Genomic Study Of Hereditary Epilepsy With Developmental Delay In Children

Epilepsy and intellectual/developmental disabilities?ID/DD?are common childhood neurological disorders.ID/DD is one of the major comorbidities of epilepsy,and the prevalence of epilepsy in children with ID/DD is about 22%-25%.In 2017,the international federation against epilepsy further identified six categories of etiology for epilepsy,including structural,hereditary,metabolic,immunological,infectious and unknown causes,highlighting the genetic etiology of epilepsy.Between 40%and 50%of epilepsy cases are unknown causes,and about 30%of them are hereditary.Epilepsy and ID/DD are both heterogeneous in clinical and genetical,which poses a challenge to the search for etiological diagnosis.The etiology of these two diseases is complex and varied,and most of them are unknown.Although frequent seizures can lead to cognitive and motor deterioration,genetic factors are considered to be underlying mechanisms in the etiology of epilepsy and ID/DD in children,especially in infants.When epilepsy occurs simultaneously with ID/DD,it is often difficult to treat and has a poor prognosis.Therefore,understanding the genetic background and pathogenesis of epilepsy and ID/DD is valuable,not only for diagnosis and prognosis,but also for genetic counseling and treatment.Advances in genomic testing have contributed to identify the genetic etiology of an increasing number of children with epilepsy and ID/DD.These genes are involved in different pathways,including ion channels and cellular signaling,DNA repair,metabolic pathways and so on.These novol genetic diagnoses provide an opportunity to characterize etiological specificity and contribute to a better understanding of the comorbidity and prognosis of epilepsy.Establishing a etiological diagnosis can also indicate prognosis or anticipated clinical processes including the risk of recurrence of epilepsy,avoid unnecessary testing,and manage the disease appropriately in the medical and family setting,with effective prenatal diagnosis,to ensure optimal health,social and medical service satisfaction for children and families.It is difficult to isolate pathogenic gene mutations from a large number of possible candidate genes by Sanger sequencing in children of epilepsy and ID/DD with different clinical phenotypes.In recent years,clinical genetic testing capabilities have expanded different techniques and strategies.Next-generation sequencing?NGS?provides the ability to analyze multiple genes simultaneously,and more accurate and efficient.12.5%to 70%of the children with epilepsy or ID/DD got the hereditary diagnosis by NGS,and the difference of the results may be related to the selection of cases,the detection method and the differences in the genes number of panels,which occupy high detection rate of detection method is usually whole exome sequencing?WES?.WES combined with chromosome microarray?CMA?and multi-junction probe amplification?MLPA?,can detect most of the pathogenic variations in epilepsy and ID/DD,including copy number variation?CNV?,providing an effective diagnostic tool for epilepsy and ID/DD patients.PCDH19 has become the second related gene of epilepsy after SCN1A.The heterozygous pathogenic variant of the gene is inherited in a rare X-linked pattern,in which heterozygous females are affected,whereas hemizygous males are not.PCDH19 related diseases are known to cause early onset epilepsy in women,which is characterized by cluster seizures induced by fever.In most cases,the onset age is within the first year of life.PCDH19 related epilepsy is characterized by incomplete penetrance and phenotypic heterogeneity.However,the detection rate of PCDH19 in children with fever sensitive epilepsy and the specific clinical phenotype recognition of PCDH19 related cluster epilepsy are rarely reported.In this study,we used WES to detect 240 epileptic children with ID/DD who were admitted to Qilu Hospital of Shandong University and Linyi people's Hospital from January 2017 to June 2019.We also used the Sanger sequencing to test the possible pathogenic single nucleotide mutation and small indels,and used quantitative PCR,CMA,and MLPA to verify the pre-determined CNV.These children were genetically diagnosed,and the research also help us explain the genetic background of epilepsy and ID/DD with different phenotypes.We also screened 152 epilepsy children with fever sensitive in Qilu Hospital of Shandong University and Linyi people's Hospital from January 2015 to June 2019,and found PCDH19 mutations in 10 female probands.We summarized the clinical characteristics of the family patients to further understand the clinical characteristics and mutation characteristics of PCDH19 Girls ClusteringEpilepsy.In addition,we discussed the potential impact of genetic diagnosis on treatment strategies.This study provides genetic evidence for diagnosis and treatment and guidance of eugenics counseling for children with epilepsy and ID/DD,and provides a research entry point for the pathogenesis of such children,which is helpful to explore new molecular therapy targets.Part ? Genetic etiology of epilepsy with intellectual/developmental disabilities in childrenObjectiveThe clinical phenotype and genetic heterogeneity of epilepsy with intellectual/developmental disabilities?ID/DD?in children are relatively different.In this study,we used whole exome sequencing?WES?combined with quantitative PCR,CMA,MLPA to detect the children with epilepsy and ID/DD.Our goal is to carry out genetic diagnosis of these children and find clues to help us explain the genetic background of epilepsy and ID/DD with different phenotypes,so as to further understand the genetic diversity and potential pathogenesis of epilepsy with ID/DD in children,and to provide evidence for accurate diagnosis and treatment and genetic guidance of eugenics counseling.MethodsChildren with epilepsy and ID/DD were collected from the Department of Pediatrics,Qilu Hospital of Shandong University and Linyi people's Hospital from January 2017 to June 2019.To inquire the medical history in detail,record the age,gender,age of seizures onset,seizure types,psychomotor development,family history,maternal and pregnant history,previous special medical history,diagnosis and treatment process and physical examination,and further collect the results of video EEG,brain MRI,intelligence test,etc.The informations were collected every three months through outpatient and telephone follow-up.The blood samples of patients and their parents were collected and sequenced by WES,and Sanger sequencing was used to verify the detected possible pathogenic single nucleotide mutations and indels.The predicted CNV was verified by quantitative PCR,CMA,MLPA,and then analyzed comprehensively.The pathogenicity of the mutation was evaluated according to the guidelines of the American Society of medical genetics and genomics?ACMG?.The data were analyzed by SPSS 19.0 to compare the detection rate of pathogenic mutations in children with different clinical phenotypes.Results1.A total of 240 epileptic children with ID/DD were enrolled in this study.There were 122 males and 118 females,including 58 West syndrome,31 Dravet syndrome,12 Otahara syndrome,three West syndrome converted to Lennox Gastaut syndrome,four Lennox Gastaut syndrome,two Doose syndrome,two epilepsy of infancy with migrating focal seizures,26 unclassified early epileptic encephalopathy,and 102 children diagnosed with unclassified epilepsy patients with ID/DD because of nonspecific clinical manifestations.Dravet syndrome,epilepsy of infancy with migrating focal seizures and unclassified early epileptic encephalopathy were the most common ones.The detection rate of pathogenic mutation is high in the children who have earlier onset age and fever sensitive.2.113 pathogenic or likely pathogenic mutations were detected in 240 epileptic children with ID/DD,among which ten were CNV mutations,103 were gene mutations,and 81.55%mutations were denovo.SCN1A,PCDH19,STXBP1,MECP2,ALDH7A1,KCNQ2,CHD2,KCNT1,CDKL5,FOXG1,GABRA1,MEF2C,SCN8A and TBC1D24 are common genes of epilepsy with ID/DD.The gene with the highest mutation frequency was SCN1A.3.Some genes with low diagnostic rate of epilepsy with ID/DD were identified,some of which were rare causes of epilepsy?ALG14,ANKRD11,BRWD3,CACNA1A,CASK,CNNM2,CUX2,DEPDC5,DNM1,EEF1A2,EIF2S3,FTSJ1,GABRB3,KCNQ3,NEXMIF,OFD1,PACS1,PAFAH1BI,PHF21A,PIGA,SYNCAP1,WDR62,SLC6A1,SLC6A8,SLC9A6,etc.?,and many rare diseases were diagnosed.4.41.75% of pathogenic or likely pathogenic mutations were genes which encoding ion channels,and voltage-gated sodium ion and potassium ion channel gene mutations accounted for the highest proportion.Genes encoding chromatin remodeling and transcriptional regulators,enzyme/enzyme regulators,cell adhesion proteins,synaptic support proteins,transporter/receptor,and mTOR channel regulatory proteins also participate in the pathogenesis of epilepsy with ID/DD.5.Ten cases?10/240,4.17%?had pathogenic CNV mutations,accounting for 8.85%of the total positive samples.Four cases of angleman syndrome and one case of Xp11.23-p11.22 repetitive syndrome were diagnosed.6.This study evaluated the clinical benefits of genetic and molecular diagnosis of epilepsy with ID/DD,including diagnosis,precise treatment,genetic consultation and establishment of long-term monitoring and follow-up.Conclusion1.The detection rate of pathogenic or likely pathogenic mutations in epilepsy patients with ID/DD was 47.08%,91.15%of them were gene related mutations,8.85%were CNV mutations,most of them were denovo.2.SCN1A,PCDH19,STXBP1,MECP2,ALDH7A1,KCNQ2,CHD2,KCNT1,CDKL5,FOXG1,GABRA1,MEF2C,SCN8A and TBC1D24 are common genes of epilepsy and ID/DD.Ion channel gene mutations were the most important type of gene mutations.3.60.19%of the mutations were novol mutations.They had identified a variety of rare pathogenic genes and phenotypes,and expanded the genotype and clinical phenotype spectrum related to epilepsy with ID/DD.4.The importance and feasibility of gene accurate diagnosis and treatment were further confirmed through the evaluation of the clinical benefits after genetic diagnosis of epileptic patients with ID/DD.Part ? Novel and de novo mutations of PCDH19 in Girls Clustering EpilepsyObjectivePCDH19 has become the second most relevant gene in epilepsy after SCN1A.Seizures in girls cluster epilepsy?PCDH19-GCE?often provoked by fever.Further studying of the genetic characteristics and clinical phenotype spectrum of PCDH19-GCE mutation in order to understand the genetic diversity and pathogenesis of PCDH19-GCE,and provide evidence for accurate diagnosis and guidance of eugenics counseling.MethodsWES was performed on the children with fever sensitive epilepsy who were admitted to Qilu Hospital of Shandong University and Linyi people's Hospital from January 2015 to June 2019.The seizure types,onset age,treatment process,growth and development history,past history,family history,physical examination,intelligence test,cranial magnetic resonance imaging?MRI?and video EEG were analyzed and followed up.ResultsIn this study,10 girls with PCDH19 mutations were detected in 152 children with fever sensitive epilepsy.Eight PCDH19 point mutations and one deletion of the whole gene were found in 10 female probands whom had clustered seizures,four novol mutations and four reported mutations.Seven mutations were located in exon 1 and one in exon 6.Two missense mutations?c.1142A>G/p.Asn381Ser;c.790G>C/p.Asp264His?,one nonsense mutation?c.1804C>T/p.Arg602*?,four frameshift mutations?c.577delG/p.Glul93Lysfs*19c.134 135del/p.Asp45Glyfs*43;c.1091dupC/p.Tyr366Leufs*10;c.2859₂860insT/p.Gly954Trpfs*15?,one frameshift mutation?c.352₃54del/p.Glu118del?.There were 16 epilepsy patients?two dead?in 10 proband families with PCDH19 mutations.The onset age varies from five months to 20 years.Psychosis occurred in one case.There was no epileptiform discharge in interictal EEG.Ictal EEG recorded four cases originate from frontal area,two from parietal and one from occipital area.There was no obvious abnormality in brain MRI except two cases with enlargement of lateral ventricle.Almost all patients were insensitive to antiepileptic drugs.Eight patients needed midazolam to help control cluster seizures.During the recent follow-up?patients aged two years and two months to 57 years old?,one patient stopped antiepileptic drugs,four patients were treated with single drug,four patients were treated with two drugs,and five patients were treated with multi drugs.The interval of seizure free was from three months to 13 years.There was no significant difference in clinical phenotype and drug efficacy in the same family.Conclusion1.PCDH19 mutations can be hereditary or denovo.PCDH19 related epilepsy has phenotypic heterogeneity.The phenotypes of PCDH19 mutations include PCDH19-GCE with or without ID/DD,autism or psychosis,and asymptomatic male carriers.2.PCDH19-GCE is characterized by fever sensitive cluster of focal seizures or tonic clonic seizures,most of which don't have status epilepticus.3.The onset age of PCDH19-GCE can be from infants to adulthood.4.Our report expands the gene mutation spectrum and clinical phenotype of PCDH19 related epilepsy.