The Research Of New Mutations In Different Phenotypes Of Pediatric Epilepsy Patients

Epilepsy is one of the most common neurological disorders with 50 to 100 million affected worldwide,and 2 to 4 million new cases diagnosed each year.Epilepsy is a complex group of chronic brain disorders that are characterized by recurrent spontaneous seizures,and these can often begin in childhood.By rationally using anti-epilepsy drugs,most patients can be cured and have normal social ability.However,around one third of patients remain uncontrolled and become refractory epilepsy.Repeated and refractory seizures can cause long-term cognitive impairment,decreased social participation and significantly lower quality of life.Epilepsy is a heterogeneous disease with diverse clinical manifestations and causes,including altered ion channel expression,neurotransmitter signaling,synaptic structure,gliosis,and inflammation.Estimates of heritability from twin studies range from 25%to 70%.Although the range of heritability estimates is quite large,disparate studies using varied methods and studying divergent populations are all consistent in concluding that there is a substantial inherited component to epilepsy.However,in most cases encountered in the clinic practice,the underlying genetic aberrations are still difficult to interpret,which may be due to the unknown epileptic genes,complex inheritance patterns,and predisposing factors.Identifying the genetic background of a patient with epilepsy may help in diagnosis,guiding treatment,and providing genetic counseling for the patient and their family members.Next-generation sequencing(NGS),which is known for high throughput,effective,rapid,unbiased acquisition of large volumes of genetic sequence data with at ever decreasing costs,is capable of accuratly sequencing entire genomes by using small amounts of tissue.It has been instrumental in the discovery of many novel genes associated with disease.NGS has redefined the boundaries between many diseases by moving away from disease classifications based on phenotype to shared genetic categorizations.With the rapid progress of NGS techniques,our knowledge of the genetic etiology in many brain disorders such as epilepsy,autism and intellectual disability has expanded greatly.The American College of Medical Genetics and Genomics(ACMG)guidelines classify variants into pathogenic,likely pathogenic,uncertain significance,likely benign,and benign categories based on genetic information that includes population,functional,computational and segregation data,which provides scientific guidance for interpreting NGS results.In this study,we investigated epilepsy candidate genes in the pediatric epilepsy patients of Qilu hospital,Shandong University by using NGS and Sanger sequencing.Our priority was to separate common and benign genetic variants from those that are likely to be related to the cause of epilepsy,and we chose to apply the ACMG guidelines.We sought to investigate the genome in a heterogeneous set of patients with epilepsy and their parents,with the hope that we would identify novel mutations,confirm existing reports of genetic associations with epilepsy and explore the genetic etiology of pediatric epilepsy.This type of genetic information can not only provide genetic diagnoses of this patient cohort,but aslo provide an entry point into the biology of epilepsy that could eventually lead to new molecular treatment targets.Part I Researches of new mutations in different phenotypes of pediatric refractory epilepsyObjectivePediatric refractory epilepsy has a broad phenotypic spectrum with great genetic heterogeneity.Next-generation sequencing(NGS)combined with Sanger sequencing could help to understand the genetic diversity and the underlying mechanisms in pediatric epilepsy.In this study,we investigated 153 epilepsy candidate genes in a cohort of 172 pediatric refractory epilepsy patients.This study aimed to provide genetic diagnoses for this patient cohort and explore the genetic etiology of pediatric refractory epilepsy.MethodsWe collected and analyzed 172 cases of pediatric refractory epilepsy patients between the ages of 1 day to 14 years old.All patients were examined and diagnosed at the Pediatric Department in Qilu Hospital using a combination of patients’ illness history,previous history,family history,physical examinations,hematological examination,ambulatory or video electroencephalography(AEEG/VEEG)monitoring,magnetic resonance imaging(MRI)or computed tomography(CT)and developmental evaluation.Seizure types and epilepsy syndromes were diagnosed and classified according to the guidelines of International League Against Epilepsy(2014,2017).Blood samples of the patients and their parents were collected and 153 epilepsy related genes were sequenced by using Next-generation sequencing(NGS).The pathogenicity of mutations was assessed in accordance with American College of Medical Genetics and Genomics(ACMG)guideline.Statistical analysis was performed using SPSS 19.The yields of deleterious variants in patients with different onset age or family history were compared using the chi-squared test.Results1.In our 153-gene panel,51 genes show elevated expression,14 genes have low expression,and 88 of them exhibit medium levels of expression in brain.The 14 low-expression genes have been associated with epilepsy,including:ARG1,ARHGEF15,CASR,CHRNA2,DBH,DIAPH3,FOLR1,GABRA 6,GLRA 1,NID2,PROC,SLC13A5,SLC1 9A3,SRPX2.Specifically,among 51 elevated genes in brain,4 genes(GABRG2,GABBR2,GABRA1,GRIN1)show restricted brain expression.2.We identified 43 pathogenic or likely pathogenic variants in 40 patients(23.3%).Among these variants,74.4%(32/43)mutations were de novo and 60.5%(26/43)mutations were novel.Patients with onset age of seizures ≤ 12 months had higher yields of deleterious variants compared to those with onset age of seizures>12 months(P = 0.006).The family history did not affect whether or not a deleterious genetic variant was identified(P = 0.804).3.Variants in ion channel genes accounted for the greatest functional gene category(55.8%),with SCN1A coming first(16/43).81.25%(13/16)of SCN1A mutations were de novo and 68.8%(11/16)were novel in Dravet syndrome.4.Pathogenic or likely pathogenic variants were found in KCNQ2,STXBP1,SCN2A genes in Ohtahara syndrome.Deleterious variants in STXBP1,KCNT1,CDKL5,ADSL genes were found in West syndrome.Novel mutations in SYNGAP1 were found in Doose syndrome,a novel SLC2A1 mutation was found in GLUT1-DS and a de novo MECP2 mutation were found in Rett syndrome,TSC1,TSC2 variants were found in 60%patients with tuberous sclerosis complex patients.5.Other novel mutations detected in unclassified epilepsy patients involve SCN8A,CACNA1A,GABRB3,GABRA1,IQSEC2,TSC1,VRK2,ATP1A2,PCDH19,SLC9A6 and CHD2 genes.6.The clinical benefit of genetic testing in those patients with identified deleterious variants include helping diagnosis(n = 8),medication selection(n = 18),reproductive planning(n = 4),and treatment planning(n = 1).ConclusionWe identified 43 pathogenic or likely pathogenic variants,of which 26 mutations were novel and 32 were de novo.Variants in ion channel genes accounted for the largest category of gene in children with refractory epilepsy.Dravet syndrome is closely related to the SCN1A gene,which was the most frequently-appearing gene showing variants in our study.Novel and de novo mutations were found in Ohtahara syndrome,West syndrome,Doose syndrome,Rett syndrome,glucose transporter type 1 deficiency and tuberous sclerosis complex patients.Our results reinforce the importance and feasibility of precise genetic diagnosis for epilepsy,with the hope that in future,this will both aid in understanding the molecular pathophysiology and lead to new treatment targets.Part II Exome sequencing reveals new mutations in different phenotypes of pediatric epilepsy patientsObj ectiveEpilepsy is a heterogeneous disease.Disparate studies using varied methods and studying divergent populations are all consistent in concluding that there is a substantial inherited component to epilepsy.Whole-exome sequencing(WES)refers to sequencing of all protein-coding exons,has been widely used in pediatric epilepsy research.WES,which comprise around 1.0%to 1.5%of the human genome,enabes rapid and effective identification of the patient’s deleterious gene.In our research,we investigated 106 pediatric epilepsy patients with whole-exome sequencing.We provided genetic diagnoses for this patient cohort and further explored the genetic etiology of pediatric epilepsy.MethodsWe enrolled 106 pediatric epilepsy patients between ages of postnatal 1 day to 14 years old in the Department of Pediatrics of Qilu Hospital,China.Patients’ illness history,family history,previous history,physical examinations,hematological examination,developmental evaluation,24hr-ambulatory or 4hr-video electroencephalography(VEEG/AEEG)monitoring,magnetic resonance imaging(MRI)were collected.Blood samples were collected from the patients and their parents,and whole-exome sequencing were performed.The pathogenicity of mutations was assessed by the guidelines of the American College of Medical Genetics and Genomics(ACMG)guidelines.All variants identified by Illumina HiSeq X Ten sequencer were confirmed by Sanger sequencing.If copy number variations were indicated by NGS,then the samples were further tested by MS-MLPA.Results1.We identified 27 likely pathogenic or pathogenic variants in 25 patients(23.6%).92.6%(25/27)variants were de novo while 44.4%(12/27)variants were novel.Variants in ion channel genes were the most common,accounting for 66.7%,with SCNIA being the most frequent(1 5/27).2.93.3%(14/15)SCN1A mutations were de novo and 40%(6/15)were novel.Among the patients with SCN1A mutations,46.15%(6/13)had Dravet syndrome(DS)and 7.69%(1/13)were febrile seizures plus(FS+)patients.3.Four de novo PCDH19 variants that scored pathogenic or likely pathogenic were identified in four female patients,the second most common gene in this study.4.Pathogenic GNA Ol variants were first found in one progressive encephalopathy with edema,hypsarrhythmia,and optic atrophy-like syndrome(PEHO-like syndrome)in Chinese population.5.One de novo pathogenic SCN2A variant was found in an Ohtahara syndrome(OS)patient.Novel deleterious variants in CHD2 were found in one epilepsy with myoclonic astatic seizures(EMAS)patients.One deletion mutation in UBE3A was found in one Angelman syndrome patient that presented with typical Lennox-Gastauat syndrome clinical manifestations.6.Other deleterious mutations found in unclassified epileptic encephalopathy or unclassified epilepsy patients were in GABRG2,KCNQ2,STXBP1,SYNGAP1 genes.ConclusionWe identified 27 pathogenic or likely pathogenic variants,of which 44.4%were novel and 92.6%were de novo.Variants in ion channel genes accounted for the most cases in this cohort of pediatric epilepsy patients,with the SCN1A gene being the most common.One PEHO-like syndrome patient with GNAO1 mutation was first reported in Chinese population.One deletion mutation in UBE3A was found in one Angelman syndrome patient.Four PCDH19 variants were identified in four female patients.One SCN2A variant was found in an Ohtahara syndrome patient.Novel CHD2 variants were found in one epilepsy with myoclonic astatic seizures patients.Our results reinforce the importance and feasibility of precise genetic diagnosis in epilepsy,which can help clinicians improve the diagnosis and treatment of many diseases,especially rare diseases.This will improve our understanding of the molecular pathophysiology of epilepsy and eventually lead to new therapeutic methods.