Identification Of Novel Disease-causing Gene For Sleep-related Hypermotor Epilepsy And Function Study

【Background】Sleep-related hypermotor epilepsy（SHE）is a focal epilepsy syndrome characterized by clusters of brief seizures with stereotyped motor patterns occurring predominantly during non-rapid eyes movements（NREM）sleep.In many SHE patients,the interictal and ictal scalp electroencephalography（EEG）features are uninformative.Diagnosis of SHE is primarily based on clinical history,and misdiagnosis is common.Poor recognition and misdiagnosis have multiple adverse consequences.Hence,it is of great clinical significance to improve the recognition of SHE for early diagnosis and timely treatment.Clinical studies have indicated that the prognosis of SHE is closely related to the cause,and exploring its pathological mechanism may provide clues for precise treatment.Genetic factors are one of the main causes of SHE.With the development and application of gene sequencing,the identification of SHE genes has become the main insight for understanding its pathogenesis.However,the currently detected genes collectively account for only a minority of SHE patients,and the pathogenic mechanism of some genes is still unclear.Besides,the relation with the sleep-wake cycle hasn’t been explained.Thus,it is urgent to screen and identify new pathogenic genes of SHE,to explore its pathogenic mechanism,and to provide insight for a comprehensive understanding of the pathogenesis of SHE and its relationship with sleep.【Objectives】1.To screen and identify the novel causative gene for SHE.2.To study the function and pathogenic mechanisms of new SHE-associated-gene in vitro and in vivo.3.To explore the underlying mechanisms between SHE and NREM sleep.【Methods】1.From 2015 to 2019,SHE patients were recruited sequentially from the Department of neurology of the Comprehensive Epilepsy Center in Xijing Hospital.The demographic and clinical characteristics were collected and summarized.Whole exome sequencing（WES）was conducted to detect causative variants.For patients with candidate variants,trio-based WES were further performed to exclude the other underlying causative variants.The filtered variants were reviewed according to the recommendation of the American College of Medical Genetics and Genomics（ACMG）guidelines.All putative causative variants identified by WES were confirmed and tested for co-segregation by Sanger sequencing.2.The functional effects of the identified variants were analyzed in both neuronal and non-neuronal cells using a combination of electrophysiology recordings,western blot,flow cytometry,and confocal microscopy.3.The variant knock-in mouse was generated using CRISPR/Cas9.The genotypes of the offspring were detected by PCR.Nissl stained were used to examine whether expression of the variant caused neuroanatomical abnormalities.Digitally synchronized video/EEG recordings lasting at least 24h were performed to determine whether variant knock-in mice had spontaneous seizures and/or altered sleep-wake patterns.Mutant and wild-type（WT）mice underwent video/EEG recording for 1h in the untreated state and for1h following administration of theγ-amino butyric acid A receptor（GABA_AR）antagonist,pentylenetetrazole（PTZ,50mg/kg）,to evaluate seizure threshold.4.In vivo multi-channel electrophysiological technology was conducted in the WT and variant knock-in mice to monitor the local field potential（LFP）of motor thalamic nuclei（Mo TN）and secondary motor cortex（M2）during the sleep-wake period.Coherence and synchronization of both brain areas were analyzed.QPCR,western blot,and immunofluorescence were used to analyze the underlying molecular mechanism.【Results】1.Gene tests results.A total of 58 unrelated patients with SHE were enrolled in this study.Six candidate variants were detected in 6 unrelated patients（6/58,10.3%）by WES,with three variants（NM＿198904.2:c.269C>T,p.T90M;NM＿198904.2:c.950C>A,p.T317N and NM＿198903.2:c.649C>T,p.Q217X）found in GABRG2.GABRG2 variants have not been reported to be associated with SHE.And trio-based WES further confirmed the pathogenicity of GABRG2 variants.Two of the three detected GABRG2 variants were transmitted unaffected maternally（T90M）or paternally（Q217X）,whereas the T317N variant arose de novo.The mother of proband carrying the T90M variant was unaffected and being mosaicism for this variant（the mutant allele proportion:14%）.GABRG2encodesγ₂ subunit of GABA_AR.The T90 and Q217 are located in the extracellular N-terminal domain,and T317 is in the TM2 helix of theγ₂subunit.Consistently,multiple algorithms predicted that the variants of T90M,Q217X,and T317N have a deleterious consequence.2.In vitro experimental results.（1）Whole-cell recordings found that the peak current amplitudes for receptors containingγ₂（T90M）subunits orγ₂（T317N）subunits were decreased significantly.Butα₁β₂γ₂（Q217X）receptor currents were similar to those of wild-type receptors.The fractional Zn²⁺inhibition of the mutantα₁β₂γ₂（T90M）currents was significantly greater than inhibition of wide-type receptor currents.And T317N variant decreased desensitization extent and slowed the deactivation rate of the GABA_AR currents.（2）Western blots and flow cytometry results revealed that the T90M variant significantly impaired the efficiency of mutantγ₂ subunits transportation to the cell surface,which made mutantγ₂ subunit trapped in the endoplasmic reticulum（ER）,the location where the immature subunits once synthesized.（3）Immunocytochemistry and confocal microscopy results demonstrated T90M and Q217X variants reduced synaptic clustering and distribution ofγ₂ subunit-containing receptors.3.In vivo experimental results.Given T317N variant arose de novo and the patient carrying this variant with a more severe clinical phenotype,we chose the T317N variant to generate a knock-in mouse(Gabrg2^T317N/wt).（1）We found gross morphology and the cellular structure of Gabrg2^T317N/wt brains appeared to be normal.（2）Gabrg2^T317N/wt mice had spontaneous seizures,which incidence rate is about 20%.And about 75%of seizures occurred during sleep.（3）With the blockade of GABA_AR by PTZ（50mg/kg）,Gabrg2^T317N/wt mice had lowered seizure threshold compared with WT littermates as demonstrated by increased percentage of seizures and high mortality rate.（4）Chronic EEG recordings revealed a significant increase in the number of brief awakenings during NREM sleep in Gabrg2^T317N/wt mice compared with WT,suggesting T317N variant caused fragmentation of NREM sleep.4.The underlying mechanism between SHE and NREM sleep.In vivo multi-channel electrophysiological recording in Gabrg2^T317N/wt mice showed the coherence and synchronization of LFP of Mo TN and M2 in the delta frequency band（1-4Hz）were significantly increased during sleep,relative to WT mice.QPCR,immunohistochemistry,and western blot results revealed that the m RNA and total protein levels of GABA_AR subunits in Gabrg2^T317N/wt various brain regions（cortex,thalamus,hippocampus,and cerebellum）were similar to the WT mice.Compared with WT,the surface expression levels ofγ₂ subunits in various brain regions of Gabrg2^T317N/wt significantly decreased in both sleep and wake periods.Moreover,the surface expression levels ofγ₂ subunits in the cortex and thalamus regions of Gabrg2^T317N/wt decreased to a greater extent during sleep.【Conclusions】1.In the present study,we firstly identified that GABRG2 may be a new causative gene of SHE,which extended the pathogenic gene spectrum of SHE and the phenotype spectrum of GABRG2.2.The GABRG2 variants（T90M,Q217X,and T317N）impaired GABA_AR function by diverse mechanisms,including impaired surface expression,ER retention,and channel gating defects.3.Gabrg2^T317N/wt mice share several features experienced by SHE patients,which can become genetically engineered animal model available for studying the pathogenic mechanism of SHE.4.The reason why SHE predominantly occurred during sleep may be associated with pathological enhancement of the synchronized discharge of the thalamocortical motor pathway.