Abstracts

Rationale: Inherited, biallelic variants in the gene SCN1B, encoding the β1 subunit of voltage gated sodium channels, are linked to Dravet syndrome (DS) or the more severe Early Infantile Developmental and Epileptic Encephalopathy (EIDEE). Sodium channel β1 subunits are multi-functional proteins that participate in sodium current modulation, regulation of sodium channel cell surface expression, cell adhesion, and transcriptional regulation. SCN1B generates two alternative splice products: one that encodes mature, transmembrane b1 (exons 1-6) and another that contains exons 1-3 with in-frame read-through to intron 3 that includes a termination codon. The function of SCN1B intron 3 retention is not understood, although several pathogenic variants have been identified in this region, suggesting that it is functionally important. Here, we tested the hypothesis that SCN1B intron 3 can function either as a nonsense mediated decay (NMD) exon or as a nuclear detained intron that serves as a reservoir for SCN1B pre-mRNA that can be efficiently spliced into mature, transmembrane SCN1B. Understanding the mechanism of SCN1B mRNA splicing is critical to understanding the control of SCN1B expression in neurons, as well as the mechanism of disease variants identified in intron 3. Here, we investigated the mechanism of an EIDEE pathogenic splice site variant in intron 3, SCN1B c.449-2A >G, which has been identified in three pedigrees. Patients have severe developmental delay and intractable seizures.


Methods: We studied Scn1b transcripts in mouse brain, iPSC neurons from the SCN1B c.449-2A >G patient cells and nonepileptic controls, as well as three cancer lines with high levels of SCN1B intron 3 retention: K562, MCF7, and SH-SY5Y. We conducted RT-PCRs using primers specific to different regions of SCN1B.

Results: Using an SMG1 inhibitor of NMD, we showed using RT-PCR and a positive control transcript that SCN1B intron 3 containing transcript does not undergo NMD. In contrast, RT-PCR results revealed that SCN1B intron 3 containing transcript includes exons 1-6, and therefore may serve as a precursor transcript to generate mature SCN1B mRNA following the final splicing step. We found that SCN1B intron 3 containing transcripts are confined to the nuclear fraction of K562, MCF7, and SH-SY5Y cells and have a very slow rate of decay, which are key characteristics of nuclear detained intron transcripts. In SCN1B c.449-2A >G patient-derived iPSC neurons, we found high expression of SCN1B intron 3 containing transcript along with lower expression of two different mis-spliced transcripts. Importantly, patient iPSC neurons did not express mature SCN1B mRNA, suggesting that patients are functionally null for transmembrane β1 subunits.

Conclusions: Our work suggests that SCN1B intron 3 retention can function as a nuclear detained precursor RNA that serves as a reservoir for mature mRNA. Furthermore, SCN1B c.449-2A >G patient derived neurons improperly splice intron 3, resulting in the absence of transmembrane b1 expression and EIDEE.

Funding: NIH R37 NS076752