Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scn1a-associated epileptic encephalopathy

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.

Exploring the Diverse Functional and Regulatory Consequences of Alternative Splicing in Development and Disease

“…An analysis of RNA-seq data of wild type mouse cortex revealed that throughout development, the inclusion of exon 20N decreases, accompanied by the increase in overall Scn1a mRNA levels. A similar pattern was observed for sodium channel, voltage-gated, type VIII, alpha ( Scn8a ) ( Voskobiynyk et al, 2021 ). Scn8a pre-mRNA contains two alternatively spliced exons, 18A and 18N.…”

“…Importantly, the pathogenic variants, all of which have intronic lesions, promote PE (exon 20N) inclusion leading to NMD of the SCN1A transcripts, thereby lowering expression levels of the sodium channel ( Carvill et al, 2018 ). In a different study, CRISPR/Cas9 was used to knock-in (KI) a pathogenic, PE promoting, variant of sodium channel, voltage-gated, type I, alpha ( Scn1a ) to generate a Dravet Syndrome (DS) mouse model ( Voskobiynyk et al, 2021 ). Scn1a +/KI mice exhibited decreased levels of Scn1a mRNA and protein in the brain, as well as an increase in exon 20N inclusion compared to control mice.…”

“…Scn1a +/KI mice exhibited decreased levels of Scn1a mRNA and protein in the brain, as well as an increase in exon 20N inclusion compared to control mice. Moreover, truncated proteins were not detected, indicating that the transcripts likely were subjected to NMD ( Voskobiynyk et al, 2021 ). Scn1a +/KI mice display phenotypes previously reported in other DS models such as hyperactivity (increase in jumping and distance travelled), premature mortality, and spontaneous seizures ( Voskobiynyk et al, 2021 ).…”

“…Moreover, truncated proteins were not detected, indicating that the transcripts likely were subjected to NMD ( Voskobiynyk et al, 2021 ). Scn1a +/KI mice display phenotypes previously reported in other DS models such as hyperactivity (increase in jumping and distance travelled), premature mortality, and spontaneous seizures ( Voskobiynyk et al, 2021 ). This not only supports the use of Scn1a +/KI mice as a DS model, but specifically implicates PE (exon 20N) inclusion in DS pathogenesis.…”

“…Greater than 80% of DS cases are due to mutations in the sodium voltage-gated alpha subunit 1 ( SCN1A ) gene, which is a subunit of the sodium channel NA v 1.1 ( Han et al, 2020 ; Helbig and Goldberg, 2021 ). Furthermore, the mutation of SCN1A that is associated with DS is characterized by a 5-fold increase in expression of a PE, which results in a 50% reduction of Scn1a mRNA and NA v 1.1 protein levels ( Voskobiynyk et al, 2021 ). However, treatment with ASOs targeting the SCN1A (ASO-22) has been demonstrated to be effective in reducing expression of the PE containing transcript, increasing the functional transcript, and increasing NA v 1.1 protein expression in human neural progenitor cells.…”