The Clinical And Molecular Genetic Study Of 333 Children With Epilepsy

Objective: To study the clinical features and prognoses of children with genetically related epilepsy.To analyze the relationship between the mutant genes and epilepsy,as well as the relationship between genotypes and phenotypes of the disease,and to explore the molecular genetic mechanisms of epilepsy by whole genome sequencing(WGS),so as to provide evidence for definitive diagnosis,accurate treatment and genetic counseling.Methods: From October 2016 to December 2017,333 family samples of probands with genetically related epilepsy were collected in the Department of Neurology of Shenzhen Children's Hospital.The complete pedigree and clinical data of the probands were established and followed up.WGS and Sanger verification were performed for genetic testing.The clinical phenotypes,treatments,prognoses and genotypes of the patients were analyzed.Results: 1.The onset age of most children with refractory or genetically related epilepsy was relatively young,about 65% occurred within 1 year old.The ratio of male to female was 1.45:1.There were two or more types of clinical seizure in 30% of patients.Epileptic syndromes included 71 cases of West Syndrome(WS),21 cases of Ohtahara Syndrome(OS),11 cases of Dravet Syndrome(DS),10 cases of Lennox-Gastaut Syndrome(LGS),7 cases of epilepsy with continuous spike-waves during slow-wave sleep(CSWS),1 case of DOOSE Syndrome,1 case of epilepsy of infancy with migrating focal seizures(EIMFS),and 48 cases of epileptic encephalopathy of unknown causes,etc.2.25% of patients were complicated with malformations in cardiovascular system,genitourinary system,digestive system,etc.5% of them had multiple malformations in different systems.The imaging of brain showed 56% of patients had brain malformations,brain dysplasia,brain atrophy,etc.81% of patients needed two or more kinds of anti-epileptic drugs.More than 20% of patients were treated with ketogenic diet.According to Engel grade,49% of patients achieved grade?and the total effective rate was 67%.3.The results of WGS: 186 candidate causal single nucleotide variants(SNVs)were detected in 158 cases,15 copy number variants(CNVs)of exons were identified in 15 cases and 20 CNVs of large fragments were found in 20 cases.The total detection rate of candidate causal variants was 54%,while the rate of SNV was 47% and CNV was 10%,respectively.Eighty-four pathogenic or likely pathogenic variants of SNV were identified in 71 patients,with the detection rate 21%,of which 45(54%)variants were novel.The results of sanger verification: 46(55%)variants were de novo,18(21%)variants were genetic,and the origins of 20(24%)variants were unknown.There were 104(56%)missense variants,23(12%)nonsense variants,35(19%)frameshift variants,17(9%)splicing variants of the mutant genes,the other 7(4%)variants included in-frame deletion or insertion,start codon variant,stop codon deletion,intron variant,etc.4.The relatively common recurrent epilepsy-related genes SCN1A?TSC2?TSC1?GNAO1?WWOX?ZEB2?BCKDK?CDKL5?PAFAH1B1?PCDH19?STXBP1?SUOX were detected in our research.Among them,the first three genes with highest detection rate of variants were SCN1A?TSC2 and TSC1.5.We speculated TARS2 might be related to epilepsy and involved in the development of the disease,which expands the clinical spectrum of the gene.6.Multiple gene findings were noted in 9% of patients,candidate causal variants were detected in two genes or both SNVs and CNVs were identified in the same patients.7.The recurrent CNVs were 17p13.3?16p11.2?15q11.2?7q11.23 deletions in our research.8.The phenotype of epilepsy was found in the patient with 7q31 deletion.It is presumed FOXP2?IMMP2L?KCND2 in the CNV are associated with epilepsy,which could be the participating factors of epilepsy in the patient.9.Seventeen cases of lissencephaly with epilepsy were demonstrated in our study.41% of the cases were point variants or deletions of PAFAH1B1,which was the main genetic cause of the disease.10.Three cases of Mowat-Wilson Syndrome(MWS)all had nonsense variants.It is speculated that the more forward the truncated sites in the ZEB2 protein locate,the more functional domains would lose,the severer the phenotypes would be.11.KCNH1?PRKCG?CHRNA2?CTSD genes were identified to be associated with infantile spasm.12.The clinical heterogeneity of Tuberous sclerosis(TSC)was considerable.Phenotypes caused by variants of TSC2 were more serious than that caused by variants of TSC1.We should pay attention to the possibility of mosaicism in the patients with negative genetic results.13.The clinical phenotype of DS was related to the types and locations of variants in the SCN1 A gene.Truncated variants,or the missense variants located in the pore regions and voltage sensitive regions of Nav1.1 showed greater influence on the clinical phenotypes.14.One case of glucose transporter 1 deficiency syndrome(GLUT1-DS)and pyridoxine-dependent epilepsy(PDE)were identified by WGS.The prognoses of the patients were significantly improved after appropriate treatment.15.One case of WWOX homozygous variants and a case of SUOX compound heterozygous variants were detected by WGS,through which we provided accurate genetic counseling for the parents.16.The genetic detection results of 79 patients were negative by whole exome sequencing and gene panels,while 6 pathogenic or likely pathogenic SNVs and 6 CNVs were detected by WGS,which increased the detection rate of variants by 15%.Conclusion: 1.The detection rate of candidate causal variants(SNV/CNV)was 54% in our study.Forty-five novel variants were identified,which expanded the genotypical spectrum of relative diseases.2.The detection rate of CNVs was 10%,suggesting that CNV is one of the important genetic mechanisms of epilepsy.3.It is speculated that TARS2?FOXP2?IMMP2L?KCND2 might be related to epilepsy and involved in the development of the disease,which provides a study direction for the genetic etiology of epilepsy.4.The clinical spectrums of relative genes were expanded in our study,which provided clinical support for the interpretation of heterogeneity and complexity of related disease.5.The relationships were enriched between phenotypes and genotypes of lissencephaly,MWS,WS,TSC and DS,etc.6.WGS was used to diagnose some rare treatable genetic diseases,which improved the prognoses of the patients.7.WGS is of great significance for the evaluation on the prognosis of epilepsy,precise treatment and genetic counselling.8.For the cases with negative genetic detection results,the detection rate of variants could be increased by 15% through WGS in our study.