Abstract
Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Nav1.1 sodium channel encoded by SCN1A. Most known DS-causing mutations are in coding regions of SCN1A, but we recently identified several disease-associated SCN1A mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved "poison" exon, 20N, whose inclusion is predicted to lead to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A expression or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Nav1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult Scn1a +/+ animals, quantitative RT-PCR assays indicated that ~1% of Scn1a mRNA included exon 20N, while brain tissue from Scn1a +/KI mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Nav1.6, encoded by Scn8a. For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by Scn1a and Scn8a is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy.
Highlights
Dravet syndrome (DS) is a developmental and epileptic encephalopathy (DEE) characterized by intractable seizures, developmental delay, speech impairment, ataxia, hypotonia, sleep disturbances, and other health problems [1]
While most patients have a mutation in the Scn1a protein levels using antiNav1.1 (SCN1A) gene that encodes the Nav1.1 voltage-gated sodium channel, about 20% do not have a mutation identified by exome or targeted sequencing
We identified variants in intron 20, a noncoding region of SCN1A, in some DS patients. We hypothesized that these variants alter SCN1A transcript processing, decrease Nav1.1 function, and lead to DS pathophysiology via inclusion of exon 20N, a “poison” exon that leads to a premature stop codon
Summary
Dravet syndrome (DS) is a developmental and epileptic encephalopathy (DEE) characterized by intractable seizures, developmental delay, speech impairment, ataxia, hypotonia, sleep disturbances, and other health problems [1]. The most frequent cause of DS are loss-of-function mutations of SCN1A, which encodes the type I voltage-gated sodium channel (Nav1.1) alpha subunit, part of a larger family of nine sodium channel proteins (Nav1.1 –Nav1.9) that control neuronal excitability [4,5,6,7,8]. DS-associated SCN1A mutations lead to a loss of Nav1.1, which is predominantly expressed in inhibitory GABAergic interneurons, so loss of function leads to network disinhibition [5,6,7,8,9]. The molecular mechanisms for Nav1.1 loss of function differ between various SCN1A mutations; many cause nonsense-mediated RNA decay, while other missense mutations affect Nav1.1 stability or function [4]
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