Investigation of a Pathogenic Splice Site Variant in SCN1B Linked to Dravet Syndrome
Abstract number :
3.011
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2022
Submission ID :
2204324
Source :
www.aesnet.org
Presentation date :
12/5/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:24 AM
Authors :
Shreeya Bakshi, BS – University of Michigan Medical School; Chunling Chen, MD – University of Michigan Medical School; Isha Verma, PhD – University of Michigan Medical School; Larissa Robinson-Cooper, BS – University of Michigan Medical School; Christelle El Achkar, MD – Harvard Medical School; Louis Dang, MD, PhD – University of Michigan Medical School; Annapurna Poduri, MD, MPH – Harvard Medical School; Jack Parent, MD – University of Michigan Medical School; Lori Isom, PhD – University of Michigan Medical School
Rationale: Biallelic variants in the gene SCN1B, encoding the β1 and β1B subunits of voltage gated sodium channels, are linked to Dravet syndrome (DS), a devastating form of Developmental and Epileptic Encephalopathy. Sodium channel β1 subunits are multi-functional. They participate in sodium current modulation, regulation of sodium channel cell surface expression, and cell-cell and cell-matrix adhesion as immunoglobulin superfamily cell adhesion molecules. SCN1B encodes two isoforms: a transmembrane isoform, β1, and a soluble secreted isoform, β1B.
Methods: We are studying a novel biallelic DS variant that has been identified in three different pedigrees, SCN1B c.449-2A >G, located in the in the 3’ splice acceptor site of intron 3 of SCN1B. We hypothesized that this variant may cause aberrant SCN1B splicing due to its location in a splice acceptor site. We are testing this hypothesis using three different models: DS patient fibroblasts, a heterologous expression system, and transgenic mice.
Results: RT-PCR data from fibroblasts obtained from a DS patient with this variant showed the presence of three different SCN1B transcripts: (1) β1B, generated by retention of intron 3; (2) SCN1B transcript lacking exon 4, which contains the b1 transmembrane domain; and (3) SCN1B transcript with a portion of exon 4 missing. Based on the predicted amino acid sequences of these transcripts, we predict that none of the resulting proteins contain the sequence that encodes the β1 transmembrane domain, such that no full-length β1 protein, the transmembrane isoform, is produced. We generated stable heterologous cell lines expressing each of the patient transcripts and found that the two mis-spliced products undergo proteasomal degradation. To understand the physiological role of β1B, which is expressed by the patient cells, we generated a mouse line that expresses β1B tagged with Tap (a histidine affinity tag and a FLAG tag). We found that homozygous β1B-tagged mice express the soluble splice variant but not the transmembrane isoform β1. Homozygous β1B-tagged mice do not survive past postnatal day 24 and have stunted growth, similar to Scn1b -/- mice, suggesting that β1B is not sufficient to rescue the null phenotype.
Conclusions: Our work suggests that DS patients with the biallelic SCN1B c.449-2A >G variant express soluble β1B but not transmembrane β1 subunits. Our new β1B-tagged mouse line, as well as SCN1B c.449-2A >G patient-derived iPSC neurons, which are in progress, will be optimal models for understanding SCN1B c.449-2A >G DS patients.
Funding: R37 NS076752, T32 GM140223
Basic Mechanisms