Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: Neurobiol Dis. 2010 Dec 13;41(3):655–660. doi: 10.1016/j.nbd.2010.11.016

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

Nicole A Hawkins a,1, Melinda S Martin c,1, Wayne N Frankel d, Jennifer A Kearney b,*, Andrew Escayg c
PMCID: PMC3035952  NIHMSID: NIHMS259319  PMID: 21156207

Abstract

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 (Scn1aRH/+) have decreased thresholds to induced seizures and infrequent spontaneous seizures, whereas homozygotes display spontaneous seizures and premature lethality. Scn2aQ54 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 Scn2aQ54 or Kcnq2V182M/+ 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 Scn1aRH/+ heterozygotes and improved survival of Scn1aRH/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.

INTRODUCTION

During the past 15 years, research has revealed several genes underlying rare monogenic forms of idiopathic generalized epilepsy (IGE); however, there has been less progress towards the identification of genes involved in the more common, genetically complex forms of IGE (Greenberg and Pal, 2007; Heron et al, 2007; Tan et al, 2006). Many of the genes now known to cause monogenic forms of epilepsy encode neuronal ion channel subunits, including voltage-gated sodium and potassium channels. Mutations in the voltage-gated sodium channels SCN1A, SCN2A, and SCN1B result in genetic (generalized) epilepsy with febrile seizures plus (GEFS+) (Escayg et al, 2000; Sugawara et al, 2001; Wallace et al, 1998). We recently generated a mouse model of GEFS+ by introducing the human SCN1A-R1648H GEFS+ mutation, which was identified in a large pedigree with 13 affected members, into the orthologous mouse Scn1a gene (Martin et al, 2010).

Scn1aR1648H/+ heterozygous mutants (Scn1aRH/+) display a normal lifespan, reduced thresholds to flurothyl- and hyperthermia-induced seizures, and infrequent spontaneous generalized seizures as adults (Martin et al, 2010). Scn1aRH/RH homozygous mice exhibit spontaneous generalized seizures and have an average lifespan of 18.5 days. Cortical interneurons from Scn1aRH/+ and Scn1aRH/RH mice display slowed recovery from inactivation, increased use-dependence, and a reduced ability to fire action potentials. These electrophysiological abnormalities are predicted to reduce the level of GABAergic inhibition, providing a mechanism for seizure generation (Martin et al, 2010).

Mutations in the voltage-gated sodium channel SCN2A have also been associated with human epilepsy syndromes, including GEFS+ and benign familial neonatal-infantile seizures (BFNIS) (Meisler and Kearney, 2005). The transgenic mouse model Scn2aQ54 has a gain-of-function mutation in Scn2a and a progressive epilepsy phenotype characterized by partial motor seizures that begin in the second month of life, followed by the development of secondary generalized seizures and a reduced lifespan. Hippocampal pyramidal neurons from Scn2aQ54 mice exhibit increased persistent sodium current (Kearney et al, 2001).

Mutations in the voltage-gated potassium channel genes KCNQ2 and KCNQ3 are associated with benign familial neonatal convulsions (BFNC), characterized by clusters of seizures in the first days of life and remission within the first year (Biervert et al, 1998; Charlier et al, 1998; Singh et al, 1998). Kv7.2 and Kv7.3, encoded respectively by KCNQ2 and KCNQ3, heterodimerize to form a slowly activating and inactivating voltage-gated potassium channel that generates the M-current, which is important in controlling repetitive firing upon strong excitatory stimulation (Cooper and Jan, 2003; Delmas and Brown, 2005). The Kcnq2Nmf134 line was generated by ethylnitrosourea (ENU) mutagenesis and carries the amino acid substitution V182M in the third transmembrane segment of Kcnq2. Kcnq2V182M/+ heterozygous mutants (Kcnq2VM/+) exhibit reduced thresholds for minimal clonic seizures and rare spontaneous seizures as adults; however, they have a normal lifespan (Kearney et al, 2006). We previously showed genetic interaction between Scn2a and Kcnq2 in mice (Kearney et al, 2006).

SCN8A mutations are associated with ataxia and behavioral abnormalities in humans and movement disorders in mice. Mice homozygous for the Scn8amed-jo missense mutation exhibit tremor and cerebellar ataxia. Although heterozygous mutants have no visible abnormalities, they do have spontaneous spike-wave discharges characteristic of non-convulsive, absence seizures (Dick et al, 1986; Kohrman et al, 1996; Papale et al, 2009; Sidman et al, 1979). We recently found that heterozygous Scn8amed-jo/+ mutants and heterozygous Scn8amed/+ mutants, that carry a loss-of-function mutation, were more resistant to flurothyl- and kainic acid-induced seizures. We also showed that the Scn8amed-jo allele could rescue the reduced seizure threshold and premature lethality of heterozygous Scn1a knockout mice, suggesting that Scn8a may play an important role in the excitatory circuits that influence convulsive seizure thresholds (Martin et al, 2007).

Unrelated individuals with GEFS+ exhibit a wide range of epilepsy subtypes and severities that may reflect, in part, the relative effect of different SCN1A mutations on channel function. Similar variability is also seen between affected family members who carry the same SCN1A mutation (Fujiwara, 2006; Singh et al, 2001). This suggests that in addition to the primary mutation, the clinical manifestation of epilepsy can be influenced by other factors such as stochastic events during development, environmental influences or genetic modifiers. Mouse models with sodium channel mutations exhibit variable phenotypes depending on the genetic background, supporting a role for genetic modifiers (Bergren et al, 2005; Kearney et al, 2006; Ogiwara et al, 2007; Yu et al, 2006). Based on these observations, we hypothesize that genetic modifier loci may contribute to the variable clinical presentation observed in GEFS+.

To test the hypothesis that genetic modifiers can contribute to GEFS+ variability, we examined the effect of mutations in Scn2a, Kcnq2, and Scn8a on the epilepsy phenotype of the Scn1aR1648H mouse model. Here we demonstrate that mutations in Scn2a and Kcnq2 exacerbate the phenotype, whereas altered Scn8a function ameliorates it. Our results provide support for genetic modification as one mechanism by which the clinical presentation of GEFS+ can be altered underscoring that neuronal excitability is influenced by the net activity of ion channels.

MATERIALS AND METHODS

Animals

Scn1aR1648H mice were generated as previously described (Martin et al, 2010). Scn1aR1648H mice, on the 129S6.C57BL/6J(N2–3) background, were used for mating with Kcnq2VM/+ and Scn8amed-jo/+ mice. Scn1aR1648H mice on the 129S6/SvEvTac background were used for mating with Scn2aQ54 mice. The Kcnq2V182M mice were generated at The Jackson Laboratory by ENU mutagenesis (http://nmf.jax.org). Kcnq2VM/+ heterozygous mutants are maintained by continued backcrossing to C57BL/6J. Scn2aQ54 transgenic mice congenic on the C57BL/6J background were established as described (Bergren et al, 2005) and are maintained by continued backcrossing of hemizygous transgenic males to C57BL/6J females. C57BL/6J-Scn8amed-jo/J mice were purchased from The Jackson Laboratory and are maintained on the C57BL/6J background. Scn1aR1648H, C57BL/6J-Scn8amed-jo/J, Scn2aQ54, and Kcnq2V182M mice were genotyped as previously described (Kearney et al, 2001; Kearney et al, 2006; Martin et al, 2007; Martin et al, 2010).

Generation of double mutant mice

Double heterozygous mutants were generated by crossing Scn1aRH/+ females with Kcnq2VM/+, Scn8amed-jo/+, or Scn2aQ54 males. Scn1aRH/RH;Scn8amed-jo/+ mutants were generated by crossing Scn1aRH/+;Scn8amed-jo/+ males with Scn1aRH/+ females. All double mutants were obtained at expected Mendelian ratios. Littermates were used for all experiments to minimize variation due to differences in genetic background. Mice were housed in pathogen-free mouse facilities with 12-h light/dark cycles. Food and water were available ad libitum. All experimental protocols were approved by the Emory University and Vanderbilt University IACUC committees.

Flurothyl seizure induction

Mice between 8 and 12 weeks of age were placed in a clear Plexiglas chamber, and flurothyl (2,2,2-trifluroethylether) (Sigma-Aldrich) was slowly introduced into the chamber via a syringe pump at a rate of 20 μl/min and allowed to volatilize. Seizure thresholds were determined by measuring latency to the first MJ and GTCS. The MJ is the first observable behavioral response and is characterized by a brief jerk of the shoulders and/or neck. The GTCS is characterized by convulsions of the entire body and a loss of posture. Data from males and females were analyzed separately. No sex differences were observed; therefore, data from both sexes were combined. Statistical analysis between genotypes was performed using one-way analysis of variance (ANOVA) followed by Fisher’s post-hoc test.

Video-ECoG monitoring

Mice were implanted with prefabricated headmounts (Pinnacle Technology, Inc.) for video-ECoG monitoring. Briefly, mice were anesthetized with isoflurane and placed in a stereotaxic frame (Kopf), and headmounts were attached to the skull with four stainless steel screws that serve as cortical surface electrodes. Headmounts were positioned 0–0.5 mm posterior to lambda. The anterior screw electrodes were 0.5–1 mm posterior to bregma and 1 mm lateral from the midline. The posterior screws were 4.5–5 mm posterior to bregma. After ≥24 hours of recovery, mice were placed in a Plexiglas bowl (14″ h × 16″ diameter), and ECoG data were collected from freely moving mice. Digitized data were acquired and analyzed with Sirenia software (Pinnacle Technology, Inc.) along with contemporaneous video recordings. Epileptiform activity was scored manually.

RESULTS

Scn2aQ54 and Kcnq2V182M alleles reduce the lifespan of Scn1aRH/+ mutants

To model the effect of inheriting mutations in the Scn1a and Scn2a sodium channel genes, we generated Scn1aRH/+;Scn2aQ54 double mutants. Beginning at P16, Scn1aRH/+;Scn2aQ54 double mutants exhibit spontaneous partial motor seizures and generalized tonic-clonic seizures (GTCS). In 137 hours of ECoG recording, we observed 25 GTCS and 11 partial motor seizures (Table 1; Fig. 2). Generalized seizures lasted 45–100 seconds, during which time mice experienced repetitive jerking of all four limbs and neck, running and jumping, and tail clonus (Supplementary Video 1). These seizures often ended with tonic hindlimb extension, indicative of a severe seizure. During ECoG recording, we observed that two Scn1aRH/+;Scn2aQ54 mice had severe seizures with hindlimb extension followed by death. Partial motor seizures lasted <10 seconds, and were characterized by forelimb clonus (Supplementary Video 2). Scn2aQ54 littermates displayed a similar number of partial motor seizures; however, GTCS were rare, with only one observed during 168 hours of ECoG recordings (Table 1). We saw no seizures in Scn1aRH/+ littermates (Table 1, Fig. 2), as was expected from previous monitoring of 3–5 month old Scn1aRH/+ mice that detected spontaneous seizures with a low average frequency of one seizure per 64 hours of recording (Martin et al, 2010). Sporadic death of Scn1aRH/+;Scn2aQ54 double mutants began to occur at P16, with 100% mortality by P24 (Fig. 1).

Table 1. Electrographic events recorded from compound mutant mice.

Video-ECoG data were collected from freely moving mice. Digitized data along was analyzed along with contemporaneous video and epileptiform activity was manually scored

Genotype Events Total Hours of ECoG Monitoring Age Range (days) n
GTCS Partial MJ
RH/+, Q54 25 11 3 137 P17–P21 5
RH/+, VM/+ 4 1 87 330 P18–P36 7
Q54 1 14 0 168 P17–P25 3
RH/+ 0 0 0 240 P18–P42 4
VM/+ 0 0 0 350 P22–P44 3
Open in a new tab

Abbreviations used: GTCS, generalized tonic-clonic seizure; MJ, myoclonic jerk.

Figure 2.

Figure 2

Open in a new tab

Scn2a and Kcnq2 alleles exacerbate the Scn1aRH/+ phenotype. A. Normal ECoG pattern from Scn1aRH/+ heterozygote. B. Representative ECoG recording from Scn1aRH/+;Scn2aQ54 double mutant during an ictal episode. C. Representative ECoG recording from Scn1aRH/+;Kcnq2VM/+ double heterozygote during an ictal episode. Channel 1, recording from right posterior to left posterior; Channel 2, recording from right anterior to left posterior.

Figure 1.

Figure 1

Open in a new tab

Decreased survival of Scn1aRH/+ mutants when combined with the Scn2aQ54 or Kcnq2VM/+ alleles (n ≥ 24 per group). Abbreviations: RH, Scn1aRH/+; Q54, Scn2aQ54; VM, Kcnvq2VM/+; WT, wildtype.

To model the effect of inheriting mutations in Kcnq2 and Scn1a, we generated Scn1aRH/+;Kcnq2VM/+ double heterozygous mutants. At P16, Scn1aRH/+;Kcnq2VM/+ mice began to display spontaneous generalized seizures. In 330 hours of ECoG recording, we observed 87 myoclonic jerks, four GTCS, and one partial motor seizure in the double heterozygous mutants (Table 1; Fig. 2). During a generalized seizure, the mice typically experienced repetitive jerking of all four limbs and neck, running and jumping, and tail clonus (Supplementary Video 3). Generalized seizures were periodically followed by tonic extension of the hindlimbs. During ECoG recording, we observed that two Scn1aRH/+;Kcnq2VM/+ mice had severe seizures with hindlimb extension followed by death. We observed no epileptiform events in the Scn1aRH/+ or Kcnq2VM/+ littermates. At P19, sporadic death of the double heterozygous mice began to occur, and there was 42% mortality by P25 (Fig. 1), demonstrating the ability of the Kcnq2V182M allele to exacerbate the phenotype of the Scn1aRH/+ mice. Interestingly, 47% of double heterozygous mutants survived for more than 100 days (Fig. 1).

Scn8a dysfunction restores normal seizure thresholds in Scn1aRH/+ mutants

We previously demonstrated that two heterozygous Scn8a mutants, Scn8amed-jo/+ and Scn8amed/+, exhibit increased resistance to flurothyl- and kainic acid-induced seizures (Martin et al, 2007). In contrast, Scn1aRH/+mutants have reduced thresholds to flurothyl-induced GTCS (Martin et al, 2010). To investigate whether the Scn8amed-jo allele could alter convulsive seizure thresholds in Scn1aRH/+ mice, we generated double heterozygous mutants harboring both mutations. Thresholds to flurothyl-induced seizures were compared between Scn1aRH/;Scn8amed-jo/+ double heterozygotes, single heterozygotes, and wild-type (WT) littermates.

In agreement with our previous observations (Martin et al, 2007; Martin et al, 2010), when compared to WT littermates, the average latency to flurothyl-induced GTCS in Scn8amed-jo/+ mutants was 30% longer while a 21% reduction was observed in Scn1aRH/+ mutants (Fig. 3). For the Scn1aRH/+;Scn8amed-jo/+ double mutants, the average latency to GTCS was 50% longer when compared to the Scn1aRH/+ mutants (Fig. 3). One-way ANOVA detected an effect of genotype (F(3,35) = 9.858, p≤0.001). Post hoc analysis demonstrated that when compared to WT littermates, the increased latency to the GTCS shown by Scn8amed-jo/+ mutants as well as the decreased latency to the GTCS observed in Scn1aRH/+ mutants were statistically significant (p<0.05 for both comparisons, Fisher). In addition, the increased latency to GTCS observed in Scn1aRH/+;Scn8amed-jo/+ double mutants was statistically significant when compared to Scn1aRH/+ mutants, but was not statistically different from WT littermates (p≤0.001 and p≥0.05, respectively; Fisher). These results demonstrate that seizure thresholds can be restored to more normal levels in Scn1aRH/+ mice by altering the function of Scn8a.

Figure 3.

Figure 3

Open in a new tab

Restoration of normal thresholds to flurothyl-induced seizures in Scn1aRH/+mutants. Average latency in minutes to the GTCS is shown. Light grey bar, WT; black bar, Scn8amed-jo/+; white bar, Scn1aRH/+; dark grey bar, Scn1aRH/+;Scn8amed-jo/+; n = 9–10 per group. Error bars represent SEM. * indicates P < 0.05 compared with WT littermates. # indicates P < 0.001 compared with Scn1aRH/+ mutants; one-way ANOVA followed by Fisher’s post hoc test.

Scn8amed-jo mutation prolongs the lifespan of Scn1aRH/RH mutants

To determine whether the presence of the Scn8amed-jo allele could improve the survival of homozygous Scn1aRH/RH mice, we compared the lifespans of Scn1aRH/RH and Scn1aRH/RH;Scn8amed-jo/+ littermates. Similar to previous observations, Scn1aRH/RH mutants exhibited 50% mortality by P19.5 and 100% lethality by P25 (Fig. 4) (Martin et al, 2010). In contrast, only 25% mortality was observed for Scn1aRH/RH;Scn8amed-jo/+ mice at P25 (P = 1.8 × 10−4), and 47% of these mutants survived for over 100 days (Fig. 4).

Figure 4.

Figure 4

Open in a new tab

Improved survival of Scn1aRH/RH mutants with the Scn8amed-jo/+ allele (n = 11–16 per group).

DISCUSSION

One feature of GEFS+ is the wide range of seizure types and severities frequently seen among family members with the same SCN1A mutation (Fujiwara, 2006; Meisler and Kearney, 2005; Singh et al, 2001). Based on these observations, we hypothesized that the variable clinical presentation in GEFS+ is due, in part, to contributions from additional genetic modifiers.

Even though it is well recognized that genetic modifiers can influence the clinical presentation of a disorder, we know of relatively few human modifier genes. Cystic fibrosis (CF) represents one good example of a monogenic human disease with a known genetic modifier. CF results from recessive mutations in CFTR, a cAMP-dependent chloride channel (Riordan et al, 1989). Multiple studies have shown a significant association between two transforming growth factor-β (TGF-β) polymorphic variants and lung disease severity in CF (Arkwright et al, 2000; Drumm and Collins, 1993). TGF-β is an inflammatory cytokine and directly inhibits CFTR function (Howe et al, 2004). As a result, polymorphisms that increase circulating TGF-β levels are more common in patients with severe lung disease.

Although genetic interactions have been difficult to demonstrate in epilepsy patients, model organisms can help in the search for genetic modifiers of seizure severity. We previously demonstrated that mutations in other ion channels could modify spontaneous seizure activity and the lifespans of Scn1a+/− and Scn2aQ54 mutants. Moreover, in the presence of the Scn8amed-jo allele, the severe seizure phenotype of Scn1a+/− mice is dramatically ameliorated (Martin et al, 2007). In contrast, the Kcnq2 mutations V182M and Szt1 exacerbate the epilepsy phenotype of Scn2aQ54 transgenic mice (Kearney et al, 2006).

Here we demonstrate that Scn2a, Kcnq2, and Scn8a mutant alleles can modify the phenotype of Scn1aR1648H mice, supporting genetic modification as one mechanism by which the clinical presentation of GEFS+ can be altered. The genetic interactions between these ion channels imply that variants within Scn2a, Kcnq2, and possibly Kcnq3, may exacerbate the clinical presentation of GEFS+, shifting it to a more severe part of the GEFS+ spectrum. As previously observed with Scn1a+/− mutants (Martin et al, 2007), the Scn8amed-jo allele could rescue the increased susceptibility to flurothyl-induced GTCS in Scn1aRH/+ mice by raising seizure thresholds to a level comparable to WT littermates. In addition, while Scn1aRH/RH mutants do not survive more than 26 days, 47% of Scn1aRH/RH;Scn8amed-jo/+ mutants were still alive after 100 days. These results demonstrate that altered Scn8a function is capable of compensating for abnormalities in neuronal excitability caused by Scn1a mutations. Furthermore, these results suggest that selective blocking of Scn8a may be similarly protective in patients with epilepsy. Interestingly, although Scn8amed-jo/+ heterozygotes have increased resistance against convulsive seizures, we recently reported that they do have spontaneous absence seizures, characterized by hypersynchrony of the thalamocortical system (Papale et al, 2009). This underscores the idea that the net effect of sodium channel dysfunction in different neuronal circuits is highly dependent on the channel composition and synaptic function within each circuit.

In contrast, whereas Scn1aRH/+ mice have infrequent, adult-onset generalized seizures, the presence of either the Scn2aQ54 or Kcnq2V182M alleles results in severe, juvenile-onset generalized seizures and a shortened lifespan. Recordings of neurons isolated from Scn1a mutant mice suggest that there is decreased GABAergic neurotransmission in the hippocampus and cortex (Martin et al, 2010; Ogiwara et al, 2007; Yu et al, 2006). In the Scn1aRH/+;Scn2aQ54 double mutants, the combination of increased excitability of pyramidal neurons due to the Scn2a mutation and decreased inhibition from the Scn1a mutation results in severe generalized seizures and lethality. Reduced GABAergic inhibition may prevent localized seizure termination, resulting in secondary generalization of seizures and a more severe phenotype. Similarly, it has been demonstrated that loss of M-current in Kcnq2 mutants results in hyperexcitability of hippocampal pyramidal CA1 neurons, which in combination with reduced GABAergic inhibition may permit secondary generalization of seizures in double Scn1aRH/+;Kcnq2VM/+ mutants (Otto et al, 2006; Singh et al, 2008). Alternatively, because these genes have widespread expression in the brain, it is possible that the severe phenotype observed in double mutant mice may be the result of primary generalized seizures.

Given that the Scn1aRH/RH; Scn8amed-jo/+ and Scn1aRH/+; Kcnq2VM/+ compound mutants are on mixed genetic backgrounds, the 47% and 42% survival rates observed raises the possibility that genetic variants which differ between the C57BL/6J and 129S6/SvEvTac inbred strains may influence disease severity. Alternatively, the increased mortality between P20 to P40 in compound heterozygotes may reflect a more vulnerable juvenile stage due to ongoing maturation processes in the developing brain. In humans KCNQ2 dysfunction leads to BFNC, an epilepsy disorder affecting the neonate which typically remits by one year of age. The spontaneous remission of BFNC appears to correlate with brain maturation (Cooper and Jan 2003). However, it is also plausible that the observed survival rates are due to stochastic events.

Together with our previous study analyzing the Scn2aQ54;Kcnq2VM/+ mice, our observations from Scn1aRH/+;Kcnq2VM/+ mutants illustrate that M-channel dysfunction in a background of abnormal excitability promotes seizure initiation and increases seizure severity, suggesting that increasing the level of the M-current may be of therapeutic benefit in individuals with generalized epilepsy. It has already been demonstrated in Phase II and Phase III clinical trials that the M-current enhancer retigabine has therapeutic benefit as an adjunctive therapy in patients with drug-resistant partial epilepsy (Bialer et al, 2009).

Conclusions

Our findings show that voltage-gated ion channel variants can modify the phenotype of a mouse model of GEFS+, and therefore suggest that coding, and possibly noncoding, variants in Scn2a, Scn8a, and Kcnq2 may influence clinical presentation and severity in patients with SCN1A mutations. The demonstrated genetic interactions between Scn1a, Scn2a, Scn8a, and Kcnq2, together with previous reports showing a genetic interaction between Scn2a and Kcnq2 (Kearney et al, 2006) and Kcna1 and Cacna1a (Glasscock et al, 2007), support the notion that neuronal firing patterns are determined by the net sum of voltage-gated ion channel activity; hence, screening patients for mutations in a panel of selected ion channel genes may improve the utility of molecular diagnosis and risk assessment for guiding disease treatment. Traditional single gene screening approaches will probably be replaced by next-generation sequencing technologies, which will enable targeted re-sequencing of panels of selected genes, or whole exome analysis. Clinical diagnostic testing using next-generation sequencing of gene panels is already being applied to cardiology genetics (GeneDx, http://www.genedx.com/site/cardiology_genetic_testing_services). Similar molecular diagnostic approaches for epilepsy genetics are likely on the horizon.

Research Highlights.

  • Scn1aR1648H mice carry a human GEFS+ epilepsy mutation and have a mild phenotype

  • A Scn2a mutation results in severe epilepsy and early lethality in Scn1aR1648H mice

  • A subclinical Kcnq2 mutation exacerbates the phenotype of Scn1aR1648H mice

  • A seizure-resistant Scn8a mutation improves the phenotype of Scn1aR1648H mice Ion channel variants may contribute to clinical variability in monogenic epilepsy

Supplementary Material

01
Download video file (6.6MB, mpg)
02
Download video file (1.2MB, mpg)
03
Download video file (3.9MB, mpg)

Acknowledgments

We thank India Reddy and Alison Miller for technical assistance and Cheryl Strauss for editorial assistance. This work was supported by the National Institutes of Health [R01NS053792 to JK, R01NS065187 to AE, and T32-NS07491 to NH].

ABBREVIATIONS

GEFS+

Generalized epilepsy with febrile seizures plus

GTCS

Generalized tonic-clonic seizure

MJ

Myoclonic jerk

ECoG

Electrocorticogram

Footnotes

CONFLICT OF INTEREST STATEMENT

The mouse model of GEFS+ described in this manuscript has been licensed to Allergan. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policy.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Arkwright PD, Laurie S, Super M, Pravica V, Schwarz MJ, Webb AK, Hutchinson IV. TGF-beta(1) genotype and accelerated decline in lung function of patients with cystic fibrosis. Thorax. 2000;55:459–62. doi: 10.1136/thorax.55.6.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergren SK, Chen S, Galecki A, Kearney JA. Genetic modifiers affecting severity of epilepsy caused by mutation of sodium channel Scn2a. Mamm Genome. 2005;16:683–90. doi: 10.1007/s00335-005-0049-4. [DOI] [PubMed] [Google Scholar]
  3. Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T, White HS. Progress report on new antiepileptic drugs: a summary of the Ninth Eilat Conference (EILAT IX) Epilepsy Res. 2009;83:1–43. doi: 10.1016/j.eplepsyres.2008.09.005. [DOI] [PubMed] [Google Scholar]
  4. Biervert C, Schroeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ, Steinlein OK. A potassium channel mutation in neonatal human epilepsy. Science. 1998;279:403–6. doi: 10.1126/science.279.5349.403. [DOI] [PubMed] [Google Scholar]
  5. Charlier C, Singh NA, Ryan SG, Lewis TB, Reus BE, Leach RJ, Leppert M. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat Genet. 1998;18:53–5. doi: 10.1038/ng0198-53. [DOI] [PubMed] [Google Scholar]
  6. Cooper EC, Jan LY. M-channels: neurological diseases, neuromodulation, and drug development. Arch Neurol. 2003;60:496–500. doi: 10.1001/archneur.60.4.496. [DOI] [PubMed] [Google Scholar]
  7. Delmas P, Brown DA. Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nat Rev Neurosci. 2005;6:850–62. doi: 10.1038/nrn1785. [DOI] [PubMed] [Google Scholar]
  8. Dick DJ, Boakes RJ, Candy JM, Harris JB, Cullen MJ. Cerebellar structure and function in the murine mutant “jolting”. J Neurol Sci. 1986;76:255–67. doi: 10.1016/0022-510x(86)90173-5. [DOI] [PubMed] [Google Scholar]
  9. Drumm ML, Collins FS. Molecular biology of cystic fibrosis. Mol Genet Med. 1993;3:33–68. doi: 10.1016/b978-0-12-462003-2.50006-7. [DOI] [PubMed] [Google Scholar]
  10. Escayg A, MacDonald BT, Meisler MH, Baulac S, Huberfeld G, An-Gourfinkel I, et al. Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2. Nat Genet. 2000;24:343–5. doi: 10.1038/74159. [DOI] [PubMed] [Google Scholar]
  11. Fujiwara T. Clinical spectrum of mutations in SCN1A gene: severe myoclonic epilepsy in infancy and related epilepsies. Epilepsy Res. 2006;70 (Suppl 1):S223–30. doi: 10.1016/j.eplepsyres.2006.01.019. [DOI] [PubMed] [Google Scholar]
  12. Glasscock E, Qian J, Yoo JW, Noebels JL. Masking epilepsy by combining two epilepsy genes. Nat Neurosci. 2007;10:1554–8. doi: 10.1038/nn1999. [DOI] [PubMed] [Google Scholar]
  13. Greenberg DA, Pal DK. The state of the art in the genetic analysis of the epilepsies. Curr Neurol Neurosci Rep. 2007;7:320–8. doi: 10.1007/s11910-007-0049-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heron SE, Scheffer IE, Berkovic SF, Dibbens LM, Mulley JC. Channelopathies in idiopathic epilepsy. Neurotherapeutics. 2007;4:295–304. doi: 10.1016/j.nurt.2007.01.009. [DOI] [PubMed] [Google Scholar]
  15. Howe KL, Wang A, Hunter MM, Stanton BA, McKay DM. TGFbeta down-regulation of the CFTR: a means to limit epithelial chloride secretion. Exp Cell Res. 2004;298:473–84. doi: 10.1016/j.yexcr.2004.04.026. [DOI] [PubMed] [Google Scholar]
  16. Kearney JA, Plummer NW, Smith MR, Kapur J, Cummins TR, Waxman SG, et al. A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities. Neuroscience. 2001;102:307–17. doi: 10.1016/s0306-4522(00)00479-6. [DOI] [PubMed] [Google Scholar]
  17. Kearney JA, Yang Y, Beyer B, Bergren SK, Claes L, Dejonghe P, Frankel WN. Severe epilepsy resulting from genetic interaction between Scn2a and Kcnq2. Hum Mol Genet. 2006;15:1043–8. doi: 10.1093/hmg/ddl019. [DOI] [PubMed] [Google Scholar]
  18. Kohrman DC, Smith MR, Goldin AL, Harris J, Meisler MH. A missense mutation in the sodium channel Scn8a is responsible for cerebellar ataxia in the mouse mutant jolting. J Neurosci. 1996;16:5993–9. doi: 10.1523/JNEUROSCI.16-19-05993.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martin MS, Dutt K, Papale LA, Dube CM, Dutton SB, de Haan G, et al. Altered function of the SCN1A voltage-gated sodium channel leads to GABAergic interneuron abnormalities. J Biol Chem. 2010;285:9823–34. doi: 10.1074/jbc.M109.078568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Martin MS, Tang B, Papale LA, Yu FH, Catterall WA, Escayg A. The voltage-gated sodium channel Scn8a is a genetic modifier of severe myoclonic epilepsy of infancy. Hum Mol Genet. 2007;16:2892–9. doi: 10.1093/hmg/ddm248. [DOI] [PubMed] [Google Scholar]
  21. Meisler MH, Kearney JA. Sodium channel mutations in epilepsy and other neurological disorders. J Clin Invest. 2005;115:2010–7. doi: 10.1172/JCI25466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ogiwara I, Miyamoto H, Morita N, Atapour N, Mazaki E, Inoue I, et al. Na(v)1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation. J Neurosci. 2007;27:5903–14. doi: 10.1523/JNEUROSCI.5270-06.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Otto JF, Yang Y, Frankel WN, White HS, Wilcox KS. A spontaneous mutation involving Kcnq2 (Kv7.2) reduces M-current density and spike frequency adaptation in mouse CA1 neurons. J Neurosci. 2006;26:2053–9. doi: 10.1523/JNEUROSCI.1575-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Papale LA, Beyer B, Jones JM, Sharkey LM, Tufik S, Epstein M, et al. Heterozygous mutations of the voltage-gated sodium channel SCN8A are associated with spike-wave discharges and absence epilepsy in mice. Hum Mol Genet. 2009;18:1633–41. doi: 10.1093/hmg/ddp081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989;245:1066–73. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  26. Sidman RL, Cowen JS, Eicher EM. Inherited muscle and nerve diseases in mice: a tabulation with commentary. Ann N Y Acad Sci. 1979;317:497–505. doi: 10.1111/j.1749-6632.1979.tb56567.x. [DOI] [PubMed] [Google Scholar]
  27. Singh NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R, et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat Genet. 1998;18:25–9. doi: 10.1038/ng0198-25. [DOI] [PubMed] [Google Scholar]
  28. Singh NA, Otto JF, Dahle EJ, Pappas C, Leslie JD, Vilaythong A, et al. Mouse models of human KCNQ2 and KCNQ3 mutations for benign familial neonatal convulsions show seizures and neuronal plasticity without synaptic reorganization. J Physiol. 2008;586:3405–23. doi: 10.1113/jphysiol.2008.154971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Singh R, Andermann E, Whitehouse WP, Harvey AS, Keene DL, Seni MH, et al. Severe myoclonic epilepsy of infancy: extended spectrum of GEFS+? Epilepsia. 2001;42:837–44. doi: 10.1046/j.1528-1157.2001.042007837.x. [DOI] [PubMed] [Google Scholar]
  30. Sugawara T, Tsurubuchi Y, Agarwala KL, Ito M, Fukuma G, Mazaki-Miyazaki E, et al. A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction. Proc Natl Acad Sci U S A. 2001;98:6384–9. doi: 10.1073/pnas.111065098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tan NC, Mulley JC, Scheffer IE. Genetic dissection of the common epilepsies. Curr Opin Neurol. 2006;19:157–63. doi: 10.1097/01.wco.0000218232.66054.46. [DOI] [PubMed] [Google Scholar]
  32. Wallace RH, Wang DW, Singh R, Scheffer IE, George AL, Jr, Phillips HA, et al. Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B. Nat Genet. 1998;19:366–70. doi: 10.1038/1252. [DOI] [PubMed] [Google Scholar]
  33. Yu FH, Mantegazza M, Westenbroek RE, Robbins CA, Kalume F, Burton KA, et al. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci. 2006;9:1142–9. doi: 10.1038/nn1754. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
Download video file (6.6MB, mpg)
02
Download video file (1.2MB, mpg)
03
Download video file (3.9MB, mpg)

RESOURCES