Novel SCN3A variants associated with focal epilepsy in children
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.
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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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