SCN5A Research Articles

Generation of iPSC line (FMCPGHi003-A) from human PBMCs of a patient with Familial hemiplegic migraine type 3

Peripheral blood mononuclear cells (PBMCs) were obtained from a patient diagnosed with Familial Hemiplegic Migraine Type 3, who carried a heterozygous A > C mutation in the SCN1A gene and reprogrammed using CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit. The iPSC line maintained the mutation while expressing markers of pluripotency. Additionally, it exhibited a normal karyotype and demonstrated potential for in vitro differentiation into cells representing all three embryonic germ layers.

Analysis of the role of Na<sub>v</sub>1.5 slow inactivation in the development of inherited cardiac pathology

Voltage-gated cardiac sodium channels Nav1.5 are responsible for the initiation and propagation of action potentials in cardiomyocytes. Dysfunction of Nav1.5 can be caused both by pathogenic variants in the SCN5A gene itself, which encodes Nav1.5, and by genetic variants in the genes of other proteins, regulating channel activity and trafficking. The change of different phases of the action potential is determined by the strict temporal organization of activation and inactivation of various ion channels. Transitions between channel functional states (for example, to slow inactivated state) can be influenced by various factors and proteins interacting with the channel. Despite the fact that the process of slow inactivation of the channel has been known for several decades, its role in the mechanism of development of hereditary heart pathology remains unclear. In this work, using the patch clamp method in whole-cell leads, we studied changes in the process of slow Nav1.5 inactivation under the influence of various mutations in structural genes (DSP-H1684R, LMNA-R249Q, FLNC-R1267Q, FLNC-V2264M) associated with a genetically determined myocardial pathology leading to dysfunction of cardiomyocytes. The study used a model of cardiomyocytes differentiated from induced pluripotent stem cells (СM-iPSCs). We have demonstrated an increase in slow inactivation in the model of CM-iPSCs obtained from patients with a phenotype of cardiomyopathy combined with ventricular arrhythmias. Thus, this work contributes to understanding the role of the slow inactivation process in the mechanism of the development of heart pathology.

Effect of SCN1Aand SCN2A gene polymorphisms on the efficacy of valproic acid treatment in Chinese children with epilepsy.

Epilepsy patients exhibit considerable differences in their response to sodium valproate (VPA) therapy, a phenomenon that might be attributed to individual genetic variances. The role of genetic variations, specifically in sodium channels encoded by SCN1A and SCN2A genes, in influencing the effectiveness of VPA in treating epilepsy is still debated. This research focuses on examining the impact of these genetic polymorphisms on the efficacy of VPA therapy among pediatric epilepsy patients in China. Five single nucleotide polymorphisms (SNPs), including SCN1A (rs10188577, rs2298771, rs3812718) and SCN2A (rs2304016, rs17183814), were genotyped in 233 epilepsy patients undergoing VPA therapy. The associations between genotypes and the antiepileptic effects of VPA were assessed, with 128 patients categorized as VPA responders and 105 as VPA non-responders. In the context of VPA monotherapy, SCN1A rs2298771 and SCN2A rs17183814 were found to be significantly associated with VPA response (P< 0.05). Our study suggests the findings of this investigation indicate that the polymorphisms SCN1A rs2298771 and SCN2A rs17183814 could potentially act as predictive biomarkers for the responsiveness to VPA among Chinese epilepsy patients.

NaV1.8 as Proarrhythmic Target in a Ventricular Cardiac Stem Cell Model.

The sodium channel NaV1.8, encoded by the SCN10A gene, has recently emerged as a potential regulator of cardiac electrophysiology. We have previously shown that NaV1.8 contributes to arrhythmogenesis by inducing a persistent Na+ current (late Na+ current, INaL) in human atrial and ventricular cardiomyocytes (CM). We now aim to further investigate the contribution of NaV1.8 to human ventricular arrhythmogenesis at the CM-specific level using pharmacological inhibition as well as a genetic knockout (KO) of SCN10A in induced pluripotent stem cell CM (iPSC-CM). In functional voltage-clamp experiments, we demonstrate that INaL was significantly reduced in ventricular SCN10A-KO iPSC-CM and in control CM after a specific pharmacological inhibition of NaV1.8. In contrast, we did not find any effects on ventricular APD90. The frequency of spontaneous sarcoplasmic reticulum Ca2+ sparks and waves were reduced in SCN10A-KO iPSC-CM and control cells following the pharmacological inhibition of NaV1.8. We further analyzed potential triggers of arrhythmias and found reduced delayed afterdepolarizations (DAD) in SCN10A-KO iPSC-CM and after the specific inhibition of NaV1.8 in control cells. In conclusion, we show that NaV1.8-induced INaL primarily impacts arrhythmogenesis at a subcellular level, with minimal effects on systolic cellular Ca2+ release. The inhibition or knockout of NaV1.8 diminishes proarrhythmic triggers in ventricular CM. In conjunction with our previously published results, this work confirms NaV1.8 as a proarrhythmic target that may be useful in an anti-arrhythmic therapeutic strategy.

Dravet Syndrome: An Electroclinical, Genetic, Treatment, and Outcome Study of 35 Patients in Argentina

AbstractWe analyzed the electroclinical features, molecular findings, treatment, disease course, and outcomes of patients with Dravet syndrome (DS) with positive genetic markers seen at a public hospital in Argentina. A retrospective study was conducted assessing the clinical records of 44 patients who met the diagnostic criteria for DS according to the 2022 classification of epilepsy of the International League Against Epilepsy seen at our center between March 2018 and June 2023. Of 44 patients, 35 (18 males and 17 females), in whom genetic studies yielded positive results, were included. Median age was 9 years (range 4 to 16 years), and the median time of follow-up was 10 years (range 3 to 14 years). The mean age at onset was 7 months. The first seizure was associated with febrile illness in all patients, and in 11 (31.4%), seizures were immediately preceded by either infectious disease or vaccination. Heterozygous pathogenic/likely pathogenic SCN1A variants were detected in 32 of the original 44 patients (73%), of which 47% were novel. Variants in other genes related to DS (HCN1, STXB1, and SCN1B) were identified in three patients. Cognitive delay and motor impairment were found to be more severe in patients that had multiple and drug-resistant seizures and in those who had the complete phenotype with myoclonic seizures. Novel SCN1A gene variants were identified in nearly half of the patients. The prognosis for cognitive development is unfavorable. Seizures are not well controlled with antiseizure medications and early treatment with ketogenic dietary therapy as well as cannabidiol should be considered.

Single-Base Gene Variants in MIR-146A and SCN1A Genes Related to the Epileptogenic Process in Drug-Responsive and Drug-Resistant Temporal Lobe Epilepsy-A Preliminary Study in a Brazilian Cohort Sample.

The drug-resistant temporal lobe epilepsy (TLE) has recently been associated with single nucleotide variants (SNVs) in microRNA(miR)-146a (MIR-146A) (rs2910164) and Sodium Voltage-Gated Channel Alpha Subunit 1 (SCN1A) (rs2298771 and rs3812718) genes. Moreover, no studies have shown an association between these SNVs and susceptibility to drug-resistant and drug-responsive TLE in Brazil. Thus, deoxyribonucleic acid (DNA) samples from 120 patients with TLE (55 drug-responsive and 65 drug-resistant) were evaluated by real-time polymerase chain reaction (RT-PCR). A total of 1171 healthy blood donor individuals from the Online Archive of Brazilian Mutations (ABraOM, from Portuguese Arquivo Brasileiro On-line de Mutações), a repository containing genomic variants of the Brazilian population, were added as a control population for the studied SNVs. MIR-146A and SCN1A relative expression was performed by quantitative RT-PCR (qRT-PCR). The statistical analysis protocol was performed using an alpha error of 0.05. TLE patient samples and ABraOM control samples were in Hardy-Weinberg equilibrium for all studied SNVs. For rs2910164, the frequencies of the homozygous genotype (CC) (15.00% vs. 9.65%) and C allele (37.80% vs. 29.97%) were superior in patients with TLE compared to controls with a higher risk for TLE disease [odds ratio (OR) = 1.89 (95% confidence interval (95%CI) = 1.06-3.37); OR = 1.38 (95%CI = 1.04-1.82), respectively]. Drug-responsive patients also presented higher frequencies of the CC genotype [21.81% vs. 9.65%; OR = 2.58 (95%CI = 1.25-5.30)] and C allele [39.09% vs. 29.97%; OR = 1.50 (95%CI = 1.01-2.22)] compared to controls. For rs2298771, the frequency of the heterozygous genotype (AG) (51.67% vs. 40.40%) was superior in patients with TLE compared to controls with a higher risk for TLE disease [OR = 2.42 (95%CI = 1.08-5.41)]. Drug-resistant patients presented a higher AG frequency [56.92% vs. 40.40%; OR = 3.36 (95%CI = 1.04-17.30)] compared to the control group. For rs3812718, the prevalence of genotypes and alleles were similar in both studied groups. The MIR-146A relative expression level was lower in drug-resistant compared to drug-responsive patients for GC (1.6 vs. 0.1, p-value = 0.049) and CC (1.8 vs. 0.6, p-value = 0.039). Also, the SCN1A relative expression levels in samples from TLE patients were significantly higher in AG [2.09 vs. 1.10, p-value = 0.038] and GG (3.19 vs. 1.10, p-value < 0.001) compared to the AA genotype. In conclusion, the rs2910164-CC and rs2298771-AG genotypes are exerting significant risk influence, respectively, on responsive disease and resistant disease, probably due to an upregulated nuclear factor kappa B (NF-kB) and SCN1A loss of function.

Impacts of DCM-linked gating pore currents on the electrophysiological characteristics of hiPSC-CM monolayers

BackgroundVariants of the SCN5A gene, which encodes the NaV1.5 cardiac sodium channel, have been linked to arrhythmic disorders associated with dilated cardiomyopathy (DCM). However, the precise pathological mechanisms remain elusive. The present study aimed to elucidate the pathophysiological consequences of the DCM-linked Nav1.5/R219H variant, which is known to generate a gating pore current, using patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in monolayers. MethodsVentricular- and atrial-like hiPSC-CM monolayers were generated from DCM patients carrying the R219H SCN5A variant as well as from healthy control individuals. CRISPR-corrected hiPSC-CMs served as isogenic controls. Simultaneous optical mapping of action potentials (APs) and calcium transients (CaTs) was employed to measure conduction velocities (CVs) and AP durations (APDs) and served as markers of electrical excitability. Calcium handling was evaluated by assessing CaT uptake (half-time to peak), recapture (tau of decay), and durations (TD50 and TD80). A multi-electrode array (MEA) analysis was conducted on hiPSC-CM monolayers to measure field potential (FP) parameters, including corrected Fridericia FP durations (FPDc). ResultsOur results revealed that CVs were significantly reduced by more than 50 % in both ventricular- and atrial-like hiPSC-CM monolayers carrying the R219H variant compared to the control group. APDs were also prolonged in the R219H group compared to the control and CRISPR-corrected groups. CaT uptake, reuptake, and duration were also markedly delayed in the R219H group compared to the control and CRISPR-corrected groups in both the ventricular- and the atrial-like hiPSC-CM monolayers. Lastly, the MEA data revealed a notably prolonged FPDc in the ventricular- and atrial-like hiPSC-CMs carrying the R219H variant compared to the control and isogenic control groups. ConclusionsThese findings highlight the impact of the gating pore current on AP propagation and calcium homeostasis within a functional syncytium environment and offer valuable insights into the potential mechanisms underlying DCM pathophysiology.

Clinical risk factors associated with cardiac events in children with BrS-SCN5A

Abstract Introduction between 20-30% of patients with Brugada syndrome (BrS) are carriers of deleterious genetic variants in the SCN5A gene. However, the risk factors for cardiac events in these patients remain under consideration, especially in the paediatric population, with many risk factors being still undetermined. Objectives to investigate and evaluate individual clinical risk factors associated with cardiac events in children with BrS-SCN5A. Methods a Spanish retrospective single-centre paediatric cohort study was conducted including 41 BrS patients under 21 years of age carrying rare variants in the SCN5A gene. Demographic, clinical, genetic, and therapeutic variables were collected. Descriptive and univariate analyses were performed to identify independent risk factors for cardiac events. Outcomes were defined as cardiac syncope, ventricular tachycardia/fibrillation (VT/VF), sudden cardiac death (SCD), and appropriate shock in ICD. Results Nearly 20% of BrS-SCN5A patients presented with cardiac events. Five patients showing cardiogenic syncope, 4-SCD, 3-VT and 2 appropriate shocks in the ICD. The presence of bradycardia and bradyarrhythmia in baseline EKG (P&amp;lt;0.05), AV block type 1 (P&amp;lt;0.02), type 1 Brugada pattern in fever EKG (P&amp;lt;0.02), atrial fibrillation(P&amp;lt;0.05), arrhythmias in febrile episodes (P&amp;lt;0.05), progressive cardiac conduction defect (PCCD; P&amp;lt;0.001), sick sinus syndrome (SSS; P=0.001) and overlap phenotype (P&amp;lt;0.001) were predictor of cardiac events in this cohort. Conclusions We have identified several statistically significant independent risk factors associated with cardiac events and worse phenotype severity in BrS-SCN5A children from a single-centre Spanish cohort. These results may help to improve prognosis assessment, establish accurate risk stratification, and prevent SCD in children with BrS-SCN5A. Future studies in large cohorts of different ethnicities are crucial to strengthen our risk factors in paediatric cohorts at high risk of malignant arrhythmias.

A Novel Presentation: Epsilon Waves in the Electrocardiogram of a Patient with Dravet Syndrome Without Structural Heart Disease

We document for the first time epsilon waves on the electrocardiogram of a patient with Dravet syndrome (DS) who does not exhibit structural cardiac pathology. DS, a severe form of pediatric epilepsy, involves mutations in the SCN1A gene, which disrupt sodium ion flow and increase the risk of sudden unexpected death in epilepsy. The epsilon waves, typically associated with arrhythmogenic right ventricular dysplasia, may also suggest a link between neuronal and cardiac dysfunctions. In our case, the epsilon waves appear to originate from abnormalities in sodium channels rather than structural changes in the myocardium. This finding underscores the importance of assessing both neurological and cardiac manifestations in patients with severe epilepsy, opening new avenues for the research and treatment of these interactions in DS.

First description of a mixed Brugada pattern in inferior leads

Brugada Syndrome is a cardiac channelopathy closely related to lethal arrhythmias and sudden death in people without underlying structural pathology. The diagnosis is usually assumed with electrocardiographic patterns strictly limited to the right precordial leads, V1 or V2; but this is not so at all. In our article, we present the clinical case of a patient with a novel and different electrocardiographic presentation: the typical diagnostic pattern was presented in a lower lead of the electrocardiogram, and next to it, another pattern was presented in a contiguous lead. In addition, we propose the possible association of a structural pathology (non-compaction cardiomyopathy) with Brugada Syndrome, since both share a possible common genetic origin in the SCN5A gene.

Current real-world evidence of genetic testing diagnostic yield in a large cohort of long QT syndrome

Abstract Background Diagnostic yield in long QT syndrome (LQTS) ranges between 30-70%, depending on the studied population. LQTS can lead to an unexpected sudden cardiac death. For this reason and due to its wider current availability, genetic testing in patients with suspicion of LQTS has spread. Purpose The aim of this study is to investigate the genetic testing yield among a large cohort of heterogeneous index cases with LQTS phenotype. Methods Diagnostic yield was restrospectively evaluated in a cohort of index cases referred to our laboratory for genetic study with LQTS phenotype. Genetic study was performed with customized libraries through next generation sequencing (NGS), which included copy number variations (CNVs) analysis. Patients with LQTS phenotype studied both with "long QT panels" and with broader cardiovascular panels were included, but those with additional cardiac phenotype (cardiomyopathies, heart defects or catecholaminergic polymorphic ventricular tachycardia) were excluded. Positive genetic reports were considered when a likely pathogenic (LP) or pathogenic (P) variant could explain patient´s phenotype. Inconclusive reports were described when a variant considered risk factor (RF) or a variant of uncertain significance variant (VUS) or a hot-VUS was identified in a clinically relevant gene. Results 1183 index cases were genotyped for LQTS phenotype. Only 164 index cases (13,8%) were studied with broader panels which included between 77 and 368 genes. The rest (n=1019) were studied with LQTS panels (either with small panels which included KCNQ1, KCNH2, KCNE1, KCNE2, SCN5A, KCNJ2, CACNA1C and with up to 36 genes-QT panels). Around 27% (n=321) of the index cases had a positive result. They carried LP/P variants in the following genes: KCNQ1 (n=166, 52%), KCNH2 (n=112), SCN5A (n=25), RYR2 (n=5), CACNA1C (n=3), HCN4 (n=4), KCNJ2 (n=2), KCNE2 (n=2) and CALM2 (n=1). 53,3% of the reports were negative. However, there were some incidental findings: 2 index cases carried P variants in LDLR, 2 carried P variants in PKP2, 2 carried a P variant in MYBPC3 and 1 index case each carried a P variant in DES, TTN and DSG2. From the 231 inconclusive reports, 37 index cases carried the RF variant in KCNE1 (p.Asp85Asn), 2 carried the RF variant in SCN5A (p.Arg1193Gln) and 50 patients carried hot-VUS in KCNQ1, KCNH2, CACNA1C, SCN5a or RYR2 gene. Conclusions In our large cohort of index cases referred for genetic testing for LQTS, diagnostic yield was around 27% when considering only LP/P variants in LQTS or arrhythmic phenotype related genes. Even by including the inconclusive but potentially relevant genetic results (RF and hot VUS), the diagnostic yield in our cohort reaches 35%. This is lower than expected for LQTS cohort. It should be taken into consideration that our cohort consists of heterogeneous patients referred for LQTS genetic testing, which would probably reflects better the real world patients seen in every day practice.

Knockdown of SCN5A alters metabolic-associated genes and aggravates hypertrophy in the cardiomyoblast.

SCN5A mutations have been reported to cause various cardiomyopathies in humans. Most of the SCN5A mutations causes loss of function and thereby, alters the overall cellular function. Therefore, to understand the loss of SCN5A function in cardiomyocytes, we have knocked down the SCN5A gene (SCN5A-KD) in H9c2 cells and explored the cell phenotype and molecular behaviors in the presence and absence of isoproterenol (ISO), an adrenergic receptor agonist that induces cardiac hypertrophy. Expression of several genes related to hypertrophy, inflammation, fibrosis, and energy metabolism pathways were evaluated. It was found that the mRNA expression of hypertrophy-related gene, brain (B-type) natriuretic peptide (BNP) was significantly increased in SCN5A-KD cells as compared to 'control' H9c2 cells. There was a further increase in the mRNA expressions of BNP and βMHC in SCN5A-KD cells after ISO treatment compared to their respective controls. Pro-inflammatory cytokine, tumor necrosis factor-alpha expression was significantly increased in 'SCN5A-KD' H9c2 cells. Further, metabolism-related genes like glucose transporter type 4, cluster of differentiation 36, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor-gamma were significantly elevated in the SCN5A-KD cells as compared to the control cells. Upregulation of these metabolic genes is associated with increased ATP production. The study revealed that SCN5A knock-down causes alteration of gene expression related to cardiac hypertrophy, inflammation, and energy metabolism pathways, which may promote cardiac remodelling and cardiomyopathy.

Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2—8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

Prediction models of persistent taxane-induced peripheral neuropathy among breast cancer survivors using whole-exome sequencing

Persistent taxane-induced peripheral neuropathy (TIPN) is highly prevalent among early-stage breast cancer survivors (ESBCS) and has detrimental effect on quality of life. We leveraged logistic regression models to develop and validate polygenic prediction models to estimate the risk of persistent PN symptoms in a training cohort and validation cohort taking clinical risk factors into account. Based on 337 whole-exome sequenced ESBCS two of five prediction models for individual PN symptoms obtained AUC results above 60% when validated. Using the model for numbness in feet (35 SNVs) in the test cohort, 73% survivors were correctly predicted. For tingling in feet (55 SNVs) 70% were correctly predicted. Both models included SNVs from the ADAMTS20, APT6V0A2, CCDC88C, CYP2C8, EPHA5, NR1H3, PSKH2/APTV0D2, and SCN10A genes. For cramps in feet, difficulty climbing stairs and difficulty opening a jar the validation was unsuccessful. Polygenic prediction models including clinical risk factors can estimate the risk of persistent taxane-induced numbness in feet and tingling in feet in ESBCS.

IMPLANTABLE LOOP RECORDER IN BRUGADA SYNDROME: INSIGHTS FROM A SINGLE–CENTER EXPERIENCE

Abstract Brugada syndrome (BrS) is characterized by a “coved” ST segment elevation of ≥2 mm in the right precordial electrocardiographic (ECG) leads, either spontaneously or drug–induced. Risk stratification in individuals with BrS remains challenging. Currently, miniaturized and remotely monitored implantable loop recorders (ILRs) are available, providing physicians with a diagnostic tool that can help guide intervention or therapy decisions for these patients. The objective of this study was to investigate the characteristics and outcomes of patients diagnosed with BrS who underwent ICM insertion during routine clinical activity. This is a retrospective single center cohort study. We screened 147 consecutive patients who were diagnosed with BrS based on the presence of spontaneous or drug–induced type 1 ECG pattern between January 2009 and May 2023. Once this comprehensive diagnostic pathway was completed, we engaged in detailed counseling with patients to discuss the potential need for ICD implantation or ILR insertion, if deemed indicated. Compared to patients who did not receive a device, those who received an ILR had a higher prevalence of suspected arrhythmic syncope (43% vs. 22%, p=0.012) and tended to be younger (median age 38 years, IQR 30–52, vs. 43 years, IQR 35–55, p=0.044) and with a higher presence of SCN5A gene mutations (17% vs. 6%). On the other hand, compared to patients who received an ICD, those with an ICM underwent programmed ventricular stimulation less frequently (40% vs. 85%, p=0.010) and, as a result, had a lower frequency of positive result of this test (0% vs. 91%, p&amp;lt; 0.001). During a median follow–up period of 14.7 months (IQR, 4.7–44.8), no deaths occurred among the patients with ILR. Eight individuals (19%) were diagnosed with arrhythmic findings through continuous ILR monitoring. Among these findings, three patients experienced AF (including one case of new–onset AF), two patients exhibited symptomatic asystolic pauses lasting 16 seconds and 3.6 seconds respectively, two patients had non–sustained ventricular tachycardia, and one patient had episodes of paroxysmal supraventricular tachycardia. Except for a history of AF, no significant differences were observed between patients with arrhythmic findings and those without any events. In the ICD group, the median follow–up period was 32.2 months (IQR, 23.2–95.0). During this period, no deaths occurred and no appropriate or inappropriate ICD therapies were delivered.

Prevention of Drug Resistant Epilepsy and Developmental Epileptic Encephalopathy: Preventative Vigabatrin Treatment in Tuberous Sclerosis Complex and the Case for Fenfluramine Treatment of Children with Newly Diagnosed Dravet Syndrome

Tuberous sclerosis complex (TSC) is caused by mutations of hamartin (TSC1) or tuberin (TSC2) resulting in disinhibition of the mTOR pathway of cellular proliferation and differentiation and severe neurocognitive impairment, intractable epilepsy and tumors. Epilepsy develops in ~90% folllowed by drug-resistant epilepsy (DRE). Recently, prevention of DRE and developmental encephalopathy was shown to be possible in TSC using early administration of vigabatrin. For the first time, medical treatment successfully prevented epilepsy and reduced neurocognitive and behavioural co-morbidities. The crucial difference between the preventive and standard treatment groups was the timing of treatment initiation, not the type of the intervention. This paradigm can be extended to patients with other genetic or acquired conditions, including Dravet Syndrome (DS), a severe developmental epileptic encephalopathy (DEE) with DRE and high mortality, due to a de novo mutation of the gene SCN1A which codes for the sodium channel protein &lt;em&gt;a&lt;/em&gt; subunit Na&lt;sub&gt;v&lt;/sub&gt;1.1. Infants usually develop normally until their first seizure, commonly between 2-15 months. This is followed by DRE and developmental regression. Randomized controlled trials (RCTs) of adjunctive fenfluramine treatment in DS reduced median convulsive seizure frequency by 55.7% over placebo at 0.7 mg/kg/day. 50% of children had 75% seizure frequency reduction, 8% had convulsive seizure freedom versus 0% with placebo, and 18% had only one convulsive seizure versus 0% with placebo. Other pivotal studies showed similar results and efficacy was sustained in for &lt;3 years. Fenfluramine is now a first-line therapy for DS. Given the existence of a remarkably effective treatment fenfluramine for Dravet, preventive therapy presents itself as a natural extension for its application. We hypothesize that not only may seizure outcomes such as time to second seizure and evolution to DRE be positively impacted, but moreover, protective effects on mortality and cognitive outcomes may also be seen.

SCN1A-Characterization of the Gene's Variants in the Polish Cohort of Patients with Dravet Syndrome: One Center Experience.

The aim of this study was to characterize the genotype and phenotype heterogeneity of patients with SCN1A gene mutations in the Polish population, fulfilling the criteria for the diagnosis of Dravet syndrome (DRVT). Particularly important was the analysis of the clinical course, the type of epileptic seizures and the co-occurrence of additional features such as intellectual disability, autism or neurological symptoms such as ataxia or gait disturbances. Based on their results and the available literature, the authors discuss potential predictors for DRVT. Identifying these early symptoms has important clinical significance, affecting the course and disease prognosis. 50 patients of the Pediatric Neurology Clinic of the Institute of Mother and Child in Warsaw clinically diagnosed with DRVT and carriers of SCN1A pathogenic variants were included. Clinical data were retrospectively collected from caregivers and available medical records. Patients in the study group did not differ significantly in parameters such as type of first seizure and typical epileptic seizures from those described in other studies. The age of onset of the first epileptic seizure was 2-9 months. The co-occurrence of intellectual disability was confirmed in 71% of patients and autism in 18%. The study did not show a correlation between genotype and phenotype, considering the severity of the disease course, clinical symptoms, response to treatment, the presence of intellectual disability, autism symptoms or ataxia. From the clinical course, a significant problem was the differentiation between complex febrile convulsions and symptoms of DRVT. The authors suggest that parameters such as the age of the first seizure, less than one year of age, the onset of a seizure up to 72 h after vaccination and the presence of more than two features of complex febrile seizures are more typical of DRVT, which should translate into adequate diagnostic and clinical management. The substantial decrease in the age of genetic verification of the diagnosis, as well as the decline in the use of sodium channel inhibitors, underscores the growing attention of pediatric neurologists in Poland to the diagnosis of DRVT.

Cardiac Sodium Channel Nav1.5 Arg222Gln Variant Induces a Sex-Specific Arrhythmia and Alters Mitochondrial Function

Introduction: Encoded by the SCN5A gene, cardiac Nav1.5 sodium channels initiate the action potential to trigger a heartbeat. As the predominant sodium channel in the heart, variants can have devastating consequences, leading to potentially lethal arrhythmias. With the gain of function arginine to glutamine (Arg222Gln) variant in Nav1.5, patients develop intermittent arrhythmias and dilated cardiomyopathy. The stress induced by chronic arrhythmias can trigger adverse metabolic reprogramming, exacerbating the underlying condition. We have generated a murine model harboring the human Arg222Gln variant where we observe that only males produce a clear observable arrhythmia. We hypothesize that the maladaptive metabolic response to the arrhythmia caused by the Arg222Gln variant maybe be a potential link to the sex differences. Methods: The human exon containing the Arg222Gln mutation was knocked into a murine model under the endogenous SCN5A promoter. Cellular metabolism (oxygen consumption) was measured using primary adult mouse cardiomyocytes (CMs) with the Seahorse XFe analyzer. Mitochondrial membrane potential was measured using ratiometric JC-1 dye and imaged on a scanning confocal microscope. Heart tissues were diced into 1mm3 cubes and fixed to perform serial block face electron microscopy to obtain 3D structure of mitochondria and mitochondrial morphology. Western blots were used to detect protein levels. Results: Basal respiration of CMs were unchanged, however the maximal respiratory capacity of Arg222Gln males was significantly lower (116.9±13.5pmol O2/min, p&lt;0.05) compared to WT males (407.3±16.4pmol O2/min). There were no differences between WT females (451.4±22.4pmol O2/min) and Arg222Gln females (463.2±33.7pmol O2/min). JC-1 dye revealed that the Arg222Gln males (0.6213±0.0172 red/green ratio, p&lt;0.001) have significantly lowered mitochondrial polarization compared to WT males (0.7550 ±0.0257). There were no differences between WT females (0.6236±0.0154) and Arg222Gln females (0.6466±0.0139). Arg222Gln males have reduced mitochondria area (1.155±0.052μm2, p&lt;0.05) compared to WT males (1.332±0.060μm2) and are more fragmented (p&lt;0.05). Electron transport chain complex protein levels and mitochondrial biomass measured by voltage dependent anion channel protein levels remain unchanged between WT males and Arg222Gln males (P&gt;0.05). The ratio of phosphorylated dynamin related (DRP) / total DRP protein levels were significantly higher in Arg222Gln males compared to WT males, indicative of mitochondrial fission (p&lt;0.05). Conclusion: We demonstrate evidence of a mitochondrial dysfunction in a murine model of arrhythmia caused by a gain of function Nav1.5 variant, adversely affecting overall cellular metabolism and mitochondrial quality. Our data suggests that the difference in metabolic reprogramming could play a role in the cardioprotection against arrhythmia in Arg222Gln females or explain why Arg222Gln males are severely affected by the arrhythmia. Future experiments will aim to elucidate how the difference in sex affects cardiac metabolism in the context of Arg222Gln Nav1.5 variant. This work was supported by a Canadian Institutes of Health Research Project Grant, the SickKids Foundation through the Curtis Joseph and Harold Groves Chair in Anesthesia and Pain Medicine (JTM) and the Department of Anesthesiology and Pain Medicine, University of Toronto through a Merit Award (JTM). TYTL is supported by the CIHR Canadian Graduate Scholarship — Doctoral Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A c.1775C > T Point Mutation of Sodium Channel Alfa Subunit Gene (SCN4A) in a Three-Generation Sardinian Family with Sodium Channel Myotonia.

The nondystrophic myotonias are rare muscle hyperexcitability disorders caused by gain-of-function mutations in the SCN4A gene or loss-of-function mutations in the CLCN1 gene. Clinically, they are characterized by myotonia, defined as delayed muscle relaxation after voluntary contraction, which leads to symptoms of muscle stiffness, pain, fatigue, and weakness. Diagnosis is based on history and examination findings, the presence of electrical myotonia on electromyography, and genetic confirmation. Next-generation sequencing including the CLCN1 and SCN4A genes was performed in patients with clinical neuromuscular disorders. Electromyography, Short Exercise Test, in vivo and in vitro electrophysiology, site-directed mutagenesis and heterologous expression were collected. A heterozygous point mutation (c.1775C > T, p.Thr592Ile) of muscle voltage-gated sodium channel α subunit gene (SCN4A) has been identified in five female patients over three generations, in a family with non-dystrophic myotonia. The muscle stiffness and myotonia involve mainly the face and hands, but also affect walking and running, appearing early after birth and presenting a clear cold sensitivity. Very hot temperatures, menstruation and pregnancy also exacerbate the symptoms; muscle pain and a warm-up phenomenon are variable features. Neither paralytic attacks nor post-exercise weakness has been reported. Muscle hypertrophy with cramp-like pain and increased stiffness developed during pregnancy. The symptoms were controlled with both mexiletine and acetazolamide. The Short Exercise Test after muscle cooling revealed two different patterns, with moderate absolute changes of compound muscle action potential amplitude. The p.Thr592Ile mutation in the SCN4A gene identified in this Sardinian family was responsible of clinical phenotype of myotonia.

Investigation of Some Ion Channel Expressions in Cochlear Nucleus of Tinnitus Induced Rats

Aim: The aim of this study is to gain a better understanding of how certain ion channels play a role in the molecular mechanisms of salicylate- and noise-induced tinnitus. Method: The present study was conducted on thirty-two, 4-month-old, male Wistar Albino rats. Rats were equally divided into four groups; two experimental groups and two control groups. The assessment of tinnitus was based on a behavioral test which was modified from the conditional suppression method. Tinnitus was induced by sodium salicylate administration and noise exposure in rats in which the suppression ratios were zero (0). All animals in both experimental and control groups were decapitated in deep anaesthesia for 2 h after salicylate or saline administration and noise exposure, consecutively. Tissues from the left and right cochlear nucleus were dissected immediately in ice-cold RNA later (Invitrogen). Before reverse transcription, the RNA pools were arranged. Quantitative changes in HCN1, HCN2, HCN4, SCN1A, SCN2A1, SCN3A, TRPM2, TRPM7 and GAPDH mRNA expressions in the cochlear nucleus in both experimental and control groups were examined by quantitative real-time PCR method. Statistical data were analysed using the SPSS 21 program (Version 21.0, SPSS Inc., Chicago, IL, USA) with the Kruskal-Wallis and Mann-Whitney U tests. Results: Fold changes in the expression levels of SCNA1, SCN2A1, SCN3A, TRPM2, TRPM7, CACNA1B, HCN1, HCN2 and HCN4 genes in both salicylate-induces tinnitus (SAT) and noise-induced tinnitus (NT) groups compared with the control group. According to these data, it is seen that the mRNA levels of all genes are lower in the cochlear nucleus area of the rats in both SAT and NT groups than in the control group. Considering each of these genes in NT group: SCNA1, SCN3A, TRPM7 genes slightly decreased; SCN2A1, TRPM2, HCN1 and HCN4 genes slightly increased compared with the SAT group. For HCN2 gene fold changes were nearly the same in the NT and SAT groups. Conclusion: The findings of this study suggest that tinnitus generation may be closely related to alterations in several key ion channel families activity including voltage-gated calcium channels, hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, transient receptor potential (TRP) channels, voltage-gated sodium channels within the CN, specifically in response to salicylate-induced and noise-induced tinnitus models.