Background

Pathogenic genomic variations in SCN8A can cause a spectrum of neurological phenotypes characterized by developmental delay, early onset multivariate seizure types, intractable epilepsy, movement disorders and other neurological manifestations [1,2,3]. Psychomotor development varies from normal to abnormal from birth. Normal development may precede subsequent delay or regression following seizure onset. Variable degrees of intellectual disability is seen with ~ 50% having a severe form. Behavioral abnormalities are also seen in some individuals.

The expression of voltage-gated sodium channels (NaVs) is key for initiation and conduction of action potentials in excitable cells such as skeletal muscle and neurons [4]. Neurons typically express multiple NaV isoforms. Loss-of-function (LoF) and gain-of-function (GoF) of voltage-gated sodium channels can lead to a wide spectrum of phenotypes. SCN8A (NaV1.6; OMIM 600702) is one of nine human genes encoding voltage-gated sodium channel α-subunits more recently implicated in epilepsy [5]. SCN8A variants in patients with epilepsy primarily result in GoF in Nav1.6 and hyperexcitability of neurons in the central nervous system [6]. Evaluation of the phenotype and genotype spectrum in SCN8A-related disorders suggests that GoF mutations are associated with severe epileptic encephalopathy, while LoF mutations cause intellectual disability with or without seizures. Sodium channel-blocking agents are effective on different levels in the treatment of seizures in GoF mutations. Anti-sense oligonucleotide therapy is in clinical trials for GoF variants and several treatment modalities are being explored in research including transfected cell lines and mouse models [7]. Targeted and genome-wide next-generation sequencing (NGS) has significantly increased the number of families identified with SCN8A-related disorders, allowing scientists to prioritize functional studies and develop a better understanding of the phenotypic spectrum [3].

In this case series, we would like to add to the growing clinical and genetic data of over 500 individuals with SCN8A-related disorders by reporting 8 affected individuals with variable phenotypes including one family with a previously published variant associated with treatable epilepsy, as well as, novel variants in SCN8A identified by exome sequencing. We establish functional evidence for a LoF SCN8A variant by using electrophysiological analyses in a patient with intellectual disability, autism spectrum disorder, and abnormal EEG. The patient also presented a co-occurring variant of unknown significance in KCNQ3.

Methods

Six families seen at neurology clinic, British Columbia Children’s Hospital were enrolled in the study. Exome sequencing was performed on the probands. Informed consent was obtained for the use of clinical and research findings for publication. The study has the approval from Institutional Ethics Committee (protocol number H14-01531). Clinical and molecular details of patients are summarized in Table 1. Detailed case description can be found in the Additional file 1.

Table 1 Clinical and molecular features of subjects with SCN8A variations
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Exome sequencing

Exome sequencing was performed in all the families. Detailed methodology and steps followed for exome sequencing wet lab and data analysis has been previously described [8]. Sanger sequencing to validate the variants and to determine the segregation in the families was performed [9].

Functional validation of SCN8A

The functional consequence of the SCN8A, c.971G>A (p.Cys324Tyr) variant was examined in vitro by heterologous protein expression in Human Embryonic Kidney cells (HEK-293). The electrophysiological properties of the HEK-293 cells expressing the p.Cys324Tyr protein were compared to control cells expressing either the wild-type protein or empty expression vector. Functional studies were not performed for the KCNQ3 variant in Patient 4.

Results

We studied eight patients from six families (males = 3, females = 5) with SCN8A heterozygous mutations. The phenotype ranged from DEE (n = 2), treatment responsive (n = 5) and an unclassified epilepsy phenotype, with possible clinical seizures in Patient 4. The age of seizure onset ranged from 3 months to 10 years. Individuals with DEE and an unclassified epilepsy phenotype presented with profound to severe intellectual disability and severe global developmental delay. Individuals with treatment responsive epilepsy were intellectually and developmentally within normal limits. Patient 4 had GDD and autism as a primary clinical phenotype with an abnormal EEG and possible clinical seizures. Treatment with valproic acid had improved EEG characteristics in the past. Four of them are seizure-free on monotherapy of carbamazepine and one with topiramate and clobazam. Exome sequencing identified three known and three novel heterozygous missense variations in SCN8A. Patient 4 also had a heterozygous, de novo, missense VUS in KCNQ3. Functionally, we observed a LoF, two GoF and three unclassified SCN8A variants. Electrophysiological analyses of the SCN8A variant in transfected cells revealed a LoF effect in Patient 4 (Fig. 1.).

Fig. 1
figure 1

A. Simplified diagram of NaV1.6 channel showing the locations of the variants identified in our cohort (novel mutations are in red font). B. HEK-293 cells were transiently transfected with hNaV1.6 WT, hNaV1.6 C324Y, plasmid vector with no channel construct to look for functional effects of C324Y variant. C324Y peak current density (pA/pF) levels were significantly different from WT but not from Vector control

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Discussion

SCN8A variants typically result in a moderate-severe epileptic encephalopathy, and account for 1% of the childhood epileptic encephalopathies [1]. The median age of seizures onset is typically 5 months (range: postnatal day 1 to 18 months of age) with multiple seizure types. The majority of affected patients have mild to severe global developmental delay. Abnormal tone, and abnormal movements may also be present [10]. In our cohort of eight individuals from six families with SCN8A-related disorders, we observed an age of onset ranging from 3 months to 10 years with severe to no clinical seizures. Developmental outcomes varied from profound developmental delay with intellectual disability and behavioural abnormalities to normal development. Developmental delay and age of onset of seizures did not seem to have a correlation in our cohort [11]. The seizure semiology in SCN8A-related disorders is variable, including focal seizures, tonic-clonic seizures, epileptic spasms, clonic seizures, absence, and myoclonic seizures [10, 12]. Patients with SCN8A mutations also have a high incidence of Sudden Unexpected Death in Epilepsy (SUDEP) [13, 14]. We noted a seizure course ranging from self-resolving focal seizures to Lennox-Gastaut syndrome (LGS) manifesting impaired awareness seizures, atypical absence seizures, generalized tonic-clonic seizures, epileptic spasms, and non-convulsive status epilepticus. The most common seizure type has been focal seizures as observed in the earlier reported patients [15].

The three novel variants are missense substitutions located on highly conserved transmembrane domains 1 and 2 of NaV1.6 (Fig. 1.). SCN8A gene variants causing substitution of amino acid residues in the highly conserved regions are often deleterious [1]. Three variants (those of Patient 2 [16, 17], Patient 3 [18], and Patient 4 [19]) were described previously. The clinical features of patient 2, and 3 were similar to what was previously described. Patient 4’s variant although published did not have phenotype information available for comparison. Variants in Patient 5 and Patient 6 have been submitted to ClinVar [20] without any detailed phenotype descriptions. It is important to note that individual differences in clinical manifestations can occur even with the same genetic variation.

LoF variants include an early stop-gain, indel frameshift or splice-site disruption resulting in truncated protein and reduced or abolished NaV1.6 function [21]. Missense changes causing GoF is the most common pathogenic mechanism for neuronal hyperexcitability and seizures. LoF is associated with cognitive impairment, movement disorders, and autism with or without seizures [22]. The clinical manifestations of SCN8A encephalopathy are likely reliant on the degree of GoF or LoF [23, 24]. GoF phenotypes include mild to severe epileptic encephalopathy. There are a few reported cases of benign or treatment-responsive infantile seizures with mild gain of function too [25]. We identified two GoF and a LoF variant with experimental evidence and three variations with unknown functional consequences. The electrophysiological analyses performed on Patient 4, LoF SCN8A variant (p.Cys324Tyr), offer valuable insights into the pathogenesis of SCN8A-related disorders. By characterizing the functional consequences of this variant, we provide evidence supporting its role in altering neuronal excitability and ion channel function. This information could potentially inform the development of targeted therapeutic strategies aimed at modulating ion channel activity to alleviate symptoms and improve patient outcomes.

In terms of the KCNQ3 variant in Patient 4, this variant was found to be a conserved amino acid and all in-silico analyses suggest the variant has a deleterious impact; however, the variant is novel and remains a variant of uncertain significance. Functional validation has not been performed. Pathogenic variations in KCNQ3 have been associated with benign or self-limited familial neonatal and infantile seizures (OMIM 121201) [26, 27]. Individuals are typically normal and grow out of their seizures, usually without any neurological sequalae in adulthood. More recently KCNQ3 mutations are identified in patients with neurodevelopmental disorders and abnormal EEG [28]. Furthermore, alterations in this gene have been reported to act as risk factors for complex diseases including other epilepsy types and autism spectrum disorder. Sands et al. delineated an electroclinical phenotype in 11 patients with 4 different heterozygous KCNQ3 GoF variants. Most of them did not have clinical seizures [28]. Patient 4 had EEG abnormalities with only possible clinical seizures which could plausibly be due to complex underlying molecular mechanisms involving KCNQ3 and SCN8A.

Many early onset neurological diseases are now known to have a molecular basis. A genetic diagnosis can have strong implications for prognosis and treatment of epilepsy [29]. Assessments of how often a genetic diagnosis has clinically actionable implications vary from 20 to 60% [30, 31]. These comparisons highlight the variability in clinical presentations, epilepsy diagnoses, and genetic diagnoses among the patients with SCN8A pathogenic variations.

Intellectual disability, epilepsy, behavioral abnormalities, and movement disorders belong to a complex set of conditions with both monogenic and multifactorial etiologies. Clinical overlap between heterogeneous phenotypes, pleiotropy, variable penetrance, and expressivity makes genetic testing a huge challenge in these families. We describe a cohort of SCN8A-related disorders in this research work. The results of this study contribute to expanding the clinical and genotypic spectrum of SCN8A-related disorders. By identifying three novel variants in SCN8A, we have enhanced our understanding of the genetic landscape associated with these disorders. The observed variability in clinical presentation further emphasizes the complex nature of SCN8A-related disorders and highlights the need for personalized approaches to diagnosis, treatment, and genetic counseling. The functional data for p.Cys324Tyr confirms causation in SCN8A-related disorders.

Conclusions

In conclusion, our study adds to the clinical and genotypic spectrum of SCN8A-related disorders by identifying novel variants and characterizing the functional consequence of p.Cys324Tyr. These findings underscore the importance of genetic testing in the diagnosis and management of individuals with SCN8A-related disorders. The mechanistic insights gained from this study may guide the development of targeted therapeutic interventions to improve patient care and outcomes in this heterogeneous group of disorders.