Aberrant regulation of a poison exon caused by a non-coding variant inScn1a-associated epileptic encephalopathy
Authors: Voskobiynyk Yuliya , Battu Gopal , Felker Stephanie A. , Cochran J. Nicholas , Newton Megan P. , Lambert Laura , Kesterson Robert A. , Myers Richard M. , Cooper Gregory M. , Roberson Erik D. , Barsh Gregory S. 
Published: June 2020

AbstractDravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Nav1.1 sodium channel encoded bySCN1A. Most known DS-causing mutations are in coding regions ofSCN1A, but we recently identified several disease-associatedSCN1Amutations in intron 20 that are within or near to a cryptic and evolutionarily conserved “poison” exon, 20N, whose inclusion leads to transcript degradation. However, it is not clear how these intron 20 variants alterSCN1Atranscript processing 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.Scn1aheterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brainScn1amRNA and Nav1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adultScn1a+/+ animals, quantitative RT-PCR assays indicated that ~1% ofScn1amRNA included exon 20N, while brain tissue fromScn1a +/KImice 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 byScn8a. 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 byScn1aandScn8ais 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.Author SummaryDravet syndrome (DS) is a neurological disorder affecting approximately 1:15,700 Americans[1]. While most patients have a mutation in theSCN1Agene encoding Nav1.1 sodium channels, about 20% do not have a mutation identified by exome sequencing. Recently, we identified variants in intron 20N, a noncoding region ofSCN1A, in some DS patients [2]. We predicted that these variants alterSCN1Atranscript 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. In this study, we generated a knock-in mouse model,Scn1a+/KI, of one of these variants, NM_006920.4(SCN1A):c.3969+2451G>C, which resides in a genomic region that is extremely conserved across vertebrate species. We found thatScn1a+/KImice have reduced levels ofScn1atranscript and Nav1.1 protein and develop DS-related phenotypes. Consistent with the poison exon hypothesis, transcripts from brains ofScn1a+/KImice showed elevated rates ofScn1aexon 20N inclusion. SinceScn1aexpression in the brain is regulated developmentally, we next explored the developmental relationship between exon 20N inclusion andScn1aexpression. During normal embryogenesis, whenScn1aexpression was low, exon 20N inclusion was high; postnatally, asScn1aexpression increased, there was a corresponding decrease in exon 20N usage. Expression of another voltage-gated sodium channel transcript,Scn8a(Nav1.6), was similarly regulated, with inclusion of a poison exon termed as 18N early in development whenScn8aexpression was low, followed by a postnatal decrease in exon 18N inclusion and corresponding increase inScn8aexpression. Together, these data demonstrate that poison exon inclusion is a conserved mechanism to control sodium channel expression in the brain, and that an intronic mutation that disrupts the normal developmental regulation of poison exon inclusion leads to reduced Nav1.1 and DS pathophysiology.