Alternative splicing potentiates dysfunction of early-onset epileptic encephalopathy SCN2A variants

Abstract

Epileptic encephalopathies are severe forms of infantile-onset epilepsy often complicated by severe neurodevelopmental impairments. Some forms of early-onset epileptic encephalopathy (EOEE) have been associated with variants in SCN2A, which encodes the brain voltage-gated sodium channel NaV1.2. Many voltage-gated sodium channel genes, including SCN2A, undergo developmentally regulated mRNA splicing. The early onset of these disorders suggests that developmentally regulated alternative splicing of NaV1.2 may be an important consideration when elucidating the pathophysiological consequences of epilepsy-associated variants. We hypothesized that EOEE-associated NaV1.2 variants would exhibit greater dysfunction in a splice isoform that is prominently expressed during early development. We engineered five EOEE-associated NaV1.2 variants (T236S, E999K, S1336Y, T1623N, and R1882Q) into the adult and neonatal splice isoforms of NaV1.2 and performed whole-cell voltage clamp to elucidate their functional properties. All variants exhibited functional defects that could enhance neuronal excitability. Three of the five variants (T236S, E999K, and S1336Y) exhibited greater dysfunction in the neonatal isoform compared with those observed in the adult isoform. Computational modeling of a developing cortical pyramidal neuron indicated that T236S, E999K, S1336Y, and R1882Q showed hyperexcitability preferentially in immature neurons. These results suggest that both splice isoform and neuronal developmental stage influence how EOEE-associated NaV1.2 variants affect neuronal excitability.

Functional and Pharmacological Evaluation of a Novel SCN2A Variant Linked to Early-onset Epilepsy

“…Our study investigated a de novo SCN2A coding sequence Unlike other SCN2A variants associated with early-onset epilepsy, expression of the variant in the neonatal splice variant of the channel was not associated with greater dysfunction. 13 Carbamazepine partially normalized the voltage-dependent inactivation of the mutant channel, dampened resurgent current, and exhibited potent frequency-dependent block of the mutant channel. Therefore, our study demonstrates concordance of in vitro pharmacology using heterologous cells with the drug response observed clinically for the affected child.…”

“…and S4 helices). Because SCN2A variants associated with early onset epilepsy may exhibit more severe functional consequences in the splice variant expressed preferentially during early development, 13 we tested whether the substitution of the neonatal exon would affect the properties of the M1879T mutant channel. In this case, none of the key functional properties (Supplemental Figure S1) were significantly different between the adult and neonatal isoforms of the M1879T channel.…”

The neonatal SCN2A mutant channel mimics adult channel properties

“…However, the evidence that the mutations are indeed pathogenic or how they affect the protein biology and/or neuronal network has not always come hand-in-hand with the genetic discoveries. In a recent issue of JGP , Thompson et al addressed this issue by determining the electrophysiological and neuronal effects of human mutations occurring in the voltage-activated Na V 1.2 ( SCN2A ) sodium channel ( Thompson et al, 2020 ). They examined the effects of the mutations on the function of the channel itself and integrated these results into the context of neuronal firing or activity.…”

“… Thompson et al (2020) examined the biophysical and cell functional ramifications of five mutants identified in patients with OS ( Nakamura et al, 2013 ) or EIMFS ( Howell et al, 2015 ), two of the most severe forms of EOEE. The mutants were expressed in the tsA201 heterologous expression system along with the β1 ( SCN1B ) and β2 ( SCN2B ) regulatory subunits, and channel electrophysiology studied using the whole-cell voltage clamp technique.…”

“… Thompson et al (2020) observed that some effects of the mutations were only manifest in the neonatal isoform. The twofold increase in sodium current of the neonatal T236S mutant compared with its wild type was no longer observed when the same comparison was made in the adult isoform.…”