An iPSC line (FINi003-A) from a male with late-onset developmental and epileptic encephalopathy caused by a heterozygous p.E1211K variant in the SCN2A gene encoding the voltage-gated sodium channel Nav1.2.

Many developmental and epileptic encephalopathies (DEEs) result from variants in cation channel genes. Using mRNA transfection, we generated and characterised an induced pluripotent stem cell (iPSC) line from the fibroblasts of a male late-onset DEE patient carrying a heterozygous missense variant (E1211K) in Nav1.2(SCN2A) protein. The iPSC line displays features characteristic of the human iPSCs, colony morphology and expression of pluripotency-associated marker genes, ability to produce derivatives of all three embryonic germ layers, and normal karyotype without SNP array-detectable abnormalities. We anticipate that this iPSC line will aid in the modelling and development of precision therapies for this debilitating condition.

Physical and functional convergence of the autism risk genes Scn2a and Ank2 in neocortical pyramidal cell dendrites.

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel NaV1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how NaV1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding NaV1.2 to the dendritic membrane of mouse neocortical neurons and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a+/- conditions. These results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can contribute to neurodevelopmental disorder pathophysiology.

What does better look like in individuals with severe neurodevelopmental impairments? A qualitative descriptive study on SCN2A-related developmental and epileptic encephalopathy.

PURPOSE: There are limited psychometric data on outcome measures for children with Developmental Epileptic Encephalopathies (DEEs), beyond measuring seizures, and no data to describe meaningful change. This study aimed to explore parent perceptions of important differences in functional abilities that would guide their participation in clinical trials. METHODS: This was a descriptive qualitative study. Semi-structured one-on-one interviews were conducted with 10 families (15 parent participants) with a child with a SCN2A-DEE [8 male, median (range) age 7.5 (4.5-21)] years. Questions and probes sought to understand the child's functioning across four domains: gross motor, fine motor, communication, and activities of daily living. Additional probing questions sought to identify the smallest differences in the child's functioning for each domain that would be important to achieve, if enrolling in a traditional therapy clinical trial or in a gene therapy trial. Data were analyzed with directed content analysis. RESULTS: Expressed meaningful differences appeared to describe smaller developmental steps for children with more limited developmental skills and more complex developmental steps for children with less limited skills and were different for different clinical trial scenarios. Individual meaningful changes were described as important for the child's quality of life and to facilitate day-to-day caring. CONCLUSION: Meaningful change thresholds have not been evaluated in the DEE literature. This study was a preliminary qualitative approach to inform future studies that will aim to determine quantitative values of change, applicable to groups and within-person, to inform interpretation of specific clinical outcome assessments in individuals with a DEE.

Distinctive In Vitro Phenotypes in iPSC-Derived Neurons From Patients With Gain- and Loss-of-Function SCN2A Developmental and Epileptic Encephalopathy.

SCN2A encodes NaV1.2, an excitatory neuron voltage-gated sodium channel and a major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with the nature of SCN2A variant dysfunction and can be divided into gain-of-function (GoF) cases with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age. We established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrent SCN2A DEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Two male patient-derived iPSC isogenic pairs were differentiated using Neurogenin-2 overexpression yielding populations of cortical-like glutamatergic neurons. Functional properties were assessed using patch clamp and multielectrode array recordings and transcriptomic profiles obtained with total mRNA sequencing after 2-4 weeks in culture. At 3 weeks of differentiation, increased neuronal activity at cellular and network levels was observed for R1882Q iPSC-derived neurons. In contrast, R853Q neurons showed only subtle changes in excitability after 4 weeks and an overall reduced network activity after 7 weeks in vitro. Consistent with the reported efficacy in some GoF SCN2A patients, phenytoin (sodium channel blocker) reduced the excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways suggested potential shared mechanisms underlying SCN2A DEE. In summary, patient iPSC-derived neuronal models of SCN2A GoF and LoF pathogenic variants causing DEE show specific functional and transcriptomic in vitro phenotypes.

Exome sequencing of ATP1A3-negative cases of alternating hemiplegia of childhood reveals SCN2A as a novel causative gene.

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopment disorder that is typically characterized by debilitating episodic attacks of hemiplegia, seizures, and intellectual disability. Over 85% of individuals with AHC have a de novo missense variant in ATP1A3 encoding the catalytic α3 subunit of neuronal Na+/K+ ATPases. The remainder of the patients are genetically unexplained. Here, we used next-generation sequencing to search for the genetic cause of 26 ATP1A3-negative index patients with a clinical presentation of AHC or an AHC-like phenotype. Three patients had affected siblings. Using targeted sequencing of exonic, intronic, and flanking regions of ATP1A3 in 22 of the 26 index patients, we found no ultra-rare variants. Using exome sequencing, we identified the likely genetic diagnosis in 9 probands (35%) in five genes, including RHOBTB2 (n = 3), ATP1A2 (n = 3), ANK3 (n = 1), SCN2A (n = 1), and CHD2 (n = 1). In follow-up investigations, two additional ATP1A3-negative individuals were found to have rare missense SCN2A variants, including one de novo likely pathogenic variant and one likely pathogenic variant for which inheritance could not be determined. Functional evaluation of the variants identified in SCN2A and ATP1A2 supports the pathogenicity of the identified variants. Our data show that genetic variants in various neurodevelopmental genes, including SCN2A, lead to AHC or AHC-like presentation. Still, the majority of ATP1A3-negative AHC or AHC-like patients remain unexplained, suggesting that other mutational mechanisms may account for the phenotype or that cases may be explained by oligo- or polygenic risk factors.

Dynamic Foraging Behavior Performance Is Not Affected by Scn2a Haploinsufficiency.

Dysfunction in the gene SCN2A, which encodes the voltage-gated sodium channel Nav1.2, is strongly associated with neurodevelopmental disorders including autism spectrum disorder and intellectual disability (ASD/ID). This dysfunction typically manifests in these disorders as a haploinsufficiency, where loss of one copy of a gene cannot be compensated for by the other allele. Scn2a haploinsufficiency affects a range of cells and circuits across the brain, including associative neocortical circuits that are important for cognitive flexibility and decision-making behaviors. Here, we tested whether Scn2a haploinsufficiency has any effect on a dynamic foraging task that engages such circuits. Scn2a +/- mice and wild-type (WT) littermates were trained on a choice behavior where the probability of reward between two options varied dynamically across trials and where the location of the high reward underwent uncued reversals. Despite impairments in Scn2a-related neuronal excitability, we found that both male and female Scn2a +/- mice performed these tasks as well as wild-type littermates, with no behavioral difference across genotypes in learning or performance parameters. Varying the number of trials between reversals or probabilities of receiving reward did not result in an observable behavioral difference, either. These data suggest that, despite heterozygous loss of Scn2a, mice can perform relatively complex foraging tasks that make use of higher-order neuronal circuits.

Frequency of SCN2A-related disorder in the regional epilepsy centre of brescia between 2002 and 2021.

OBJECTIVE: SCN2A gene pathogenic variants are associated with a wide phenotypic spectrum, encompassing epilepsy, developmental delay, and autism spectrum disorder. Researches conducted in Denmark have revealed a disease frequency of approximately 1/78,608 (0.0012%) live births in this population. We estimated the frequency of SCN2A-related disorder in the birth cohort of Brescia and its province between 2002 and 2021. METHODS: Frequency was calculated by ratio between patients with SCN2A pathogenic variant and the total number of live births at the Regional Epilepsy Center of Brescia, between 2002 and 2021. The number of births in Brescia and province was obtained from the Italian National Institute of Statistics (ISTAT). RESULTS: A frequency of 11/23,2678 births (0.0047%) was found. In comparison with Danish data, we noticed a higher frequency of the pathogenic variant in our population, even considering the same time frame (0.0035% of subjects born between 2006 and 2014). CONCLUSION: The frequency of SCN2A pathogenic variant among live births in Brescia and its Province between 2006 and 2014 was about three times that of Danish population; this difference was about four times if we consider the period from 2002 to 2021. More studies are needed to further delineate the frequency of SCN2A pathogenic variant in Italian population.

Generation of an iPSC line (FINi001-A) from a girl with developmental and epileptic encephalopathy due to a heterozygous gain-of-function p.R1882Q variant in the voltage-gated sodium channel Nav1.2 protein encoded by the SCN2A gene.

A range of epilepsies, including the most severe group of developmental and epileptic encephalopathies (DEEs), are caused by gain-of-function variants in voltage-gated channels. Here we report the generation and characterisation of an iPSC cell line from the fibroblasts of a girl with early infantile DEE carrying heterozygous missense gain-of-function mutation (R1882Q) in Nav1.2(SCN2A) protein, using transient transfection with a single mRNA molecule. The established iPSC line displays typical human primed pluripotent stem cell characteristics: typical colony morphology and robust expression of pluripotency-associated marker genes, ability to give rise to derivatives of all three embryonic germ layers, and normal karyotype without any SNP array-detectable copy number variations. We anticipate that this iPSC line will be useful for the development of neuronal hyperactivity-caused human stem cell-based DEE models, advancing both understanding and potential therapy development for this debilitating condition.

Epilepsy-associated SCN2A (NaV1.2) variants exhibit diverse and complex functional properties.

Pathogenic variants in voltage-gated sodium (NaV) channel genes including SCN2A, encoding NaV1.2, are discovered frequently in neurodevelopmental disorders with or without epilepsy. SCN2A is also a high-confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function variants cause neonatal-onset epilepsy, whereas loss-of-function variants are associated with ASD and ID. However, this framework was derived from a limited number of studies conducted under heterogeneous experimental conditions, whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of SCN2A variants using automated patch-clamp recording to demonstrate the validity of this method and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common variants using two alternatively spliced isoforms of NaV1.2 expressed in HEK293T cells. Automated patch-clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for variants that were previously studied using manual patch clamp. Many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-functions that are difficult to classify by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of variants with greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor. This approach offers an enhanced ability to discern relationships between channel dysfunction and neurodevelopmental disorders.

Early recognition of characteristic conventional and amplitude-integrated EEG patterns of seizures in SCN2A and KCNQ3-related epilepsy in neonates.

PURPOSE: Early recognition of seizures in neonates secondary to pathogenic variants in potassium or sodium channel coding genes is crucial, as these seizures are often resistant to commonly used anti-seizure medications but respond well to sodium channel blockers. Recently, a characteristic ictal amplitude-integrated electroencephalogram (aEEG) pattern was described in neonates with KCNQ2-related epilepsy. We report a similar aEEG pattern in seizures caused by SCN2A- and KCNQ3-pathogenic variants, as well as conventional EEG (cEEG) descriptions. METHODS: International multicentre descriptive study, reporting clinical characteristics, aEEG and cEEG findings of 13 neonates with seizures due to pathogenic SCN2A- and KCNQ3-variants. As a comparison group, aEEGs and cEEGs of neonates with seizures due to hypoxic-ischemic encephalopathy (n = 117) and other confirmed genetic causes affecting channel function (n = 55) were reviewed. RESULTS: In 12 out of 13 patients, the aEEG showed a characteristic sequence of brief onset with a decrease, followed by a quick rise, and then postictal amplitude attenuation. This pattern correlated with bilateral EEG onset attenuation, followed by rhythmic discharges ending in several seconds of post-ictal amplitude suppression. Apart from patients with KCNQ2-related epilepsy, none of the patients in the comparison groups had a similar aEEG or cEEG pattern. DISCUSSION: Seizures in SCN2A- and KCNQ3-related epilepsy in neonates can usually be recognized by a characteristic ictal aEEG pattern, previously reported only in KCNQ2-related epilepsy, extending this unique feature to other channelopathies. Awareness of this pattern facilitates the prompt initiation of precision treatment with sodium channel blockers even before genetic results are available.

Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing.

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.

Missense mutations in the membrane domain of PRRT2 affect its interaction with Nav1.2 voltage-gated sodium channels.

PRRT2 is a neuronal protein that controls neuronal excitability and network stability by modulating voltage-gated Na+ channel (Nav). PRRT2 pathogenic variants cause pleiotropic syndromes including epilepsy, paroxysmal kinesigenic dyskinesia and episodic ataxia attributable to loss-of-function pathogenetic mechanism. Based on the evidence that the transmembrane domain of PRRT2 interacts with Nav1.2/1.6, we focused on eight missense mutations located within the domain that show expression and membrane localization similar to the wild-type protein. Molecular dynamics simulations showed that the mutants do not alter the structural stability of the PRRT2 membrane domain and preserve its conformation. Using affinity assays, we found that the A320V and V286M mutants displayed respectively decreased and increased binding to Nav1.2. Accordingly, surface biotinylation showed an increased Nav1.2 surface exposure induced by the A320V mutant. Electrophysiological analysis confirmed the lack of modulation of Nav1.2 biophysical properties by the A320V mutant with a loss-of-function phenotype, while the V286M mutant displayed a gain-of-function with respect to wild-type PRRT2 with a more pronounced left-shift of the inactivation kinetics and delayed recovery from inactivation. The data confirm the key role played by the PRRT2-Nav interaction in the pathogenesis of the PRRT2-linked disorders and suggest an involvement of the A320 and V286 residues in the interaction site. Given the similar clinical phenotype caused by the two mutations, we speculate that circuit instability and paroxysmal manifestations may arise when PRRT2 function is outside the physiological range.

Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons.

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. In contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations.

Inhibitory effect of atomoxetine on Nav1.2 voltage-gated sodium channel currents.

BACKGROUND: Atomoxetine (ATX), a norepinephrine reuptake inhibitor (NRI), is used to attenuate the symptoms of Attention Deficit/Hyperactivity Disorder (AD/HD) by increasing neurotransmitter concentrations at the synaptic cleft. Although Nav1.2 voltage-gated sodium channels (VGSCs) are thought to play a role in monoamine transmitter release in the synaptic junction, it is unclear how atomoxetine affects Nav1.2 VGSCs. METHODS: In this study, we investigated the effect of ATX on Nav1.2 VGSC-transfected HEK293 cells with the whole-patch clamp technique. RESULTS: Nav1.2 VGSC current decreased by 51.15 ± 12.75% under treatment with 50 µM ATX in the resting state (holding membrane potential at - 80 mV). The IC50 of ATX against Nav1.2 VGSC current was 45.57 µM. The activation/inactivation curve of Nav1.2 VGSC currents was shifted toward hyperpolarization by 50 µM ATX. In addition, the inhibitory effect of ATX increased with membrane depolarization (holding membrane potential at - 50 mV) and its IC50 was 10.16 µM. Moreover, ATX showed the time-dependent interaction in the inactivation state. CONCLUSION: These findings suggest that ATX interacts with Nav1.2 VGSCs producing the inhibition of current and the modification of kinetic properties in the state-dependent manner.

Cryo-EM structure of human voltage-gated sodium channel Nav1.6.

Voltage-gated sodium channel Nav1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Nav1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Nav1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Nav1.6 in the presence of auxiliary subunits ß1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the ß1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Nav1.6-specific blockers. Structural mapping of disease-related Nav1.6 mutations provides insights into their pathogenic mechanism.

A Push-Pull Mechanism Between PRRT2 and ß4-subunit Differentially Regulates Membrane Exposure and Biophysical Properties of NaV1.2 Sodium Channels.

Proline-rich transmembrane protein 2 (PRRT2) is a neuron-specific protein implicated in the control of neurotransmitter release and neural network stability. Accordingly, PRRT2 loss-of-function mutations associate with pleiotropic paroxysmal neurological disorders, including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. PRRT2 is a negative modulator of the membrane exposure and biophysical properties of Na+ channels NaV1.2/NaV1.6 predominantly expressed in brain glutamatergic neurons. NaV channels form complexes with ß-subunits that facilitate the membrane targeting and the activation of the α-subunits. The opposite effects of PRRT2 and ß-subunits on NaV channels raises the question of whether PRRT2 and ß-subunits interact or compete for common binding sites on the α-subunit, generating Na+ channel complexes with distinct functional properties. Using a heterologous expression system, we have observed that ß-subunits and PRRT2 do not interact with each other and act as independent non-competitive modulators of NaV1.2 channel trafficking and biophysical properties. PRRT2 antagonizes the ß4-induced increase in expression and functional activation of the transient and persistent NaV1.2 currents, without affecting resurgent current. The data indicate that ß4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of NaV channels and the intrinsic neuronal excitability.

A Complex Genomic Rearrangement Resulting in Loss of Function of SCN1A and SCN2A in a Patient with Severe Developmental and Epileptic Encephalopathy.

Complex genomic rearrangements (CGRs) are structural variants arising from two or more chromosomal breaks, which are challenging to characterize by conventional or molecular cytogenetic analysis (karyotype and FISH). The integrated approach of standard and genomic techniques, including optical genome mapping (OGM) and genome sequencing, is crucial for disclosing and characterizing cryptic chromosomal rearrangements at high resolutions. We report on a patient with a complex developmental and epileptic encephalopathy in which karyotype analysis showed a de novo balanced translocation involving the long arms of chromosomes 2 and 18. Microarray analysis detected a 194 Kb microdeletion at 2q24.3 involving the SCN2A gene, which was considered the likely translocation breakpoint on chromosome 2. However, OGM redefined the translocation breakpoints by disclosing a paracentric inversion at 2q24.3 disrupting SCN1A. This combined genomic high-resolution approach allowed a fine characterization of the CGR, which involves two different chromosomes with four breakpoints. The patient's phenotype resulted from the concomitant loss of function of SCN1A and SCN2A.