Epileptic Encephalopathy Patients Research Articles

Exome data of developmental and epileptic encephalopathy patients reveals de novo and inherited pathologic variants in epilepsy-associated genes

PurposeIn Developmental and Epileptic Encephalopathies (DEEs), identifying the precise genetic factors guides the clinicians to apply the most appropriate treatment for the patient. Due to high locus heterogeneity, WES analysis is a promising approach for the genetic diagnosis of DEE. Therefore, the aim of the present study is to evaluate the utility of WES in the diagnosis and treatment of DEE patients. MethodsThe exome data of 29 DEE patients were filtrated for destructive and missense mutations in 1896 epilepsy-related genes to detect the causative variants and examine the genotype-phenotype correlations. We performed Sanger sequencing with the available DNA samples to follow the co-segregation of the variants with the disease phenotype in the families. Also, the structural effects of p.Asn1053Ser, p.Pro120Ser and p.Glu1868Gly mutations on KCNMA1, NPC2, and SCN2A proteins, respectively, were evaluated by molecular dynamics (MD) and molecular docking simulations. ResultsOut of 29, nine patients (31%) harbor pathological (P) or likely pathological (LP) mutations in SCN2A, KCNQ2, ATP1A2, KCNMA1, and MECP2 genes, and three patients have VUS variants (10%) in SCN1A and SCN2A genes. Sanger sequencing results indicated that three of the patients have de novo mutations while eight of them carry paternally and/or maternally inherited causative variants. MD and molecular docking simulations supported the destructive effects of the mutations on KCNMA1, NPC2, and SCN2A protein structures. ConclusionHerein we demonstrated the effectiveness of WES for DEE with high locus heterogeneity. Identification of the genetic etiology guided the clinicians to adjust the proper treatment for the patients.

Insights from Spatial Measures of Intolerance to Identifying Pathogenic Variants in Developmental and Epileptic Encephalopathies

Developmental and epileptic encephalopathies (DEEs) are a group of epilepsies with early onset and severe symptoms that sometimes lead to death. Although previous work successfully discovered several genes implicated in disease outcomes, it remains challenging to identify causative mutations within these genes from the background variation present in all individuals due to disease heterogeneity. Nevertheless, our ability to detect possible pathogenic variants has continued to improve as in silico predictors of deleteriousness have advanced. We investigate their use in prioritising likely pathogenic variants in epileptic encephalopathy patients' whole exome sequences. We showed that the inclusion of structure-based predictors of intolerance improved upon previous attempts to demonstrate enrichment within epilepsy genes.

Generation of a human induced pluripotent stem cell line (CIPi002-A) from an early infantile epileptic encephalopathy patient with a heterozygous mutation in SCN8A

SCN8A is linked to early infantile epileptic encephalopathy type 13 (EIEE13). We generate a human induced pluripotent stem cell (iPSC) line from a child diagnosed with EIEE, caused by SCN8A variation. The iPSC line expresses high pluripotency markers, retains SCN8A variation and is able to differentiate into three germ layers in vitro. The iPSC lines will provide useful resources for studying the pathogenesis and drug screening of SCN8A-related epilepsy.

Time‐limited alterations in cortical activity of a knock‐in mouse model of KCNQ2‐related developmental and epileptic encephalopathy

De novo missense variants in the KCNQ2 gene encoding the Kv7.2 subunit of voltage-gated potassium Kv7/M channels are the main cause of developmental and epileptic encephalopathy with neonatal onset. Although seizures usually resolve during development, cognitive/motor deficits persist. To gain a better understanding of the cellular mechanisms underlying network dysfunction and their progression over time, we investigated in vivo, using local field potential recordings of freely moving animals, and ex vivo in layers II/III and V of motor cortical slices, using patch-clamp recordings, the electrophysiological properties of pyramidal cells from a heterozygous knock-in mouse model carrying the Kv7.2 p.T274M pathogenic variant during neonatal, postweaning and juvenile developmental stages. We found that knock-in mice displayed spontaneous seizures preferentially at postweaning rather than at juvenile stages. At the cellular level, the variant led to a reduction in M ​​current density/conductance and to neuronal hyperexcitability. These alterations were observed during the neonatal period in pyramidal cells of layers II/III and during the postweaning stage in pyramidal cells of layer V. Moreover, there was an increase in the frequency of spontaneous network-driven events mediated by GABA receptors, suggesting that the excitability of interneurons was also increased. However, all these alterations were no longer observed in layers II/III and V of juvenile mice. Thus, our data indicate that the action of the variant is regulated developmentally. This raises the possibility that the age-related seizure remission observed in KCNQ2-related developmental and epileptic encephalopathy patients results from a time-limited alteration of Kv7 channel activity and neuronal excitability. KEY POINTS: The electrophysiological impact of the pathogenic c.821C>T mutation of the KCNQ2 gene (p.T274M variant in Kv7.2 subunit) related to developmental and epileptic encephalopathy has been analysed both in vivo and ex vivo in layers II/III and V of motor cortical slices from a knock-in mouse model during development at neonatal, postweaning and juvenile stages. Mcurrent density and conductance are decreased and the excitability of layer II/III pyramidal cells is increased in slices from neonatal and postweaning knock-in mice but not from juvenile knock-in mice. Mcurrent and excitability of layer V pyramidal cells are impacted in knock-in mice only at the postweaning stage. Spontaneous GABAergic network-driven events can be recorded until the postweaning stage, and their frequency is increased in layers II/III of the knock-in mice. Knock-in mice display spontaneous seizures preferentially at postweaning rather than at juvenile stages.

Detection of Deregulated miRNAs in Childhood Epileptic Encephalopathies.

The term "epileptic encephalopathy" is used to describe a possible relationship between epilepsy and developmental delay. The pathogenesis of developmental encephalopathies, independent of epilepsy, can be defined by genetic control mechanisms. The aim of this study was to investigate the use of miRNAs as serum biomarkers for the determination and discrimination of epileptic encephalopathies. Whole blood samples obtained from 54 individuals in 2 groups designated as epileptic encephalopathy patients' group (n = 24) and healthy controls (n = 30) were included in this study. The expression levels of 10 miRNAs were determined using qRT-PCR. After the determination of expression levels, the correlation of upregulated miRNA levels and Ki67 index was calculated using Pearson correlation test. The comparison of epileptic encephalopathy patients' group with healthy controls revealed the upregulation of one miRNAs (hsa-miR-324-5p) and downregulation of three miRNAs (hsa-miR-146a-5p, hsa-miR-138-5p, hsa-miR-187-3p). It has been determined that miRNAs with altered expression are an important factor in the formation of epileptic seizures and seizure-induced neuronal death. The fact that processes that play a key role in epiloptogenesis are under the control of miRNAs causes miRNAs to become meta-controllers of gene expression in the brain. We thought that further studies are needed to prove that especially hsa-miR-146a-5p, hsa-miR-138-5p, and hsa-miR-187-3p can be used as epileptic encephalopathy biomarkers. The detection of disease-specific miRNAs could contribute to the development of precision treatments.

Epileptic encephalopathy patients with SCN2A variant initiated by neonatal seizure.

Epileptic encephalopathy patients with SCN2A variant initiated by neonatal seizure.

Functional Connectivity Derived From Electroencephalogram in Pharmacoresistant Epileptic Encephalopathy Using Cannabidiol as Adjunctive Antiepileptic Therapy.

To explore brain function using functional connectivity and network topology derived from electroencephalogram (EEG) in patients with pharmacoresistant epileptic encephalopathy with cannabidiol as adjunctive antiepileptic treatment. Sixteen epileptic patients participated in the study, six of whom had epileptic encephalopathy with a stable dose of cannabidiol Epidiolex (CBD) as adjunctive therapy. Functional connectivity derived from EEG was analyzed based on the synchronization likelihood (SL). The analysis also included reconstructing graph-theoretic measures from the synchronization matrix. Comparison of functional connectivity data between each pathological group with the control group was carried out using a nonparametric permutation test applied to SL values between pairs of electrodes for each frequency band. To compare the association patterns between graph-theoretical properties of each pathological group with the control group, Z Crawford was calculated as a measure of distance. There were differences between pairs of electrodes in all frequency bands evaluated in encephalopathy epileptic patients with CBD adjunctive therapy compared with the control (p < 0.05, permutation test). In the epileptic encephalopathy group without CBD therapy, the SL values were higher than in the control group for the beta, theta, and delta EEG frequency bands, and lower for the alpha frequency band. Interestingly, patients who had CBD as adjunctive therapy demonstrated greater synchronization for all frequency bands, showing less spatial distribution for alpha frequency compared with the control. When comparing both epileptic groups, those patients who had adjunctive CBD treatment also showed increased synchronization for all frequency bands. In epileptic encephalopathy with adjunctive CBD therapy, the pattern of differences for graph-theoretical measures according to Z Crawford indicated less segregation and greater integration suggesting a trend towards the random organization of the network principally for alpha and beta EEG bands. This exploratory study revealed a tendency to an overconnectivity with a random network topology mainly for fast EEG bands in epileptic encephalopathy patients using CBD adjunctive therapy. It can therefore be assumed that the CBD treatment could be related to inhibition of the transition of the interictal to ictal state and/or to the improvement of EEG organization and brain function.

Pathogenic variants identified by whole-exome sequencing in 43 patients with epilepsy

BackgroundEpilepsy is a group of neurological disorders characterized by recurrent epileptic seizures. Epilepsy is affected by many factors, approximately 20–30% of cases are caused by acquired conditions, but in the remaining cases, genetic factors play an important role. Early establishment of a specific diagnosis is important to treat and manage this disease.MethodsIn this study, we have recruited 43 epileptic encephalopathy patients and the molecular genetic analysis of those children was performed by whole-exome sequencing (WES).ResultsFourteen patients (32.6%, 14/43) had positive genetic diagnoses, including fifteen mutations in fourteen genes. The overall diagnostic yield was 32.6%. A total of 9 patients were diagnosed as pathogenic mutations, including 4 variants had been reported as pathogenic previously and 6 novel variants that had not been reported previously. Therefore, WES heralds promise as a tool for clinical diagnosis of patients with genetic disease.ConclusionEarly establishment of a specific diagnosis, on the one hand, is necessary for providing an accurate prognosis and recurrence risk as well as optimizing management and treatment options. On the other hand, to unveil the genetic architecture of epilepsy, it is of vital importance to investigate the phenotypic and genetic complexity of epilepsy.

Generation of a human iPSC line from an epileptic encephalopathy patient with electrical status epilepticus during sleep carrying KCNA2 (p.P405L) mutation.

Mutations in KCNA2 gene, encoding for the voltage-gated K+ channel Kv1.2, has been reported to be associated with epilepsy disorders. This channel is mainly expressed in the central nervous system and plays an important role in neuronal excitability and neurotransmitter release. Herein, we generated an induced pluripotent stem cell (iPSC) line from the peripheral blood mononuclear cells of an eight-year-old girl with epileptic encephalopathy and electrical status epilepticus during sleep carrying a mutation (c.1214C>T, p.Pro405Leu) in KCNA2. These iPSCs exhibited stable amplification, expressed pluripotent markers, and differentiated spontaneously into three germ layers in vitro.

Epilepsy subtype-specific copy number burden observed in a genome-wide study of 17 458 subjects.

Cytogenic testing is routinely applied in most neurological centres for severe paediatric epilepsies. However, which characteristics of copy number variants (CNVs) confer most epilepsy risk and which epilepsy subtypes carry the most CNV burden, have not been explored on a genome-wide scale. Here, we present the largest CNV investigation in epilepsy to date with 10 712 European epilepsy cases and 6746 ancestry-matched controls. Patients with genetic generalized epilepsy, lesional focal epilepsy, non-acquired focal epilepsy, and developmental and epileptic encephalopathy were included. All samples were processed with the same technology and analysis pipeline. All investigated epilepsy types, including lesional focal epilepsy patients, showed an increase in CNV burden in at least one tested category compared to controls. However, we observed striking differences in CNV burden across epilepsy types and investigated CNV categories. Genetic generalized epilepsy patients have the highest CNV burden in all categories tested, followed by developmental and epileptic encephalopathy patients. Both epilepsy types also show association for deletions covering genes intolerant for truncating variants. Genome-wide CNV breakpoint association showed not only significant loci for genetic generalized and developmental and epileptic encephalopathy patients but also for lesional focal epilepsy patients. With a 34-fold risk for developing genetic generalized epilepsy, we show for the first time that the established epilepsy-associated 15q13.3 deletion represents the strongest risk CNV for genetic generalized epilepsy across the whole genome. Using the human interactome, we examined the largest connected component of the genes overlapped by CNVs in the four epilepsy types. We observed that genetic generalized epilepsy and non-acquired focal epilepsy formed disease modules. In summary, we show that in all common epilepsy types, 1.5-3% of patients carry epilepsy-associated CNVs. The characteristics of risk CNVs vary tremendously across and within epilepsy types. Thus, we advocate genome-wide genomic testing to identify all disease-associated types of CNVs.

Frequently Identified Genetic Developmental and Epileptic Encephalopathy: A Review Focusing on Precision Medicine

In this article, we reviewed current knowledge regarding gene-specific therapies for some developmental and epileptic encephalopathy caused by genes with high diagnostic yields, and which are therefore, also more frequently encountered by physicians during treatment, including ALDH7A1, CDKL5, KCNQ2, KCNT1, SCN2A, SCN8A, STXBP1, and SYNGAP1. Among these therapies, the ones directly targeting causative mutations are retigabine in KCNQ2 encephalopathy and quinidine in KCNT1 encephalopathy. However, despite promising results in vitro, the outcomes related to these therapies were disappointing when administered to patients. Considering the pathologic mechanisms of causative mutations, sodium channel blockers are recommended for patients with KCNQ2 mutations, infantile epileptic encephalopathy patients with SCN2A mutations, and patients with SCN8A mutations. Levetiracetam can be considered for patients with STXBP1 mutations. Keywords: Epilepsy; Precision medicine

De novo variants in the alternative exon 5 of SCN8A cause epileptic encephalopathy

PurposeAs part of the Epilepsy Genetics Initiative, we re-evaluated clinically generated exome sequence data from 54 epilepsy patients and their unaffected parents to identify molecular diagnoses not provided in the initial diagnostic interpretation.MethodsWe compiled and analyzed exome sequence data from 54 genetically undiagnosed trios using a validated analysis pipeline. We evaluated the significance of the genetic findings by reanalyzing sequence data generated at Ambry Genetics, and from a number of additional case and control cohorts.ResultsIn 54 previously undiagnosed trios, we identified two de novo missense variants in SCN8A in the highly expressed alternative exon 5 A—an exon only recently added to the Consensus Coding Sequence database. One additional undiagnosed epilepsy patient harboring a de novo variant in exon 5 A was found in the Ambry Genetics cohort. Missense variants in SCN8A exon 5 A are extremely rare in the population, further supporting the pathogenicity of the de novo alterations identified.ConclusionThese results expand the range of SCN8A variants in epileptic encephalopathy patients and illustrate the necessity of ongoing reanalysis of negative exome sequences, as advances in the knowledge of disease genes and their annotations will permit new diagnoses to be made.

GABBR2 mutations determine phenotype in rett syndrome and epileptic encephalopathy.

Rett syndrome (RTT) and epileptic encephalopathy (EE) are devastating neurodevelopmental disorders with distinct diagnostic criteria. However, highly heterogeneous and overlapping clinical features often allocate patients into the boundary of the two conditions, complicating accurate diagnosis and appropriate medical interventions. Therefore, we investigated the specific molecular mechanism that allows an understanding of the pathogenesis and relationship of these two conditions. We screened novel genetic factors from 34 RTT-like patients without MECP2 mutations, which account for ∼90% of RTT cases, by whole-exome sequencing. The biological function of the discovered variants was assessed in cell culture and Xenopus tropicalis models. We identified a recurring de novo variant in GABAB receptor R2 (GABBR2) that reduces the receptor function, whereas different GABBR2 variants in EE patients possess a more profound effect in reducing receptor activity and are more responsive to agonist rescue in an animal model. GABBR2 is a genetic factor that determines RTT- or EE-like phenotype expression depending on the variant positions. GABBR2-mediated γ-aminobutyric acid signaling is a crucial factor in determining the severity and nature of neurodevelopmental phenotypes. Ann Neurol 2017;82:466-478.

Variable expressivity of a likely pathogenic variant in KCNQ2 in a three-generation pedigree presenting with intellectual disability with childhood onset seizures.

KCNQ2 has been reported as a frequent cause of autosomal dominant benign familial neonatal seizures. De novo likely pathogenic variants in KCNQ2 have been described in neonatal or early infantile onset epileptic encephalopathy patients. Here, we report a three-generation family with six affected patients with a novel likely pathogenic variant (c.628C>T; p.Arg210Cys) in KCNQ2. Four family members, three adults and a child, presented with a childhood seizure onset with variability in the severity of seizures and response to treatment, intellectual disability (ID) as well as behavioral problems. The two youngest affected patients had a variable degree of global developmental delay with no seizures at their current age. This three-generation family with six affected members expands the phenotypic spectrum of KCNQ2 associated encephalopathy to KCNQ2 associated ID and or childhood onset epileptic encephalopathy. We think that KCNQ2 associated epileptic encephalopathy should be included in the differential diagnosis of childhood onset epilepsy and early onset global developmental delay, cognitive dysfunction, or ID. Furthermore, whole exome sequencing in families with ID and history of autosomal dominant inheritance pattern with or without seizures, may further broaden the phenotypic spectrum of KCNQ2 associated epileptic encephalopathy or encephalopathy.