Genotype-phenotype associations in 1018 individuals with SCN1A-related epilepsies.

OBJECTIVE: SCN1A variants are associated with epilepsy syndromes ranging from mild genetic epilepsy with febrile seizures plus (GEFS+) to severe Dravet syndrome (DS). Many variants are de novo, making early phenotype prediction difficult, and genotype-phenotype associations remain poorly understood. METHODS: We assessed data from a retrospective cohort of 1018 individuals with SCN1A-related epilepsies. We explored relationships between variant characteristics (position, in silico prediction scores: Combined Annotation Dependent Depletion (CADD), Rare Exome Variant Ensemble Learner (REVEL), SCN1A genetic score), seizure characteristics, and epilepsy phenotype. RESULTS: DS had earlier seizure onset than other GEFS+ phenotypes (5.3 vs. 12.0 months, p < .001). In silico variant scores were higher in DS versus GEFS+ (p < .001). Patients with missense variants in functionally important regions (conserved N-terminus, S4-S6) exhibited earlier seizure onset (6.0 vs. 7.0 months, p = .003) and were more likely to have DS (280/340); those with missense variants in nonconserved regions had later onset (10.0 vs. 7.0 months, p = .036) and were more likely to have GEFS+ (15/29, χ2 = 19.16, p < .001). A minority of protein-truncating variants were associated with GEFS+ (10/393) and more likely to be located in the proximal first and last exon coding regions than elsewhere in the gene (9.7% vs. 1.0%, p < .001). Carriers of the same missense variant exhibited less variability in age at seizure onset compared with carriers of different missense variants for both DS (1.9 vs. 2.9 months, p = .001) and GEFS+ (8.0 vs. 11.0 months, p = .043). Status epilepticus as presenting seizure type is a highly specific (95.2%) but nonsensitive (32.7%) feature of DS. SIGNIFICANCE: Understanding genotype-phenotype associations in SCN1A-related epilepsies is critical for early diagnosis and management. We demonstrate an earlier disease onset in patients with missense variants in important functional regions, the occurrence of GEFS+ truncating variants, and the value of in silico prediction scores. Status epilepticus as initial seizure type is a highly specific, but not sensitive, early feature of DS.

Drug resistant epilepsy and associated factors among children with epilepsies in tanzania: a cross-sectional study.

BACKGROUND: Epilepsy contributes to high morbidity among children and adolescents in developing countries. A quarter of all children with epilepsy will be resistant to anti-seizure medications (ASMs), with associated neurocognitive impairments and risk of higher mortality. This study aimed to estimate and characterize drug-resistant epilepsy (DRE) (defined as failure to achieve sustained remission after adequate trials of two tolerated and appropriately chosen ASMs) and its associated factors among children and adolescents with epilepsies attending the pediatric neurology clinic at Muhimbili National Hospital (MNH), Dar es Salaam Tanzania. METHODS: This cross-sectional study was conducted from June 2020 to June 2021. Children with epilepsies and who had been treated with ASMs for at least 3 months were eligible for inclusion. Exclusion criteria included children whose caregivers denied consent and those who exhibited acute medical conditions necessitating admission on the scheduled visit day. Data on demographic characteristics, perinatal history, detailed history of the seizures semiology, drug history, magnetic resonance imaging (MRI), and electroencephalography (EEG) results were obtained from caregivers and medical records available during recruitment. Seizures and epilepsies were classified using the 2017 International League Against Epilepsy (ILAE) classification. Logistic regression was used to determine factors associated with DRE. RESULTS: A total of 236 children and adolescents aged between 4 months and 15 years (Median age 72 months (IQR = 42-78)) were enrolled in this study. We found the proportion of DRE to be 14.8% in this cohort. Of the thirty-five patients with DRE, 60% had generalized epilepsy and almost 25% had a diagnosis of an epilepsy syndrome, the most common being Lennox-Gastaut syndrome (LGS). Structural abnormalities on brain MRI were seen in almost 80% of all patients with DRE, the most prevalent being cystic encephalomalacia, which was observed in 34% of patients. Patients using both ASMs and alternative therapies accounted for 9% of this cohort. The onset of seizures during the first month of life (aOR = 1.99; 95%CI 1.7-4.6; p = 0.031) and high initial seizure frequency (aOR = 3.6; 95%CI 1.6-8;p = 0.002) were found to be independently associated with DRE. CONCLUSION: The proportion of DRE in Tanzania is high. Patients with neonatal onset seizures and high initial seizure frequency should be followed up closely to ensure early diagnosis of DRE.

Phenotypic and functional assessment of two novel KCNQ2 gain-of-function variants Y141N and G239S and effects of amitriptyline treatment.

While loss-of-function (LoF) variants in KCNQ2 are associated with a spectrum of neonatal-onset epilepsies, gain-of-function (GoF) variants cause a more complex phenotype that precludes neonatal-onset epilepsy. In the present work, the clinical features of three patients carrying a de novo KCNQ2 Y141N (n â€‹= â€‹1) or G239S variant (n â€‹= â€‹2) respectively, are described. All three patients had a mild global developmental delay, with prominent language deficits, and strong activation of interictal epileptic activity during sleep. Epileptic seizures were not reported. The absence of neonatal seizures suggested a GoF effect and prompted functional testing of the variants. In vitro whole-cell patch-clamp electrophysiological experiments in Chinese Hamster Ovary cells transiently-transfected with the cDNAs encoding Kv7.2 subunits carrying the Y141N or G239S variants in homomeric or heteromeric configurations with Kv7.2 subunits, revealed that currents from channels incorporating mutant subunits displayed increased current densities and hyperpolarizing shifts of about 10 â€‹mV in activation gating; both these functional features are consistent with an in vitro GoF phenotype. The antidepressant drug amitriptyline induced a reversible and concentration-dependent inhibition of current carried by Kv7.2 Y141N and G239S mutant channels. Based on in vitro results, amitriptyline was prescribed in one patient (G239S), prompting a significant improvement in motor, verbal, social, sensory and adaptive behavior skillsduring the two-year-treatment period. Thus, our results suggest that KCNQ2 GoF variants Y141N and G239S cause a mild DD with prominent language deficits in the absence of neonatal seizures and that treatment with the Kv7 channel blocker amitriptyline might represent a potential targeted treatment for patients with KCNQ2 GoF variants.

From diagnosis to treatment in genetic epilepsies: Implementation of precision medicine in real-world clinical practice.

The implementation of whole exome sequencing (WES) has had a major impact on the diagnostic yield of genetic testing in individuals with epilepsy. The identification of a genetic etiology paves the way to precision medicine: an individualized treatment approach, based on the disease pathophysiology. The aim of this retrospective cohort study was to: (1) determine the diagnostic yield of WES in a heterogeneous cohort of individuals with epilepsy referred for genetic testing in a real-world clinical setting, (2) investigate the influence of epilepsy characteristics on the diagnostic yield, (3) determine the theoretical yield of treatment changes based on genetic diagnosis and (4) explore the barriers to implementation of precision medicine. WES was performed in 247 individuals with epilepsy, aged between 7 months and 68 years. In 34/247 (14 %) a (likely) pathogenic variant was identified. In 7/34 (21 %) of these individuals the variant was found using a HPO-based filtering. Diagnostic yield was highest for individuals with an early onset of epilepsy (39 %) or in those with a developmental and epileptic encephalopathy (34 %). Precision medicine was a theoretical possibility in 20/34 (59 %) of the individuals with a (likely) pathogenic variant but implemented in only 11/34 (32 %). The major barrier to implementation of precision treatment was the limited availability or reimbursement of a given drug. These results confirm the potential impact of genetic analysis on treatment choices, but also highlight the hurdles to the implementation of precision medicine. To optimize precision medicine in real-world practice, additional endeavors are needed: unifying definitions of precision medicine, establishment of publicly accessible databases that include data on the functional effect of gene variants, increasing availability and reimbursement of precision therapeutics, and broadening access to innovative clinical trials.

Resting-State Functional MRI and PET Imaging as Noninvasive Tools to Study (Ab)Normal Neurodevelopment in Humans and Rodents.

Neurodevelopmental disorders (NDDs) are a group of complex neurologic and psychiatric disorders. Functional and molecular imaging techniques, such as resting-state functional magnetic resonance imaging (rs-fMRI) and positron emission tomography (PET), can be used to measure network activity noninvasively and longitudinally during maturation in both humans and rodent models. Here, we review the current knowledge on rs-fMRI and PET biomarkers in the study of normal and abnormal neurodevelopment, including intellectual disability (ID; with/without epilepsy), autism spectrum disorder (ASD), and attention deficit hyperactivity disorder (ADHD), in humans and rodent models from birth until adulthood, and evaluate the cross-species translational value of the imaging biomarkers. To date, only a few isolated studies have used rs-fMRI or PET to study (abnormal) neurodevelopment in rodents during infancy, the critical period of neurodevelopment. Further work to explore the feasibility of performing functional imaging studies in infant rodent models is essential, as rs-fMRI and PET imaging in transgenic rodent models of NDDs are powerful techniques for studying disease pathogenesis, developing noninvasive preclinical imaging biomarkers of neurodevelopmental dysfunction, and evaluating treatment-response in disease-specific models.

Pathogenic variants in SMARCA1 cause an X-linked neurodevelopmental disorder modulated by NURF complex composition.

Pathogenic variants in ATP-dependent chromatin remodeling proteins are a recurrent cause of neurodevelopmental disorders (NDDs). The NURF complex consists of BPTF and either the SNF2H (SMARCA5) or SNF2L (SMARCA1) ISWI-chromatin remodeling enzyme. Pathogenic variants in BPTF and SMARCA5 were previously implicated in NDDs. Here, we describe 40 individuals from 30 families with de novo or maternally inherited pathogenic variants in SMARCA1. This novel NDD was associated with mild to severe ID/DD, delayed or regressive speech development, and some recurrent facial dysmorphisms. Individuals carrying SMARCA1 loss-of-function variants exhibited a mild genome-wide DNA methylation profile and a high penetrance of macrocephaly. Genetic dissection of the NURF complex using Smarca1, Smarca5, and Bptfsingle and double mouse knockouts revealed the importance of NURF composition and dosage for proper forebrain development. Finally, we propose that genetic alterations affecting different NURF components result in a NDD with a broad clinical spectrum.

First report of Tunisian patients with CDKL5-related encephalopathy.

OBJECTIVE: Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) are associated with a wide spectrum of clinical presentations. Early-onset epileptic encephalopathy (EOEE) is the most recognized phenotype. Here we describe phenotypic features in 8 Tunisian patients with CDKL5-related encephalopathy. METHODS: We included all cases with clinical features consistent with CDKL5-related encephalopathy: infantile epileptic spasm, acquired microcephaly, movement disorders and visual impairment. We collected data about seizure types, electroencephalogram, magnetic resonance imaging and metabolic analysis. The diagnosis of CDKL5 mutation was made thanks to Sanger sequencing with an ABI PRISM 3100-Avant automated DNA sequencer using a Big Dye Terminator Cycle Sequencing Reaction Kit v1.1. and Next Generation Sequencing (NGS) since the development of a gene panel responsible for DEE within the framework of "Strengthening the Sfax University Expertise for diagnosis and management of epileptic encephalopathies". RESULTS: We collected 4 boys and 4 girls aged meanly 6-years-old with confirmed mutation on CDKL5 gene. Overall, we identified 5 de novo CDKL5 mutations including three Frameshift mutations; one missense mutation and a splicing variant. The mean age at first seizure onset was 4 months. The first seizure type was infantile epileptic spasm (4/8) followed by tonic (2/8) and myoclonic seizures (2/8). Out of 8 cases, 4 exhibited two stages epileptic course while epilepsy in 3 other patients progressed on three stages. Regarding development, most cases (6/8) had psychomotor retardation from the start whilst the two others showed psychomotor regression with the onset of seizures. Additional clinical features included visual impairment (7/8), tone abnormalities (7/8), stereotypies (7/8) and acquired microcephaly (6/8). SIGNIFICANCE: Our present report delineates an unusual phenotype of CDKL5-related encephalopathy with male gender predominance and delayed onset epilepsy. It interestingly described new phenotypic features and uncommon benign developmental profiles in boys, different patterns of CDKL5-epilepsy, neuroimaging findings and CDKL5 mutational spectru.

Solving the unsolved genetic epilepsies: Current and future perspectives.

Many patients with epilepsy undergo exome or genome sequencing as part of a diagnostic workup; however, many remain genetically unsolved. There are various factors that account for negative results in exome/genome sequencing for patients with epilepsy: (1) the underlying cause is not genetic; (2) there is a complex polygenic explanation; (3) the illness is monogenic but the causative gene remains to be linked to a human disorder; (4) family segregation with reduced penetrance; (5) somatic mosaicism or the complexity of, for example, a structural rearrangement; or (6) limited knowledge or diagnostic tools that hinder the proper classification of a variant, resulting in its designation as a variant of unknown significance. The objective of this review is to outline some of the diagnostic options that lie beyond the exome/genome, and that might become clinically relevant within the foreseeable future. These options include: (1) re-analysis of older exome/genome data as knowledge increases or symptoms change; (2) looking for somatic mosaicism or long-read sequencing to detect low-complexity repeat variants or specific structural variants missed by traditional exome/genome sequencing; (3) exploration of the non-coding genome including disruption of topologically associated domains, long range non-coding RNA, or other regulatory elements; and finally (4) transcriptomics, DNA methylation signatures, and metabolomics as complementary diagnostic methods that may be used in the assessment of variants of unknown significance. Some of these tools are currently not integrated into standard diagnostic workup. However, it is reasonable to expect that they will become increasingly available and improve current diagnostic capabilities, thereby enabling precision diagnosis in patients who are currently undiagnosed.

GABRA1-Related Disorders: From Genetic to Functional Pathways.

OBJECTIVE: Variants in GABRA1 have been associated with a broad epilepsy spectrum, ranging from genetic generalized epilepsies to developmental and epileptic encephalopathies. However, our understanding of what determines the phenotype severity and best treatment options remains inadequate. We therefore aimed to analyze the electroclinical features and the functional effects of GABRA1 variants to establish genotype-phenotype correlations. METHODS: Genetic and electroclinical data of 27 individuals (22 unrelated and 2 families) harboring 20 different GABRA1 variants were collected and accompanied by functional analysis of 19 variants. RESULTS: Individuals in this cohort could be assigned into different clinical subgroups based on the functional effect of their variant and its structural position within the GABRA1 subunit. A homogenous phenotype with mild cognitive impairment and infantile onset epilepsy (focal seizures, fever sensitivity, and electroencephalographic posterior epileptiform discharges) was described for variants in the extracellular domain and the small transmembrane loops. These variants displayed loss-of-function (LoF) effects, and the patients generally had a favorable outcome. A more severe phenotype was associated with variants in the pore-forming transmembrane helices. These variants displayed either gain-of-function (GoF) or LoF effects. GoF variants were associated with severe early onset neurodevelopmental disorders, including early infantile developmental and epileptic encephalopathy. INTERPRETATION: Our data expand the genetic and phenotypic spectrum of GABRA1 epilepsies and permit delineation of specific subphenotypes for LoF and GoF variants, through the heterogeneity of phenotypes and variants. Generally, variants in the transmembrane helices cause more severe phenotypes, in particular GoF variants. These findings establish the basis for a better understanding of the pathomechanism and a precision medicine approach in GABRA1-related disorders. Further studies in larger populations are needed to provide a conclusive genotype-phenotype correlation. ANN NEUROL 2023.

Long-read sequencing and profiling of RNA-binding proteins reveals the pathogenic mechanism of aberrant splicing of an SCN1A poison exon in epilepsy.

Pathogenic loss-of-function SCN1A variants cause a spectrum of seizure disorders. We previously identified variants in individuals with SCN1A -related epilepsy that fall in or near a poison exon (PE) in SCN1A intron 20 (20N). We hypothesized these variants lead to increased PE inclusion, which introduces a premature stop codon, and, therefore, reduced abundance of the full-length SCN1A transcript and Na v 1.1 protein. We used a splicing reporter assay to interrogate PE inclusion in HEK293T cells. In addition, we used patient-specific induced pluripotent stem cells (iPSCs) differentiated into neurons to quantify 20N inclusion by long and short-read sequencing and Na v 1.1 abundance by western blot. We performed RNA-antisense purification with mass spectrometry to identify RNA-binding proteins (RBPs) that could account for the aberrant PE splicing. We demonstrate that variants in/near 20N lead to increased 20N inclusion by long-read sequencing or splicing reporter assay and decreased Na v 1.1 abundance. We also identified 28 RBPs that differentially interact with variant constructs compared to wild-type, including SRSF1 and HNRNPL. We propose a model whereby 20N variants disrupt RBP binding to splicing enhancers (SRSF1) and suppressors (HNRNPL), to favor PE inclusion. Overall, we demonstrate that SCN1A 20N variants cause haploinsufficiency and SCN1A -related epilepsies. This work provides insights into the complex control of RBP-mediated PE alternative splicing, with broader implications for PE discovery and identification of pathogenic PE variants in other genetic conditions.

Widespread genomic influences on phenotype in Dravet syndrome, a 'monogenic' condition.

Dravet syndrome is an archetypal rare severe epilepsy, considered 'monogenic', typically caused by loss-of-function SCN1A variants. Despite a recognizable core phenotype, its marked phenotypic heterogeneity is incompletely explained by differences in the causal SCN1A variant or clinical factors. In 34 adults with SCN1A-related Dravet syndrome, we show additional genomic variation beyond SCN1A contributes to phenotype and its diversity, with an excess of rare variants in epilepsy-related genes as a set and examples of blended phenotypes, including one individual with an ultra-rare DEPDC5 variant and focal cortical dysplasia. The polygenic risk score for intelligence was lower, and for longevity, higher, in Dravet syndrome than in epilepsy controls. The causal, major-effect, SCN1A variant may need to act against a broadly compromised genomic background to generate the full Dravet syndrome phenotype, whilst genomic resilience may help to ameliorate the risk of premature mortality in adult Dravet syndrome survivors.

ILAE Genetic Literacy Series: Self-limited familial epilepsy syndromes with onset in neonatal age and infancy.

The self-limited (familial) epilepsies with onset in neonates or infants, formerly called benign familial neonatal and/or infantile epilepsies, are autosomal dominant disorders characterized by neonatal- or infantile-onset focal motor seizures and the absence of neurodevelopmental complications. Seizures tend to remit during infancy or early childhood and are therefore called "self-limited". A positive family history for epilepsy usually suggests the genetic etiology, but incomplete penetrance and de novo inheritance occur. Here, we review the phenotypic spectrum and the genetic architecture of self-limited (familial) epilepsies with onset in neonates or infants. Using an illustrative case study, we describe important clues in recognition of these syndromes, diagnostic steps including genetic testing, management, and genetic counseling.

Case report: Functional analysis of the p.Arg507Trp variant of the PIGT gene supporting the moderate epilepsy phenotype of mutations in the C-terminal region.

Pathogenic germline variants in the PIGT gene are associated with the "multiple congenital anomalies-hypotonia-seizures syndrome 3" (MCAHS3) phenotype. So far, fifty patients have been reported, most of whom suffer from intractable epilepsy. Recently, a comprehensive analysis of a cohort of 26 patients with PIGT variants has broadened the phenotypical spectrum and indicated that both p.Asn527Ser and p.Val528Met are associated with a milder epilepsy phenotype and less severe outcomes. Since all reported patients are of Caucasian/Polish origin and most harbor the same variant (p.Val528Met), the ability to draw definitive conclusions regarding the genotype-phenotype correlation remains limited. We report a new case with a homozygous variant p.Arg507Trp in the PIGT gene, detected on clinical exome sequencing. The North African patient in question displays a predominantly neurological phenotype with global developmental delay, hypotonia, brain abnormalities, and well-controlled epileptic seizures. Homozygous and heterozygous variants in codon 507 have been reported to cause PIGT deficiency without biochemical confirmation. In this study, FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the p.Arg507Trp variant leads to mildly reduced activity. Our result confirm the pathogenicity of this variant and strengthen recently reported evidence on the genotype-phenotype correlation of the PIGT variant.

Clinical and Neurophysiologic Phenotypes in Neonates With BRAT1 Encephalopathy.

BACKGROUND AND OBJECTIVES: BRAT1 encephalopathy is an ultra-rare autosomal recessive neonatal encephalopathy. We delineate the neonatal electroclinical phenotype at presentation and provide insights for early diagnosis. METHODS: Through a multinational collaborative, we studied a cohort of neonates with encephalopathy associated with biallelic pathogenic variants in BRAT1 for whom detailed clinical, neurophysiologic, and neuroimaging information was available from the onset of symptoms. Neuropathologic changes were also analyzed. RESULTS: We included 19 neonates. Most neonates were born at term (16/19) from nonconsanguineous parents. 15/19 (79%) were admitted soon after birth to a neonatal intensive care unit, exhibiting multifocal myoclonus, both spontaneous and exacerbated by stimulation. 7/19 (37%) had arthrogryposis at birth, and all except 1 progressively developed hypertonia in the first week of life. Multifocal myoclonus, which was present in all but 1 infant, was the most prominent manifestation and did not show any EEG correlate in 16/19 (84%). Video-EEG at onset was unremarkable in 14/19 (74%) infants, and 6 (33%) had initially been misdiagnosed with hyperekplexia. Multifocal seizures were observed at a median age of 14 days (range: 1-29). During the first months of life, all infants developed progressive encephalopathy, acquired microcephaly, prolonged bouts of apnea, and bradycardia, leading to cardiac arrest and death at a median age of 3.5 months (range: 20 days to 30 months). Only 7 infants (37%) received a definite diagnosis before death, at a median age of 34 days (range: 25-126), and almost two-thirds (12/19, 63%) were diagnosed 8 days to 12 years postmortem (median: 6.5 years). Neuropathology examination, performed in 3 patients, revealed severely delayed myelination and diffuse astrogliosis, sparing the upper cortical layers. DISCUSSION: BRAT1 encephalopathy is a neonatal-onset, rapidly progressive neurologic disorder. Neonates are often misdiagnosed as having hyperekplexia, and many die undiagnosed. The key phenotypic features are multifocal myoclonus, an organized EEG, progressive, persistent, and diffuse hypertonia, and an evolution into refractory multifocal seizures, prolonged bouts of apnea, bradycardia, and early death. Early recognition of BRAT1 encephalopathy allows for prompt workup, appropriate management, and genetic counseling.

Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Downregulation of PMP22 ameliorates myelin defects in iPSC-derived human organoid cultures of CMT1A.

Charcot-Marie-Tooth disease is the most common inherited disorder of the PNS. CMT1A accounts for 40-50% of all cases and is caused by a duplication of the PMP22 gene on chromosome 17, leading to dysmyelination in the PNS. Patient-derived models to study such myelination defects are lacking as the in vitro generation of human myelinating Schwann cells has proved to be particularly challenging. Here, we present an induced pluripotent stem cell-derived organoid culture, containing various cell types of the PNS, including myelinating human Schwann cells, which mimics the human PNS. Single-cell analysis confirmed the PNS-like cellular composition and provides insight into the developmental trajectory. We used this organoid model to study disease signatures of CMT1A, revealing early ultrastructural myelin alterations, including increased myelin periodic line distance and hypermyelination of small axons. Furthermore, we observed the presence of onion-bulb-like formations in a later developmental stage. These hallmarks were not present in the CMT1A-corrected isogenic line or in a CMT2A iPSC line, supporting the notion that these alterations are specific to CMT1A. Downregulation of PMP22 expression using short-hairpin RNAs or a combinatorial drug consisting of baclofen, naltrexone hydrochloride and D-sorbitol was able to ameliorate the myelin defects in CMT1A-organoids. In summary, this self-organizing organoid model can capture biologically meaningful features of the disease and capture the physiological complexity, forms an excellent model for studying demyelinating diseases and supports the therapeutic approach of reducing PMP22 expression.

Epilepsy and sudden unexpected death in epilepsy in a mouse model of human SCN1B-linked developmental and epileptic encephalopathy.

Voltage-gated sodium channel ß1 subunits are essential proteins that regulate excitability. They modulate sodium and potassium currents, function as cell adhesion molecules and regulate gene transcription following regulated intramembrane proteolysis. Biallelic pathogenic variants in SCN1B, encoding ß1, are linked to developmental and epileptic encephalopathy 52, with clinical features overlapping Dravet syndrome. A recessive variant, SCN1B-c.265C>T, predicting SCN1B-p.R89C, was homozygous in two children of a non-consanguineous family. One child was diagnosed with Dravet syndrome, while the other had a milder phenotype. We identified an unrelated biallelic SCN1B-c.265C>T patient with a clinically more severe phenotype than Dravet syndrome. We used CRISPR/Cas9 to knock-in SCN1B-p.R89C to the mouse Scn1b locus (Scn1bR89/C89). We then rederived the line on the C57BL/6J background to allow comparisons between Scn1bR89/R89 and Scn1bC89/C89 littermates with Scn1b+/+ and Scn1b-/- mice, which are congenic on C57BL/6J, to determine whether the SCN1B-c.265C>T variant results in loss-of-function. Scn1bC89/C89 mice have normal body weights and ∼20% premature mortality, compared with severely reduced body weight and 100% mortality in Scn1b-/- mice. ß1-p.R89C polypeptides are expressed in brain at comparable levels to wild type. In heterologous cells, ß1-p.R89C localizes to the plasma membrane and undergoes regulated intramembrane proteolysis similar to wild type. Heterologous expression of ß1-p.R89C results in sodium channel α subunit subtype specific effects on sodium current. mRNA abundance of Scn2a, Scn3a, Scn5a and Scn1b was increased in Scn1bC89/C89 somatosensory cortex, with no changes in Scn1a. In contrast, Scn1b-/- mouse somatosensory cortex is haploinsufficient for Scn1a, suggesting an additive mechanism for the severity of the null model via disrupted regulation of another Dravet syndrome gene. Scn1bC89/C89 mice are more susceptible to hyperthermia-induced seizures at post-natal Day 15 compared with Scn1bR89/R89 littermates. EEG recordings detected epileptic discharges in young adult Scn1bC89/C89 mice that coincided with convulsive seizures and myoclonic jerks. We compared seizure frequency and duration in a subset of adult Scn1bC89/C89 mice that had been exposed to hyperthermia at post-natal Day 15 versus a subset that were not hyperthermia exposed. No differences in spontaneous seizures were detected between groups. For both groups, the spontaneous seizure pattern was diurnal, occurring with higher frequency during the dark cycle. This work suggests that the SCN1B-c.265C>T variant does not result in complete loss-of-function. Scn1bC89/C89 mice more accurately model SCN1B-linked variants with incomplete loss-of-function compared with Scn1b-/- mice, which model complete loss-of-function, and thus add to our understanding of disease mechanisms as well as our ability to develop new therapeutic strategies.

Natural History Study of STXBP1-Developmental and Epileptic Encephalopathy Into Adulthood.

BACKGROUND AND OBJECTIVES: Pathogenic STXBP1 variants cause a severe early-onset developmental and epileptic encephalopathy (STXBP1-DEE). We aimed to investigate the natural history of STXBP1-DEE in adults focusing on seizure evolution, the presence of movement disorders, and the level of functional (in)dependence. METHODS: In this observational study, patients with a minimum age of 18 years carrying a (likely) pathogenic STXBP1 variant were recruited through medical genetics departments and epilepsy centers. Treating clinicians completed clinical questionnaires and performed semistructured video examinations while performing tasks from the (modified) Unified Parkinson Disease Rating Scale when possible. RESULTS: Thirty adult patients were included for summary statistics, with video recordings available for 19 patients. The median age at last follow-up was 24 years (range 18-58 years). All patients had epilepsy, with a median onset age of 3.5 months. At last follow-up, 80% of adults had treatment-resistant seizures despite long periods of seizure freedom in 37%. Tonic-clonic, focal, and tonic seizures were most frequent in adults. Epileptic spasms, an unusual feature beyond infancy, were present in 3 adults. All individuals had developmental impairment. Periods of regression were present in 59% and did not always correlate with flare-ups in seizure activity. Eighty-seven percent had severe or profound intellectual disability, 42% had autistic features, and 65% had significant behavioral problems. Video examinations showed gait disorders in all 12 patients able to walk, including postural abnormalities with external rotation of the feet, broad-based gait, and asymmetric posture/dystonia. Tremor, present in 56%, was predominantly of the intention/action type. Stereotypies were seen in 63%. Functional outcome concerning mobility was variable ranging from independent walking (50%) to wheelchair dependence (39%). Seventy-one percent of adults were nonverbal, and all were dependent on caregivers for most activities of daily living. DISCUSSION: STXBP1-DEE warrants continuous monitoring for seizures in adult life. Periods of regression are more frequent than previously established and can occur into adulthood. Movement disorders are often present and involve multiple systems. Although functional mobility is variable in adulthood, STXBP1-DEE frequently leads to severe cognitive impairments and a high level of functional dependence. Understanding the natural history of STXBP1-DEE is important for prognostication and will inform future therapeutic trials.

KCNQ2 R144 variants cause neurodevelopmental disability with language impairment and autistic features without neonatal seizures through a gain-of-function mechanism.

BACKGROUND: Prior studies have revealed remarkable phenotypic heterogeneity in KCNQ2-related disorders, correlated with effects on biophysical features of heterologously expressed channels. Here, we assessed phenotypes and functional properties associated with KCNQ2 missense variants R144W, R144Q, and R144G. We also explored in vitro blockade of channels carrying R144Q mutant subunits by amitriptyline. METHODS: Patients were identified using the RIKEE database and through clinical collaborators. Phenotypes were collected by a standardized questionnaire. Functional and pharmacological properties of variant subunits were analyzed by whole-cell patch-clamp recordings. FINDINGS: Detailed clinical information on fifteen patients (14 novel and 1 previously published) was analyzed. All patients had developmental delay with prominent language impairment. R144Q patients were more severely affected than R144W patients. Infantile to childhood onset epilepsy occurred in 40%, while 67% of sleep-EEGs showed sleep-activated epileptiform activity. Ten patients (67%) showed autistic features. Activation gating of homomeric Kv7.2 R144W/Q/G channels was left-shifted, suggesting gain-of-function effects. Amitriptyline blocked channels containing Kv7.2 and Kv7.2 R144Q subunits. INTERPRETATION: Patients carrying KCNQ2 R144 gain-of-function variants have developmental delay with prominent language impairment, autistic features, often accompanied by infantile- to childhood-onset epilepsy and EEG sleep-activated epileptiform activity. The absence of neonatal seizures is a robust and important clinical differentiator between KCNQ2 gain-of-function and loss-of-function variants. The Kv7.2/7.3 channel blocker amitriptyline might represent a targeted treatment. FUNDING: Supported by FWO, GSKE, KCNQ2-Cure, Jack Pribaz Foundation, European Joint Programme on Rare Disease 2020, the Italian Ministry for University and Research, the Italian Ministry of Health, the European Commission, the University of Antwerp, NINDS, and Chalk Family Foundation.