SCN1A/NaV1.1 channelopathies: Mechanisms in expression systems,animal models,and human iPSC models

Pathogenic SCN1A/Na_V1.1 mutations cause well‐defined epilepsies, including genetic epilepsy with febrile seizures plus (GEFS+) and the severe epileptic encephalopathy Dravet syndrome. In addition, they cause a severe form of migraine with aura, familial hemiplegic migraine. Moreover, SCN1A/Na_V1.1 variants have been inferred as risk factors in other types of epilepsy. We review here the advancements obtained studying pathologic mechanisms of SCN1A/Na_V1.1 mutations with experimental systems. We present results gained with in vitro expression systems, gene‐targeted animal models, and the induced pluripotent stem cell (iPSC) technology, highlighting advantages, limits, and pitfalls for each of these systems. Overall, the results obtained in the last two decades confirm that the initial pathologic mechanism of epileptogenic SCN1A/Na_V1.1 mutations is loss‐of‐function of Na_V1.1 leading to hypoexcitability of at least some types of γ‐aminobutyric acid (GABA)ergic neurons (including cortical and hippocampal parvalbumin‐positive and somatostatin‐positive ones). Conversely, more limited results point to Na_V1.1 gain‐of‐function for familial hemiplegic migraine (FHM) mutations. Behind these relatively simple pathologic mechanisms, an unexpected complexity has been observed, in part generated by technical issues in experimental studies and in part related to intrinsically complex pathophysiologic responses and remodeling, which yet remain to be fully disentangled.     

Dravet syndrome or genetic (generalized) epilepsy with febrile seizures plus?

Dravet syndrome and genetic epilepsy with febrile seizures plus (GEFS+) can both arise due to mutations of SCN1A, the gene encoding the alpha 1 pore-forming subunit of the sodium channel. GEFS+ refers to a familial epilepsy syndrome where at least two family members have phenotypes that fit within the GEFS+ spectrum. The GEFS+ spectrum comprises a range of mild to severe phenotypes varying from classical febrile seizures to Dravet syndrome. Dravet syndrome is a severe infantile onset epilepsy syndrome with multiple seizure types, developmental slowing and poor outcome. More than 70% of patients with Dravet syndrome have mutations of SCN1A; these include both truncation and missense mutations. In contrast, only 10% of GEFS+ families have SCN1A mutations and these comprise missense mutations. GEFS+ has also been associated with mutations of genes encoding the sodium channel beta 1 subunit, SCN1B, and the GABA_A receptor gamma 2 subunit, GABRG2. The phenotypic heterogeneity that is characteristic of GEFS+ families is likely to be due to modifier genes. Interpretation of the significance of a SCN1A missense mutation requires a thorough understanding of the phenotypes in the GEFS+ spectrum whereas a de novo truncation mutation is likely to be associated with a severe phenotype. Early recognition of Dravet syndrome is important as aggressive control of seizures may improve developmental outcome.     

A homozygous mutation of voltage‐gated sodium channel βI gene SCN1B in a patient with Dravet syndrome

Dravet syndrome is a severe form of epileptic encephalopathy characterized by early onset epileptic seizures followed by ataxia and cognitive decline. Approximately 80% of patients with Dravet syndrome have been associated with heterozygous mutations in SCN1A gene encoding voltage‐gated sodium channel (VGSC) α_I subunit, whereas a homozygous mutation (p.Arg125Cys) of SCN1B gene encoding VGSC β_I subunit was recently described in a patient with Dravet syndrome. To further examine the involvement of homozygous SCN1B mutations in the etiology of Dravet syndrome, we performed mutational analyses on SCN1B in 286 patients with epileptic disorders, including 67 patients with Dravet syndrome who have been negative for SCN1A and SCN2A mutations. In the cohort, we found one additional homozygous mutation (p.Ile106Phe) in a patient with Dravet syndrome. The identified homozygous SCN1B mutations indicate that SCN1B is an etiologic candidate underlying Dravet syndrome.     

Confirming an expanded spectrum of SCN2A mutations: a case series

Mutations in sodium channel genes are highly associated with epilepsy. Mutation of SCN1A, the gene encoding the voltage gated sodium channel (VGSC) alpha subunit type 1 (Nav1.1), causes Dravet syndrome spectrum disorders. Mutations in SCN2A have been identified in patients with benign familial neonatal‐infantile epilepsy (BFNIE), generalised epilepsy with febrile seizures plus (GEFS+), and a small number of reported cases of other infantile‐onset severe intractable epilepsy. Here, we report three patients with infantile‐onset severe intractable epilepsy found to have de novo mutations in SCN2A. While a causal role for these mutations cannot be directly established, these findings contribute to growing evidence that mutation of SCN2A is associated with a range of epilepsy phenotypes including severe infantile‐onset epilepsy.     

Mutations of the SCN1A gene in acute encephalopathy

Purpose: Acute encephalopathy is the most serious complication of pediatric viral infections, such as influenza and exanthema subitum. It occurs worldwide, but is most prevalent in East Asia. Recently, there have been sporadic case reports of epilepsy/febrile seizure and acute encephalopathy with a neuronal sodium channel alpha 1 subunit (SCN1A) mutation. To determine whether SCN1A mutations are a predisposing factor of acute encephalopathy, we sought to identify SCN1A mutations in a large case series of acute encephalopathy including various syndromes. Methods: We analyzed the SCN1A gene in 87 patients with acute encephalopathy, consisting of 20 with acute necrotizing encephalopathy (ANE), 61 with acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), and six with nonspecific (unclassified) acute encephalopathy. Key Findings: Three patients had distinct point mutations. Two of them had epileptic seizures prior to acute encephalopathy. Clinical and neuroradiologic findings of acute encephalopathy were diverse among the three patients, although all had a prolonged and generalized seizure at its onset. The first patient with V982L had partial epilepsy and AESD. The second patient with M1977L had febrile seizures and nonspecific acute encephalopathy. The third patient with R1575C had no seizures until the onset of ANE. M1977L was a novel mutation, whereas the remaining two, V982L and R1575C, have previously been reported in cases of Dravet syndrome and acute encephalopathy, respectively. Significance: These findings provide further evidence that SCN1A mutations are a predisposing factor for the onset of various types of acute encephalopathy.     

The incidence of SCN1A‐related Dravet syndrome in Denmark is 1:22,000: A population‐based study from 2004 to 2009

Dravet syndrome is a severe infantile‐onset epileptic encephalopathy associated with mutations in the sodium channel alpha‐1 subunit gene SCN1A. We aimed to describe the incidence of Dravet syndrome in the Danish population. Based on a 6‐year birth cohort from 2004 to 2009, we propose an incidence of 1:22,000, which is higher than what has been established earlier. We identified 17 cases with SCN1A mutation–positive Dravet syndrome. Fifteen patients were found, by conventional Sanger sequencing. Two additional patients with clinical Dravet syndrome, but without a detectable SCN1A mutation by Sanger sequencing, were diagnosed with a SCN1A mutation after using a targeted next‐generation sequencing gene panel.     

De novo SCN1A pathogenic variants in the GEFS+ spectrum: Not always a familial syndrome

Genetic epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome characterized by heterogeneous phenotypes ranging from mild disorders such as febrile seizures to epileptic encephalopathies (EEs) such as Dravet syndrome (DS). Although DS often occurs with de novo SCN1A pathogenic variants, milder GEFS+ spectrum phenotypes are associated with inherited pathogenic variants. We identified seven cases with non‐EE GEFS+ phenotypes and de novo SCN1A pathogenic variants, including a monozygotic twin pair. Febrile seizures plus (FS+) occurred in six patients, five of whom had additional seizure types. The remaining case had childhood‐onset temporal lobe epilepsy without known febrile seizures. Although early development was normal in all individuals, three later had learning difficulties, and the twin girls had language impairment and working memory deficits. All cases had SCN1A missense pathogenic variants that were not found in either parent. One pathogenic variant had been reported previously in a case of DS, and the remainder were novel. Our finding of de novo pathogenic variants in mild phenotypes within the GEFS+ spectrum shows that mild GEFS+ is not always inherited. SCN1A screening should be considered in patients with GEFS+ phenotypes because identification of pathogenic variants will influence antiepileptic therapy, and prognostic and genetic counseling.     

Role of the sodium channel SCN9A in genetic epilepsy with febrile seizures plus and Dravet syndrome

Mutations of the SCN1A subunit of the sodium channel is a cause of genetic epilepsy with febrile seizures plus (GEFS⁺) in multiplex families and accounts for 70–80% of Dravet syndrome (DS). DS cases without SCN1A mutation inherited have predicted SCN9A susceptibility variants, which may contribute to complex inheritance for these unexplained cases of DS. Compared with controls, DS cases were significantly enriched for rare SCN9A genetic variants. None of the multiplex febrile seizure or GEFS⁺ families could be explained by highly penetrant SCN9A mutations.     

Vaccination and Occurrence of Seizures in SCN1A Mutation–Positive Patients: A Multicenter Italian Study

BackgroundThe relation between epileptic seizures and vaccinations is sometimes debated. In the present work, the impact of vaccination on seizure onset and clinical outcome of SCN1A mutation-positive patients is addressed.MethodsSeventy-two patients diagnosed with Dravet syndrome or generalized epilepsy with febrile seizure plus, carrying SCN1A mutations or not, were included. Details on vaccination type, temporal relationship between vaccination and seizure occurrence, seizure type at onset and during development, cognitive functioning, and vaccination completion was obtained by reviewing clinical records. Patients were divided into two groups based on the temporal window between vaccination and seizure onset (proximate group: <48 hours; distant group: >48 hours).ResultsVaccination-related seizures occurred in 25% of patients with SCN1A mutation and 18% of patients without the mutation (no significant difference). The proximate group showed an earlier age at seizure onset and a higher frequency of status epilepticus during development than did the distant group. No other significant differences were found. Subsequent vaccinations did not significantly alter the evolution of the disease.ConclusionsResults from this relatively small series provide evidence that vaccinations do not significantly affect clinical and cognitive evolution of Dravet syndrome and generalized epilepsy with febrile seizure plus patients even if they carry SCN1A mutations.     

De novo truncating mutation in SCN1A as a cause of febrile seizures plus (FS+)

SCN1A is one of the most relevant epilepsy genes. In general, de novo severe mutations, such as truncating mutations, lead to a classic form of Dravet syndrome (DS), while missense mutations are associated with both DS and milder phenotypes within the GEFS+ spectrum, however, these phenotype‐genotype correlations are not entirely consistent. Case report. We report an 18‐year‐old woman with a history of recurrent febrile generalized tonic‐clonic seizures (GTCS) starting at age four months and afebrile asymmetric GTCS and episodes of arrest, suggestive of focal impaired awareness seizures, starting at nine months. Her psychomotor development was normal. Sequencing of SCN1A revealed a heterozygous de novo truncating mutation (c.5734C>T, p.Arg1912X) in exon 26. Conclusion. Truncating mutations in SCN1A may be associated with milder phenotypes within the GEFS+ spectrum. Accordingly, SCN1A gene testing should be performed as part of the assessment for sporadic patients with mild phenotypes that fit within the GEFS+ spectrum, since the finding of a mutation has diagnostic, therapeutic and genetic counselling implications.     

Does a SCN1A gene mutation confer earlier age of onset of febrile seizures in GEFS+?

SCN1A is the most clinically relevant epilepsy gene and is associated with generalized epilepsy and febrile seizure plus (GEFS+) and Dravet syndrome. We postulated that earlier onset of febrile seizures in the febrile seizure (FS) and febrile seizure plus (FS+) phenotypes may occur in the presence of a SCN1A mutation. This was because of the age-related onset of Dravet syndrome, which typically begins in the first year of life. We found that patients with FS and FS+ with SCN1A mutations had earlier median onset of febrile seizures compared to the population median. Patients with GABRG2 mutations had a similar early onset in contrast to patients with SCN1B mutations where onset was later. This study is the first to demonstrate that a specific genetic abnormality directly influences the FS and FS+ phenotype in terms of age of onset.     

Investigating the genetic basis of fever‐associated syndromic epilepsies using copy number variation analysis

Fever‐associated syndromic epilepsies ranging from febrile seizures plus (FS+) to Dravet syndrome have a significant genetic component. However, apart from SCN1A mutations in >80% of patients with Dravet syndrome, the genetic underpinnings of these epilepsies remain largely unknown. Therefore, we performed a genome‐wide screening for copy number variations (CNVs) in 36 patients with SCN1A‐negative fever‐associated syndromic epilepsies. Phenotypes included Dravet syndrome (n = 23; 64%), genetic epilepsy with febrile seizures plus (GEFS+) and febrile seizures plus (FS+) (n = 11; 31%) and unclassified fever‐associated epilepsies (n = 2; 6%). Array comparative genomic hybridization (CGH) was performed using Agilent 4 × 180K arrays. We identified 13 rare CNVs in 8 (22%) of 36 individuals. These included known pathogenic CNVs in 4 (11%) of 36 patients: a 1q21.1 duplication in a proband with Dravet syndrome, a 14q23.3 deletion in a proband with FS+, and two deletions at 16p11.2 and 1q44 in two individuals with fever‐associated epilepsy with concomitant autism and/or intellectual disability. In addition, a 3q13.11 duplication in a patient with FS+ and two de novo duplications at 7p14.2 and 18q12.2 in a patient with atypical Dravet syndrome were classified as likely pathogenic. Six CNVs were of unknown significance. The identified genomic aberrations overlap with known neurodevelopmental disorders, suggesting that fever‐associated epilepsy syndromes may be a recurrent clinical presentation of known microdeletion syndromes.     

When should clinicians order genetic testing for Dravet syndrome?

The role of neuronal voltage-gated sodium channel, α-1 subunit (SCN1A) gene mutations in Dravet syndrome is well-established. With a broader phenotype than initially described, some patients lack features of Dravet syndrome as defined by the International League Against Epilepsy. We evaluated the predictive value of International League Against Epilepsy criteria for a positive mutation in a cohort of consecutively tested children. Mutations of SCN1A were evident in 16 of 69 children. Exhibiting ≥4 International League Against Epilepsy criteria demonstrated 100% sensitivity. Seven criteria (resistance to multiple antiepileptic drugs, multiple seizure types, abnormal electroencephalogram features, exacerbation with hyperthermia, normal development before seizure onset, seizures beginning before age 1 year, and psychomotor retardation) were present in ≥85% of mutation-positive cases. The three criteria that best predicted a mutation in SCN1A included exacerbation with hyperthermia, normal development before seizure onset, and the appearance of ataxia, pyramidal signs, or interictal myoclonus. We have demonstrated a high-sensitivity testing strategy for detecting mutations of SCN1A in children with suspected Dravet syndrome.     

A novel SCN1A mutation in a cytoplasmic loop in intractable juvenile myoclonic epilepsy without febrile seizures

Generalised (genetic) epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome with various phenotypes. The majority of individuals with GEFS+ have generalised seizure types, in addition to febrile seizures (FS) or febrile seizures plus (FS+), defined as either continued FS after 6 years of age or afebrile seizures following FS. A 27‐year‐old man with no history of FS/FS+ experienced intractable generalised convulsive seizures. The patient's father had a history of similar seizures during puberty and the patient's siblings had only FS. No individual in the family had both generalised seizures and FS/FS+, although GEFS+ might be considered to be present in the family. Analysis of SCN1A, a sodium channel gene, revealed a novel mutation (c.3250A>T [S1084C]) in the cytoplasmic loop 2 of SCN1A in both the patient and his father. Most previously reported SCN1A mutations in GEFS+ patients are located in the conserved homologous domains of SCN1A, whereas mutations in the cytoplasmic loops are very rare. SCN1A gene analysis is not commonly performed in subjects with generalised seizures without FS. SCN1A mutation may be a clinically‐useful genetic marker in order to distinguish GEFS+ patients from those with classic idiopathic generalised epilepsy, even if they present an atypical clinical picture.     

Missense mutation of the sodium channel gene SCN2A causes Dravet syndrome

Mutations of the gene encoding the α2 subunit of the neuronal sodium channel, SCN2A, have been found in benign familial neonatal-infantile seizures (BFNIS). In Dravet syndrome, only one nonsense mutation of SCN2A was identified, while hundreds of mutations were found in the paralogue gene, SCN1A, which encodes the α1 subunit. This study examines whether SCN2A mutations are associated with Dravet syndrome. We screened for mutations of SCN1A, SCN2A and GABRG2 (the gene encoding γ2 subunit of the GABA_A receptor) in 59 patients with Dravet syndrome and found 29 SCN1A mutations and three missense SCN2A mutations. Among the three, one de novo SCN2A mutation (c.3935G>C: R1312T) identified in a patient was thought to affect an arginine residue in a voltage sensor of the channel and hence, to be pathogenic. This finding suggests that both nonsense mutations and missense SCN2A mutations cause Dravet syndrome.     

Sodium channel SCN1A and epilepsy: Mutations and mechanisms

Mutations in a number of genes encoding voltage‐gated sodium channels cause a variety of epilepsy syndromes in humans, including genetic (generalized) epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS, severe myoclonic epilepsy of infancy). Most of these mutations are in the SCN1A gene, and all are dominantly inherited. Most of the mutations that cause DS result in loss of function, whereas all of the known mutations that cause GEFS+ are missense, presumably altering channel activity. Family members with the same GEFS+ mutation often display a wide range of seizure types and severities, and at least part of this variability likely results from variation in other genes. Many different biophysical effects of SCN1A‐GEFS+ mutations have been observed in heterologous expression systems, consistent with both gain and loss of channel activity. However, results from mouse models suggest that the primary effect of both GEFS+ and DS mutations is to decrease the activity of GABAergic inhibitory neurons. Decreased activity of the inhibitory circuitry is thus likely to be a major factor contributing to seizure generation in patients with GEFS+ and DS, and may be a general consequence of SCN1A mutations.     

SCN1A testing for epilepsy: Application in clinical practice

This report is a practical reference guide for genetic testing of SCN1A, the gene encoding the α1 subunit of neuronal voltage‐gated sodium channels (protein name: Na_v1.1). Mutations in this gene are frequently found in Dravet syndrome (DS), and are sometimes found in genetic epilepsy with febrile seizures plus (GEFS+), migrating partial seizures of infancy (MPSI), other infantile epileptic encephalopathies, and rarely in infantile spasms. Recommendations for testing: (1) Testing is particularly useful for people with suspected DS and sometimes in other early onset infantile epileptic encephalopathies such as MPSI because genetic confirmation of the clinical diagnosis may allow optimization of antiepileptic therapy with the potential to improve seizure control and developmental outcome. In addition, a molecular diagnosis may prevent the need for unnecessary investigations, as well as inform genetic counseling. (2) SCN1A testing should be considered in people with possible DS where the typical initial presentation is of a developmentally normal infant presenting with recurrent, febrile or afebrile prolonged, hemiclonic seizures or generalized status epilepticus. After age 2, the clinical diagnosis of DS becomes more obvious, with the classical evolution of other seizure types and developmental slowing. (3) In contrast to DS, the clinical utility of SCN1A testing for GEFS+ remains questionable. (4) The test is not recommended for children with phenotypes that are not clearly associated with SCN1A mutations such as those characterized by abnormal development or neurologic deficits apparent at birth or structural abnormalities of the brain. Interpreting test results: (1) Mutational testing of SCN1A involves both conventional DNA sequencing of the coding regions and analyses to detect genomic rearrangements within the relevant chromosomal region: 2q24. Interpretation of the test results must always be done in the context of the electroclinical syndrome and often requires the assistance of a medical geneticist, since many genomic variations are possible and it is essential to differentiate benign polymorphisms from pathogenic mutations. (2) Missense variants may have no apparent effect on the phenotype (benign polymorphisms) or may represent mutations underlying DS, MPSI, GEFS+, and related syndromes and can provide a challenge in interpretation. (3) Conventional methods do not detect variations in introns or promoter or regulatory regions; therefore, a negative test does not exclude a pathogenic role of SCN1A in a specific phenotype. (4) It is important to note that a negative test does not rule out the clinical diagnosis of DS or other conditions because genes other than SCN1A may be involved. Obtaining written informed consent and genetic counseling should be considered prior to molecular testing, depending on the clinical situation and local regulations.     

Partial epilepsy with antecedent febrile seizures and seizure aggravation by antiepileptic drugs: Associated with loss of function of Nav1.1

Purpose: Generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy in infancy (SMEI) are associated with sodium channel α‐subunit type‐1 gene (SCN1A) mutations. Febrile seizures and partial seizures occur in both GEFS+ and SMEI; sporadic onset and seizure aggravation by antiepileptic drugs (AEDs) are features of SMEI. We thus searched gene mutations in isolated cases of partial epilepsy with antecedent FS (PEFS+) that showed seizure aggravations by AEDs. Methods: Genomic DNA from four patients was screened for mutations in SCN1A, SCN2A, SCN1B, and GABRG2 using denaturing high‐performance liquid chromatography (dHPLC) and sequencing. Whole‐cell patch clamp analysis was used to characterize biophysical properties of two newly defined mutants of Na_v1.1 in tsA201 cells. Results: Two heterozygous de novo mutations of SCN1A (R946H and F1765L) were detected, which were proven to cause loss of function of Na_v1.1. When the functional defects of mutants reported previously are compared, it is found that all mutants from PEFS+ have features of loss of function, whereas GEFS+ shows mild dysfunction excluding loss of function, coincident with mild clinical manifestations. PEFS+ is similar to SMEI clinically with possible AED‐induced seizure aggravation and biophysiologically with features of loss of function, and different from SMEI by missense mutation without changes in hydrophobicity or polarity of the residues. Conclusions: Isolated milder PEFS+ may associate with SCN1A mutations and loss of function of Na_v1.1, which may be the basis of seizure aggravation by sodium channel–blocking AEDs. This study characterized phenotypes biologically, which may be helpful in understanding the pathophysiologic basis, and further in management of the disease.     

Early clinical features and diagnosis of Dravet syndrome in 138 Chinese patients with SCN1A mutations

Objective: To summarize the early clinical features of Dravet syndrome (DS) patients with SCN1A gene mutations before the age of one. Methods: SCN1A gene mutation screening was performed by PCR–DNA sequencing and multiple ligation-dependent probe amplication (MLPA). The early clinical features of DS patients with SCN1A mutations were reviewed with attention to the seizures induced by fever and other precipitating factors before the first year of life. Results: The clinical data of 138 DS patients with SCN1A gene mutations were reviewed. The median seizure onset age was 5.3 months. Ninety-nine patients (71.7%) experienced seizures with duration more than 15 min in the first year of life. Two or more seizures induced by fever within 24 h or the same febrile illness were observed in 93 patients (67.4%). 111 patients (80.4%) had hemi-clonic and (or) focal seizures. Seizures had been triggered by fever of low degree (T < 38 °C) in 62.3% (86/138) before the first year of life. Vaccine-related seizures were observed in 34.8% (48/138). Seizures in 22.5% (31/138) of patients were triggered by hot bath. Carbamazepine, oxcarbazepine, lamotrigine, phenobarbital and phenytoin showed either no effect or exacerbating the seizures in our group. Conclusion: The seizure onset age in DS patients was earlier than that was in common febrile seizures. When a baby exhibits two or more features of complex febrile seizures in the first year of life, a diagnosis of DS should be considered, and SCN1A gene mutation screening should be performed as early as possible. Early diagnosis of DS will help clinicians more effectively prescribe antiepileptic drugs for stronger prognosis.