Identification And Molecular Mechanism Of Pathogenic Genes In Two Hereditary Epilepsy Families

Epilepsy is one of the most common and serious brain disorders,affecting over 70 million people worldwide.Epilepsy is a disorder of the brain characterized by an enduring predisposition to generate abnormal excessive or synchronous neuronal activity in the brain.The disease is clinically characterized by recurrent spontaneous epileptic seizures,which can severely affect patients’ neurological,cognitive and psychological systems.Epilepsy has a complex etiology,multiple risk factors,and strong genetic predisposition.However,there are still a large proportion of epilepsy patients who have unknown causes.Exploring the causative genes and pathogenesis of epilepsy will facilitate clinical molecular diagnosis,drug use and development of novel therapeutic targets.In this study,we collected and identified two large epilepsy families based on high-throughput whole exome sequencing(WES)technology and other means of genetic analysis to identify genetic mutations and used molecular,cellular experiments and animal models to explore the pathogenesis.The main research results are as follows.(1)Identification of pathogenesis in a family with familial focal epilepsy with variable foci.A large five-generation familial focal epilepsy with variable foci(FFEVF)pedigree comprising 26 individuals was collected in the study.Clinical heterogeneity was found in patients within the family,with two patients exhibiting focal nonmotor seizures,and the remaining patients exhibiting generalized seizures.A novel heterozygous mutation NPRL3 c.937＿945del/p.Ala313＿315Hisdel was screened and identified using WES,leading to a loss of in-frame three amino acids.Further Sanger sequencing revealed that the mutation co-segregated with disease in the family.The mutation in 100 unrelated healthy controls was absent by created restriction site PCR-RFLP analysis.In addition,with our collaborators,two novel NPRL3 mutations,16p13.3 NC＿000016.10: g.(110143＿110181)＿(116848＿116886)del and c.1514 dup C were identified in two other focal families,respectively.NPRL3 is one member of the GATOR1 complex(NPRL3,NPRL2 and DEPDC5),which plays a negative role in regulating the mTORC1 signal pathway.All three above mutations had truncated protein expression with different sizes,and co-immunoprecipitation assays found the mutations did not affect the formation of NPRL3-NPRL2 dimer but almost lost the ability to interact with DEPDC5.In addition,Western blot assays revealed that mutant NPRL3 proteins impaired inhibition of activated mTORC1 signaling under leucinestimulated conditions,suggesting the mutations are partial loss-of-function.These results showed a partial loss of mTORC1 inhibitory function by GATOR1 complexes formed with NPRL3 mutants.Knockdown of the Drosophila homolog nprl3 by RNA interference(RNAi)induced an epilepsy-like behavior and abnormal synaptic development,suggesting that NPRL3-associated FFEVF may be related to abnormal synaptic development.(2)Genetic analysis and functional studies of a family with temporal lobe epilepsy and psychiatric diseasesThis study conducted a clinical diagnosis of a seven-generation pedigree with temporal lobe epilepsy accompanied by psychiatric diseases,among which four patients had epilepsy,and five patients had mental disorders.WES analysis identified two in cis mutations of disease-causing gene CHRNA7,including c.698A>G/p.Y233 C and c.370G>A/p.A124 T.Both mutations co-segregated with the phenotype.CHRNA7 encodes nicotinic acetylcholine receptor,which is localized presynaptically,postsynaptically,and extrasynaptically,and is important for mediating rapid signal transduction at synapses.Expression analysis revealed that the expression of both CHRNA7 p.Y233 C and CHRNA7-p.A124T/Y233 C decreased in the whole lysate and membrane,while the expression of CHRNA7 p.A124 T was not altered.Moreover,the membrane/total ratio of CHRNA7 mutant proteins was not affected,suggesting the mutations did not appear to be defective in their membrane trafficking efficiency.Electrophysiological studies of Xenopus oocytes showed that the voltage clamp did not record the current of CHRNA7 p.Y233 C or CHRNA7-p.A124T/Y233 C under the stimulation of acetylcholine and allosteric agonist 4BP-TQS,and the p.A124 T mutant caused a significant decrease in current density and a reduced concentration of response to acetylcholine.The above results suggest that CHRNA7 mutations cause dysfunction of the CHRNA7 channel protein,which may induce the excitation-inhibition imbalance of hippocampal neurons in the brain and lead to epilepsy.In summary,our results found a novel variant in an FFEVF family,NPRL3 c.937＿945del,and NPRL3 mutations leading to impaired interaction of NPRL3/NPRL2 with DEPDC5,and then the dysregulation of the mTORC1 pathway,which may be the cause of FFEVF.The abnormal synaptic development and excitatory glutamate receptor expression imbalance in nprl3 RNAi flies confirm the pathological basis of neuronal excitationinhibition imbalance in NPRL3-associated epilepsy.In cis mutations c.698A>G/p.Y233 C and c.370G>A/p.A124 T in CHRNA7 were identified in a family with temporal lobe epilepsy accompanied by psychiatric diseases,and the double mutation caused downregulation of protein expression and channel dysfunction.These findings broaden the mutation and phenotype spectrum of epilepsy,providing a theoretical basis for further understanding epileptogenesis.