Impaired neuronal activity and differential gene expression in STXBP1 encephalopathy patient iPSC-derived GABAergic neurons.

Syntaxin-binding protein 1 (STXBP1; also called MUNC18-1), encoded by STXBP1, is an essential component of the molecular machinery that controls synaptic vesicle docking and fusion. De novo pathogenic variants of STXBP1 cause a complex set of neurological disturbances, namely STXBP1 encephalopathy (STXBP1-E) that includes epilepsy, neurodevelopmental disorders and neurodegeneration. Several animal studies have suggested the contribution of GABAergic dysfunction in STXBP1-E pathogenesis. However, the pathophysiological changes in GABAergic neurons of these patients are still poorly understood. Here, we exclusively generated GABAergic neurons from STXBP1-E patient-derived induced pluripotent stem cells (iPSCs) by transient expression of the transcription factors ASCL1 and DLX2. We also generated CRISPR/Cas9-edited isogenic iPSC-derived GABAergic (iPSC GABA) neurons as controls. We demonstrated that the reduction in STXBP1 protein levels in patient-derived iPSC GABA neurons was slight (approximately 20%) compared to the control neurons, despite a 50% reduction in STXBP1 mRNA levels. Using a microelectrode array-based assay, we found that patient-derived iPSC GABA neurons exhibited dysfunctional maturation with reduced numbers of spontaneous spikes and bursts. These findings reinforce the idea that GABAergic dysfunction is a crucial contributor to STXBP1-E pathogenesis. Moreover, gene expression analysis revealed specific dysregulation of genes previously implicated in epilepsy, neurodevelopment and neurodegeneration in patient-derived iPSC GABA neurons, namely KCNH1, KCNH5, CNN3, RASGRF1, SEMA3A, SIAH3 and INPP5F. Thus, our study provides new insights for understanding the biological processes underlying the widespread neuropathological features of STXBP1-E.

Genetics and gene therapy in Dravet syndrome.

Dravet syndrome is a well-established electro-clinical condition first described in 1978. A main genetic cause was identified with the discovery of a loss-of-function SCN1A variant in 2001. Mechanisms underlying the phenotypic variations have subsequently been a main topic of research. Various genetic modifiers of clinical severities have been elucidated through many rigorous studies on genotype-phenotype correlations and the recent advances in next generation sequencing technology. Furthermore, a deeper understanding of the regulation of gene expression and remarkable progress on genome-editing technology using the CRISPR-Cas9 system provide significant opportunities to overcome hurdles of gene therapy, such as enhancing NaV1.1 expression. This article reviews the current understanding of genetic pathology and the status of research toward the development of gene therapy for Dravet syndrome. This article is part of the Special Issue "Severe Infantile Epilepsies".

Application of induced pluripotent stem cells in epilepsy.

Epilepsy is among the most common neurological disorders, affecting approximately 50 million people worldwide. Importantly, epilepsy is genetically and etiologically heterogenous, but several epilepsy types exhibit similar clinical presentations. Epilepsy-associated genes are being identified. However, the molecular pathomechanisms remain largely unknown. Approximately one-third of epilepsy is refractory to multiple conventional anti-epileptic drugs (AEDs). Induced pluripotent stem cells (iPSCs) provide an excellent tool to study the pathomechanisms underlying epilepsy and to develop novel treatments. Indeed, disease-specific iPSCs have been established for several genetic epilepsies. In particular, the molecular mechanisms underlying certain developmental and epileptic encephalopathies, such as Dravet syndrome, have been revealed. Modeling epilepsy with iPSCs enables new drug development based on the elucidated pathomechanisms. This can also be used to evaluate conventional AEDs and drug repurposing. Furthermore, transplanting neuronal cells derived from iPSCs into the brain has great potential to treat refractory epilepsies. Recent advances in iPSC technology have enabled the generation of neuronal organoids, or "mini brains." These organoids demonstrate electrophysiological activities similar to those of the brain and have the potential for extensive epilepsy research opportunities. Thus, the application of iPSCs in epilepsy provides insight into novel treatments based on the molecular pathomechanisms of epilepsy. In this review, we comprehensively discuss the studies conducted on iPSCs established for genetic epilepsy or epilepsies without major structural dysmorphic features.

Operational classification of seizure types by the International League Against Epilepsy: Position Paper of the ILAE Commission for Classification and Terminology.

The International League Against Epilepsy (ILAE) presents a revised operational classification of seizure types. The purpose of such a revision is to recognize that some seizure types can have either a focal or generalized onset, to allow classification when the onset is unobserved, to include some missing seizure types, and to adopt more transparent names. Because current knowledge is insufficient to form a scientifically based classification, the 2017 Classification is operational (practical) and based on the 1981 Classification, extended in 2010. Changes include the following: (1) "partial" becomes "focal"; (2) awareness is used as a classifier of focal seizures; (3) the terms dyscognitive, simple partial, complex partial, psychic, and secondarily generalized are eliminated; (4) new focal seizure types include automatisms, behavior arrest, hyperkinetic, autonomic, cognitive, and emotional; (5) atonic, clonic, epileptic spasms, myoclonic, and tonic seizures can be of either focal or generalized onset; (6) focal to bilateral tonic-clonic seizure replaces secondarily generalized seizure; (7) new generalized seizure types are absence with eyelid myoclonia, myoclonic absence, myoclonic-atonic, myoclonic-tonic-clonic; and (8) seizures of unknown onset may have features that can still be classified. The new classification does not represent a fundamental change, but allows greater flexibility and transparency in naming seizure types.

Instruction manual for the ILAE 2017 operational classification of seizure types.

This companion paper to the introduction of the International League Against Epilepsy (ILAE) 2017 classification of seizure types provides guidance on how to employ the classification. Illustration of the classification is enacted by tables, a glossary of relevant terms, mapping of old to new terms, suggested abbreviations, and examples. Basic and extended versions of the classification are available, depending on the desired degree of detail. Key signs and symptoms of seizures (semiology) are used as a basis for categories of seizures that are focal or generalized from onset or with unknown onset. Any focal seizure can further be optionally characterized by whether awareness is retained or impaired. Impaired awareness during any segment of the seizure renders it a focal impaired awareness seizure. Focal seizures are further optionally characterized by motor onset signs and symptoms: atonic, automatisms, clonic, epileptic spasms, or hyperkinetic, myoclonic, or tonic activity. Nonmotor-onset seizures can manifest as autonomic, behavior arrest, cognitive, emotional, or sensory dysfunction. The earliest prominent manifestation defines the seizure type, which might then progress to other signs and symptoms. Focal seizures can become bilateral tonic-clonic. Generalized seizures engage bilateral networks from onset. Generalized motor seizure characteristics comprise atonic, clonic, epileptic spasms, myoclonic, myoclonic-atonic, myoclonic-tonic-clonic, tonic, or tonic-clonic. Nonmotor (absence) seizures are typical or atypical, or seizures that present prominent myoclonic activity or eyelid myoclonia. Seizures of unknown onset may have features that can still be classified as motor, nonmotor, tonic-clonic, epileptic spasms, or behavior arrest. This "users' manual" for the ILAE 2017 seizure classification will assist the adoption of the new system.

[A patient with an early diagnosis of PCDH19-related epilepsy].

An abnormality in PCDH19 causes intractable early-onset epilepsy limited to females, and its significance in pediatric epilepsy is currently increasing. We report the case of a girl with an early diagnosis of PCDH19-related epilepsy. Focal seizures, consisting of eye deviation and asymmetrical tonic posturing, first appeared in clusters at the age of 5 months. Although each seizure was brief (less than a few minutes), seizures occurred in clusters. Cluster was observed at ages of 7, 10, 11, 14, and 19 months, respectively, and all were intractable to multiple treatments. Each cluster continued for 3 days to 2 weeks. However, no seizures occurred outsides the clusters. The pattern of seizure occurrences was characteristic of PCDH19-related epilepsy, which we first suspected when the patient was 11 months old. Genetic analysis of PCDH19 revealed two novel missense substitutions: c.1294G≥C (p.D417H) and c.1786G≥T (p.D596Y). Her psychomotor development was normal at the last follow-up at age of 1 year and 9 months. Currently, the pathogenesis and best treatments of PCDH19-related epilepsy remain unclear. However, to provide correct diagnosis and genetic counseling, and to avoid overtreatments, the possibility of this disease should be considered early in girls with intractable seizure clusters which starting during infancy to early childhood.

Prophylactic antiseizure medications for recurrent status epilepticus in nonsyndromic childhood epilepsy.

PURPOSE: The management of status epilepticus (SE) has mainly focused on the termination of ongoing SE episodes. However, long-term therapeutic strategies for the prevention of SE are lacking. This study aimed to investigate the effectiveness of prophylactic antiseizure medications (ASMs) for SEs in nonsyndromic childhood epilepsy. METHODS: This retrospective study was conducted at Jikei University Hospital. Patients <18 years of age, diagnosed with epilepsy, and experiencing three or more SE episodes within 1 year between April 1, 2017, and October 1, 2021, were included. ASMs introduced for seizure types that developed into SE were evaluated. The effectiveness of ASMs was determined by using the "Rule of Three": An ASM was determined effective if patients were free of SE for a duration at least three times that of their longest SE interval in 12 months prior to intervention. RESULTS: The investigation included a total of 32 ASMs administered to 13 patients. The longest interval between SE episodes before ASM administration was 28-257 d. The first SE interval after ASM administration was 12-797 d. Levetiracetam (LEV) and clobazam (CLB) showed effectiveness in 2/10 and 5/6 patients, respectively. Other ASMs were ineffective. The leading etiology of epilepsy was perinatal brain injury, identified in four patients, and CLB was effective in all of them. CONCLUSIONS: The present study suggests that CLB and LEV may prolong the SE interval in some cases of nonsyndromic childhood epilepsy. CLB may be beneficial, particularly in patients with perinatal brain injury.

Developmental changes in brain activity of heterozygous Scn1a knockout rats.

Introduction: Dravet syndrome (DS) is an infantile-onset developmental and epileptic encephalopathy characterized by an age-dependent evolution of drug-resistant seizures and poor developmental outcomes. Functional impairment of gamma-aminobutyric acid (GABA)ergic interneurons due to loss-of-function mutation of SCN1A is currently considered the main pathogenesis. In this study, to better understand the age-dependent changes in the pathogenesis of DS, we characterized the activity of different brain regions in Scn1a knockout rats at each developmental stage. Methods: We established an Scn1a knockout rat model and examined brain activity from postnatal day (P) 15 to 38 using a manganese-enhanced magnetic resonance imaging technique (MEMRI). Results: Scn1a heterozygous knockout (Scn1a +/-) rats showed a reduced expression of voltage-gated sodium channel alpha subunit 1 protein in the brain and heat-induced seizures. Neural activity was significantly higher in widespread brain regions of Scn1a +/- rats than in wild-type rats from P19 to P22, but this difference did not persist thereafter. Bumetanide, a Na+-K+-2Cl- cotransporter 1 inhibitor, mitigated hyperactivity to the wild-type level, although no change was observed in the fourth postnatal week. Bumetanide also increased heat-induced seizure thresholds of Scn1a +/- rats at P21. Conclusions: In Scn1a +/- rats, neural activity in widespread brain regions increased during the third postnatal week, corresponding to approximately 6 months of age in humans, when seizures most commonly develop in DS. In addition to impairment of GABAergic interneurons, the effects of bumetanide suggest a possible contribution of immature type A gamma-aminobutyric acid receptor signaling to transient hyperactivity and seizure susceptibility during the early stage of DS. This hypothesis should be addressed in the future. MEMRI is a potential technique for visualizing changes in basal brain activity in developmental and epileptic encephalopathies.

Long-term efficacy of low-dose perampanel for progressive myoclonus epilepsy in a patient with Gaucher disease type 3.

PURPOSE: We report the case of a patient with progressive myoclonus epilepsy due to Gaucher disease type 3 whose seizures and ability to perform activities of daily living were significantly improved after starting low-dose perampanel therapy. CASE: Our patient's generalized tonic-clonic seizures and myoclonus did not improve despite the administration of multiple antiseizure medications and enzyme replacement therapy. The myoclonus reduced following pharmacological chaperone therapy, but this effect was temporary, and the generalized tonic-clonic seizures continued to occur. However, the generalized tonic-clonic seizures disappeared following treatment with 2 mg/day of perampanel. In addition, the decrease in myoclonus dramatically improved motor function such as talking, eating, and walking and stabilized the patient's mental status. These effects have been sustained for more than 4 years. CONCLUSION: Perampanel is expected to be effective in the treatment of progressive myoclonus epilepsy associated with Gaucher disease type 3 and should be considered the drug of choice for this condition.

GFAP variant p. Tyr366Cys demonstrated widespread brain cavitation in neonatal Alexander disease.

Alexander disease is a rare form of leukodystrophy caused by heterozygous mutations in the gene encoding glial fibrillary acidic protein (GFAP). Brain cavitation in the white matter, predominantly distributed in the frontal periventricular area, has been described in some cases. Here, we present a case of a 1-year-old boy with neonatal Alexander disease caused by the p. Tyr366Cys GFAP variant, with rapid and widespread white matter cavitation. This case broadens the radiological spectrum of Alexander disease and suggests a possible genotype-phenotype correlation between the p. Tyr366Cys variant and cavitation.

Body temperature predicts recurrent febrile seizures in the same febrile illness.

BACKGROUND: The incidence of recurrent febrile seizures during the same febrile illness (RFS) is 14-24%. A pilot study found that body temperature and male sex were predictors of RFS. This study sought to validate body temperature as a predictor of RFS, calculate the optimal cut-off body temperature for predicting RFS, and identify the other predictors of RFS. METHODS: This prospective cohort study enrolled children with febrile seizures aged 6-60 months who visited the emergency department at Atsugi City Hospital, Japan, between March 1, 2019, and February 29, 2020. Children who had multiple seizures, diazepam administration before the emergency department visit, seizures lasting >15 min, underlying diseases, or who could not be followed up were excluded. The optimal cut-off body temperature was determined using a receiver-operating characteristic curve. RESULTS: A total of 109 children were enrolled, of whom 13 (11.9%) had RFS. A lower body temperature was significantly associated with RFS (P = 0.02). The optimal cut-off body temperature for predicting RFS was 39.2 °C. Children with RFS also had significantly lower C-reactive protein and blood glucose levels (P = 0.01 and 0.047, respectively), but none of the other factors considered were significantly associated with RFS. CONCLUSIONS: This large prospective study confirmed that body temperature is a predictor of RFS. The optimal cut-off body temperature for predicting RFS was 39.2 °C. Low C-reactive protein level and blood glucose level might be predictors of RFS, but this needs to be confirmed in prospective multicenter studies.

Interictal cerebral blood flow abnormality in cryptogenic West syndrome.

PURPOSE: To elucidate the abnormality of interictal regional cerebral blood flow (rCBF) of West syndrome at the onset. METHODS: Quantitative measurement of rCBF with an autoradiography method using N-isopropyl-((123)I) p-iodoamphetamine single photon emission computed tomography (SPECT) was performed on 14 infants with cryptogenic West syndrome. Regions of interest (ROIs) for rCBF were placed automatically using an automated ROI analysis software (three-dimensional stereotactic ROI template), and were grouped into 12 segments: callosomarginal, precentral, central, parietal, angular, temporal, posterior cerebral, pericallosal, lenticular nucleus, thalamus, hippocampus, and cerebellum. We compared rCBF between the patients and seven age-matched infants with cryptogenic focal epilepsy as a control group. The patients were divided into two groups according to the duration from onset to SPECT, to compare rCBF. RESULTS: Quantitative analysis revealed cerebral hypoperfusion in cryptogenic West syndrome with normal SPECT images under visual inspection. In bilateral central, posterior cerebral, pericallosal, lenticular nucleus, and hippocampus, and in the left parietal, temporal, and cerebellum, and in the right angular and thalamus segments there were statistical differences (p < 0.05). Compared with the duration from onset to SPECT, there were no significant differences of rCBF in all segments. DISCUSSION: Broad cerebral hypoperfusion with posterior predominance involving the hippocampus and lenticular nucleus implies that even cryptogenic West syndrome has a widespread cerebral dysfunction at least transiently, which would correspond to clinical manifestations of hypsarrhythmia and epileptic spasms. Hippocampal hypoperfusion suggests the dysfunction of hippocampal circuitry in the brain adrenal axis, and may contribute to subsequent cognitive impairment of cryptogenic West syndrome.

Tatton-Brown-Rahman syndrome with a novel DNMT3A mutation presented severe intellectual disability and autism spectrum disorder.

Tatton-Brown-Rahman syndrome is a congenital anomaly syndrome that manifests with overgrowth, macrocephaly, and characteristic facial features. This autosomal dominant disease is caused by a germline mutation in DNMT3A. Some patients with this syndrome develop mild to severe intellectual disability, which is sometimes accompanied by autism spectrum disorder or other developmental disorders. We report a Japanese patient with severe intellectual disability and autism spectrum disorder with a de novo mutation in the active domain of DNMT3A.

Establishment of human induced pluripotent stem cells derived from skin cells of a patient with Dravet syndrome.

Dravet syndrome is known as an intractable infantile epilepsy caused by a heterozygous de novo mutation in SCN1A, with mutations being reported globally. In this study, we established 2 human induced pluripotent stem cell lines by expressing reprogramming factors, OCT3/4, SOX2, KLF4, L-MYC, LIN28 and p53 shRNA in the fibroblast skin cells of a patient with Dravet syndrome harboring the Y1102X pathogenic mutation in SCN1A. These cell lines showed pluripotency, ability for differentiation to the 3 germ layers, and normal karyotype.

Predictors of recurrent febrile seizures during the same febrile illness in children with febrile seizures.

Febrile seizures (FS) are common in childhood. Of children who experience an FS, 14-24% experience recurrence within 24 h, during the same febrile illness (RFS). The aim of this pilot study was to identify the predictors of RFS among children who experience FS. We conducted a retrospective cohort study of children aged 6-60 months, who visited the emergency department (ED) at Atsugi City Hospital in Japan for treatment of an FS between December 1, 2018 and February 28, 2019. Exclusion criteria included multiple seizures before visiting the ED, diazepam administration before visiting the ED or on departure, seizures lasting >15 min, underlying diseases such as epilepsy, and absence of laboratory test results. The primary outcome was RFS. Fifty-one patients fulfilled the inclusion criteria, of whom nine (17.6%) had RFS. The incidence of RFS was significantly higher in children with a body temperature ≤ 39.8 °C during the ED visit (P = .01). The combination of male sex and a body temperature ≤ 39.8 °C had a sensitivity, specificity and negative predictive value of 88.9%, 76.2%, and 97.0%, respectively. In conclusion, the incidence of RFS was 17.6%. The major predictors of RFS were male sex and a body temperature ≤ 39.8 °C.

[Epileptic spasms without hypsarrhythmia showing astatic seizures in clusters].

It is difficult to differentiate astatic seizures among atonic, myoclonic and tonic seizures without the help of ictal polygraphic recordings. We described a girl with epilepsy presenting periodic astasia caused by epileptic spasms. Her seizures had occurred in clusters since 1-year and 7-month of age. Her interictal electroencephalogram (EEG) showed intermittent diffuse (poly) spike (s) and wave discharges, without a trace of hypsarrhythmia. She had a diagnosis of epilepsy with myoclonic-astatic seizures at a previous hospital, and her seizures had been resistant to multiple antiepileptic agents. After she was referred to our hospital, the ictal video-EEG recordings were made before and after ACTH administration, which revealed that her astatic seizures were epileptic spasms with presumed cortical origin. We should carefully evaluate the astatic seizures during early childhood which may be an atypical presentation of epileptic spasms.

Establishment of a human induced stem cell line (FUi002-A) from Dravet syndrome patient carrying heterozygous R1525X mutation in SCN1A gene.

De novo mutations in SCN1A are the most common cause of Dravet syndrome (DS), an infantile-onset epileptic encephalopathy. In this study, human induced pluripotent stem cell (hiPSC) line FUi002-A was generated from skin fibroblasts obtained from a clinically diagnosed 26-year-old male DS patient with the R1525X variant of the SCN1A gene. Skin fibroblasts were reprogrammed using OriP/EBNA-1 based episomal plasmids expressing reprogramming factors expressing OCT4, SOX2, KLF-4, L-MYC, LIN28, and p53 shRNA. The transgene-free FUi002-A showed pluripotency, three germ layer differentiation capacity in vitro, and a normal karyotype. The resulting hiPSCs were heterozygous for the mutation in the SCN1A gene.