Authors :
Presenting Author: Antonella Riva, MD – University of Genoa
Loretta Ferrera, PhD – IRCCS Istituto Giannina Gaslini; Martina Albini, PhD – University of Genoa; Alessandra Ludovico, PhD – University of Genoa; Lisastella Morinelli, PhD – University of Genoa; Giulia Lombardo, MD – University of Catania; Bruno Sterlini, PhD – University of Genoa; Gaetan Lesca, MD, PhD – Lyon University Hospitals; Francesca Madia, PhD – IRCCS Isitituto Giannina Gaslini; Raffaele Falsaperla, MD – University of Catania; Pasquale Striano, MD, PhD – University of Genoa; Anna Corradi, PhD – University of Genoa; Federico Zara, PhD – University of Genoa
Rationale:
Mutations in the voltage-gated sodium channel Nav1.2 cause a phenotypic spectrum including epileptic encephalopathy and intellectual disability or autism without epilepsy. Early infantile onset encephalopathies are generally well respondent to sodium channel blockers, assuming a gain-of-function mutation. We evaluated aSCN2A: c.4976C >T (p.A1659V) pathogenic variant found in two infants non-respondent to carbamazepine.
Methods:
The variant was identified through Next Generation Sequencing and the mutation was inserted in the Pir cmv SCN2A plasmid encoding for Nav1.2. HEK293 cells transfected with WT and mutated (A1659V) SCN2A were stained with an anti-PanNaV antibody for immunofluorescence or lysated for Western blot assay. The immunofluorescences were acquired both at the epifluorescence and confocal microscopy.
For functional characterization, Nav1.2 WT and A1659V mutant were expressed in HEK293 cells and membrane currents were evaluated by whole-cell patch-clamp technique. Cells were stimulated with constant pulse potentials ranging from -60 to +30 mV, Δ=10 mV, where holding potential was -90 mV (n≥12 experiments).
Results:
SCN2A A1659V mutation does not impact on channel expression. Whole-cell conductance (GNa) was calculated as GNa = I/(V – Erev), where I is the measured peak current, V is the step potential, and Erev is the calculated sodium reversal potential predicted by linear regression of the I-V curve for each cell. To calculate voltage dependence of activation, normalized GNa was plotted against voltage and fitted with the Boltzmann function G/Gmax= (1 + exp[(V – V1/2)/k])−1 , where V1/2 indicates the voltage at half-maximal activation and k is a slope factor describing voltage sensitivity of the channel. Expression of A1659V induced a smaller current respect to WT. The quantitative analysis of A1659V activation properties show a shift of V1/2 about 10 mV towards more negative potentials and a time constant slower than the WT channel.
Conclusions:
SCN2A loss-of-function mutations may cause a severe, early infantile, phenotype. Functional characterization may direct clinical interventions and expand funotype-phenotype correlations.
Funding: No fundings to be reported