastaut 综合征相关的GABRB3基因新生错义突变的功能研究

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.

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。

本研究通过收集癫痫患者及其父母的血液，抽提全基因组DNA，通过全外显子测序技术获取基因变异位点、方式及来源，在两个表型轻重不一的癫痫患者中发现编码γ-氨基丁酸A型受体（gamma-aminobutyric acid receptor type A, GABAAR）β3亚基（GABRB3）基因两个新的错义突变，其中严重癫痫类型Lennox-Gastaut综合征（Lennox-Gastaut syndrome, LGS）携带新生的N328D突变；轻型癫痫类型青少年失神癫痫（juvenile absence epilepsy, JAE）携带E357K突变。通过流式细胞技术发现了9个与癫痫及神经发育疾病相关的GABRB3基因突变（包括N328D和E357K）会引起β3亚基在HEK293细胞的表面表达降低，其中大多数同时引起γ2亚基表达降低。通过细胞实验及膜片钳技术发现了N328D和E357K突变体均导致GABAAR激活电流幅度降低，前者降低更为显著；而对阻滞剂锌离子的敏感性，E357K高于N328D。两个突变降低β3亚基在HEK293细胞膜上的表达，同时降低γ2亚基的表达。在大鼠神经细胞中，两个突变导致β3亚基在细胞中、细胞膜和抑制性突触的表达量均下降，同时导致γ2亚基上膜及在突触后膜上表达减少，突触后膜上GABAAR数量减少。LGS相关的突变N328D与JAE相关的突变E357K比较，前者引起β3亚基表达降低更为显著，突变蛋白基本聚集在神经元胞体内，无法上膜；后者的突变蛋白有少量可以上膜。前者突触后膜上GABAAR数量减少更为显著。在β3亚基敲除小鼠上同样发现抑制性突触后膜上γ2亚基及GABAAR数量减少。首次发现了GABRB3基因突变可以降低β3亚基的表达及上膜，同时可以降低γ2亚基的表达及上膜，从而减少了抑制性突触后膜GABAAR的数量，损害功能，这可能是GABRB3基因突变导致癫痫及神经发育疾病的共同机制；GABRB3基因突变通过不同的细胞机制引起亚基在神经细胞内聚集程度不一，导致突触后膜上GABAAR数量存在差异，从而导致抑制功能损害程度出现差异，引发表型轻重不一的癫痫发生。本研究提出了β3亚基突变损害GABAAR抑制功能的新机制，同时为理解GABRB3突变如何导致严重的癫痫及表型轻重不一的癫痫提供了关键性的实验依据。