Expanding the genotype-phenotype spectrum in SCN8A-related disorders

Background SCN8A-related disorders are a group of variable conditions caused by pathogenic variations in SCN8A. Online Mendelian Inheritance in Man (OMIM) terms them as developmental and epileptic encephalopathy 13, benign familial infantile seizures 5 or cognitive impairment with or without cerebellar ataxia. Methods In this study, we describe clinical and genetic results on eight individuals from six families with SCN8A pathogenic variants identified via exome sequencing. Results Clinical findings ranged from normal development with well-controlled epilepsy to significant developmental delay with treatment-resistant epilepsy. Three novel and three reported variants were observed in SCN8A. Electrophysiological analysis in transfected cells revealed a loss-of-function variant in Patient 4. Conclusions This work expands the clinical and genotypic spectrum of SCN8A-related disorders and provides electrophysiological results on a novel loss-of-function SCN8A variant.


Abstract
Background SCN8A-related disorders are a group of variable conditions caused by pathogenic variations in SCN8A. Online Mendelian Inheritance in Man (OMIM) terms them as developmental and epileptic encephalopathy 13, benign familial infantile seizures 5 or cognitive impairment with or without cerebellar ataxia.

Methods
In this study, we describe clinical and genetic results on eight individuals from six families with SCN8A pathogenic variants identi ed via exome sequencing.

Results
Clinical ndings ranged from normal development with well-controlled epilepsy to signi cant developmental delay with treatment-resistant epilepsy. Three novel and three reported variants were observed in SCN8A. Electrophysiological analysis in transfected cells revealed a loss-of-function variant in Patient 4.

Conclusions
This work expands the clinical and genotypic spectrum of SCN8A-related disorders and provides electrophysiological results on a novel loss-of-function SCN8A variant.

Background
Pathogenic genomic variations in SCN8A can cause a spectrum of neurological phenotype characterized by developmental delay, early onset multivariate seizure types, intractable epilepsy, movement disorders and other neurological manifestations.(1-3) Psychomotor development varies from normal to abnormal since 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 nextgeneration sequencing (NGS) has signi cantly increased the number of families identi ed with SCN8Arelated 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 (8, 9) 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 identi ed 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 signi cance 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 ndings 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 le 1.

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.

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 unclassi ed phenotype with possible clinical seizures in Patient 4. The age of seizure onset ranged from 3 months to 10 years.
Individuals with DEE and unknown phenotypes presented with profound to severe intellectual disability and severe global developmental delay. Individuals with treatment responsive phenotype were intellectually and developmentally within normal limits. Patient 4 had GDD and autism as a primary clinical phenotype with a characteristic EEG abnormality, with possible clinical seizures, treated with valproic acid which 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 identi ed 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 unclassi ed SCN8A variants. Electrophysiological analyses of the SCN8A variant in transfected cells revealed a LoF effect in Patient 4 ( Fig. 1.).

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 be present. (12) 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. (13) The seizure semiology in SCN8A-related disorders is variable, including focal seizures, tonic-clonic seizures, epileptic spasms, clonic seizures, absence, and myoclonic seizures. (12,14) Patients with SCN8A mutations also have a high incidence of Sudden Unexpected Death in Epilepsy (SUDEP). (15,16) We noted a seizure course ranging from self-resolving focal seizures to Lennox-Gastaut syndrome (LGS) manifesting impaired awareness seizures, atypical absence seizures, generalized tonicclonic seizures, epileptic spasms, and non-convulsive status epilepticus. The most common seizure type has been focal seizures as observed in the earlier reported patients. (17) The three novel mutations 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 mutations (those of Patient 2(18, 19), Patient 3 (20), and Patient 4(21)) were described previously. The clinical features of patient 2, and 3 were quite similar as the characterization of patients with each same mutation previously described. Patient 4's mutation although published did not have phenotype information for comparison. Variants in Patient 5 and Patient 6 have been submitted to ClinVar (22) 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. (23) 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. (24) The clinical manifestations of SCN8A encephalopathy are likely reliant on the degree of GoF or LoF. (25,26) GoF phenotypes include mild to severe epileptic encephalopathy. There are a few reported cases of benign infantile seizures with mild gain of function too. (27) We identi ed 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 deleterious impact; however, the variant is novel and remains a variant of uncertain signi cance. Functional validation has not been performed. Pathogenic variations in KCNQ3 have been associated with benign familial neonatal and infantile seizures (OMIM 121201). 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 con rms 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 ndings 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. Further research is needed to elucidate the precise mechanisms underlying SCN8A-related disorders and to identify potential therapeutic targets for intervention. Tables Table 1 is available in the Supplementary Files section.