CLCN4基因编码氯通道ClC-4，在脑区高表达，参与胞内体酸化和物质转运等，其缺失可影响GABA摄取和树棘突分枝。既往报道CLCN4突变主要与智力障碍密切相关。我们前期研究在癫痫性脑病患者或家系中发现了6例CLCN4突变，其基因型和临床表型各异且均为新发现，提示该基因与癫痫性脑病有关，突变的功能差异有可能是表型差异的基础。其基因型-功能改变-表型之间的关系、以及致病机制有待明确，可能与ClC-4通道异常所致的微抑制性突触后电流改变有关。本项目拟利用HEK细胞和原代培养神经元研究各突变体的亚细胞定位、对氯通道属性、神经元兴奋性的影响，分析突变的功能差异与临床表型的关系；在此基础上遴选致病性不等的突变位点构建点突变大鼠，研究突变对树突棘发育、GABA能突触传递、微抑制性突触后电流及行为学的影响。本项目将明确CLCN4突变在癫痫性脑病中的作用及发病机理，为临床精准诊疗及遗传咨询提供理论依据。

CLCN4 resides on Xp22.2 and encodes voltage-gated chloride channel ClC-4, which is highly expressed in the brain. The channel ClC-4 plays important roles in the endosomal acidification, endocytosis and intracellular trafficking. Primary neurons derived from Clcn4 knock-out mice showed a significant effect on GABA uptake and dendritic branching. In clinic, CLCN4 mutations have been reported to be associated with X-linked intellectual disability. Our previous studies identified six novel CLCN4 mutations in sporadic and familial patients with epileptic encephalopathies, suggesting a pathogenic role of CLCN4 mutations in epileptic encephalopathy. These mutations contain nonsense, missense and splicing mutations, which are related to different clinical phenotypes of epileptic encephalopathy with varying severity. It suggests that the functional defects of ClC-4 channel caused by CLCN4 mutations are possible one of the key determinants for the heterogeneous phenotypes. The pathogenic mechanisms of CLCN4 mutations in epileptic encephalopathy, particularly in phenotypic variants, remain elusive. This project will analyze the electrophysiological properties of mutant ClC-4 channels in HEK293 cells, and compare the subcellular localization, synaptic responses and neuronal firing patterns between rat primary cultured hippocampal neurons with different Clcn4 mutations. It will reveal the genotype-functional alteration-phenotype associations of CLCN4 mutations. Further, we will analyze the alteration of dendritic spine development, GABAergic synaptic transmission and miniature inhibitory postsynaptic currents (mIPSC) in Clcn4 mutation-specific rats. This project will define the role of CLCN4 mutations in epileptic encephalopathy and shed light on the underling pathogenic mechanism, which will help in clinical or genetic counseling practice.