Abstracts

Rationale:
SCN2A variants are associated with early-onset epilepsy or neurodevelopmental disorders with or without seizures. SCN2A encodes the voltage-gated sodium channel NaV1.2, which is highly expressed in glutamatergic neurons and is responsible for the generation and propagation of action potentials. SCN2A undergoes developmentally-regulated alternative mRNA splicing, which can modify the functional properties of certain epilepsy-associated variants. Currently, there are >350 published SCN2A variants, which presents a formidable challenge for interrogating functional consequences that may be affected by alternative splicing.
Method:
We employed a high throughput strategy using automated patch-clamp electrophysiology to survey the functional properties of several SCN2A variants. We expressed recombinant human NaV1.2 channels in a HEK293T cell line stably expressing the sodium channel β1 and β2 subunits using high-efficiency electroporation. NaV1.2 plasmids included the coding sequence of a fluorescent protein (mScarlet) that enabled quantification of transfection efficiency by flow cytometry. Whole-cell sodium currents were recorded by using the Nanion Syncropatch 768PE system.
Results:
We investigated the functional properties of 28 SCN2A variants. Twenty-two were associated with early onset of seizures (< 2 months of age) and were studied in the neonatal and canonical (adult expressed) splice isoforms. Five were studied only in the adult splice isoform because they were associated with later onset disease ( >6 months of age). One variant, present exclusively in the neonatal expressed alternative exon, was only studied in that isoform. Among the 50 variant constructs studied we observed a range of functional differences relative to WT channels including variability in current density, altered voltage-dependence of activation and/or inactivation, differences in the kinetics of inactivation, or enhanced persistent current. Importantly, we observed concordant patterns of dysfunction for a subset of variants that were studied previously by manual patch clamp, thus validating our automated patch clamp assay. Certain variants exhibited predominantly gain-of-function properties (R1626Q, M1879T, R1882Q, R1882L) whereas others had changes associated with loss-of-function (G882E, F978L, R1319L, K1422E). However, many variants exhibited constellations of functional properties that could not be easily summarized as either gain or loss of function, or that had mild functional perturbations. The functional properties of some variants associated with early-onset epilepsy were different in the neonatal SCN2A splice isoform compared with the adult isoform. Investigations into correlations between clinical features and response to antiseizure medications with variant functional attributes are ongoing.
Conclusion:
We demonstrated that automated patch-clamp recording can be successfully applied to the functional annotation of SCN2A variants associated with epilepsy.
Funding:
:NIH U54-NS108874