Key result
Disease-associated SCN8A variants exhibited significant isoform-dependent functional effects, highlighting the necessity of using the appropriate developmental splice isoform to accurately assess variant pathogenicity.
Why the study?
Previous approaches to determine SCN8A variant function may be confounded by using a neonatal-expressed splice isoform and engineered TTX-resistance mutations.
The functional effects of SCN8A variants are highly dependent on the molecular and cellular context, specifically alternative splicing, highlighting the need for context-specific functional assays.
SCN8A variant assays using neonatal isoforms or TTX-resistant constructs may misclassify adult channel effects; leaves open splicing impact on epileptic encephalopathy and potential cardiac excitability.
Pathogenic variants in SCN8A , which encodes the voltage-gated sodium (Na V ) channel Na V 1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of Na V 1.6 (Na V 1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (Na V 1.6N, Na V 1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous Na V current to obviate the need for TTX-resistance mutations. Expression of Na V 1.6N or Na V 1.6A in ND7/LoNav cells generated Na V currents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.
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Vanoye et al. (2023) studied SCN8A-related neurodevelopmental disorders and epilepsy (n=3,265). SCN8A variants expressed in NaV1.6N or NaV1.6A isoforms vs. Wild-type (WT) NaV1.6 channels was evaluated on Channel functional properties (voltage-dependence of activation and inactivation, current density, persistent current). Disease-associated SCN8A variants exhibited significant isoform-dependent functional effects, highlighting the necessity of using the appropriate developmental splice isoform to accurately assess variant pathogenicity.
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