Nav1.1 channels with mutations of severe myoclonic epilepsy in infancy display attenuated currents

doi:10.1016/S0920-1211(03)00084-6

Epilepsy Research

Volume 54, Issues 2–3, May 2003, Pages 201-207

https://doi.org/10.1016/S0920-1211(03)00084-6 Get rights and content

Abstract

Severe myoclonic epilepsy in infancy (SMEI) is characterized by intractable febrile and afebrile seizures, severe mental decline, and onset during the first year of life. Nonsense, frameshift, and missense mutations of SCN1A gene encoding the voltage-gated Na⁺ channel α-subunit type I (Na_v1.1) have been identified in patients with SMEI. Here, we performed whole-cell patch-clamp analyses on HEK293 cells expressing human Na_v1.1 channels bearing SMEI nonsense and missense mutations. The mutant channels showed remarkably attenuated or barely detectable inward sodium currents. Our findings indicate that SMEI mutations lead to loss-of-function and may contribute to the development of SMEI phenotypes.

Introduction

Severe myoclonic epilepsy in infancy (SMEI) (Dravet, 1978, Dravet et al., 1982) is classified as “syndrome undetermined as to whether it is focal or generalized,” and characterized by various signs including conspicuous fever-sensitive generalized tonic-clonic, alternative hemiclonic, myoclonic, atypical absence, complex partial seizures, and photosensitivity (Commission on Classification and Terminology of the International League Against Epilepsy, 1989). SMEI has an onset during the first year of life and is followed by severe mental decline. Frequent seizures are often prolonged and resistant to most anti-epileptic drugs (Oguni et al., 2001).

Recently, mutations of SCN1A, the gene encoding voltage-gated Na⁺ channel α-subunit type I (Na_v1.1), were reported in SMEI patients (Claes et al., 2001). Mutations were identified in all seven cases studied: four frameshift, one nonsense, one splice-donor, one missense, and all were heterozygous and de novo mutations. Subsequently, we reported frequent truncation mutations of SCN1A in Japanese SMEI patients (Sugawara et al., 2002). Nonsense or frameshift mutations resulting in truncated channels are a major component in typical SMEI patients, however recently we and other groups reported that missense mutations also play roles in the SMEI phenotype (Ohmori et al., 2002, Fujiwara et al., 2003). Mutations of SCN1A have also been known to be responsible for generalized epilepsy with febrile seizures plus (GEFS+) that is a milder idiopathic epilepsy (Escayg et al., 2000, Escayg et al., 2001, Wallace et al., 2001, Sugawara et al., 2001a). A term autosomal dominant epilepsy with febrile seizures plus (ADEFS+) has also been proposed instead of GEFS+ because of the occasional association of partial seizures (Ito et al., 2002). In contrast to the SMEI truncation mutations, the GEFS+ mutations are exclusively missense type.

Na_v1.1 has four homologous repeats (DI–DIV) each consisting of six transmembrane helices (S1–S6). Pore region (S5, S6 and loop between them), voltage sensor (S4), phosphorylation sites (intracellular loop), and inactivation gate (DIII–DIV loop) have been identified and characterized by the site-directed mutagenesis (Catterall, 2000). Na_v1.1 is primarily expressed at the soma of neuronal cells in the CNS (Gong et al., 1999) and is responsible for the rising phase of the action potential. The functional effects of GEFS+ missense mutations of SCN1A have been investigated by three groups (Alekov et al., 2001, Spampanato et al., 2001, Lossin et al., 2002). Biophysical characterization of the mutant channels has revealed alterations of the inactivation process as a major factor contributing to persistent Na⁺ influx.

GEFS+ and SMEI are caused by gene deficits in the same gene, whereas there are many differences between their phenotypes (Scheffer et al., 2001, Singh et al., 2001). Functional analyses of channel proteins with those mutations may elucidate the phenotypic diversity. Here we present a biophysical study of channels with six SMEI-associated mutations. Both nonsense and missense SCN1A mutations resulted in loss-of-function and lead to a reduction of the sodium current which may contribute to the pathogenesis of SMEI.

Section snippets

SMEI mutations

Clinical data of patients with SMEI harboring mutations studied here were reported elsewhere (Fujiwara et al., 2003). All patients but one (G979R) met the criteria of SMEI according to the proposal of the Commission on Classification and Terminology of the International League Against Epilepsy (1989). The patient with G979R mutation did not have a history of myoclonic seizures but all other clinical features are same as SMEI (Fujiwara et al., 2003).

Molecular cloning of human SCN1A cDNA and mutagenesis

5′- and 3′-RACE reactions were carried out

Results

The gating kinetics of channels with SMEI-associated nonsense mutations of SCN1A (R712X, R1407X, and R1892X) (Sugawara et al., 2001a) and missense mutations (G979R, N985I, and F1831S) (Fujiwara et al., 2003) were analyzed. The locations of these mutations are illustrated in Fig. 1. All mutant channels were transiently expressed in HEK293 cells, and expression levels and molecular sizes of the transcripts for the WT and all mutant channels were assessed by immuno-blot analysis (data not shown).

Discussion

The biophysical analysis of human Na_v1.1 channels bearing SMEI-associated nonsense and missense mutations described here indicates that such mutations would lead to loss-of-function with a profound decrement or absence of Na⁺ currents. It is conceivable that the truncated channels with R712X and R1407X mutations generate non-functional channels given the large missing domains (Fig. 1). By contrast, the channel with the R1892X mutation produced small yet detectable sodium currents, presumably

Acknowledgements

We are thankful to Dr. Makoto Kaneda (Department of Physiology, Keio University) for his helpful comment. This study was supported in part by grants from the Ministry of Education, Science and Culture of Japan. Research at the University of California is supported by NIH Grant GM-49711.

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Impact of variant subtype on electro-clinical phenotype of Dravet syndrome- a South Indian cohort study
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We aimed to compare the electroclinical correlates of truncating and missense variants of SCN1A variants in children with Dravet syndrome (DS) and to determine phenotypic features in relation to variants identified and seizure outcomes.
A single center prospective study was carried out on a South Indian cohort. Patients below 18 years of age who met the clinical criteria for DS who had undergone genetic testing and completed a minimum of one year follow up were included. We compared the differences in clinical profile, seizure outcome, developmental characteristics and anti-seizure medication (ASM) responsiveness profiles between patients with missense and truncating variants.
Out of a total of 3967 children with drug-resistant epilepsy during the period 2015–2021, 49 patients who fulfilled the inclusion criteria were studied. Thirty-seven had positive genetic tests, out of which 29 were SCN1A variants and 9 were other novel variants. The proportion of missense (14; 48.3%) and truncating SCN1A variants (15; 51.7%) was similar. A significant trend for developing multiple seizure types was noted among children with truncating variants (p = 0.035) and seizure freedom was more likely among children with missense variants (p = 0.042). All patients with truncating variants had ASM resistant epilepsy (p = 0.020). Developmental outcomes did not differ between the variant subtypes.
Our results show that children harbouring missense variants demonstrated a significantly lower propensity for multiple seizure subtypes and a higher proportion with seizure freedom. However developmental implications appear to be independent of variant subtype.
Novel variants in established epilepsy genes in focal epilepsy
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Next generation sequencing (NGS) has greatly expanded our understanding of genetic contributors in multiple epilepsy syndromes, including focal epilepsy. Describing the genetic architecture of common syndromes promises to facilitate the diagnostic process as well as aid in the identification of patients who stand to benefit from genetic testing, but most studies to date have been limited to examining children or adults with intellectual disability. Our aim was to determine the yield of targeted sequencing of 5 established epilepsy genes (DEPDC5, LGI1, SCN1A, GRIN2A, and PCHD19) in an extensively phenotyped cohort of focal epilepsy patients with normal intellectual function or mild intellectual disability, as well as describe novel variants and determine the characteristics of variant carriers.
Targeted panel sequencing was performed on 96 patients with a strong clinical suspicion of genetic focal epilepsy. Patients had previously gone through a comprehensive diagnostic epilepsy evaluation in The Neurology Clinic, University Clinical Center of Serbia. Variants of interest (VOI) were classified using the American College of Medical Genetics and the Association for Molecular Pathology criteria.
Six VOI in eight (8/96, 8.3%) patients were found in our cohort. Four likely pathogenic VOI were determined in six (6/96, 6.2%) patients, two DEPDC5 variants in two patients, one SCN1A variant in two patients and one PCDH19 variant in two patients. One variant of unknown significance (VUS) was found in GRIN2A in one (1/96, 1.0%) patient. Only one VOI in GRIN2A was classified as likely benign. No VOI were detected in LGI1.
Sequencing of only five known epilepsy genes yielded a diagnostic result in 6.2% of our cohort and revealed multiple novel variants. Further research is necessary for a better understanding of the genetic basis in common epilepsy syndromes in patients with normal intellectual function or mild intellectual disability.
Severe epilepsy phenotype with SCN1A missense variants located outside the sodium channel core region: Relationship between functional results and clinical phenotype
2022, Seizure
Citation Excerpt :
The screening process is shown in Figure S2. Seven articles were included in the analysis, and twelve missense SCN1A variants were finally included, as shown in Table 2 [15–21]. All variants were located outside the sodium channel core region.
Most SCN1A missense variants located outside the sodium channel core region show a mild phenotype. However, there are exceptions, because of which it is challenging to determine the correlation between genotype and phenotype. In this study, we aimed to determine whether functional study could be used to determine disease severity in cases with such variants, and elucidate possible genotype-phenotype relationships.
Forty-seven patients with SCN1A missense variants were recruited, and one with a Dravet syndrome phenotype with an SCN1A missense variant (c.3811T>C/ p.W1271R) located outside the core region was screened with electrophysiological tests. We also reviewed functional SCN1A studies on patients with inconsistent phenotypes and genotypes, and studied the relationship between electrophysiological measurements and clinical phenotype.
Patch clamp experiments showed that the W1271R variant caused significantly reduced sodium current, decreased channel voltage sensitivity, loss of channel availability, and prolonged recovery time from inactivation compared with wild type (WT), which ultimately caused a change in loss of function (LOF). Twelve cases of severe SCN1A-related epilepsy with missense variants located outside the channel core region were also included from the functional studies. Nine patients with missense SCN1A variants showed complete (3/9) or partial (6/9) physiological LOF. Two missense SCN1A variants caused physiological gain-and-loss of function (G-LOF), and one caused decreased excitability (DE).
Not all missense variants located outside the core region cause a mild phenotype. Although current functional studies in heterologous expression systems do not accurately reflect disease severity caused by SCN1A missense variants, they could be an effective model for generation of data to study the initial effects of SCN1A missense variants.
Mutations in the sodium channel genes SCN1A, SCN3A, and SCN9A in children with epilepsy with febrile seizures plus(EFS+)
2021, Seizure
Purpose: To explore disease-causing gene mutations of epilepsy with febrile seizures plus (EFS+) in Southern Chinese Han population.
Methods: Blood samples and clinical data were collected from 49 Southern Han Chinese patients with EFS+. Gene screening was performed using whole-exome sequencing and panel sequencing for 485 epilepsy-related genes. The pathogenicity of variants was evaluated based on ACMG scoring and assessment of clinical concordance.
Results: We identified 10 putatively causative sodium channel gene variants in 49 patients with EFS+, including 8 variants in SCN1A (R500Q appeared twice), one in SCN3A and one in SCN9A. All these missense mutations were inherited from maternal or paternal and were evaluated to be of uncertain significance according to ACMG. The clinical features of patients were in concordance with the EFS+ phenotype of the mutated SCN1A, SCN3A and SCN9A gene. The clinical phenotypes of 11 probands with these gene variants included febrile seizures plus (FS+, n=7), Dravet Syndrome (n=3), FS+ with focal seizures (n=1). Three probands with SCN1A variants (R500Q located in the non-voltage areas, or G1711D in the pore-forming domain) developed severe Dravet syndrome. The affected individuals with the other 6 SCN1A variants located outside the pore-forming domain showed mild phenotypes. Novel SCN3A variant ((D1688Y) and SCN9A variant (R185H) were identified in two probands respectively and both of the probands had FS+.
Conclusion: The SCN1A, SCN3A, and SCN9A gene mutations might be a pathogenic cause of EFS+ in Southern Chinese Han population.
A more efficient conditional mouse model of Dravet syndrome: Implications for epigenetic selection and sex-dependent behaviors
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Common co-morbidities in human DS patients include hyperactivity, cognitive and memory impairment, and an autistic-like phenotype (Dravet, 2011). In most cases, DS is caused by a heterozygous truncation or loss-of-function missense mutation in SCN1A, the gene which encodes the brain Type-I voltage-gated sodium channel Nav1.1 (Bechi et al., 2011; Sugawara et al., 2003; Claes et al., 2001). We have previously developed a mouse line with deletion of Scn1a exon 26, leading to global haploinsufficiency of Nav1.1 in heterozygotes (here termed “DS mice”).
Dravet Syndrome (DS) is an epileptic disorder characterized by spontaneous and thermally-induced seizures, hyperactivity, cognitive deficits, autistic-like behaviors, and Sudden Unexpected Death in Epilepsy (SUDEP). DS is caused by de novo loss-of-function mutations in the SCN1A gene. Selective loss of GABAergic interneuron excitability is the primary cause of the disease. Up to 60% of Scn1a^+/− mice die from SUDEP before sexual maturity.
We used Cre-Lox technology to conditionally delete Scn1a in all epiblast-derived somatic cells by crossing a floxed Scn1a mouse with a mouse expressing Cre under the Meox2 promoter.
Parental Scn1a flox (F) mice, parental Meox2 Cre⁺ mice, and their F/+:Meox2-Cre⁻ offspring were phenotypically normal and did not prematurely die. In contrast, F/+:Meox2-Cre⁺ offspring recapitulated DS seizure and behavioral phenotypes. Unexpectedly, male F/+:Meox2-Cre⁺ mice demonstrated impaired social interaction, while females did not.
In the previous models, colony maintenance required breeding SUDEP survivors, which greatly increased colony size required to sustain experimental animal production, and raised the concern that surviving breeders have epigenetic traits that impart new phenotypes to their offspring. Our method greatly facilitates breeding, recapitulates DS phenotypes, eliminates concerns about parents that are survivors, and provides initial evidence for unexpected sex-dependent social interaction impairment.
We introduce a more efficient mouse model of human DS that demonstrates an efficient breeding strategy free from potential inherited epigenetic changes and reveals an unexpected sex-specific impairment of social interaction in DS. This new model should have great value to investigators of DS.
Two mild cases of Dravet syndrome with truncating mutation of SCN1A
2017, Brain and Development
Citation Excerpt :
Later, generalized spikes and waves occur, with photosensitivity and focal abnormalities [2]. It is generally considered that an SCN1A truncating mutation causes the severe phenotype of Dravet syndrome because of the loss of SCN1A function [2–4]. We report two mild cases of Dravet syndrome with a truncating mutation of SCN1A.
SCN1A is the gene that codes for the neuronal voltage-gated sodium-channel alpha-subunit 1. It is generally considered that an SCN1A truncating mutation causes the severe phenotype of Dravet syndrome.
We describe 11- and 4-year-old male patients presenting with mild Dravet syndrome with a truncating mutation of SCN1A. The former patient showed moderate mental retardation; however, seizure was controlled to almost one incident a year by levetiracetam and topiramate. Carbamazepine was also effective, which is atypical of Dravet syndrome. The latter patient showed a borderline developmental quotient and did not have episodes of afebrile seizure.
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Nav1.1 channels with mutations of severe myoclonic epilepsy in infancy display attenuated currents

Abstract

Introduction

Section snippets

SMEI mutations

Molecular cloning of human SCN1A cDNA and mutagenesis

Results

Discussion

Acknowledgements

Neuron

Am. J. Hum. Genet.

J. Biol. Chem.

Am. J. Hum. Genet.

Epilepsy Res.

Neuron

Biochem. Biophys. Res. Commun.

Brain Dev.

J. Biol. Chem.

Am. J. Hum. Genet.

Cell

Enhanced inactivation and acceleration of activation of the sodium channel associated with epilepsy in man

Eur. J. Neurosci.

Genetic predisposition to severe myoclonic epilepsy in infancy

Epilepsia

A single Na+ channel mutation causing both long-QT and Brugada syndromes

Circ. Res.

Na_v1.1 channels with mutations of severe myoclonic epilepsy in infancy display attenuated currents

A single Na⁺ channel mutation causing both long-QT and Brugada syndromes