Elsevier

Neurobiology of Disease

Volume 62, February 2014, Pages 313-322
Neurobiology of Disease

Novel SCN3A variants associated with focal epilepsy in children

https://doi.org/10.1016/j.nbd.2013.10.015Get rights and content

Highlights

  • Identified four novel SCN3A missense variants in pediatric focal epilepsy patients.

  • Variant characterization in heterologously expressed NaV1.3 revealed functional defects.

  • All variants exhibited increased current in response to depolarizing voltage ramps.

  • Increased ramp currents may be a common biophysical defect in SCN3A-associated epilepsy.

Abstract

Voltage-gated sodium (NaV) channels are essential for initiating and propagating action potentials in the brain. More than 800 mutations in genes encoding neuronal NaV channels including SCN1A and SCN2A have been associated with human epilepsy. Only one epilepsy-associated mutation has been identified in SCN3A encoding the NaV1.3 neuronal sodium channel. We performed a genetic screen of pediatric patients with focal epilepsy of unknown cause and identified four novel SCN3A missense variants: R357Q, D766N, E1111K and M1323V. We determined the functional consequences of these variants along with the previously reported K354Q mutation using heterologously expressed human NaV1.3. Functional defects were heterogeneous among the variants. The most severely affected was R357Q, which had a significantly smaller current density and slower activation than the wild-type (WT) channel as well as depolarized voltage dependences of activation and inactivation. Also notable was E1111K, which evoked a significantly greater level of persistent sodium current than WT channels. Interestingly, a common feature shared by all variant channels was increased current activation in response to depolarizing voltage ramps revealing a functional property consistent with conferring neuronal hyper-excitability. Discovery of a common biophysical defect among variants identified in unrelated pediatric epilepsy patients suggests that SCN3A may contribute to neuronal hyperexcitability and epilepsy.

Introduction

Voltage-gated sodium (NaV) channels are essential for initiating and propagating action potentials in the brain. The channels exist in a native protein complex composed of a pore-forming α subunit and two smaller accessory β subunits (Catterall, 1988, Isom et al., 1994). More than 800 sodium channel mutations, mostly in SCN1A and a few in SCN2A, have been associated with epilepsy (Lossin, 2009). SCN3A is clustered with SCN1A and SCN2A on human chromosome 2q24, but only one mutation in SCN3A, encoding NaV 1.3, is known to be associated with epilepsy (Holland et al., 2008). The involvement of NaV1.3 in epilepsy is supported by studies showing that SCN3A mRNA is expressed at higher levels in human CA4 hilar cells in the epileptic hippocampus (Whitaker et al., 2001) and in rat neurons of CA1–CA3 and in the dentate granule cell layer after the induction of status epilepticus (Aronica et al., 2001, Bartolomei et al., 1997).

NaV1.3 channels possess several properties that may cause neuronal hyper-responsiveness. For example, NaV1.3 channels recover from inactivation rapidly and sustain high-frequency firing (Cummins et al., 2001), activate during slow ramp depolarizations, and produce persistent sodium current (Chen et al., 2000, Cummins and Waxman, 1997, Estacion et al., 2010, Sun et al., 2007). The NaV1.3 epilepsy-associated mutation K354Q was previously demonstrated to enhance persistent current and ramp current (Estacion et al., 2010, Holland et al., 2008). Several studies indicate that NaV persistent current participates in spontaneous neuronal firing in a variety of cell types including hippocampal neurons (Agrawal et al., 2001, Kearney et al., 2001) and subicular neurons isolated from patients with temporal lobe epilepsy (Vreugdenhil et al., 2004). Moreover, several epilepsy-associated NaV1.1 mutations are known to enhance persistent current (Kahlig et al., 2008, Lossin et al., 2002, Rhodes et al., 2004, Spampanato et al., 2004).

We performed a genetic screen of pediatric patients with cryptogenic focal epilepsy and identified four novel SCN3A missense variants: R357Q, D766N, E1111K and M1323V. Electrophysiological studies revealed a diversity of functional defects, but interestingly, all mutant channels exhibited abnormal current activation during a slow depolarizing voltage ramp. This common defect among the novel alleles could explain neuronal hyperexcitability.

Section snippets

Study subjects

Subjects were ascertained from the St. Louis Children's Hospital Pediatric Epilepsy Center and the Cincinnati Children's Hospital Comprehensive Epilepsy Center. Diagnoses were classified as focal epilepsy of unknown cause according to the International League Against Epilepsy guidelines (Berg et al., 2010). Additional inclusion criteria used at the Cincinnati Children's Hospital Comprehensive Epilepsy Center were: i) treatment with carbamazepine or oxcarbamazepine; and ii) have classifiable

Results

We identified four novel SCN3A missense variants (R357Q, D766N, E1111K, M1323V) in a screen of 179 pediatric patients with focal epilepsy who were SCN1A-mutation negative. None of the variants were detected in up to 590 chromosomes from control subjects (Table 1). Three of the variants (R357Q, D766N, M1323V) were not detected by the NHLBI Exome Sequencing Project (Exome Variant Server, 2013) in 4300 EuropeanAmerican or 2203 AfricanAmerican subjects. One heterozygous E1111K carrier was reported

Discussion

Among the myriad epilepsy-associated NaV mutations described to date, only one SCN3A mutation has been reported previously in a patient with focal epilepsy (Holland et al., 2008, Lossin, 2009). In this study we report four new SCN3A variants that were identified in pediatric focal epilepsy. We further demonstrated that all four novel SCN3A variants confer significant functional defects to the encoded NaV1.3 sodium channel suggesting these as putative epilepsy-associated mutations. Two

Acknowledgments

This work was supported by NIH grants NS032387 (A.L.G.) and NS053792 (J.A.K.). The authors would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010).

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