Jennifer A. Kearney

Sodium channel mutations in epilepsy and other neurological disorders

Since the first mutations of the neuronal sodium channel SCN1A were identified 5 years ago, more than 150 mutations have been described in patients with epilepsy. Many are sporadic mutations and cause loss of function, which demonstrates haploinsufficiency of SCN1A. Mutations resulting in persistent sodium current are also common. Coding variants of SCN2A, SCN8A, and SCN9A have also been identified in patients with seizures, ataxia, and sensitivity to pain, respectively. The rapid pace of discoveries suggests that sodium channel mutations are significant factors in the etiology of neurological disease and may contribute to psychiatric disorders as well.

Sodium channels SCN1A, SCN2A and SCN3A in familial autism.

Autism is a psychiatric disorder with estimated heritability of 90%. One-third of autistic individuals experience seizures. A susceptibility locus for autism was mapped near a cluster of voltage-gated sodium channel genes on chromosome 2. Mutations in two of these genes, SCN1A and SCN2A, result in the seizure disorder GEFS+. To evaluate these sodium channel genes as candidates for the autism susceptibility locus, we screened for variation in coding exons and splice sites in 117 multiplex autism families. A total of 27 kb of coding sequence and 3 kb of intron sequence were screened. Only six families carried variants with potential effects on sodium channel function. Five coding variants and one lariat branchpoint mutation were each observed in a single family, but were not present in controls. The variant R1902C in SCN2A is located in the calmodulin binding site and was found to reduce binding affinity for calcium-bound calmodulin. R542Q in SCN1A was observed in one autism family and had previously been identified in a patient with juvenile myoclonic epilepsy. The effect of the lariat branchpoint mutation was tested in cultured lymphoblasts. Additional population studies and functional tests will be required to evaluate pathogenicity of the coding and lariat site variants. SNP density was 1/kb in the genomic sequence screened. We report 38 sodium channel SNPs that will be useful in future association and linkage studies.

A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities

The GAL879-881QQQ mutation in the cytoplasmic S4-S5 linker of domain 2 of the rat brain IIA sodium channel (Na(v)1.2) results in slowed inactivation and increased persistent current when expressed in Xenopus oocytes. The neuron-specific enolase promoter was used to direct in vivo expression of the mutated channel in transgenic mice. Three transgenic lines exhibited seizures, and line Q54 was characterized in detail. The seizures in these mice began at two months of age and were accompanied by behavioral arrest and stereotyped repetitive behaviors. Continuous electroencephalogram monitoring detected focal seizure activity in the hippocampus, which in some instances generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic Q54 mice exhibited increased persistent sodium current which may underlie hyperexcitability in the hippocampus. During the progression of the disorder there was extensive cell loss and gliosis within the hippocampus in areas CA1, CA2, CA3 and the hilus. The lifespan of Q54 mice was shortened and only 25% of the mice survived beyond six months of age. Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities. The transgenic Q54 mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction. The human ortholog, SCN2A, is a candidate gene for seizure disorders mapped to chromosome 2q22-24.

A Novel Epilepsy Mutation in the Sodium Channel SCN1A Identifies a Cytoplasmic Domain for β Subunit Interaction

A mutation in the sodium channel SCN1A was identified in a small Italian family with dominantly inherited generalized epilepsy with febrile seizures plus (GEFS+). The mutation, D1866Y, alters an evolutionarily conserved aspartate residue in the C-terminal cytoplasmic domain of the sodium channel α subunit. The mutation decreased modulation of the α subunit by β1, which normally causes a negative shift in the voltage dependence of inactivation in oocytes. There was less of a shift with the mutant channel, resulting in a 10 mV difference between the wild-type and mutant channels in the presence of β1. This shift increased the magnitude of the window current, which resulted in more persistent current during a voltage ramp. Computational analysis suggests that neurons expressing the mutant channels will fire an action potential with a shorter onset delay in response to a threshold current injection, and that they will fire multiple action potentials with a shorter interspike interval at a higher input stimulus. These results suggest a causal relationship between a positive shift in the voltage dependence of sodium channel inactivation and spontaneous seizure activity. Direct interaction between the cytoplasmic C-terminal domain of the wild-typeα subunit with the β1or β3 subunit was first demonstrated by yeast two-hybrid analysis. The SCN1A peptide K1846-R1886 is sufficient for β subunit interaction. Coimmunoprecipitation from transfected mammalian cells confirmed the interaction between the C-terminal domains of the α and β1 subunits. The D1866Y mutation weakens this interaction, demonstrating a novel molecular mechanism leading to seizure susceptibility.

Mutation of sodium channel SCN3A in a patient with cryptogenic pediatric partial epilepsy

Mutations in the sodium channel genes SCN1A and SCN2A have been identified in monogenic childhood epilepsies, but SCN3A has not previously been investigated as a candidate gene for epilepsy. We screened a consecutive cohort of 18 children with cryptogenic partial epilepsy that was classified as pharmacoresistant because of nonresponse to carbamazepine or oxcarbazepine, antiepileptic drugs that bind sodium channels. The novel coding variant SCN3A-K354Q was identified in one patient and was not present in 295 neurological normal controls. Twelve novel SNPs were also detected. K354Q substitutes glutamine for an evolutionarily conserved lysine residue in the pore domain of SCN3A. Functional analysis of this mutation in the backbone of the closely related gene SCN5A demonstrated an increase in persistent current that is similar in magnitude to epileptogenic mutations of SCN1A and SCN2A. This observation of a potentially pathogenic mutation of SCN3A (Nav1.3) indicates that this gene should be further evaluated for its contribution to childhood epilepsy.

De novo KCNB1 mutations in epileptic encephalopathy

Numerous studies have demonstrated increased load of de novo copy number variants or single nucleotide variants in individuals with neurodevelopmental disorders, including epileptic encephalopathies, intellectual disability, and autism.

Sodium Channels and Neurological Disease: Insights from Scn8a Mutations in the Mouse

The human genome contains 10 voltage-gated sodium channel genes, 7 of which are expressed in neurons of the CNS and PNS. The availability of human genome sequences and high-throughput mutation screening methods make it likely that many human disease mutations will be identified in these genes in the near future. Mutations of Scn8a in the mouse demonstrate the broad spectrum of neurological disease that can result from different alleles of the same sodium channel gene. Null mutations of Scn8a produce motor neuron failure, loss of neuromuscular transmission, and lethal paralysis. Less severe mutations result in ataxia, tremor, muscle weakness, and dystonia. The effects of Scn8a mutations on channel properties have been studied in the Xenopusoocyte expression system and in neurons isolated from the mutant mice. The Scn8a mutations provide insight into the mode of inheritance, effect on neuronal sodium currents, and role of modifier genes in sodium channel disease, highlighting the ways in which mouse models of human mutations can be used in the future to understand the pathophysiology of human disease.

Genetic modifiers affecting severity of epilepsy caused by mutation of sodium channel Scn2a.

Mutations in the voltage-gated sodium channels SCN1A and SCN2A are responsible for several types of human epilepsy. Variable expressivity among family members is a common feature of these inherited epilepsies, suggesting that genetic modifiers may influence the clinical manifestation of epilepsy. The transgenic mouse model Scn2aQ54 has an epilepsy phenotype as a result of a mutation in Scn2a that slows channel inactivation. The mice display progressive epilepsy that begins with short-duration partial seizures that appear to originate in the hippocampus. The partial seizures become more frequent and of longer duration with age and often induce secondary generalized seizures. Clinical severity of the Scn2aQ54 phenotype is influenced by genetic background. Congenic C57BL/6J.Q54 mice exhibit decreased incidence of spontaneous seizures, delayed seizure onset, and longer survival in comparison with [C57BL/6J × SJL/J]F1.Q54 mice. This observation indicates that strain SJL/J carries dominant modifier alleles at one or more loci that determine the severity of the epilepsy phenotype. Genome-wide interval mapping in an N2 backcross revealed two modifier loci on Chromosomes 11 and 19 that influence the clinical severity of of this sodium channel-induced epilepsy. Modifier genes affecting clinical severity in the Scn2aQ54 mouse model may contribute to the variable expressivity seen in epilepsy patients with sodium channel mutations.

Neuronal voltage-gated ion channels are genetic modifiers of generalized epilepsy with febrile seizures plus.

Mutations in the neuronal voltage-gated sodium channel genes SCN1A and SCN2A are associated with inherited epilepsies, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (severe myoclonic epilepsy of infancy). The clinical presentation and severity of these epilepsies vary widely, even in people with the same mutation, suggesting the action of environmental or genetic modifiers. To gain support for the hypothesis that genetic modifiers can influence clinical presentation in patients with SCN1A-derived GEFS+, we used mouse models to study the effect of combining the human GEFS+ mutation SCN1A-R1648H with SCN2A, KCNQ2, and SCN8A mutations. Knock-in mice heterozygous for the R1648H mutation (Scn1a(RH/+)) have decreased thresholds to induced seizures and infrequent spontaneous seizures, whereas homozygotes display spontaneous seizures and premature lethality. Scn2a(Q54) transgenic mice have a mutation in Scn2a that results in spontaneous, adult-onset partial motor seizures, and mice carrying the Kcnq2-V182M mutation exhibit increased susceptibility to induced seizures, and rare spontaneous seizures as adults. Combining the Scn1a-R1648H allele with either Scn2a(Q54) or Kcnq2(V182M/+) results in early-onset, generalized tonic-clonic seizures and juvenile lethality in double heterozygous mice. In contrast, Scn8a mutants exhibit increased resistance to induced seizures. Combining the Scn1a-R1648H and Scn8a-med-jo alleles restores normal thresholds to flurothyl-induced seizures in Scn1a(RH/+) heterozygotes and improved survival of Scn1a(RH/RH) homozygotes. Our results demonstrate that variants in Scn2a, Kcnq2, and Scn8a can dramatically influence the phenotype of mice carrying the Scn1a-R1648H mutation and suggest that ion channel variants may contribute to the clinical variation seen in patients with monogenic epilepsy.

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.