SCN5A Research Articles

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< 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 <em>a</em> subunit Na<sub>v</sub>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 <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<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<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<0.05) compared to WT males (1.332±0.060μm2) and are more fragmented (p<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>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<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.

Genetic exploration of Dravet syndrome: two case report

BackgroundDravet syndrome is an infantile-onset developmental and epileptic encephalopathy (DEE) characterized by drug resistance, intractable seizures, and developmental comorbidities. This article focuses on manifestations in two Indonesian children with Javanese ethnicity who experienced Dravet syndrome with an SCN1A gene mutation, presenting genetic analysis findings using next-generation sequencing.Case presentationWe present a case series involving two Indonesian children with Javanese ethnicity whom had their first febrile seizure at the age of 3 months, triggered after immunization. Both patients had global developmental delay and intractable seizures. We observed distinct genetic findings in both our cases. The first patient revealed heterozygous deletion mutation in three genes (TTC21B, SCN1A, and SCN9A). In our second patient, previously unreported mutation was discovered at canonical splice site upstream of exon 24 of the SCN1A gene. Our patient’s outcomes improved after therapeutic evaluation based on mutation findings When comparing clinical manifestations in our first and second patients, we found that the more severe the genetic mutation discovered, the more severe the patient’s clinical manifestations.ConclusionThese findings emphasize the importance of comprehensive genetic testing beyond SCN1A, providing valuable insights for personalized management and tailored therapeutic interventions in patients with Dravet syndrome. Our study underscores the potential of next-generation sequencing in advancing genotype–phenotype correlations and enhancing diagnostic precision for effective disease management.

Machine learning models reveal distinct disease subgroups and improve diagnostic and prognostic accuracy for individuals with pathogenic SCN8A gain-of-function variants.

Distinguishing clinical subgroups for patients suffering with diseases characterized by a wide phenotypic spectrum is essential for developing precision therapies. Patients with gain-of-function (GOF) variants in the SCN8A gene exhibit substantial clinical heterogeneity, viewed historically as a linear spectrum ranging from mild to severe. To test for hidden clinical subgroups, we applied two machine-learning algorithms to analyze a dataset of patient features collected by the International SCN8A Patient Registry. We used two research methodologies: a supervised approach that incorporated feature severity cutoffs based on clinical conventions, and an unsupervised approach employing an entirely data-driven strategy. Both approaches found statistical support for three distinct subgroups and were validated by correlation analyses using external variables. However, distinguishing features of the three subgroups within each approach were not concordant, suggesting a more complex phenotypic landscape. The unsupervised approach yielded strong support for a model involving three partially ordered subgroups rather than a linear spectrum. Application of these machine-learning approaches may lead to improved prognosis and clinical management of individuals with SCN8A GOF variants and provide insights into the underlying mechanisms of the disease.

Correlation between clinical phenotypes and genotypes among 46 children with SCN1A-related developmental epileptic encephalopathy

To explore the correlation between clinical phenotypes and genotypes among 46 children with SCN1A-related developmental epileptic encephalopathy (DEE). Clinical data of 46 children with DEE and SCN1A variants identified at the Guangzhou Women and Children's Medical Center between January 2018 and June 2022 were collected. The children were grouped based on their age of onset, clinical manifestations, neurodevelopmental status, and results of genetic testing. The correlation between SCN1A genotypes and clinical phenotypes was analyzed. Among the 46 patients, 2 children (4.35%) had developed the symptoms before 3 months of age, 42 (91.30%) were between 3 to 9 months, and 2 cases (4.35%) were after 10 months. Two cases (4.35%) presented with epilepsy of infancy with migrating focal seizures (EIMFS), while 44 (95.7%) had presented with Dravet syndrome (DS), including 28 cases (63.6%) with focal onset (DS-F), 13 cases (29.5%) with myoclonic type (DS-M), 1 case (2.27%) with generalized type (DS-G), and 2 cases (4.55%) with status epilepticus type (DS-SE). Both of the two EIMFS children had severe developmental delay, and among the DS patients, 7 cases had normal development, while the remaining had developmental delay. A total of 44 variants were identified through genetic sequencing, which included 16 missense variants and 28 truncating variants. All EIMFS children had carried the c.677C>T (p.Thr226Met) missense variant. In the DS group, there was a significant difference in the age of onset between the missense variants group and the truncating variants group (P < 0.05). Missense variants were more common in D1 (7/15, 46.7%) and pore regions (8/15, 53.3%), while truncating variants were more common in D1 (12/28, 42.9%). Children with variants outside the pore region were more likely to develop myoclonic seizures. The clinical phenotypes of DEE are diverse. There is a difference in the age of onset between individuals with truncating and missense variants in the SCN1A gene. Missense variants outside the pore region are associated with a higher incidence of myoclonic seizures.

Idiopathic generalized epilepsy in a family with SCN4A-related myotonia.

Myotonia is a clinical sign typical of a group of skeletal muscle channelopathies, the non-dystrophic myotonias. These disorders are electrophysiologically characterized by altered membrane excitability, due to specific genetic variants in known causative genes (CLCN1 and SCN4A). Juvenile Myoclonic Epilepsy (JME) is an epileptic syndrome identified as idiopathic generalized epilepsy, its genetics is complex and still unclarified. The co-occurrence of these two phenotypes is rare and the causes likely have a genetic background. In this study, we have genetically investigated an Italian family in which co-segregates myotonia, JME, or abnormal EEG without seizures was observed. All six individuals of the family, 4 affected and 2 unaffected, were clinically evaluated; EMG and EEG examinations were performed. For genetic testing, Exome Sequencing was performed for the six family members and Sanger sequencing was used to confirm the candidate variant. Four family members, the mother and three siblings, were affected by myotonia. Moreover, EEG recordings revealed interictal generalized sharp-wave discharges in all affected individuals, and two siblings were affected by JME. All four affected members share the same identified variant, c.644 T > C, p.Ile215Thr, in SCN4A gene. Variants that could account for the epileptic phenotype alone, separately from the myotonic one, were not identified. These results provide supporting evidence that both myotonic and epileptic phenotypes could share a common genetic background, due to variants in SCN4A gene. SCN4A pathogenic variants, already known to be causative of myotonia, likely increase the susceptibility to epilepsy in our family. This study analyzed all members of an Italian family, in which the mother and three siblings had myotonia and epilepsy. Genetic analysis allowed to identify a variant in the SCN4A gene, which appears to be the cause of both clinical signs in this family.

Variant-specific invitro neuronal network phenotypes and drug sensitivity in SCN2A developmental and epileptic encephalopathy.

De novo variants in the NaV1.2 voltage-gated sodium channel gene SCN2A are among the major causes of developmental and epileptic encephalopathies (DEE). Based on their biophysical impact on channel conductance and gating, SCN2A DEE variants can be classified into gain-of-function (GoF) or loss-of-function (LoF). Clinical and functional data have linked early seizure onset DEE to the GoF SCN2A variants, whereas late seizure onset DEE is associated with the loss of SCN2A function. This study aims to assess the impact of GoF and LoF SCN2A variants on cultured neuronal network activity and explore their modulation by selected antiseizure medications (ASM). To this end, primary cortical cultures were generated from two knock-in mouse lines carrying variants corresponding to human GoF SCN2A p.R1882Q and LoF p.R853Q DEE variant. Invitro neuronal network activity and responses to ASM were analyzed using multielectrode array (MEA) between 2 and 4 weeks in culture. The SCN2A p.R1882Q neuronal cultures showed significantly greater mean firing and burst firing. Their network synchronicity was also higher. In contrast, the SCN2A p.R853Q cultures showed lower mean firing rate, and burst firing events were less frequent. The network synchronicity was also lower. Phenytoin and levetiracetam reduced the excitability of GoF cultures, while retigabine showed differential and potentially beneficial effects on cultures with both GoF and LoF variants. We conclude that invitro neuronal networks harboring SCN2A GoF or LoF DEE variants present with distinctive phenotypes and responses to ASM.

EEG band power and phase‐amplitude coupling in patients with Dravet syndrome

AbstractObjectiveDravet syndrome (DS) is an epileptic encephalopathy caused by haploinsufficiency of the SCN1A gene. SCN1A gene deficiency limits the firing rates of fast‐spiking inhibitory interneurons, which should reflect in abnormal aggregate network oscillatory electroencephalography (EEG) activity that can be measured by spectral power and phase‐amplitude coupling (PAC) analysis. In this retrospective pilot study, we tested whether spectral EEG frequency band power and PAC metrics distinguish children with DS from age‐matched controls, an early step toward establishing EEG markers of target engagement by gene or drug therapy.MethodsEEG data were collected from patients with DS (N = 6) and age‐matched control pediatric participants (N = 11) and analyzed for cumulative spectral power and PAC and classification capacity of these metrics, by logistic regression analysis. For this initial spectral and PAC analysis, we focused on sleep EEG, where myogenic artifact is minimal and where δ–γ and θ–γ coupling is otherwise expected to be robust.ResultsCumulative δ (1– &lt;4 Hz) and θ (4–7 Hz) power was significantly reduced in the DS group, compared with age‐matched controls (p = 0.001 and p = 0.02, respectively). The δ power was a stronger classifier of separating DS from controls than θ power, with 87% and 83% accuracy, respectively. The γ power trended toward significant reduction (p = 0.08) in the DS group. We found significantly lower PAC between 1–2 Hz phase and 63–80 Hz amplitude in patients with DS compared with the age‐matched controls (p = 0.003), with 78% classification accuracy between groups for PAC.InterpretationIn this pilot study assessing EEG patterns during sleep, we found lower δ–θ power and PAC in patients with DS versus controls, which may reflect abnormal aggregate macroscale network communication patterns resulting from SCN1A deficiency. These measures may be useful metrics of therapeutic target engagement, particularly if the therapy restores the underlying DS pathophysiology. The sorting capacity of these metrics distinguished patients with DS from patients without DS and may in turn facilitate near‐future development of disease and therapy target engagement biomarkers in this syndrome.

Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Analysis of three Chinese pedigrees affected with Genetic epilepsy with febrile seizures plus due to variants of SCN1A gene

To analyze the clinical and genetic characteristics of three Chinese pedigrees affected with Genetic epilepsy with febrile seizures plus (GEFS+). Three GEFS+ probands and their pedigree members presented at the Children's Hospital of Zhengzhou University from January 2020 to December 2021 were selected as the study subjects. Clinical data of the pedigrees were collected. Whole exome sequencing was carried out for the probands, and Sanger sequencing was used to verify the candidate variants. Proband 1 was a 3-year-and-2-month-old male with febrile seizure plus. His father, two aunts, grandmother, aunt grandmother, uncle grandfather, and paternal great-grandmother also had onset of febrile seizures at 1 ~ 2 years of age with remission before 6 years old. Proband 2 was a 1-year-and-4-month-old male with complex febrile seizure. His mother, maternal uncle, and maternal grandmother also had febrile seizures before 5 ~ 6 years of age. Proband 3 was a 3-year-and-11-month-old male with febrile seizure plus. His father and grandfather also had febrile seizures plus with remission at 7 ~ 8 years of age. Genetic testing revealed that proband 1 had harbored a paternally derived heterozygous SCN1A: c.1613T>C variant, proband 2 had harbored a maternally derived heterozygous SCN1A: c.2804A>G variant, and proband 3 had harbored a paternally derived heterozygous SCN1A: c.1271T>C variant. All of the three variants were predicted as likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (PM1+PM2_Supporting+PP1+PP3+PP4). The c.1613T>C, c.2804A>G and c.1271T>C variants probably underlay the pathogenesis of GEFS+ in these pedigrees.

Novel Gain-of-Function Mutation in the Kv11.1 Channel Found in the Patient with Brugada Syndrome and Mild QTc Shortening.

Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST-segment elevation in the right precordial leads on electrocardiograms (ECG), and high risk of life-threatening ventricular arrhythmia and sudden cardiac death (SCD). Mutations in the responsible genes have not been fully characterized in the BrS patients, except for the SCN5A gene. We identified a new genetic variant, c.1189C>T (p.R397C), in the KCNH2 gene in the asymptomatic male proband diagnosed with BrS and mild QTc shortening. We hypothesize that this variant could alter IKr-current and may be causative for the rare non-SCN5A-related form of BrS. To assess its pathogenicity, we performed patch-clamp analysis on IKr reconstituted with this KCNH2 mutation in the Chinese hamster ovary cells and compared the phenotype with the wild type. It appeared that the R397C mutation does not affect the IKr density, but facilitates activation, hampers inactivation of the hERG channels, and increases magnitude of the window current suggesting that the p.R397C is a gain-of-function mutation. In silico modeling demonstrated that this missense mutation potentially leads to the shortening of action potential in the heart.

Biophysical mechanisms of myocardium sodium channelopathies.

Genetic variants of gene SCN5A encoding the alpha-subunit of cardiac voltage-gated sodium channel Nav1.5 are associated with various diseases, including long QT syndrome (LQT3), Brugada syndrome (BrS1), and progressive cardiac conduction disease (PCCD). In the last decades, the great progress in understanding molecular and biophysical mechanisms of these diseases has been achieved. The LQT3 syndrome is associated with gain-of-function of sodium channels Nav1.5 due to impaired inactivation, enhanced activation, accelerated recovery from inactivation or the late current appearance. In contrast, BrS1 and PCCD are associated with the Nav1.5 loss-of-function, which in electrophysiological experiments can be manifested as reduced current density, enhanced fast or slow inactivation, impaired activation, or decelerated recovery from inactivation. Genetic variants associated with congenital arrhythmias can also disturb interactions of the Nav1.5 channel with different proteins or drugs and cause unexpected reactions to drug administration. Furthermore, mutations can affect post-translational modifications of the channels and their sensitivity to pH and temperature. Here we briefly review the current knowledge on biophysical mechanisms of LQT3, BrS1 and PCCD. We focus on limitations of studies that use heterologous expression systems and induced pluripotent stem cells (iPSC) derived cardiac myocytes and summarize our understanding of genotype-phenotype relations of SCN5A mutations.

An iPSC line (FINi003-A) from a male with late-onset developmental and epileptic encephalopathy caused by a heterozygous p.E1211K variant in the SCN2A gene encoding the voltage-gated sodium channel Nav1.2

Many developmental and epileptic encephalopathies (DEEs) result from variants in cation channel genes. Using mRNA transfection, we generated and characterised an induced pluripotent stem cell (iPSC) line from the fibroblasts of a male late-onset DEE patient carrying a heterozygous missense variant (E1211K) in Nav1.2(SCN2A) protein. The iPSC line displays features characteristic of the human iPSCs, colony morphology and expression of pluripotency-associated marker genes, ability to produce derivatives of all three embryonic germ layers, and normal karyotype without SNP array-detectable abnormalities. We anticipate that this iPSC line will aid in the modelling and development of precision therapies for this debilitating condition.

Implantable loop recorder in Brugada syndrome: Insights from a single-center experience

BackgroundThis study aimed to investigate the characteristics and outcomes of patients diagnosed with Brugada syndrome (BrS) who underwent implantable loop recorder (ILR) insertion during routine clinical activity. MethodsWe conducted a comprehensive screening of all consecutive patients diagnosed with BrS at our institution. We analyzed baseline clinical characteristics, arrhythmic findings, and outcomes. ResultsOut of 147 BrS patients, 42 (29 %) received an ILR, 13 (9 %) underwent implantable cardioverter-defibrillator (ICD) placement, and 92 patients (63 %) continued regular cardiological follow-up. Patients 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, interquartile range 30–52, vs. 43 years, 35–55, p = 0.044) with a higher presence of SCN5A gene mutations (17 % vs. 6 %, p = 0.066) compared to those who continued regular follow-up. Additionally, compared to patients with an ICD, those with an ILR had a significantly lower frequency of positive programmed ventricular stimulation (0 % vs. 91 %, p < 0.001). During a median follow-up period of 14.7 months (4.7–44.8), no deaths occurred among the patients with ILR. Eight individuals (19 %) were diagnosed with arrhythmic findings through continuous ILR monitoring, primarily atrial fibrillation, and asystolic pauses. The median time from insertion to the occurrence of these events was 8.7 months (3.6–46.4). No adverse events related to ILR were reported. ConclusionContinuous monitoring with ILR may facilitate the timely detection of non-malignant rhythm disorders in BrS patients with risk factors but without an indication for primary prevention ICD implantation.