Molecular Mechanism Of Childhood Absence Epilepsy Caused By CLCN2 Gene Variants

BackgroundChildhood absence epilepsy（CAE）is a common generalized epilepsy syndrome,which is characterized by typical absence seizures.The pathogenesis of CAE is complex,and its molecular mechanism is not clear.The flow of ions on the membrane is the center of many physiological processes.The function of ion channel requires a continuous water hole to pass through the protein to ensure the migration of ions through electro diffusion.CIC transporters and channels are widely expressed in various organs and tissues of prokaryotes and eukaryotes,and play an important role in normal physiology and diseases.CIC transporter family includes Cl^-ion channel,H⁺/Cl^-and H⁺/NO^3-exchange channel.At present,it is found that CLCN2 gene variation can lead to protein misfolding,and the misfolded protein will be degraded by endoplasmic reticulum and endolysosomal related proteins,resulting in intracellular Cl^-concentration imbalance.In the whole exome sequencing of patients with typical absence attack,we found that four CAE patients carried four different missense mutations of CLCN2 gene.This subject will use bioinformatics,genetics,molecular genetics and electrophysiology technology to clarify the molecular pathogenesis of CAE at the molecular level.ObjectivesCLCN2 gene encodes chloride channel protein 2（CIC-2）.CIC-2 is a chloride channel existing in plasma membrane and widely expressed in human body.It plays an indispensable role in chloride migration,ion transmembrane transport,regulation of aldosterone biosynthesis and regulation of ion transmembrane transport.Previous studies have found that four patients with CAE carry four variants of CLCN2 gene.This project aims to clarify the molecular mechanism of CAE caused by CLCN2 gene mutation,which will help to uncover the pathogenesis of CAE and provide a theoretical basis for the development of new treatment methods.MethodsClinical follow-up of typical CAE patients:collect the clinical data and medication of typical CAE patients,and analyze the EEG of CAE patients.Screening genes after whole exome sequencing:explicit recessive genetic model,homozygous analysis,etc;Remove synonymous mutations;The mutation sites with minor allele frequency（MAF）<0.0001were retained to analyze the harmfulness of variation.After Sanger sequencing verified the mutation,CLCN2 expression plasmid was constructed:CLCN2 wild-type and variant expression vectors were constructed.Prove the molecular mechanism of CAE caused by CLCN2 variation at the cellular level:CLCN2 variation affects protein expression:CLCN2wild-type/variant plasmid was transfected into HEK293T cells,and the protein was extracted.The effect of CAE candidate related gene（CLCN2）variation on its protein function was studied at the molecular level by Western blot and other experiments.CLCN2subcellular localization:the mutant plasmid and the control plasmid with luminous group were transfected into He La cells,and the subcellular membrane localization was found by immunofluorescence to judge the effect of its mutation on the plasma membrane.MG-132/CHX can affect the expression level of CLCN2 variant protein:transfect wild-type and variant plasmids into HEK293T cells,extract cell protein,and study the protein expression after treatment by Western blot and other experiments.Co IP detection interaction:To study the interaction between ubiquitin activating enzyme E1 and CLCN2 variant,so as to explore the pathogenesis of CAE.Result1.10 patients were screened for clinical EEG,and all showed extensive 3 Hz spike slow wave explosive discharge2.Four mutation sites of CAE related gene（CLCN2）were screened and verified by Sanger sequencing;The four mutation loci were:（C73T/C1141G/G1705A/C1885T）.3.Evolutionary conservation analysis of CLCN2:according to the analysis of biological evolutionary conservation,it is concluded that the screened genes are highly conserved in biological evolution.4.The molecular mechanism of CAE is at the level of evidence.CLCN2 variants can lead to changes in total protein:compared with variant CLCN2,the expression of variant CLCN2（C73T/C1141G/G1705A/C1885T）is lower than that of wild-type CLCN2.CLCN2variants can lead to changes in membrane proteins:compared with variant CLCN2,the expression of variant CLCN2 is lower than that of wild-type CLCN2.CLCN2 variant can lead to abnormal localization of membrane protein:the localization of wild-type and variant was observed by immunofluorescence technique,and its subcellular localization had no change.MG-132/CHX can inhibit the degradation of CLCN2 variants:MG-132 can inhibit the degradation of CLCN2 variants;CHX can prove that there is no change in protein half-life.Conclusion1.The onset age of typical CAE is 2～10 years old.Extensive 3 Hz spike slow wave explosive discharge can be seen,and it is most obvious in the awake period.2.CLCN2 variant can reduce the expression of total protein and membrane protein.3.CLCN2 variants may be degraded by ubiquitin proteasome system.