Screening Of Candidate Pathogenic Gene And Identifying Of Its Functional Role In Focal Cortical Dysplasia-related Refractory Epilepsy

Epilepsy,a chronic neurologic disease,is associated with multiple factors and causes and is characterized by abnormal discharge synchronization in cortical neurons and followed by transitory dysfunction of central nervous system,mostly affecting the juveniles and children.Though most of the patients have better clinical relief after treatment of first-line anti-seizure drugs,near 30%patients still progress to drug-resistant epilepsy（DRE）with poor prognosis.Thus,demonstrating the epileptogenesis of DRE will provide better understanding of the mechanism and treatment of intractable epilepsy.Recent evidences showed that malformations of cortical development（MCD）was identified as the main cause of DRE.There is about 37.6%⁵7.6%of patients who undergo epilepsy surgery are caused by MCD.Moreover,MCD accounted for near 80%in patients less than 3 years old with intractable epilepsy.MCD is composed of a wide spectrum of developmental disorders such as focal cortical dysplasia（FCD）,tuberous sclerosis complex（TSC）and polymicrogyria et al.The most typical type of MCD is FCD,which presents typical pathological changes in MCD-affected lesions with blur of the gray-white matter border and destruction of cortical lamellar structure.Moreover,FCD-related epilepsy is usually drug-resistant belonging to DRE.FCD is preliminary divided into simple and mixed types and can be further classified as FCD I and FCD II subtypes,which characterized by gray-white matter border abnormalities and lamellar structure malformations（FCD I and II）and sporadic malformed cells including balloon cells（BCs）（FCD IIb）and dysmorphic neurons（DNs）（FCD IIa and IIb）according to the FCD classification from ILAE in 2011.As a rare multisystem autosomal dominant disease,TSC can cause similar pathological changes in brain tissue such as large amount of DNs and giant cells（GCs）in FCD IIb patients.Although many research hypotheses recently attempted to demonstrate the epileptogenesis and mechanism of DRE such as Glutamate receptor hypothesis,GABA receptor hypothesis and Pacemaker GABA synaptic hypothesis et al,these hypotheses are difficult to clarify the epileptogenesis of FCD due to lacking enough evidences.Therefore,further exploring molecular genetics on the epileptogenesis of FCD will be assistant to improve the diagnosis and treatment of intractable epilepsy.Whole exome sequencing（WES）is the most widely targeted sequencing method with high throughput,accuracy and reliability and is across a wide range of applications including population genetics,genetic disease and cancer studies.As a delineated approach,WES presents an attractive advantage to whole-genome sequencing（WGS）for cost-effective alternatives.It provides coverage of more than 95%of the exons harboring the majority of the genetic variants and also facilitates the discovery and validation of common and rare variants such as single-nucleotide variants and de novo mutations associated with human neurologic diseases including epilepsy.To this end,four parts of experiments were conducted in our current study.First,preoperative EEG evaluation was performed between stereo-electroencephalography（SEEG）and Subdural EEG in DRE cases including FCD.Next,by WES and bioinformatics methods in FCD lesions,we aimed to detect the potential mutations in the gene exon region involved in FCD epileptogenic zone and further to screen potential candidate pathogenic genes associated with FCD.Third,we sought to validate these mutations of candidate genes in a larger FCD lesions and analyze the clinical correlation between these mutations and surgical prognosis to identify potential major pathogenic genes associated with FCD.Fourth,we further attempted to investigate the potential role of the candidate genes in the epileptic seizure using induced epileptic mice model and candidate gene knockout mice.The results in this study are presented as following:Part I.Preoperative EEG evaluation of DREBy preoperative EEG evaluation analysis in 100 DRE cases who underwent SEEG（n=48）and subdural EEG（n=52）,we found that SEEG is a safe,accurate and effective method for intracranial monitoring with low-associated complications,particularly regarding hemorrhage and infection.Part II.Screening of FCD associated gene mutations using WES1.By WES and bioinformatics analysis,we found that FCD IIa and FCD IIb cases shared 62 common mutations with level I-III risk.These common and specific gene mutations mainly distributed in the mTOR pathway-related genes,development-related genes,inflammation&tumors-related genes and metabolism-related genes.2.Using mass spectrometry SNP analysis,we detected suspicious pathogenic mutations in SCARB1、PMS1 and LTBP1 genes from peripheral blood and cortical epileptogenic zone of FCD II patients（2 cases of FCD IIa and 1 case of FCD IIb）.Furthermore,sanger DNA sequencing showed that seminary heterozygous mutations were present in the first-generation immediate family and proband from 2 cases family with FCD IIa.3.Through literatures review and analysis,we found that SCARB1 was a homo-oligomeric plasma membrane glycoprotein receptor for high density lipoprotein cholesterol and has been implicated in the mechanism of numerous central nervous system diseases including epilepsy.Therefore,we assume that SCARB1 is a potential pathogenic gene involved in the pathogenesis and epileptogenesis of FCD II.Part III.WES analysis of expanded FCD-associated DRE,focusing on SCARB1gene,focusing on SCARB1 geneThis section will focus on SCARB1 gene mutations in a larger number of DRE cases including patients with FCD and will investigate the clinical correlation between the mutations and surgical prognosis.DRE cases were divided into five groups including normal control（obtained from Database,n=200）,FCD group（n=26）,TSC group（n=8）,MRI negative group（n=27）and Acquired Etiology group（Encephalomalacia/Encephalatrophy/Tumor,n=14）.The results are obtained as following:1.WES and bioinformatics analysis showed that some pathogenic gene mutations were detected in 35 cases of 76 DRE cases（46%）.Meanwhile,positive pathogenic mutation rates of SCARB1 gene in five groups were as following:TSC group 100%（8/8）,MRI negative group 55.6%（15/27）,FCD group 30.7%（8/26）and acquired etiology group 7.1%（1/14）.These results suggest that SCARB1 mutation might be correlated with DRE.2.WES and bioinformatics analysis showed that some unknown pathogenic SCARB1mutations with I-III variants including frameshift mutation,splice variants and nonsynonymous mutation were detected in 7 cases of 76 DRE cases.The 7 cases are consisting of 5 cases with FCD（71.4%）and 2 cases with negative MRI（28.6%）.Pathogenic SCARB1 mutations were not detected in other three groups.These results further indicate that SCARB1 mutation is correlated with DRE.3.Bioinformatics analysis showed that III level mutation rate of SCARB1 gene in 200cases normal control data is 8.5%（17/200）but no I and II level mutation was detected.Meanwhile,in 76 DRE cases,level III mutation rate of SCARB1 gene is 9.2%（7/76）and level II mutation rate is 1.3%（1/76）.In 26 FCD cases,level III mutation rate of SCARB1gene is 19.2%（5/26）and level II mutation rate is 3.85%（1/26）.However,no level I mutation of SCARB1 gene was detected in both 76 DRE cases and 26 FCD cases.（P<0.01）.There is no significant difference in level III mutation rate between different groups（P>0.05）.As for level II mutation of SCARB1 gene,there was obviously increased mutation rate in DRE case without pathogenic mutation compared with normal controls（P<0.05）while there was significantly increased level II mutation rate in FCD group compared with normal controls（P<0.01）.Thus,these data suggested that SCARB1 mutation is correlated with FCD.4.By analysis of clinical correlation between gene mutations and surgical prognosis,we found that post-operative efficiency（Engel’s I-III level）is 96.6%and the postoperative excellent rate（Engel’s I-II level）is 79.3%in 29 FCD patients underwent surgical treatment.In post-operative 3 FCD patients with SCARB1 mutations,curative effect achieved at III level compared with I level（None）（P<0.05）.These data suggested that SCARB1 mutation is correlated with surgical prognosis of FCD.5.Clinical analysis indicates that FCD patients with others gene mutations rather than SCARB1 mutations still can act as ideal candidates for surgical treatment of FCD-associated epilepsy after removing the high-risk factors like mutations of ion channels gene SCN1A,KCNQ2 et al.Our finding suggests that SCARB1 gene may play critical role in epileptogenesis of refractory epilepsy,specifically in FCD-related DRE.Thus,the functional role of SCARB1 gene in pathogenesis of DRE need to be further investigated.Part IV.Exploring the role of SCARB1 in epileptic seizures based on mice modelsIn this section,we aimed to detect the SCARB1 expression in epileptic mice models and to determine whether SCARB1 deficiency is associated with neuronal hyperexcitability,thereby causing epileptic seizures.1.Here,we established an epileptic mouse model using wild-type C57BL/6 mice via kainic acid induction.Western blotting showed that SCARB1 protein was significantly up-regulated in CTX tissues upon kainic acid（KA）induction compared with the normal saline（NS）treatment.Immunofluorescence analysis showed that SCARB1 was mainly expressed on the membrane surface of neurons in the CTX tissues of acute epileptic mice model.These results revealed that SCARB1 could play an antiepileptic role,providing a potential target for antiepileptic treatment.2.To explore the role of SCARB1 in neuronal excitability,we established another epileptic mouse model using SCARB1^-/-mice by KA induction to test whether SCARB1deficiency can mitigate the hyperexcitable state in the epileptic brain.Electrophysiological recording analysis showed that SCARB1 knockout decreased the latent period of epileptic waves discharges and increased total duration of epileptic waves discharges,indicating that SCARB1 deficiency increased neuronal excitability in epilepsy.The results observed for LFPs in the KA-induced acute epilepsy model further indicated that SCARB1 may exert a protective role against seizures.In summary,our study suggests that:1.SEEG is a safe,accurate and effective method for DRE intracranial monitoring for its low-associated complications,particularly regarding hemorrhage and infection.2.In cortical lesions of FCD patients,we detected potential pathogenic gene mutations distributed in ion channels-related genes,NMDA receptor-related genes and development related genes and also detected suspicious pathogenic mutations of SCARB1、PMS1 and LTBP1 genes in FCD patients.Because SCARB1 is a homo-oligomeric plasma membrane cell surface glycoprotein receptor for high density lipoprotein cholesterol,which is involving in central nervous system diseases like epilepsy.Therefore,we suggest that SCARB1 may participate in the pathogenesis of FCD.3.By focusing on SCARB1 gene in a larger number of DRE cases with multiple etiology including FCD,we detected some unknown pathogenic SCARB1 mutations with I-III variants including frameshift mutation,splice variants and nonsynonymous mutation.Meanwhile,we found that FCD patients with SCARB1 mutation presented poor surgical prognosis.Therefore,SCARB1 gene might be a candidate of the major pathogenic genes,whose mutation may be involved in the pathogenesis of FCD and epileptic seizures by disturbing metabolic program of central nervous system.4.Furthermore,SCARB1 was mainly upregulated on the membrane surface of neurons in the CTX tissues of KA-induced acute epileptic seizures in WT mice.Moreover,we found that SCARB1 deficiency increased KA-induced neuronal hyperexcitability and epileptic seizures in SCARB1^-/-mice.In conclusion,SCARB1 gene may play critical role in pathogenesis and epileptogenesis of FCD-associated epilepsy as a potential candidate pathogenic gene.Meanwhile,SCARB1might be a potential target for FCD associated DRE treatment.