Characterization of a de novo SCN8A mutation in a patient with epileptic encephalopathy

Abstract

Objective Recently, de novo SCN8A missense mutations have been identified as a rare dominant cause of epileptic encephalopathies. Functional studies on the first described case demonstrated gain-of-function effects of the mutation. We describe a novel de novo mutation of SCN8A in a patient with epileptic encephalopathy, and functional characterization of the mutant protein. Design Whole exome sequencing was used to discover the variant. We generated a mutant cDNA, transfected HEK293 cells, and performed Western blotting to assess protein stability. To study channel functional properties, patch-clamp experiments were carried out in transfected neuronal ND7/23 cells. Results The proband exhibited seizure onset at 6 months of age, diffuse brain atrophy, and more profound developmental impairment than the original case. The mutation p.Arg233Gly in the voltage sensing transmembrane segment D1S4 was present in the proband and absent in both parents. This mutation results in a temperature-sensitive reduction in protein expression as well as reduced sodium current amplitude and density and a relative increased response to a slow ramp stimulus, though this did not result in an absolute increased current at physiological temperatures. Conclusion The new de novo SCN8A mutation is clearly deleterious, resulting in an unstable protein with reduced channel activity. This differs from the gain-of-function attributes of the first SCN8A mutation in epileptic encephalopathy, pointing to heterogeneity of mechanisms. Since Nav1.6 is expressed in both excitatory and inhibitory neurons, a differential effect of a loss-of-function of Nav1.6 Arg223Gly on inhibitory interneurons may underlie the epilepsy phenotype in this patient.

Complex SCN8A DNA-abnormalities in an individual with therapy resistant absence epilepsy

“…One of the three functional analyses of SCN8A mutations in people with EE showed an unstable protein with reduced channel activity. The individual carrying this loss of function allele exhibited a severe phenotype with seizure onset at 6 months old, tonic clonic seizures, diffuse brain atrophy and profound developmental impairment (de Kovel et al, 2014).…”

Pathogenic mechanism of recurrent mutations of SCN8A in epileptic encephalopathy

“…A contrasting mechanism, premature channel activation, was observed for two mutations located in transmembrane segments of domain II, pThr767Ile and p.Asn984Lys 6, 9. The other two functionally characterized SCN8A mutations caused reduction in peak current density (p.Gly1451Ser)6 and decreased protein stability (p.Arg223Gly)7; the role of these effects in neuronal hyperexcitability is unclear.…”

“…Clinical features include seizure onset before 18 months of age, intellectual disability, and developmental delay 1, 2, 3, 4, 5. Movement disorders are common and 50% of affected individuals are nonambulatory 1, 3, 6, 7, 8, 9, 10, 11, 12. SUDEP (sudden unexpected death in epilepsy) is reported in 10% of cases 1, 2, 4, 5…”

Neuronal hyperexcitability in a mouse model of SCN8A epileptic encephalopathy

“…Ten of these mutations have been tested functionally in transfected cells, and eight were found to introduce changes in the biophysical properties of Na v 1.6 that are predicted to result in neuronal hyperexcitability, including elevated persistent sodium current (I Na,P ), hyperpolarizing shifts in the voltage dependence of activation, and impaired current inactivation (10,16,(18)(19)(20)(21). It is important to confirm these observations in multiple classes of neurons expressing the mutation at a constitutive level, because different types of neurons respond differently to mutations of Scn8a (1) and effects in brain may differ from those in transfected cells (e.g., ref.…”