SCN5A Mutation Research Articles

Sinus node dysfunction in children: different aetiologies, similar clinical course in two-centre experience.

This study aims to evaluate the clinical characteristics and outcomes of children diagnosed with sinus node dysfunction. This was a retrospective review of patients diagnosed with sinus node dysfunction in two tertiary paediatric cardiology centres in Turkey from January 2011 to June 2022. In all, 77 patients (50, 64.9% males) were included, with a mean age of 8.2 ± 6.3 years and a mean weight of 28.2 ± 18.8 kg. While age-incompatible bradycardia and pauses were the most common rhythm disturbances, syncope, presyncope, and dizziness (n:33, 43%) were the most frequent initial symptoms. Structural heart disease was present in 58 (75.3%) of the 77 patients, 47 (61%) of whom were congenital. The most commonly associated CHDs were transposition of the great arteries (n:8), atrial septal defect (n:7), and atrioventricular septal defect (n:5). Seven of them also had left atrial isomerism. The remaining 19 patients were isolated. Four patients had SCN5A mutation (two of them were siblings) and two of them had Emery-Dreifuss muscular dystrophy. Although sinus node dysfunction is rare in children, it has been diagnosed with increasing frequency with structural heart disease, especially in patients who have undergone corrective cardiac surgery related to atrial tissue. Since sinus node dysfunction can occur at any time postoperatively, these patients should be kept under constant control. If symptomatic sinus node dysfunction is confirmed, permanent pacing is an effective therapeutic modality.

Human iPSC-derived microglia sense and dampen hyperexcitability of cortical neurons carrying the epilepsy-associated SCN2A-L1342P mutation.

Neuronal hyperexcitability is a hallmark of epilepsy. It has been recently shown in rodent models of seizures that microglia, the brain's resident immune cells, can respond to and modulate neuronal excitability. However, how human microglia interact with human neurons to regulate hyperexcitability mediated by an epilepsy-causing genetic mutation found in patients is unknown. The SCN2A gene is responsible for encoding the voltage-gated sodium channel Nav1.2, one of the leading contributors to monogenic epilepsies. Previously, we demonstrated that the recurring Nav1.2-L1342P mutation leads to hyperexcitability in a male donor (KOLF2.1) hiPSC-derived cortical neuron model. Microglia originate from a different lineage (yolk sac) and are not naturally present in hiPSCs-derived neuronal cultures. To study how microglia respond to neurons carrying a disease-causing mutation and influence neuronal excitability, we established a co-culture model comprising hiPSC-derived neurons and microglia. We found that microglia display increased branch length and enhanced process-specific calcium signal when co-cultured with Nav1.2-L1342P neurons. Moreover, the presence of microglia significantly lowered the repetitive action potential firing and current density of sodium channels in neurons carrying the mutation. Additionally, we showed that co-culturing with microglia led to a reduction in sodium channel expression within the axon initial segment of Nav1.2-L1342P neurons. Furthermore, we demonstrated that Nav1.2-L1342P neurons release a higher amount of glutamate compared to control neurons. Our work thus reveals a critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing mutation.Significance Statement Seizure studies in mouse models have highlighted the role of microglia in modulating neuronal activity, particularly in the promotion or suppression of seizures. However, a gap persists in comprehending the influence of human microglia on intrinsically hyperexcitable neurons carrying epilepsy-associated pathogenic mutations. This research addresses this gap by investigating human microglia and their impact on neuronal functions. Our findings demonstrate that microglia exhibit dynamic morphological alterations and calcium fluctuations in the presence of neurons carrying an epilepsy-associated SCN2A mutation. Furthermore, microglia suppressed the excitability of hyperexcitable neurons, suggesting a potential beneficial role. This study underscores the role of microglia in the regulation of abnormal neuronal activity, providing insights into therapeutic strategies for neurological conditions associated with hyperexcitability.

Characteristic spatial and frequency distribution of mutations in SCN1A

BackgroundSCN1A is the most well-recognized and commonly mutated gene related to epilepsy. This study analyzed the characteristic spatial and frequency distributions of SCN1A mutations, aiming to provide important insight into the mutagenesis etiopathology of SCN1A-associated epilepsy.MethodsEpilepsy-associated SCN1A variants were retrieved from the SCN1A mutation database, the HGMD database, and literature reviews. The base substitutions, mutation frequencies in CpG dinucleotides, and spatial distributions of mutations in terms of exons and structural domains were analyzed.ResultsA total of 2621 SCN1A variants were identified in 5106 unrelated cases. The most common type was missense mutation, followed by frameshift mutations and splice site mutations. Among the missense mutations, transitions within CpG dinucleotides were much more recurrently identified than transitions within non-CpG dinucleotides, and the most common type was the G > A transition. Among the nonsense mutations, the most predominant type of single-base substitution was the C > T transition, among which 75.3% (235/312) were within CpG sites. The most common “hotspot” codons for missense mutations were codons 101, 946, and 1783; while for nonsense mutations it was codon 712. One-base deletion or insertion was the most common type of frameshift mutation, causing protein truncation. The three most common frameshift mutations were c.5536_5539delAAAC, c.4554dupA, and c.5010_5013delGTTT. Splice mutations were the most frequently identified in exon 4 with a hotspot site c.602 + 1G > A. The spatial distribution of missense mutations showed that exons 22 and 4 had the highest mutation density (111 and 84 mutations per 100 bp, respectively), and exon 12 had the lowest mutation density, with 4 mutations per 100 bp. Further distribution analysis of the protein domains revealed that missense mutations were more common in the pore region and voltage sensor (231 mutations per 100 amino acids, respectively), and the protein truncation mutations were distributed evenly among the domains.ConclusionsSCN1A mutations tend to cluster at distinct sites, depending on the characteristic CpG dinucleotides, exons, and functional domains. Higher mutation density in particular regions, such as exon 22 and exon 4, offers promising targets for therapeutic genetic interventions.

Phenotype heterogeneity of SCN5A mutations in Brugada syndrome

Abstract Introduction Although SCN5A is the main gene associated with Brs syndrome (Brs), rare variants in this gene only affect 20 to 30 % of patients. Additionally, associated phenotype may differ even among relatives sharing the same mutation emphasizing a phenotype heterogeneity that is poorly understood for now. Objective Here we aim to investigate the association between the presence of SCN5A mutations and the expression of a BrS phenotype in large families. Methods Among Brugada patients enrolled in our prospective cohort, we included patients from families with affected proband carrier of a SCN5A mutation and at least 4 relatives carriers of the same SCN5A mutation. Patient without a spontaneous type 1 ECG and no pharmacological provocative test were excluded. Results A total of 463 individuals from 45 large families were enrolled. Among them, 258 (56%) were mutation carriers of one of the 33 identified SCN5A mutations. After pharmacological test, 219 (47%) individuals were diagnosed with BrS, including 32 not carriers of the familial mutation. Seventy-five patients (30%), carriers of the familial SCN5A mutation, remain negative for Brugada syndrome after pharmacological test. The prevalence of genotype-phenotype mismatch was 24%, including 32 BrS patients non carrier of the familial mutation (phenocopy) and 75 patients with the familial mutation but without BrS (30% of uncomplete penetrance). Conclusion Our data strongly suggest that SCN5A mutation is not either necessary nor sufficient to explain the occurrence of BrS phenotype. We now aim to identify which associated factors might interfere, focusing first on the genetic background of phenotype/genotype mismatched individuals.

Identification of genetic variants by using a whole exome sequencing approach in Taiwanese patients with Brugada Syndrome

Abstract Background Brugada Syndrome (BrS) is a life-threatening arrhythmia disease primarily affecting young men. Many genetic studies have been conducted mainly in Caucasian BrS patients to identify potential genetic drivers. SCN5A mutations are less common in Asian BrS patients (around 10%) compared to Caucasian patients (20-30%). This means the pathogenic process of more than 70% of BrS patients remains unclear. With the advancement in NGS technologies, we can conduct genome-wide screening of DNA variants in BrS patients efficiently. Purpose Since less than 30% of the BrS patients have known genetic drivers for the disease, we aimed to identify more DNA variants in BrS patients by using the whole exome sequencing (WES) approach. Furthermore, we evaluated the landscape of DNA variants in the two groups of BrS patients according to whether they have SCN5A mutations or not. Methods A total of 201 BrS patients were recruited for the WES experiments. After obtaining the raw sequencing data, we performed the quality check procedures using the multiQC and Trimmomatic softwares (Figure 1). For reads passing the quality check, the BWA algorithm was used for the alignment. Next, the samtools, bamtools, and Picard methods were used to remove duplicates and perform the sorting. Subsequently, those sorted sequencing files were used to call the DNA variants by using the GATK approach and annotate them using the ANNOVAR method. The 201 BrS patients were classified into two different groups based on their mutation status of SCN5A, presentation of sudden cardiac death (SCD), and SCD along with syncope. The gene-based algorithm, SKAT, was used to analyze the summarized effects of DNA variants located in a single gene. For each DNA variant, Fisher's exact test was used to compare the allele frequency in the BrS patients under their groupings. Results The WES approach in the 201 BrS patients revealed a total of 205,454 variants, which were further categorized into two groups, common variants (51,867) and rare variants (153,587), based on their allele frequencies (5%). The SKAT algorithm was performed in the 30 genes that have been previously reported to be associated with BrS. No significant differences were observed in any gene in BrS patients for the common variants. Intriguingly, KCNJ8, SCN1B, ZFPM2, MAPRE2, and KCNE5 showed significant differences in allele frequencies between the SCN5A+ and SCN5A- patients (Figure 2). Fisher's exact test further revealed that three DNA variants (chr19:35524980, chr12:21926440, chr6:126210346) were significantly enriched in SCN5A+ patients. The 5 DNA variants located in ZFPM2 showed a linkage disequilibrium block and were enriched in SCN5A+ patients. Conclusions The WES approach in BrS patients revealed DNA variants that were reported in previous studies. Several DNA variants were significantly enriched in SCN5A+ patients, suggesting that the genome-wide mutation landscape was different in SCN5A+ and SCN5A- BrS patients.Figure 1Figure 2

Characterisation of the novel SCN5A-P1891A variant, implicated in left ventricular noncompaction cardiomyopathy in a Finnish family, using human induced pluripotent stem cell-derived cardiomyocytes

Abstract Introduction Left ventricular noncompaction (LVNC) is a heterogenous cardiomyopathy characterised by an extensively trabeculated left ventricle (LV). Several genetic variants that perturb trabeculae compaction, and thus LV maturation, have been reported [1]. However, the precise mechanisms underpinning this have proven contentious with, for example, differentially aberrant cardiomyocyte proliferation implicated in the development of LVNC [2,3]. Purpose In this study, we investigate a novel SCN5A variant, P1891A, which is associated with LVNC in a Finnish family. We ascertain the effects of this variant through the characterisation of variant-carrying human induced pluripotent stem cell-derived cardiomyocytes (P1891A-hiPSC-CMs). Methods We isolated dermal fibroblasts from a symptomatic member of a Finnish family carrying the SCN5A-P1891A variant; derived P1891A-hiPSCs via nucleofection with reprogramming plasmids [4]; and differentiated these into hiPSC-CMs alongside healthy control-hiPSCs. We determined the action potential (AP) and sodium- (INa) and calcium-current (ICa) densities of hiPSC-CMs via patch-clamp. We investigated gene expression in hiPSC-CMs through qPCR and employed Multiple Approaches Combined (MCA) proteomics [5] in SCN5A-transfected HEK293-like cells to elucidate protein-protein interactions (PPI). We subjected hiPSC-CMs to mechanical stretch [6] to examine their stress response and assessed their proliferation capacity via high-content analysis for the percentage of BrdU positive hiPSC-CMs following mitogenic stimulation (5 µM CHIR99021 and 1 µM SB203580). Finally, we ascertained contractile kinetics and apparent contractile force using fibrin hydrogel-based engineered heart tissues (EHTs) [7]. Results The P1891A-hiPSC-CMs demonstrated elevated INa density alongside enhanced arrhythmogenicity relative to healthy control hiPSC-CMs although, AP kinetics and ICa density were unperturbed (Fig. 1). Under baseline conditions, P1891A-hiPSC-CMs exhibited higher SCN5A gene expression and proteomics revealed a reduction in PPI. Baseline proliferation was unchanged; however, aged P1891A-hiPSC-CMs had enhanced proliferative capacity following mitogenic stimulation. Further, the P1891A-hiPSC-CMs displayed impaired tolerance to mechanical stretch as evidenced by upregulated NPPB and ACTA1 expression. In the context of the EHT, P1891A-hiPSC-CMs yielded disparate phenotypes. Whilst a subset compacted fully and yielded weaker contractile parameters relative to healthy control EHTs, the majority of P1891A-hiPSC-CM EHTs failed to fully compact thereby recapitulating the LVNC phenotype (Fig. 2). Conclusions This study presents a unique aetiology of LVNC and links, for the first time, SCN5A mutations with enhanced human cardiomyocyte proliferation. The ability to recapture the noncompaction phenotype in EHTs presents a powerful model for future mechanistic research and drug development.

Parvalbumin interneuron impairment causes synaptic transmission deficits and seizures in SCN8A developmental and epileptic encephalopathy.

SCN8A developmental and epileptic encephalopathy (DEE) is a severe epilepsy syndrome resulting from mutations in the voltage-gated sodium channel Nav1.6, encoded by the gene SCN8A. Nav1.6 is expressed in excitatory and inhibitory neurons, yet previous studies primarily focus on how SCN8A mutations affect excitatory neurons, with limited studies on the importance of inhibitory interneurons. Parvalbumin (PV) interneurons are a prominent inhibitory interneuron subtype that expresses Nav1.6. To assess PV interneuron function within SCN8A DEE, we used 2 mouse models harboring patient-derived SCN8A gain-of-function variants, Scn8aD/+, where the SCN8A variant N1768D is expressed globally, and Scn8aW/+-PV, where the SCN8A variant R1872W is selectively expressed in PV interneurons. Expression of the R1872W SCN8A variant selectively in PV interneurons led to development of spontaneous seizures and seizure-induced death. Electrophysiology studies showed that Scn8aD/+ and Scn8aW/+-PV interneurons were susceptible to depolarization block and exhibited increased persistent sodium current. Evaluation of synaptic connections between PV interneurons and pyramidal cells showed synaptic transmission deficits in Scn8aD/+ and Scn8aW/+-PV interneurons. Together, our findings indicate that PV interneuron failure via depolarization block along with inhibitory synaptic impairment likely elicits an overall inhibitory reduction in SCN8A DEE, leading to unchecked excitation and ultimately resulting in seizures and seizure-induced death.

Neuropathologic Findings and Electrolyte Abnormalities in a Case of Lennox-Gastaut Syndrome (SCN2a- related) Encountered at Autopsy

Abstract Introduction/Objective Lennox-Gastaut syndrome is a severe form of childhood epilepsy which may be due to several conditions, including sodium channelopathies which alter neuronal firing patterns. We present a case of a 6-year-old female with Lennox-Gastaut syndrome (SCN2A-related) complicated by developmental delay, spastic quadriplegia, and gastrojejunostomy tube dependence who was referred for medicolegal autopsy due to death subsequent to becoming unresponsive during a supervised bath. Methods/Case Report A complete autopsy was performed, including gross and microscopic examination of all primary organ systems, formal neuropathologic examination, and postmortem microbiology, toxicology, and vitreous electrolyte studies. The decedent showed evidence of developmental delay and flexion contractures of the extremities. No significant traumatic injuries were identified. A postmortem nasopharyngeal swab was positive for Rhinovirus/Enterovirus and Influenza A H3. A postmortem vitreous electrolyte panel demonstrated a dehydration pattern, including markedly elevated sodium and chloride and elevated urea nitrogen. Gross examination of the brain showed a thin corpus callosum, small hippocampi, as well as abnormal cerebellar dentate and inferior olivary nuclei. Microscopic examination of the brain tissue supported the gross findings, including dentato-olivary dysplasia. The cause of death was certified as dehydration complicating Lennox-Gastaut syndrome (SCN2a-related). Rhinovirus/Enterovirus and Influenza A H3 infections were considered contributory to death. The manner of death remained undetermined, since it was unknown if the decedent’s natural diseases, issues following a scheduled gastrojejunostomy feeding regimen, neglect, or some combination thereof caused the dehydration. Results (if a Case Study enter NA) NA Conclusion This case highlights unique and rarely documented neuropathologic findings and electrolyte abnormalities that may be seen in Lennox-Gastaut syndrome associated with an SCN2A mutation in addition to demonstrating the challenges of cause and manner death certification in cases of severe developmental disorders of childhood related to sodium channelopathies.

Persistent Na + current couples spreading depolarization to seizures in Scn8a gain of function mice.

Spreading depolarization (SD) is a slowly propagating wave of massive cellular depolarization that transiently impairs the function of affected brain regions. While SD typically arises as an isolated hemispheric event, we previously reported that reducing M-type potassium current (I KM ) by ablation of Kcnq2 in forebrain excitatory neurons results in tightly coupled spontaneous bilateral seizure-SD complexes in the awake mouse cortex. Here we find that enhanced persistent Na + current due to gain-of-function (GOF) mutations in Scn8a (N1768D/+, hereafter D/+) produces a similar compound cortical excitability phenotype. Chronic DC-band EEG recording detected spontaneous bilateral seizure-SD complexes accompanied by seizures with a profound tonic component, which occurs predominantly during the light phase and were detected in the mutant mice across ages between P40-100. Laser speckle contrast imaging of cerebral blood flow dynamics resolved SD as bilateral wave of hypoperfusion and subsequent hour-lasting hypoperfusion in Scn8a D/+ cortex in awake head-restrained mice subjected to a subconvulsive PTZ. Subcortical recordings in freely moving mice revealed that approximately half of the spontaneous cortical seizure-SD complexes arose with concurrent SD-like depolarization in the thalamus and delayed depolarization in the striatum. In contrast, SD-like DC potential shifts were rarely detected in the hippocampus or upper pons. Consistent with the high spontaneous incidence in vivo , cortical slices from Scn8a D/+ mice showed a raised SD susceptibility, and pharmacological inhibition of persistent Na + current (I NaP ), which is enhanced in Scn8a D/+ neurons, inhibited SD generation in cortical slices ex vivo , indicating that I NaP contributes to SD susceptibility. Ex vivo Ca 2+ imaging studies using acute brain slices expressing genetic Ca 2+ sensor (Thy1-GCAMP6s) demonstrated that pharmacological activation of I KM suppressed Ca 2+ spikes and SD, whereas I KM inhibitor drastically increased the frequency of Ca 2+ spikes in the hippocampus of Scn8a D/+ mice, but not in WT, suggesting that I KM restrains the hyperexcitability resulting from Scn8a GOF mutation. Together, our study identifies a cortical SD phenotype in Scn8a GOF mice shared with the Kcnq2 - cKO model of developmental epileptic encephalopathy and reveals that an imbalance of non-inactivating inward and outward membrane currents bidirectionally modulates spatiotemporal SD susceptibility.

Nonmodifiable Risk Factors Predict Outcomes in Brugada Syndrome

BackgroundRisk stratification in Brugada syndrome (BrS) is based on the occurrence of dynamic factors, such as unexplained syncope and documentation of spontaneous type 1 pattern. At odds with other channelopathies, the role of nonmodifiable risk factors such as sex or genetics remains uncertain. ObjectivesThis study aims to identify nonmodifiable risk factors for the occurrence of life-threatening arrhythmic events (LAEs) and define their clinical utility. MethodsClinical and genetic data from consecutive, unrelated Italian patients with Brugada syndrome screened on the sodium voltage-gated channel alpha subunit 5 (SCN5A) gene and 3 pivotal single-nucleotide variations (formerly single-nucleotide polymorphisms) associated with BrS (rs11708996, rs10428132, and rs9388451) were analyzed using multivariable Cox proportional hazards model. ResultsIn 2,182 unrelated patients with BrS (81% males; median age at diagnosis: 41.6 years [Q1-Q3: 33.4-50.3 years]), male sex (HR: 3.6; 95% CI: 1.9-6.9; P = 0.0001), missense SCN5A mutations in BrS-enriched domains (HR: 2.3; 95% CI: 1.2-4.3; P = 0.008), nonmissense SCN5A mutations (HR: 3.2; 95% CI: 1.8-5.7; P < 0.001), and polygenic risk score for BrS (HR: 1.3; 95% CI: 1.0-1.6; P = 0.041) were all independently associated with a significantly higher risk of a first LAE since birth. Based on these results, we derived the nonmodifiable risk of each patient with BrS, and the division of nonmodifiable risk into tertiles identified 3 distinct risk profiles. In an analysis at follow-up, nonmodifiable risk was independently associated with LAE at follow-up (HR: 1.8; 95% CI: 1.1-2.7; P = 0.014), alongside classical predictors including: history of LAE before diagnosis (HR: 13.8; 95% CI: 8.1-23.7; P < 0.0001), history of unexplained syncope before diagnosis (HR: 4.1; 95% CI: 2.4-6.8; P < 0.0001), and spontaneous type 1 pattern at diagnosis (HR: 2.1; 95% CI: 1.2-3.8; P = 0.010). The model was internally validated, and we derived the equation permitting to calculate the granular 5-year risk of experiencing an LAE at follow-up for each patient with BrS, which may be used to facilitate clinical decision-making. ConclusionsOur data show that male sex, type of SCN5A mutation, and polygenic risk score for BrS define the nonmodifiable risk of each patient with BrS. Nonmodifiable risk is independently associated with LAE, regardless of symptoms or pattern type.

Early developmental alterations of CA1 pyramidal cells in Dravet syndrome

Dravet Syndrome (DS) is most often caused by heterozygous loss-of-function mutations in the voltage-gated sodium channel gene SCN1A (Nav1.1), resulting in severe epilepsy and neurodevelopmental impairment thought to be cause by reduced interneuron excitability. However, recent studies in mouse models suggest that interneuron dysfunction alone does not completely explain all the cellular and network impairments seen in DS. Here, we investigated the development of the intrinsic, synaptic, and network properties of CA1 pyramidal cells in a DS model prior to the appearance of overt seizures. We report that CA1 pyramidal cell development is altered by heterozygous reduction of Scn1a, and propose that this is explained by a period of reduced intrinsic excitability in early postnatal life, during which Scn1a is normally expressed in hippocampal pyramidal cells. We also use a novel ex vivo model of homeostatic plasticity to show an instability in homeostatic response during DS epileptogenesis. This study provides evidence for the early effects of Scn1a haploinsufficiency in pyramidal cells in contributing to the pathophysiology of DS.

Sex differences in cardiac mitochondrial respiration and reactive oxygen species production may predispose Scn1a−/+ mice to cardiac arrhythmias and Sudden Unexpected Death in Epilepsy

Dravet Syndrome (DS) is a pediatric-onset epilepsy with an elevated risk of Sudden Unexpected Death in Epilepsy (SUDEP). Most individuals with DS possess mutations in the voltage-gated sodium channel gene Scn1a, expressed in both the brain and heart. Previously, mutations in Scn1a have been linked to arrhythmia. We used a Scn1a−/+ DS mouse model to investigate changes to cardiac mitochondrial function that may underlie arrhythmias and SUDEP. We detected significant alterations in mitochondrial bioenergetics that were sex-specific. Mitochondria from male Scn1a−/+ hearts had deficits in maximal (p = 0.02) and Complex II-linked respiration (p = 0.03). Male Scn1a−/+ mice were also more susceptible to cardiac arrhythmias under increased workload. When isolated cardiomyocytes were subjected to diamide, cardiomyocytes from male Scn1a−/+ hearts were less resistant to thiol oxidation. They had decreased survivability compared to Scn1a+/+ (p = 0.02) despite no whole-heart differences. Lastly, there were no changes in mitochondrial ROS production between DS and wild-type mitochondria at basal conditions, but Scn1a−/+ mitochondria accumulated more ROS during hypoxia/reperfusion. This study determines novel sex-linked differences in mitochondrial and antioxidant function in Scn1a-linked DS. Importantly, we found that male Scn1a−/+ mice are more susceptible to cardiac arrhythmias than female Scn1a−/+ mice. When developing new therapeutics to address SUDEP risk in DS, sex should be considered.

Characterization of a novel SCN5A mutation associated with long QT syndrome and arrhythmogenic right ventricular cardiomyopathy in a family.

Sudden cardiac death represents a significant diagnostic challenge for forensic pathologists, particularly in inherited arrhythmia syndromes or cardiomyopathies resulting from genetic defects. Molecular autopsies can reveal the underlying molecular etiology in such cases. In this study, we investigated a family with a history of sudden cardiac death to elucidate the molecular basis responsible for sudden cardiac death. The proband underwent a comprehensive forensic examination. Family members received thorough clinical evaluations, including electrocardiogram, Holter monitoring, echocardiography, and cardiac magnetic imaging. Whole exome sequencing and genetic analysis were performed on the deceased and her parents. In addition, Western blotting and patch-clamp recordings were employed to evaluate the expression and function of the mutant protein in vitro. Forensic examination diagnosed arrhythmogenic right ventricular cardiomyopathy (ARVC) as the cause of sudden death. Genetic analysis identified a novel missense mutation in SCN5A (p.V1323L), which was assessed as likely pathogenic by the ACMG guideline. Another family member carrying the mutation manifested long QT syndrome and mild cardiac fibrosis. The cellular electrophysiological study demonstrated that the mutation resulted in an enhanced late sodium current, suggesting it was a gain-of-function mutation. This study characterizes a novel SCN5A mutation that putatively causes long QT syndrome and may contribute to the development of ARVC. Our work expands the pathogenic spectrum of SCN5A variants and underscores the importance of molecular autopsy in sudden death cases, especially in those with suspected genetic disorders.

Abstract Mo134: Restoring NaV1.5 Mutant Functionality in Arrhythmia with ManNAc Supplementation

The SCN5A gene encodes the human cardiac sodium channel NaV1.5, essential for cardiac excitability and action potential propagation. Mutations in SCN5A are linked to various cardiac disorders, including Brugada Syndrome (BrS), typically leading to channel loss-of-function. Understanding alterations in NaV1.5 expression and sodium current density is vital for comprehending arrhythmogenesis and formulating therapeutic strategies. Post-translational modifications, especially sialylation, significantly impact channel functionality. We previously demonstrated that BrS patients show decreased protein sialylation, correlating with reduced sodium channel activity. This study aimed to investigate sialic acid's role in regulating NaV1.5 activity, testing if sialylation induction via mannosamine (ManNAc) supplementation could restore channel activity in-vitro. Preliminary tests with exogenous sialidases and ManNAc supplementation affirmed our hypothesis. We examined ManNAc's effect on NaV1.5 mutants common in BrS, which typically exhibit partial functional loss. We overexpressed mutations p.A344S, p.K1479A, and p.G1406R in HEK293A cells and measured sodium currents before and after ManNAc supplementation to stimulate sialic acid synthesis. ManNAc effectively restored sodium currents in mutants with intracellular and extracellular loop mutations. However, the p.1406G&gt;A mutant, affecting the pore structure, showed no improvement, suggesting that sialic acid mainly influences channel membrane trafficking rather than direct glycosylation. This underscores the complexity of sialic acid's role and points to trafficking pathways as potential therapeutic targets. Notably, the treatment neither increased nor decreased sodium currents in wild-type NaV1.5 channels or in the CaV3.2 calcium channel, underscoring its specificity. In summary, our findings demonstrate that ManNAc supplementation can selectively restore the function of certain NaV1.5 mutants without affecting wild-type channels, highlighting the potential of targeting sialylation pathways for SCN5A-related arrhythmia treatments. Future research should aim to unravel these mechanisms further, providing insights into novel treatments for SCN5A-related arrhythmias.

Comparative Analysis of Lennox-Gastaut Syndrome With Different Subtypes of Tonic Seizures: A Single-Center Retrospective Cohort Study

BackgroundLennox-Gastaut syndrome (LGS) is one of the most severe childhood-onset epileptic encephalopathies, primarily characterized by tonic seizures. In clinical practice, we have identified various subtypes of tonic seizures in LGS. This study aimed to analyze the clinical characteristics, electrographic features, treatment responses, and prognosis across different subtypes of LGS. MethodsThis retrospective cohort study included 46 patients diagnosed with LGS at our center between January 2017 and January 2020. Patients were classified into four groups based on tonic seizure subtypes: Group A (tonic), Group B (spasm-tonic), Group C (myoclonic-tonic), and Group D (combination of spasm-tonic and myoclonic-tonic). Comprehensive clinical data were collected and analyzed. ResultsOf the 46 patients, 33 were male. The mean age of onset for Group B (12.38 ± 7.85 months) was significantly less than those of the other three groups (P = 0.02). No significant differences in etiology were found among the groups. Genetic analysis identified mutations in SCN8A, MCCC2, STXBP1, GABRB3, and CACNA1H. After a minimum follow-up of 24 months, the treatment outcomes were more favorable in Groups A and C, whereas psychomotor development was notably poorer in Groups B and D. ConclusionsThe findings of this study suggest that LGS may present with distinct subtypes of tonic seizures, with spasm-tonic seizures presenting at an earlier age. Patients with LGS experiencing spasm-tonic seizures, with or without myoclonic-tonic seizures, exhibited poorer treatment responses and psychomotor development than those with other subtypes.

Correlations of ventricular fibrillation and monomorphic ventricular tachycardia with SCN5A mutations and other clinical variables in Brugada syndrome

Correlations of ventricular fibrillation and monomorphic ventricular tachycardia with SCN5A mutations and other clinical variables in Brugada syndrome

Insights into clinical phenotypes and treatment responses in a Small cohort of Taiwanese patients with SCN1A variants: A Preliminary study

BackgroundSCN1A channelopathy is the most well-known cause for epileptic encephalopathies and contributes to a wide phenotypic spectrum. The variable expressivity is troublesome for the interpretation of clinical significance and prognoses. To investigate the clinical manifestations, medications and outcomes of patients with SCN1A channelopathies, we conducted this observation retrospective study in Taiwan. MethodsA cohort consisting of 16 patients (5 males and 11 females) from multiple centers with identified SCN1A variants was investigated and phenotypically relevant factors were recorded. The variants were identified using NGS and confirmed by Sanger sequencing. A panel of 90 epileptic-related genes was used to identify SCN1A variants and to evaluate some of the potential SCN1A modifier genes. ResultsThe mean age of seizure onset was 10.4 months. Twelve of the sixteen patients (75%) had different degrees of neurocognitive sequela and psychobehavioral comorbidity in our cohort. Cognitive impairment was noted in all ten patients with Dravet syndrome (DS) and in two of the patients with non-DS phenotypes. A lower response rate to medications was also noted in patients with DS. Notably, a medication-specific tendency towards valproic acid (VPA), clobazam (CLB), and levetiracetam (LEV) was observed, revealing the effective pharmacotherapies for SCN1A-related seizures. An asymptomatic carrier with a reported pathogenic SCN1A variant was reviewed along with her monozygotic twin sister with DS. Nine novel SCN1A mutations are herein reported, eight of which being classified as pathogenic. ConclusionOur study revealed unfavorable outcomes for patients with SCN1A variants. Some patients with SCN1A channelopathy showed specific responsiveness to the pharmacotherapies previously either recommended or contraindicated for these patients. Our study also expands the genotype and provides valuable prognostic insights in patients with SCN1A channelopathy.

NaV1.5 autoantibodies in Brugada syndrome: pathogenetic implications.

Patients suffering from Brugada syndrome (BrS) are predisposed to life-threatening cardiac arrhythmias. Diagnosis is challenging due to the elusive electrocardiographic (ECG) signature that often requires unconventional ECG lead placement and drug challenges to be detected. Although NaV1.5 sodium channel dysfunction is a recognized pathophysiological mechanism in BrS, only 25% of patients have detectable SCN5A variants. Given the emerging role of autoimmunity in cardiac ion channel function, this study explores the presence and potential impact of anti-NaV1.5 autoantibodies in BrS patients. Using engineered HEK293A cells expressing recombinant NaV1.5 protein, plasma from 50 BrS patients and 50 controls was screened for anti-NaV1.5 autoantibodies via western blot, with specificity confirmed by immunoprecipitation and immunofluorescence. The impact of these autoantibodies on sodium current density and their pathophysiological effects were assessed in cellular models and through plasma injection in wild-type mice. Anti-NaV1.5 autoantibodies were detected in 90% of BrS patients vs. 6% of controls, yielding a diagnostic area under the curve of .92, with 94% specificity and 90% sensitivity. These findings were consistent across varying patient demographics and independent of SCN5A mutation status. Electrophysiological studies demonstrated a significant reduction specifically in sodium current density. Notably, mice injected with BrS plasma showed Brugada-like ECG abnormalities, supporting the pathogenic role of these autoantibodies. The study demonstrates the presence of anti-NaV1.5 autoantibodies in the majority of BrS patients, suggesting an immunopathogenic component of the syndrome beyond genetic predispositions. These autoantibodies, which could serve as additional diagnostic markers, also prompt reconsideration of the underlying mechanisms of BrS, as evidenced by their role in inducing the ECG signature of the syndrome in wild-type mice. These findings encourage a more comprehensive diagnostic approach and point to new avenues for therapeutic research.

Genetic and clinical characteristics of single and compound types of patients with long QT syndrome type 3

Objective: To explore the genetic background and clinical features of patients with long QT syndrome type 3 (LQT3). Methods: This retrospective cohort included patients diagnosed with LQT3 at the Department of Cardiology, Renmin Hospital of Wuhan University from January 1998 to December 2022. Patients were categorized into compound type group and single type group based on the presence of a single SCN5A mutation. The two groups were followed up and the differences in baseline characteristics, electrocardiograms, and clinical events between the two groups and probands were compared. Kaplan-Meier curves were used for survival analysis, and the log-rank test was employed to compare the event-free survival rates of first cardiac events between the groups and probands. Results: A total of 97 LQT3 patients were enrolled, including 59 probands. The age at diagnosis was (23.45±19.86) years, with 46 patients (47.4%) being male. Among them, 89 patients were classified as single type group, while 8 patients were classified as compound type group. Genetic testing identified 49 SCN5A mutations, with missense mutations being the majority (91.8%), primarily located in transmembrane regions (40.8%, n=20), interdomain linker regions (28.6%, n=14), and C-terminus (22.4%, n=11). The first cardiac event occurred in 44 patients (45.4%), with an onset age of (13.82±12.50) years. The main trigger was identified as rest or sleep (54.5%, n=24). Compared with patients in single type group, patients in compound type group were younger at diagnosis ((10.35±10.28) years vs. (24.63±20.13) years, P=0.040), had a significantly higher proportion of syncope (87.5% (7/8) vs. 33.7% (30/89), P=0.009), aborted cardiac arrest (62.5% (5/8) vs. 11.2% (10/89), P=0.001), and a lower incidence of event-free survival rates of first cardiac events (12.5% (1/8) vs.58.4% (52/89), log-rank P=0.001). The probands in compound type group had a significantly higher proportion of aborted cardiac arrest comparing to probands in single type group (62.5% (5/8) vs. 17.6% (9/51), P=0.020), while the difference in the incidence rate of event-free survival rates of first cardiac events between the probands in two groups was not statistically significant (12.5% (1/8) vs. 39.2% (20/51), log-rank P=0.08). Conclusion: Compound type LQT3 patients are not uncommon. Such patients are diagnosed at a younger age and exhibit more severe phenotypes, requiring close follow-up and proactive intervention strategies.

Dentate gyrus granule cells are a locus of pathology in Scn8a developmental encephalopathy

Gain-of-function mutations in SCN8A cause developmental and epileptic encephalopathy (DEE), a disorder characterized by early-onset refractory seizures, deficits in motor and intellectual functions, and increased risk of sudden unexpected death in epilepsy. Altered activity of neurons in the corticohippocampal circuit has been reported in mouse models of DEE. We examined the effect of chronic seizures on gene expression in the hippocampus by single-nucleus RNA sequencing in mice expressing the patient mutation SCN8A-p.Asn1768Asp (N1768D). One hundred and eighty four differentially expressed genes were identified in dentate gyrus granule cells, many more than in other cell types. Electrophysiological recording from dentate gyrus granule cells demonstrated an elevated firing rate. Targeted reduction of Scn8a expression in the dentate gyrus by viral delivery of an shRNA resulted in doubling of median survival time from 4 months to 8 months, whereas delivery of shRNA to the CA1 and CA3 regions did not result in lengthened survival. These data indicate that granule cells of the dentate gyrus are a specific locus of pathology in SCN8A-DEE.