? PROJECT 1 Channelopathies, particularly those involving voltage-gated sodium (NaV) and potassium (KV) channels, are responsible for a diverse group of epilepsy syndromes. Collectively, genes encoding NaV and KV channels have the greatest cumulative variant burden among all epilepsy-associated genes, representing >30% of all reported variants in genetic epilepsies, and variants in just four genes (SCN1A, KCNQ2, SCN2A, SCN8A) account for 40% of variant-positive cases tested for epilepsy by one large commercial laboratory (GeneDx). Functional assessments of ion channel function using patch clamp electrophysiological recording are the cornerstone of research determining the pathogenicity of channel variants and establishing genotype- phenotype relationships. However, the technique in its typical embodiment has limited throughput, is extremely time- and labor-intensive, and suffers from a lack of standardization that hampers reproducibility. In Project 1, we will exploit high throughput automated patch clamp recording platforms available at two academic centers (Northwestern University, Broad Institute) to determine the functional consequences of ion channel variants associated with epilepsy at an unprecedented scale.
For Aim 1, specific human NaV and KV channel variants will be prioritized for functional analyses by the Variant Prioritization and Curation Core (Core A), and cells expressing the variants will be provided by the Mutagenesis and Cell Expression Core (Core B). The enormous capacity of the combined automated patch clamp facilities at Northwestern and Broad Institute will enable functional evaluation of up to 1,000 epilepsy-associated variants over 5 years. Additional functional studies of prototypical variants will be performed with orthologous murine channels to validate and prioritize variants for generating mouse models of channelopathy-associated epilepsy (Project 3).
In Aim 2, prototypical variants (e.g., those with functional properties representative of a larger cohort of variants) will be tested against panels of approved and investigational anticonvulsant drugs/compounds to determine which agents are best able to correct the observed functional abnormality.
In Aim 3, we will develop and test an orthogonal strategy to predetermine which amino acid substitutions in SCN1A lead to loss of function (LOF) in a high- throughput pooled screen. This strategy will couple saturation mutagenesis of a genetic ?hot spot? and next- generation sequencing with an assay to determine the ability of cells transfected with a missense variant in SCN1A to survive when exposed to a potent NaV channel activator. The goal of this pilot study is to demonstrate proof-of-principle that we can create a comprehensive database for all possible LOF substitutions in SCN1A. If successful, this approach will generate an allele characterization framework that can be scaled to accomplish genome-guided genotype-phenotype mapping.
