Expanded clinical phenotype spectrum correlates with variant function in SCN2A-related disorders.

SCN2A-related disorders secondary to altered function in the voltage-gated sodium channel NaV1.2 are rare with clinically heterogeneous expressions that include epilepsy, autism, and multiple severe to profound impairments and other conditions. To advance understanding of the clinical phenotypes and their relation to channel function, 81 patients (36, 44% female, median age 5.4 years) with 69 unique SCN2A variants were systematically phenotyped and their NaV1.2 channel function systematically assessed. Participants were recruited through the FamileSCN2A Foundation. Primary phenotype (epilepsy of neonatal-onset, N=27; infant onset, N=18; and later onset N=24; and autism without seizures, (N=12) was strongly correlated with a non-seizure severity index (p=0.002), which was based on presence of severe impairments in gross motor, fine motor, communication abilities, gastrostomy tube dependence, and diagnosis of cortical visual impairment and scoliosis. Non-seizure severity was greatest in the neonatal-onset group and least in the autism group (p=0.002). Children with the lowest severity indices were still severely impaired, as reflected by an average Vineland adaptive behavior composite score of 49.5 (>3 SD below the test's norm-referenced mean). Epileptic spasms were significantly more common in infant onset (67%) than in neonatal (22%) or later-onset (29%) epilepsy (p=0.007). Primary phenotype also strongly correlated with variant function (p<0.0001); gain of function and mixed function variants predominated in neonatal-onset epilepsy, shifting to moderate loss of function in infant-onset epilepsy, and severe and complete loss of function in later-onset epilepsy and autism groups. Exploratory cluster analysis identified five groups representing (1) primarily later-onset epilepsy with moderate loss of function variants and low severity indices, (2) mostly infant-onset epilepsy with moderate loss of function variants but higher severity indices, (3) late-onset and autism only with the lowest severity indices (mostly 0) and severe/complete loss of function variants. Two exclusively neonatal clusters were distinguished from each other largely on non-seizure severity scores and secondarily on variant function. The relation between primary phenotype and variant function emphasizes the role of developmental factors in the differential clinical expression of SCN2A variants based on their effects on NaV1.2 channel function. The non-seizure severity of SCN2A disorders depends on a combination of the age at seizure onset (primary phenotype) and variant function. As precision therapies for SCN2A-related disorders advance toward clinical trials, knowledge of the relationship between variant function and clinical disease expression will be valuable for identifying appropriate patients for these trials and in selecting efficient clinical outcomes.

Altered neurological and neurobehavioral phenotypes in a mouse model of the recurrent KCNB1-p.R306C voltage-sensor variant.

Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1-p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described altered sensitivity and cooperativity of the voltage sensor and impaired capacity for repetitive firing of neurons. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1-p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1. Using CRISPR/Cas9 genome editing, we generated the Kcnb1R306C mouse model and characterized the molecular and phenotypic effects. Consistent with the in vitro studies, neurons from Kcnb1R306C mice showed altered excitability. Heterozygous and homozygous R306C mice exhibited hyperactivity, altered susceptibility to chemoconvulsant-induced seizures, and frequent, long runs of slow spike wave discharges on EEG, reminiscent of the slow spike and wave activity characteristic of Lennox Gastaut syndrome. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies.

Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice.

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to developmental and epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to intellectual disability and/or autism spectrum disorder (ASD) with or without co-morbid seizures. One unique case less easily classified using this framework is the de novo missense variant SCN2A-p.K1422E, associated with infant-onset developmental delay, infantile spasms and features of ASD. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model incorporating variant channels that predicted reductions in peak action potential (AP) speed. We generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Cultured cortical neurons from heterozygous Scn2aK1422E/+ mice exhibited lower current density with a TTX-resistant component and reversal potential consistent with mixed ion permeation. Recordings from Scn2aK1442E/+ cortical slices demonstrated impaired AP initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the AP, suggesting complex effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal electroencephalogram abnormalities, altered induced seizure thresholds, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from other Scn2a models, consistent with complex effects of K1422E on NaV1.2 channel function.

Cryptic prokaryotic promoters explain instability of recombinant neuronal sodium channels in bacteria.

Mutations in genes encoding the human-brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, and other neurodevelopmental disorders. A major obstacle hindering investigations of the functional consequences of brain NaV channel mutations is an unexplained instability of the corresponding recombinant complementary DNA (cDNA) when propagated in commonly used bacterial strains manifested by high spontaneous rates of mutation. Here, using a combination of in silico analysis, random and site-directed mutagenesis, we investigated the cause for instability of human NaV1.1 cDNA. We identified nucleotide sequences within the NaV1.1 coding region that resemble prokaryotic promoter-like elements, which are presumed to drive transcription of translationally toxic mRNAs in bacteria as the cause of the instability. We further demonstrated that mutations disrupting these elements mitigate the instability. Extending these observations, we generated full-length human NaV1.1, NaV1.2, and NaV1.6 plasmids using one or two introns that interrupt the latent reading frames along with a minimum number of silent nucleotide changes that achieved stable propagation in bacteria. Expression of the stabilized sequences in cultured mammalian cells resulted in functional NaV channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human NaV channels, and we describe re-engineered plasmids that attenuate this problem.

Enhanced slow inactivation contributes to dysfunction of a recurrent SCN2A mutation associated with developmental and epileptic encephalopathy.

KEY POINTS: The recurrent SCN2A mutation R853Q is associated with developmental and epileptic encephalopathy with typical onset after the first months of life. Heterologously expressed R853Q channels exhibit an overall loss-of-function as a result of multiple defects in time- and voltage-dependent channel properties. A previously unrecognized enhancement of slow inactivation is conferred by the R853Q mutation and is a major driver of loss-of-function. Enhanced slow inactivation is potentiated in the canonical splice isoform of the channel and this may explain the later onset of symptoms associated with R853Q. ABSTRACT: Mutations in voltage gated sodium (NaV ) channel genes, including SCN2A (encoding NaV 1.2), are associated with diverse neurodevelopmental disorders with or without epilepsy that present clinically with varying severity, age-of-onset and pharmacoresponsiveness. We examined the functional properties of the most recurrent SCN2A mutation (R853Q) to determine whether developmentally-regulated alternative splicing impacts dysfunction severity and to investigate effects of the mutation on slow inactivation. We engineered the R853Q mutation into neonatal and adult NaV 1.2 splice isoforms. Channel constructs were heterologously co-expressed in HEK293T cells with human ß1 and ß2 subunits. Whole-cell patch clamp recording was used to compare time- and voltage-dependent properties of mutant and wild-type channels. The R853Q mutation exhibits an overall loss-of-function attributed to multiple functional defects including a previously undiscovered enhancement of slow inactivation. The mutation exhibited altered voltage dependence of activation and inactivation, slower recovery from inactivation and decreased channel availability during high-frequency depolarizations. More notable were effects on slow inactivation, including a 10-fold slower rate of recovery from slow inactivation exhibited by mutant channels. The impairments in fast inactivation properties were more severe in the neonatal splice isoform, whereas slow inactivation was more pronounced in the splice isoform of the channel expressed predominantly in later childhood. Enhanced later-onset slow inactivation may be a primary driver of the later onset of neurological features associated with this mutation.

SCN3A-Related Neurodevelopmental Disorder: A Spectrum of Epilepsy and Brain Malformation.

OBJECTIVE: Pathogenic variants in SCN3A, encoding the voltage-gated sodium channel subunit Nav1.3, cause severe childhood onset epilepsy and malformation of cortical development. Here, we define the spectrum of clinical, genetic, and neuroimaging features of SCN3A-related neurodevelopmental disorder. METHODS: Patients were ascertained via an international collaborative network. We compared sodium channels containing wild-type versus variant Nav1.3 subunits coexpressed with ß1 and ß2 subunits using whole-cell voltage clamp electrophysiological recordings in a heterologous mammalian system (HEK-293T cells). RESULTS: Of 22 patients with pathogenic SCN3A variants, most had treatment-resistant epilepsy beginning in the first year of life (16/21, 76%; median onset, 2 weeks), with severe or profound developmental delay (15/20, 75%). Many, but not all (15/19, 79%), exhibited malformations of cortical development. Pathogenic variants clustered in transmembrane segments 4 to 6 of domains II to IV. Most pathogenic missense variants tested (10/11, 91%) displayed gain of channel function, with increased persistent current and/or a leftward shift in the voltage dependence of activation, and all variants associated with malformation of cortical development exhibited gain of channel function. One variant (p.Ile1468Arg) exhibited mixed effects, with gain and partial loss of function. Two variants demonstrated loss of channel function. INTERPRETATION: Our study defines SCN3A-related neurodevelopmental disorder along a spectrum of severity, but typically including epilepsy and severe or profound developmental delay/intellectual disability. Malformations of cortical development are a characteristic feature of this unusual channelopathy syndrome, present in >75% of affected individuals. Gain of function at the channel level in developing neurons is likely an important mechanism of disease pathogenesis. ANN NEUROL 2020;88:348-362.

CaMKII modulates sodium current in neurons from epileptic Scn2a mutant mice.

Monogenic epilepsies with wide-ranging clinical severity have been associated with mutations in voltage-gated sodium channel genes. In the Scn2aQ54 mouse model of epilepsy, a focal epilepsy phenotype is caused by transgenic expression of an engineered NaV1.2 mutation displaying enhanced persistent sodium current. Seizure frequency and other phenotypic features in Scn2aQ54 mice depend on genetic background. We investigated the neurophysiological and molecular correlates of strain-dependent epilepsy severity in this model. Scn2aQ54 mice on the C57BL/6J background (B6.Q54) exhibit a mild disorder, whereas animals intercrossed with SJL/J mice (F1.Q54) have a severe phenotype. Whole-cell recording revealed that hippocampal pyramidal neurons from B6.Q54 and F1.Q54 animals exhibit spontaneous action potentials, but F1.Q54 neurons exhibited higher firing frequency and greater evoked activity compared with B6.Q54 neurons. These findings correlated with larger persistent sodium current and depolarized inactivation in neurons from F1.Q54 animals. Because calcium/calmodulin protein kinase II (CaMKII) is known to modify persistent current and channel inactivation in the heart, we investigated CaMKII as a plausible modulator of neuronal sodium channels. CaMKII activity in hippocampal protein lysates exhibited a strain-dependence in Scn2aQ54 mice with higher activity in F1.Q54 animals. Heterologously expressed NaV1.2 channels exposed to activated CaMKII had enhanced persistent current and depolarized channel inactivation resembling the properties of F1.Q54 neuronal sodium channels. By contrast, inhibition of CaMKII attenuated persistent current, evoked a hyperpolarized channel inactivation, and suppressed neuronal excitability. We conclude that CaMKII-mediated modulation of neuronal sodium current impacts neuronal excitability in Scn2aQ54 mice and may represent a therapeutic target for the treatment of epilepsy.

Direct evidence of impaired neuronal Na/K-ATPase pump function in alternating hemiplegia of childhood.

Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current ('pump current'). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to -80 mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential impact of this genetic defect on cellular excitability.

The novel sodium channel modulator GS-458967 (GS967) is an effective treatment in a mouse model of SCN8A encephalopathy.

OBJECTIVE: De novo mutations of SCN8A, encoding the voltage-gated sodium channel NaV 1.6, have been associated with a severe infant onset epileptic encephalopathy. Individuals with SCN8A encephalopathy have a mean age of seizure onset of 4-5 months, with multiple seizure types that are often refractory to treatment with available drugs. Anecdotal reports suggest that high-dose phenytoin is effective for some patients, but there are associated adverse effects and potential for toxicity. Functional characterization of several SCN8A encephalopathy variants has shown that elevated persistent sodium current is one of several common biophysical defects. Therefore, specifically targeting elevated persistent current may be a useful therapeutic strategy in some cases. METHODS: The novel sodium channel modulator GS967 has greater preference for persistent as opposed to peak current and nearly 10-fold greater potency than phenytoin. We evaluated the therapeutic effect of GS967 in the Scn8aN1768D/+ mouse model carrying an SCN8A patient mutation that results in elevated persistent sodium current. We also performed patch clamp recordings to assess the effect of GS967 on peak and persistent sodium current and excitability in hippocampal neurons from Scn8aN1768D/+ mice. RESULTS: GS967 potently blocked persistent sodium current without affecting peak current, normalized action potential morphology, and attenuated excitability in neurons from heterozygous Scn8aN1768D/+ mice. Acute treatment with GS967 provided dose-dependent protection against maximal electroshock-induced seizures in Scn8aN1768D/+ and wild-type mice. Chronic treatment of Scn8aN1768D/+ mice with GS967 resulted in lower seizure burden and complete protection from seizure-associated lethality observed in untreated Scn8aN1768D/+ mice. Protection was achieved at a chronic dose that did not cause overt behavioral toxicity or sedation. SIGNIFICANCE: Persistent sodium current modulators like GS967 may be an effective precision targeting strategy for SCN8A encephalopathy and other functionally similar channelopathies when elevated persistent sodium current is the primary dysfunction.

SCN1A variants associated with sudden infant death syndrome.

We identified SCN1A variants in 2 infants who died of sudden infant death syndrome (SIDS) with hippocampal abnormalities from an exome sequencing study of 10 cases of SIDS but no history of seizures. One harbored SCN1A G682V, and the other had 2 SCN1A variants in cis: L1296M and E1308D, a variant previously associated with epilepsy. Functional evaluation in a heterologous expression system demonstrated partial loss of function for both G682V and the compound variant L1296M/E1308D. Our cases represent a novel association between SCN1A and SIDS, extending the SCN1A spectrum from epilepsy to SIDS. Our findings provide insights into SIDS and support genetic evaluation focused on epilepsy genes in SIDS.

Strain- and age-dependent hippocampal neuron sodium currents correlate with epilepsy severity in Dravet syndrome mice.

Heterozygous loss-of-function SCN1A mutations cause Dravet syndrome, an epileptic encephalopathy of infancy that exhibits variable clinical severity. We utilized a heterozygous Scn1a knockout (Scn1a(+/-)) mouse model of Dravet syndrome to investigate the basis for phenotype variability. These animals exhibit strain-dependent seizure severity and survival. Scn1a(+/-) mice on strain 129S6/SvEvTac (129.Scn1a(+/-)) have no overt phenotype and normal survival compared with Scn1a(+/-) mice bred to C57BL/6J (F1.Scn1a(+/-)) that have severe epilepsy and premature lethality. We tested the hypothesis that strain differences in sodium current (INa) density in hippocampal neurons contribute to these divergent phenotypes. Whole-cell voltage-clamp recording was performed on acutely-dissociated hippocampal neurons from postnatal days 21-24 (P21-24) 129.Scn1a(+/-) or F1.Scn1a(+/-) mice and wild-type littermates. INa density was lower in GABAergic interneurons from F1.Scn1a(+/-) mice compared to wild-type littermates, while on the 129 strain there was no difference in GABAergic interneuron INa density between 129.Scn1a(+/-) mice and wild-type littermate controls. By contrast, INa density was elevated in pyramidal neurons from both 129.Scn1a(+/-) and F1.Scn1a(+/-) mice, and was correlated with more frequent spontaneous action potential firing in these neurons, as well as more sustained firing in F1.Scn1a(+/-) neurons. We also observed age-dependent differences in pyramidal neuron INa density between wild-type and Scn1a(+/-) animals. We conclude that preserved INa density in GABAergic interneurons contributes to the milder phenotype of 129.Scn1a(+/-) mice. Furthermore, elevated INa density in excitatory pyramidal neurons at P21-24 correlates with age-dependent onset of lethality in F1.Scn1a(+/-) mice. Our findings illustrate differences in hippocampal neurons that may underlie strain- and age-dependent phenotype severity in a Dravet syndrome mouse model, and emphasize a contribution of pyramidal neuron excitability.

Antiepileptic activity of preferential inhibitors of persistent sodium current.

OBJECTIVE: Evidence from basic neurophysiology and molecular genetics has implicated persistent sodium current conducted by voltage-gated sodium (NaV ) channels as a contributor to the pathogenesis of epilepsy. Many antiepileptic drugs target NaV channels and modulate neuronal excitability, mainly by a use-dependent block of transient sodium current, although suppression of persistent current may also contribute to the efficacy of these drugs. We hypothesized that a drug or compound capable of preferential inhibition of persistent sodium current would have antiepileptic activity. METHODS: We examined the antiepileptic activity of two selective persistent sodium current blockers ranolazine, a U.S. Food and Drug Administration (FDA)-approved drug for treatment of angina pectoris, and GS967, a novel compound with more potent effects on persistent current, in the epileptic Scn2a(Q54) mouse model. We also examined the effect of GS967 in the maximal electroshock model and evaluated effects of the compound on neuronal excitability, propensity for hilar neuron loss, development of mossy fiber sprouting, and survival of Scn2a(Q54) mice. RESULTS: We found that ranolazine was capable of reducing seizure frequency by approximately 50% in Scn2a(Q54) mice. The more potent persistent current blocker GS967 reduced seizure frequency by >90% in Scn2a(Q54) mice and protected against induced seizures in the maximal electroshock model. GS967 greatly attenuated abnormal spontaneous action potential firing in pyramidal neurons acutely isolated from Scn2a(Q54) mice. In addition to seizure suppression in vivo, GS967 treatment greatly improved the survival of Scn2a(Q54) mice, prevented hilar neuron loss, and suppressed the development of hippocampal mossy fiber sprouting. SIGNIFICANCE: Our findings indicate that the selective persistent sodium current blocker GS967 has potent antiepileptic activity and that this compound could inform development of new agents.

Chloride channels: often enigmatic, rarely predictable.

Until recently, anion (Cl(-)) channels have received considerably less attention than cation channels. One reason for this may be that many Cl(-) channels perform functions that might be considered cell-biological, like fluid secretion and cell volume regulation, whereas cation channels have historically been associated with cellular excitability, which typically happens more rapidly. In this review, we discuss the recent explosion of interest in Cl(-) channels, with special emphasis on new and often surprising developments over the past five years. This is exemplified by the findings that more than half of the ClC family members are antiporters, and not channels, as was previously thought, and that bestrophins, previously prime candidates for Ca(2+)-activated Cl(-) channels, have been supplanted by the newly discovered anoctamins and now hold a tenuous position in the Cl(-) channel world.

Molecular and cellular context influences SCN8A variant function.

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

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.

Nontruncating SCN1A mutations associated with severe myoclonic epilepsy of infancy impair cell surface expression.

Mutations in SCN1A, encoding the voltage-gated sodium channel Na(V)1.1, are the most common cause of severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome. SMEI is most often associated with premature truncations of Na(V)1.1 that cause loss of function, but nontruncating mutations also occur. We hypothesized that some nontruncating mutations might impair trafficking of Na(V)1.1 to the plasma membrane. Here we demonstrated that seven nontruncating missense or in-frame deletion mutations (L986F, delF1289, R1648C, F1661S, G1674R, and G1979E) exhibited reduced cell surface expression relative to wild type (WT) Na(V)1.1 consistent with impaired trafficking. We tested whether two commonly prescribed antiepileptic drugs (phenytoin, lamotrigine), as well as the cystic fibrosis transmembrane conductance regulator (CFTR) trafficking corrector VRT-325, could rescue cell surface and functional expression of two representative Na(V)1.1 mutants (R1648C, G1674R). Treatment of cells with phenytoin increased cell surface expression of WT-Na(V)1.1 and both mutant channels, whereas lamotrigine only increased surface expression of R1648C. VRT-325 did not alter surface expression of WT-Na(V)1.1 or mutant channels. Although phenytoin increased surface expression of G1674R, channel function was not restored, suggesting that this mutation also causes an intrinsic loss of function. Both phenytoin and lamotrigine increased functional expression of R1648C, but lamotrigine also increased persistent sodium current evoked by this mutation. Our findings indicate that certain nontruncating SCN1A mutations associated with SMEI have impaired cell surface expression and that some alleles may be amenable to pharmacological rescue of this defect. However, rescue of dysfunctional Na(V)1.1 channels to the plasma membrane could contribute to exacerbating rather than ameliorating the disease.

Epilepsy-associated SCN2A (Na V 1.2) Variants Exhibit Diverse and Complex Functional Properties.

Pathogenic variants in neuronal voltage-gated sodium (Na V ) channel genes including SCN2A , which encodes Na V 1.2, are frequently discovered in neurodevelopmental disorders with and 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 (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A variants using automated patch clamp recording to assess the analytical validity of this approach 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 population variants using two distinct alternatively spliced forms of Na V 1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of Na V 1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of Na V channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.

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.

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.

Molecular and Cellular Context Influences SCN8A Variant Function.

Pathogenic variants in SCN8A , which encodes the voltage-gated sodium (Na V ) channel Na V 1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of Na V 1.6 (Na V 1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (Na V 1.6N, Na V 1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous Na V current to obviate the need for TTX-resistance mutations. Expression of Na V 1.6N or Na V 1.6A in ND7/LoNav cells generated Na V currents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.