Pathogenesis And Targeted Therapy Of Severe Epilepsy Phenotypes Caused By SCN1A Missense Variants Outside The Sodium Channel Core Region

PART Ⅰ ANALYSIS OF RISK FACTORS FOR PREDICTING SEVERE EPILEPSY PHENOTYPES ASSOCIATED WITH SCN1 A VARIANTSObjective: To summarize the clinical and genetic characteristics of patients with epilepsy associated with SCN1 A gene variants,analyze the risk factors leading to severe phenotypes,and evaluate its predictive value.Methods: The clinical data of 111 children with epilepsy associated with SCN1 A variants treated in the Affiliated Children’s Hospital of Chongqing Medical University from January 2015 to January 2022 were included.The development,treatment and prognosis of the children were followed up through outpatient follow-up and telephone.The main risk factors affecting severe phenotypes were analyzed by univariate analysis and multivariate logistic regression.Calculate the area under the receiver operating characteristic(ROC)curve(AUC)of risk factors and prognosis,and compare the predictive value of various risk factors through diagnostic test evaluation indicators.Results:(1)Among the 111 children,there were 51 males and 60 females.The median age of seizure onset was 6 months(2 months to 3years old),and 98 patients(88.3%)started within 1 year old.There were 65 patients with severe phenotypes(58.6%),mainly including 47 patients with Dravet syndrome(42.3%),16 patients with SCN1 A related developmental and epileptic encephalopathy(14.4%),and 2 patients with SCN1 A related refractory epilepsy(1.8%);there were 17 patients with intermediate epilepsy(15.3%);29 patients(26.1%)had mild phenotypes,including 14patients(12.6%)of genetic epilepsy with febrile seizures plus,10 patients(9.0%)of febrile seizures plus and 5 patients(4.5%)of febrile seizures.(2)There were at least 6 seizures types in this group,including 70 patients with unilateral or focal onset motor seizures(63.1%),38 patients with myoclonic seizures(38.8%),35 patients with absence or atypical absence seizures(31.5%),and 8 patients with atonic seizures(7.2%).76 patients(69.1%)had status epilepticus during the course of the disease.(3)Gene detection showed that the types of SCN1 A gene variants included: missense variants in 63 cases(56.8%),of which 30 cases(46.9%)were located in the sodium channel core region(S4-S6)and 26 cases(40.6%)were located outside the core region;there were 19 cases of nonsense variants(17.1%),13 cases of frameshift variants(11.7%),10 cases of splice site variants(9.0%),3 cases of microdeletion/ duplication(2.7%),2 cases of large deletions(1.8%),and 1 case of intron variants(0.9%).(4)Among the 111 patients,except 3(2.7%)were not treated with antiseizure medications(ASMs),the other 108 patients were treated with 1 to 8 kinds of ASMs,including 18 patients(16.2%)treated with single drug and 72 patients(64.8%)treated with three or more kinds of ASMs;16 cases(14.4%)had been treated with ketogenic diet.After treatment,the seizures were completely free in 11 cases(10.2%),markedly effective in 31 cases(28.7%),effective in 30 cases(27.8%),and invalid in 36 cases(33.3%);among them,37 patients(33.3%)used sodium channel blockers,and 36 patients were invalid or aggravated.(5)Multivariate logistic regression analysis showed that status epilepticus(OR=5.155)and severe variant types(OR=5.042)were the risk factors of severe phenotypes;the results of ROC analysis showed that when the patient’s seizure onset age was less than 7.5 months,presented status epilepticus,and variant types were missense variants in the core region,nonsense variants,frameshift variants,splice site variants and deletion/ duplication,the sensitivity,specificity and area under the ROC curve were 75.6%,87.1% and 0.833 respectively.(6)The missense variants of SCN1 A located in the core region of sodium channel,74.1% showed severe epilepsy phenotypes;83.3% of the missense mutations located outside the core region showed non-severe epilepsy phenotypes,and the difference was statistically significant(P<0.001).However,the relationship between the position of missense variants and clinical phenotype is not completely corresponding.Conclusion: The clinical phenotypes of epilepsy associated with SCN1 A variants are highly heterogeneous.The characteristic clinical manifestations are helpful to early identifying epileptic patients with SCN1 A variants.When the seizure onset age of patients is earlier than 7.5months,status epilepticus occurs and the type of SCN1 A variants is severe,we should be highly vigilant about the possibility of severe epilepsy phenotypes associated with SCN1 A variants.PART Ⅱ THE MECHANISM OF SEVERE EPILEPSY PHENOTYPES CAUSED BY SCN1 A MISSENSE VARIANTS LOCATED OUTSIDE THE SODIUM CHANNEL CORE REGIONObjective: To confirm the relationship between SCN1 A missense variants located outside the sodium channel core region and severe epilepsy phenotypes,and to explore the pathogenesis of severe epilepsy phenotypes caused by one SCN1 A missense variant(p.W1271R)located outside the core region.Methods:(1)Collect the clinical data of 111 children with epilepsy associated with SCN1 A variants,and analyze the clinical phenotypes;SCN1A missense variants located outside the sodium channel core region related to severe phenotypes were screened.(2)The species conservation of variant sites was compared and analyzed by T-Coffee online software.Through UCSF chimera 1.15 software,simulate the changes of protein three-dimensional structure before and after SCN1 A variants,and predict the effect of SCN1 A variants on the structure and function of Nav1.1.(3)The plasmid carrying wild-type SCN1 A and SCN1 A with p.W1271 R variant were constructed and transfected into HEK293 T cells respectively.The effects of this variant on electrophysiological parameters such as Nav1.1 current density,voltage dependent activation,voltage dependent steady-state inactivation and recovery after inactivation were performed by patch clamp experiment.(4)Extract Nav1.1 total protein and membrane protein,and the effect of variant on Nav1.1 expression was detected by western blot.Results:(1)In this group,4 patients showed severe epilepsy phenotypes while SCN1 A missense variants were located outside the core region,and one patient considered as Dravet syndrome was screened out.The patient had an early seizure onset,fever sensitivity,and myoclonic seizures after 1 year old,accompanied by cognitive and language retardation and abnormal gait.Extensive sharp-slow waves were observed in the bilateral brain in EEG.After treatment with 5 kinds of ASMs,epilepsy was controlled.(2)The SCN1 A gene displayed a missense variant in the non-core region in this patient(p.W1271R).The conservation analysis between species revealed that the heterotopic point of this variant was extremely conservative.Three-dimensional protein analysis revealed that the variant resulted in the transition of non-polar tryptophan into polar arginine.According to the Grantham formula,the D value of amino acid change was 101,and the physical and chemical properties of amino acid changed.(3)Patch clamp results showed that the W1271 R variant significantly reduced the peak current density of Nav1.1 in HEK293 T cells transfected with the variant plasmid(P<0.001);The half activation voltage of the W1271 R variant was significantly lower than that of wild type(P<0.001),and the slope factor was significantly higher than that of wild type(P<0.001);The half inactivation voltage of the W1271 R variant was significantly higher than that of wild type(P<0.001),and the slope factor was significantly higher than that of wild type(P<0.001).The fast time constant and slow time constant of the W1271 R variant after inactivation were greater than those of the wild type(P=0.024 and P<0.001).(4)Western blot showed that compared with the wild type,the expression of Nav1.1 total protein of the W1271 R variant decreased slightly,but the expression of Nav1.1 membrane protein of the W1271 R variant decreased significantly(P< 0.001).Conclusion: The SCN1 A W1271R variant located outside the core region resulted in the decrease of Nav1.1 overall current,the decrease of sodium channel sensitivity and availability,and the prolongation of recovery time;the expression of Nav1.1 membrane protein decreased.Therefore,it is speculated that the severe epilepsy phenotypes of this missense variant(W1271R)of SCN1 A may affect the transport and degradation of Nav1.1,resulting in the decrease of Nav1.1 expression on cell membrane;at the same time,it affects the electrophysiological characteristics of sodium channel,which eventually leads to the loss of Nav1.1 channel function.PART Ⅲ EXPLORATION OF TARGETED THERAPY FOR SEVERE EPILEPSY PHENOTYPES CAUSED BY NAV1.1 LOSS OF FUNCTIONObjective: To investigate the role of ubiquitination in the regulation of post-translational modification of Nav1.1 and the possibility of precise treatment through this target,and to study the possible mechanism of fenfluramine in improving the damage of Nav1.1 caused by SCN1 A variants.Methods: Proteasome inhibitors MG-132 and fenfluramine were used to intervene HEK293 T cells transfected with SCN1 A W1271R missense variant plasmid.The changes of Nav1.1 function and expression were detected by patch clamp and western blot.The eukaryotic expression plasmid of Nedd4-2 was constructed and co-transfected with wild-type SCN1 A plasmid into HEK293 T cells.The changes of Nav1.1 function and expression caused by overexpression of Nedd4-2 were analyzed by patch clamp and western blot.The interaction between Nav1.1 and Nedd4-2 was analyzed by immunoprecipitation technology,so as to explore the relationship between them.The total proteins of HEK293 T cells expressing wild-type and variant Nav1.1,and intervened by fenfluramine and MG-132 were extracted,and the expression of Nedd4-2 was detected by western blot.Results:(1)Patch clamp results showed that 8 μ M MG-132 intervention can increase the sodium current density of Nav1.1 W1271 R by about 30%,but the three concentrations of 6 μ M,8 μ M and 10 μ M MG-132 intervention did not significantly increase the peak current density of Nav1.1(P=0.389;P=0.124;P=0.124),also the half activation voltage(P=0.594;P=0.061;P=0.733)and slope factor(P=0.542;P=0.624;P=0.665)were not significantly different from those in the DMSO control group;the results of western blot showed that after 8 μ M MG-132 intervention,the expression of Nav1.1 W1271 R total protein did not change significantly(P=0.690),but the expression of membrane protein increased(P=0.020).(2)Patch clamp results showed that overexpression of Nedd4-2 significantly reduced the peak current density of Nav1.1(P=0.008)and did not affect the half activation voltage(P=0.108),but the slope factor was higher than that of transfected with empty plasmid group(P=0.046);Overexpression of Nedd4-2 did not affect the half inactivation voltage(P=0.159)and slope factor(P=0.666);western blot showed that overexpression of Nedd4-2 did not affect the expression of Nav1.1 total protein(P=0.401),but the expression of membrane protein decreased significantly(P=0.041).(3)Immunocoprecipitation test showed that Nav1.1 antibody could pull down Nedd4-2 protein,and there was an interaction between them.(4)Patch-clamp results showed that both 20 μM and 30 μ M fenfluramine intervention significantly increased the peak current density of Nav1.1(P=0.047;P=0.023),but the half activation voltage(P=0.529;P=0.640)and slope factor(P=0.650;P=0.261)were not significantly different from those in the DMSO control group;while western blot results indicated that the expression of Nav1.1 W1271 R total protein did not change significantly after 25 μM fenfluramine intervention(P=0.600),but the expression of membrane protein increased(P=0.034).(5)Western blot results showed that Nedd4-2 had no significant difference in HEK293 T cells expressing wild-type and W1271 R variant Nav1.1,also had no significant changes after fenfluramine and MG-132 intervention(P=0.111;P=0.874;P=0.937).Conclusion: Ubiquitination modification is involved in the transport and degradation of Nav1.1,which may be a potential target for new drug research and development or new use of old drugs.The intervention of this target is helpful to realize the precise treatment of patients with SCN1 A variant;Fenfluramine may alleviate the severity of seizures in SCN1 A related Dravet syndrome by increasing the expression of Nav1.1 membrane protein and increasing sodium current.