Efficacy Of Methylprednisolone Pulse Therapy For Electrical Status Epilepticus During Sleep In Children Andanalysis Of The Related Key Genes

Electrical status epilepticus during sleep(ESES)is known as a special EEG pattern,which shows diffuse and persistent spike-waves during slow-wave sleep.It can be seen in a variety of epileptic syndromes,the most of which is so-called ESES related syndrome.This syndrome is a spectrum of diseases involving cognitive and behavioral impairment.From the most benign end to the most severe end,it is followed by benign epilepsy with centrotemporal spikes(BECT),BECT variant,Landau-Kleffner syndrome(LKS)and epilepsy with continues spike-wave during slow-wave sleep(CSWS).In clinical practice,ESES related syndrome is not rare,which can show different types of epileptic seizures and abnormal cognitive development or degeneration(including language impairment,reading disability,attention impairment,memory deficits and so on).Some patients have refractory epilepsy,and the others have permanent cognitive disorders.Their social skills and life quality are both affected,which also brings about more severe burdens to the society and family.ESES related syndrome has complex etiology,including structural,immunological and genetic factors.The etiology of genetics is a hot topic,with monogenic mutations and copy number variations been reported.Based on the reports of discordant monozygotic twins and the low consistency of several genetic research results,it is suggested that the disease spectrum has complex genetic patterns and genetic heterogeneity.The development of nextgeneration sequencing is helpful to discover the unknown genes related to the diseases.With the help of bioinformatics methods,it is also helpful to further screen candidate genes.Through the study of patients’ genetic etiology,we can explore the pathogenesis,develop new drugs and implement individualized treatment.This study will analyze the factors influencing the curative effect from the aspects of clinical characteristics,laboratory indicators and candidate genes of the disease,so as to further explore the precision treatment.Part 1 Effect and prognosis of methylprednisolone pulse therapy for electrical status epilepticus during sleep in childrenObjective:To observe the curative effect of methylprednisolone pulse therapy(MPT)on electrical status epilepticus during sleep in children and to explore the related factors affecting the curative effect.Methods:From January 2016 to December 2019,a total of 52 clinical cases were recruited,including 34 cases of BECT variant,14 cases of CSWS and 4 cases of LKS.The average follow-up time was 2 years and 8 months.The seizure control and improvement of ESES in EEG were observed after methylprednisolone pulse therapy,and the adverse reactions and long-term recurrence rates were recorded.Patients were divided into the effective group and ineffective group according to the efficacy of MPT in the treatment of ESES.The age of onset,time interval from onset to methylprednisolone pulse therapy,gender,seizure type,ASM use,method of methylprednisolone pulse therapy,SWI before methylprednisolone pulse therapy,serum cytokine level and other indicators were compared between the two groups,and the possible factors affecting the efficacy were analyzed.Results:1.The effective rates of methylprednisolone pulse therapy on seizure control of BECT variant,CSWS and LKS were 73.5%,64.3%and 75%respectively.2.The effective rates of methylprednisolone pulse therapy in reducing SWI of EEG for BECT variant,CSWS and LKS were 70.5%,50%and 50%respectively.3.During the methylprednisolone pulse therapy,some children had adverse reactions such as hyperglycemia,hypertension,mental symptoms,headache,infection,Cushing’s syndrome,but most of the adverse reactions were mild and transient.After a follow-up of more than one year,33.3%of the children with effective treatment had recurrence.4.There was no significant difference in clinical indexes and serum cytokine level between the effective group and the ineffective group before treatment(P>0.05).Conclusion:Methylprednisolone pulse therapy is beneficial for children with electrical status epilepticus during sleep.But there are still some adverse reactions and recurrence.At present,the common clinical indicators and serum cytokine can not well predict the efficacy of the therapy.Part 2 Analysis of key genes related with the efficacy of methylprednisolone pulse therapy in patients with electrical status epilepticus during sleepObjective:To screen candidate genes of patients with ESES related syndrome,and to explore the key gene related with the efficacy of methylprednisolone pulse therapy.Methods:From January 2016 to December 2019,32 children with ESES related syndrome,who were treated with methylprednisolone pulse therapy were recruited,including 1 LKS,6 CSWS and 25 BECT variants.The clinical history,personal history,family history,physical examination,video EEG monitoring,magnetic resonance imaging,cognitive evaluation,treatment and follow-up of all patients were recorded,and the clinical biological information database was established.The venous blood of the patients and their parents were collected.All patients were detected by whole exome sequencing(WES)and copy number variations(CNV).A series of screening criteria were used to identify the possible candidate genes and mutations.Sanger sequencing was used to verify the variant and analyze the origin of parents.The venous blood of the affected grandfather of one patient was also collected for WES and CNV.The possible pathogenic gene was identified by comparative analysis among relatives.All kinds of gene related information were analyzed and standardized scores were gained.The candidate genes screened from patients who responded to methylprednisolone pulse therapy in the study were enriched by KEGG pathway.Results:1.A total of 132 mutations were detected in 102 genes,including 111 missense mutations,2 nonsense mutations,10 frameshift mutations,4 deletion mutations,3 insertion mutations and 2 classical splice site mutations.Among them,106(80.3%)were novel mutations.Six de novo mutations were indentified in gene KCNA2,KCND3,TSC2,NPRL3,TRIO and CACNA1H.2.Among these 102 genes,14 were consistent with previously reported pathogenic/candidate genes,including KCNA2,KCNQ2,KCNB1,KCNT1,SCN3A,SCN9A,PRRT2,BSN,SLC6A1,NID2,SRPX2,TSC2,RBFOX3 and RELN.However,no identical mutations exited between our study and the previous researches.3.Among the 132 gene mutations screened out,16 from 15 patients were rated as pathogenic or likely pathogenic according to ACMG guidelines.14 of them were rated as likely pathogenic,2 of them were rated as pathogenic.According to the statistical analysis,the detection rate of the pathogenic or likely pathogenic mutations according to the criteria of ACMG was not related to the severity of epilepsy.4.Among these 102 genes,the most commonly affected gene was CACNA1A,which was present in 4 patients.Other genes mutated in more than one patient included six genes(ADGRV1、CLCN2、SHANK3、BSN、TSC2、HECW2)in 3 patients,twelve genes(AUTS2、GJB2、CDH2、CDH8、DIAPH1、INTS1、KIF1A、PRRT2、RELN、SMARCA4、SYNGAP1、TAS2R19)in 2 patients.5.CNV results of all patients were negative.6.Several mutated genes were screened in the epilepsy family.By comparison,the gene related to epilepsy was only PRRT2(c.236C>T).The father of the patient was not affected but had carried the mutated gene.The mother was wild type.7.KEGG analysis showed that the most significant enrichment was GABAergic pathway,and seven genes were enriched in this pathway:SLC6A1,NSF CACNA1B,CACNA1A,CACNA1D,CACNA1C,GABRD.8.Some patients with GABAergic pathway related gene mutations,such as SLC6A1,NSF,CACNA1A and CACNA1C were sensitive to methylprednisolone pulse therapy.As to patients with RELN,KCNA2,KCND3 gene mutation,methylprednisolone pulse therapy is also effective.Conclusion:By sequencing the whole exome of 32 children with ESES related syndrome,we identified that a hot spot mutation c.649dupc of PRRT2 was related to the pathogenesis of ESES,as well as the novel mutations of c.236C>T and c.1238T>A in gene PRRT2 and KCNA2.In addition,we identified some additional candidate genes,including HECW2,KIF1A,TSC2,NPRL3,NSF,CHD2,CACNA1A and CLCN2.Patients with mutations in GABAergic pathway related genes responded to methylprednisolone pulse therapy.These discoveries will help to explore the pathogenesis and therapeutic targets of ESES.