Aberrant regulation of a poison exon caused by a non-coding variant inScn1a-associated epileptic encephalopathy

Abstract

Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Na v 1.1 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 leads to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A transcript 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. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Na v 1.1 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 Na v 1.6, 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.Correspondence to Gregory Barsh (gbarsh@hudsonalpha.org | hudsonalpha.org/faculty/greg-barsh | @GregBarsh ) Correspondence to Erik Roberson (eroberson@uabmc.edu | robersonlab.net | @RobersonLabUAB) Author SummaryDravet syndrome (DS) is a neurological disorder affecting approximately 1:15,700 Americans[1]. While most patients have a mutation in the SCN1A gene encoding Na v 1.1 sodium channels, about 20% do not have a mutation identified by exome sequencing. Recently, we identified variants in intron 20N, a noncoding region of SCN1A, in some DS patients [2]. We predicted that these variants alter SCN1A transcript processing, decrease Na v 1.1 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 that Scn1a+/KI mice have reduced levels ...

The dose makes the poison—Novel insights into Dravet syndrome and SCN1A regulation through nonproductive splicing

“…Voskobiynyk and colleagues [ 10 ] then proceed to assess poison exon inclusion and found that up to 70% of SCN1A transcripts included exon 20N during embryonal development, which decreased over time to 10% postnatally. This suggests that poison exon inclusion is not just an epiphenomenon, but an active regulatory process to suppress SCN1A expression during early development.…”

“…In the study by Voskobiynyk and colleagues [ 10 ], the authors provide a deep dive into regulation of SCN1A through poison exons. The SCN1A gene contains the naturally occurring poison exon 20N, and inclusion of this exon results in nonproductive splicing and nonsense-mediated RNA decay, effectively reducing the amount of Nav1.1-containing sodium channels made available to neurons.…”

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing

“…Probands A, B, and C have variants within intron 20 of SCN1A and each has seizure phenotypes that are consistent with SCN1A-associated epilepsy (see Case Reports in Supplemental Data 4). This intron harbors the 20N poison exon 9 variants in or near to 20N have been previously associated with epilepsy in both humans and mice 8,11 . The phenotype of Proband B also included developmental delay and spastic dystonic cerebral palsy with left triplegia.…”

“…The inclusion of PTCs cause translation to stall, thereby triggering nonsense-mediated decay (NMD) of the transcript and reducing protein levels in the cell. Some PEs are spliced into transcripts at relatively high levels during mammalian brain development in early stages and are hypothesized to regulate protein levels of the genes in which they reside 7,8,9,10 . Crucially, since PE usage leads to NMD, and therefore reduction of protein, genetic variation that alters PE inclusion can lead to a loss-of-function effect.…”

Gene Editing and Modulation: the Holy Grail for the Genetic Epilepsies?

“…If technology for allelespecific targeting were to be improved [16], however, then NHEJ might be harnessed to destroy a gene variant that acts in a dominant or dominant negative manner, in a context where haploinsufficiency of the gene product results in a less severe phenotype [17]. A possibility for genetic epilepsy, and an as of yet untested strategy, could be to use CRISPR to destroy the splice-site of the recently discovered poison exon (20 N) in SCN1A, in order to permanently upregulate SCN1A expression in Dravet syndrome [18,19]. Proof-of-principle of such an approach has been demonstrated using Targeted Augmentation of Nuclear Gene Output (TANGO) technology, which uses an antisense oligonucleotide targeted to the poison exon of Scn1a to reduce a non-productive alternative splicing event [20].…”