GABRG2突变导致遗传性癫痫伴热性惊厥附加症（GEFS+）的分子遗传学特征及机制研究

GABRG2是氯离子通道的重要组成部分，其突变是遗传性癫痫伴热性惊附加症（GEFS+）发生的重要因素，但确切分子机制所知甚少。课题组前期发现GABRG2敲除后，GABRA1/EFHC1显著下调，随着温度升高GABRA1表达降低，提示其可能通过下调上述因子促进GEFS+发生。项目拟开展：①针对GABRG2，利用CRISPR/Cas9技术构建基因敲除小鼠②GABRG2可位于胞膜，应用免疫印迹、蛋白共定位及膜片钳等手段，寻找在海马神经元胞膜上与GABRG2相互作用的蛋白、离子及调控疾病发生的靶蛋白③GABRG2可入核，采用免疫印迹/荧光、ChIP-Seq和基因测序分析GABRG2代谢途径中蛋白-DNA作用及主要信号通路，探讨GABRG2敲除后是否在海马神经元胞核内影响组蛋白的修饰并由此影响GEFS+发生的靶蛋白表达。这将有助于阐明GEFS+发生的分子机制，为GEFS+的治疗提供有利证据。

GABRG2 is an important compponent of chloride channel in mammal CNS, and its mutations are critical factors in the occurrence of the genetic epilepsy with febrile seizures plus (GEFS+) . However, the exact molecular mechanism is poorly understand. Our previous research found that GABRA1/EFHC1 was significantly down-regulated after GABRG2 knockout, and the expression of GABRA1 decreased with the increase of temperature. The preliminary results implied that GABRG2 may promote GEFS+ by down-regulating those above factors. The project to be carried out: ① For GABRG2, the gene knockout mice were constructed by CRISPR/Cas9 genome editing technology; ② GABRG2 could be located in the cell-membrane, Western blotting, protein co-localization and patch clamp technologies were used to find the proteins, ions and target proteins inserting with GABRG2 on the cell membrane of hippocampal neurons; ③ GABRG2 could be inserted into the nucleus, using immunoblotting/immunofluorescence, ChIP-Seq and RNA-seq technologies to analyze the role of protein-DNA and its main signaling pathway in the GABRG2 metabolic process , and to explore whether GABRG2 knockout affects histone modification in the nucleus of hippocampal neurons, and thus affect the target protein expression of GEFS+ occurrence. This will contribute to elucidate the molecular mechanism of GEFS+ occurrence, and provide favorable evidence for the treatment of GEFS+.