Mutations In SCN5A Gene Research Articles

Diverse biophysical mechanisms in voltage-gated sodium channel Nav1.4 variants associated with myotonia.

Mutations in SCN4A gene encoding Nav1.4 channel α-subunit, are known to cause neuromuscular disorders such as myotonia or paralysis. Here, we study the effect of two amino acid replacements, K1302Q and G1306E, in the DIII-IV loop of the channel, corresponding to mutations found in patients with myotonia. We combine clinical, electrophysiological, and molecular modeling data to provide a holistic picture of the molecular mechanisms operating in mutant channels and eventually leading to pathology. We analyze the existing clinical data for patients with the K1302Q substitution, which was reported for adults with or without myotonia phenotypes, and report two new unrelated patients with the G1306E substitution, who presented with severe neonatal episodic laryngospasm and childhood-onset myotonia. We provide a functional analysis of the mutant channels by expressing Nav1.4 α-subunit in Xenopus oocytes in combination with β1 subunit and recording sodium currents using two-electrode voltage clamp. The K1302Q variant exhibits abnormal voltage dependence of steady-state fast inactivation, being the likely cause of pathology. K1302Q does not lead to decelerated fast inactivation, unlike several other myotonic mutations such as G1306E. For both mutants, we observe increased window currents corresponding to a larger population of channels available for activation. To elaborate the structural rationale for our experimental data, we explore the contacts involving K/Q1302 and E1306 in the AlphaFold2 model of wild-type Nav1.4 and Monte Carlo-minimized models of mutant channels. Our data provide the missing evidence to support the classification of K1302Q variant as likely pathogenic and may be used by clinicians.

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.

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.

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.

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.

Brugada syndrome in Iran: Insights from a 12-year longitudinal study.

Brugada syndrome (BrS) is characterized by ST-segment elevation in the right precordial leads, which is not explained by ischemia, electrolyte disturbances, or obvious structural heart disease. In present study, we aim to evaluate presentation, long-term outcome, genetic findings, and therapeutic interventions in patients with BrS. Between September 2001 and June 2022, all consecutive patients with diagnosis of BrS were enrolled in the present study. All patients gave written informed consent for the procedure, and the local ethical committee approved the study. Of the 76 cases, 79% were proband and 21% were detected during screening after diagnosis of BrS in a family member. Thirty-three (43%) patients had a typical spontaneous electrocardiogram (ECG) pattern. Thirty percent of the patients were symptomatic; symptomatic patients were more likely to have spontaneous type 1 Brugada ECG pattern in their ECGs (p = .01), longer PR interval (p = .03), and SCN5A mutation (p = .01) than asymptomatic patients. The mean PR interval was considerably longer in men than women (p = .034). SCN5A mutation was found in 9 out of 50 (18%) studied patients. Fifteen percent received appropriate implantable cardioverter-defibrillator (ICD) therapy and inappropriate ICD interventions were observed in 17%. Presentation with aborted SCD or arrhythmic syncope was the only predictor of adverse outcome in follow-up (odds ratio: 3.1, 95% confidence interval: 0.7-19.6, p = .001). Symptomatic patients with BrS are more likely to present with spontaneous type 1 Brugada ECG pattern, longer PR interval, and pathogenic mutation in SCN5A gene. Appropriate ICD interventions are more likely in symptomatic patients and those with SCN5A mutation.

Switching from zonisamide to perampanel improved the frequency of seizures caused by hyperthermia in Dravet syndrome: a case report

BackgroundDravet syndrome is a severe epilepsy disorder characterized by drug-resistant seizures and cognitive dysfunction, often caused by SCN1A gene mutations. It leads to neurodevelopmental delays and motor, behavioral, and cognitive impairments, with a high mortality rate. Treatment options include sodium valproate, clobazam, and newer agents such as cannabidiol and fenfluramine. Zonisamide, which is used in some cases, can cause hyperthermia and oligohydrosis. Herein, we present a case of a patient with Dravet syndrome whose seizures were controlled by treating infections and switching from zonisamide to perampanel.Case presentationA 24-year-old Japanese man with Dravet syndrome presented to our department with aspiration pneumonia. The patient had been treated with valproate, sodium bromide, and zonisamide for a long time. His seizures were triggered by hyperthermia. The patient was experiencing a sustained pattern of hyperthermia caused by infection, zonisamide, and persistent convulsions, which caused a vicious cycle of further seizures. In this case, the control of infection and switching from zonisamide to perampanel improved seizure frequency.ConclusionDravet syndrome usually begins with generalized clonic seizures in its infancy because of fever and progresses to various seizure types, often triggered by fever or seizure-induced heat due to mutations in the SCN1A gene that increases neuronal excitability. Seizures usually diminish with age, but the heat sensitivity remains. In this case, seizures were increased by repeated infections, and hyperthermia was induced by zonisamide, resulting in status epilepticus. Perampanel, an aminomethylphosphonic acid receptor antagonist, decreased seizures but caused psychiatric symptoms. It was effective in suppressing seizures of Dravet syndrome in this patient.

Brugada syndrome: variability of clinical and genetic characteristics

AIM: To evaluate the clinical characteristics of patients with diverse genetic variants of Brugada syndrome.&#x0D; MATERIALS AND METHODS: 24 patients (17 male and 7 female) aged 18 to 55 years (median age 32.5 [20; 42] years) with a pattern of Brugada syndrome on electrocardiogram were observed for 3 years. From their ECGs, a type 1 pattern was found in 9 (37.5%) of these patients, type 2 pattern in 14 (58.3%) and type 3 pattern only in 1 patient. The clinical and instrumental study included 12-lead electrocardiogram, 24-hour Holter electrocardiogram monitoring, provocative drug test with intravenous administration of sodium channel blockers (novocainamide), electrophysiologic study according to indications, genealogical history collection and family history of sudden cardiac death, transthoracic echocardiography and cardiac magnetic resonance imaging to detect structural myocardial changes. High-throughput sequencing was utilized to search for mutations in genes linked to the onset of channelopathies and other inherited rhythm disorders.&#x0D; RESULTS: In 15 (62.5%) of the 24 probands included in the study, variants of the nucleotide sequence of pathogenicity classes III–V according to The American College of Medical Genetics and Genomics criteria (2015) were found in genes encoding sodium (SCN5A, SCN10A) and potassium (KCNE3, KCNJ2, KCNJ8, KCNA5) channels, as well as in HCN4 and SNTA1 genes linked with these channels. Moreover, 3 variants were identified in ANK2 gene associated with ankyrinopathies, and 3 variants in DSP and DES genes connected with arrhythmogenic right ventricular cardiomyopathy. Four genetic variants in SCN5A gene were of pathogenicity classes IV and V, the rest were variants of uncertain clinical significance (class III). Six (40.0%) of the 15 genotype-positive patients had several genetic variants. The most severe form of the disease, manifested by the development of ventricular fibrillation with successful resuscitation and subsequent cardioverter-defibrillator implantation, was observed in patients with mutations in SCN5A, SCN10A genes. Recurrent syncope, polymorphic ventricular tachycardia induced by programmed ventricular stimulation during electrophysiologic study, followed by cardioverter-defibrillator implantation were observed in patients with variants KCNJ8 and HCN4, DES and MYH11. In 2 patients with clinical manifestations, no mutations were identified. 13 (54.2%) patients were asymptomatic, while 3 of them had pathogenic and likely pathogenic mutations in SCN5A gene, as well as variants of uncertain clinical significance.&#x0D; CONCLUSION: Thus, this study examined various genetic variants in patients with Brugada syndrome based on their clinical manifestation. The impact of the genotype on the Brugada syndrome phenotype is not unambiguous. The most severe form of the disease with the development of ventricular fibrillation and successful resuscitation with subsequent cardioverter-defibrillator implantation was observed mainly in patients with variants in several genes (SCN5A and JUP, KCNJ8 and HCN4, DES and MYH11). This substantiate the idea that Brugada syndrome, along with monogenic, may also have a polygenic nature of the disease, in which the clinical phenotype is determined by variants in respective genes linked to the onset of cardiovascular disorders.

Bilateral and synchronous “dents de scie” spikes: A highly specific EEG pattern of young adult Dravet syndrome

BackgroundIn Dravet syndrome (DS), EEGs evolve over time. ObjectiveTo describe a peculiar EEG pattern in two adults with a de novo SCN1A gene mutation, in exon 5 (case 1) and 9 (case 2). MethodsTwo female patients underwent a prolonged video EEG (24 h) as part of their epilepsy assessment. ResultsIn both cases, the EEG showed a very peculiar and stereotypical pattern of bilateral synchronous spikes at about 5–6 Hz. This activity was present during wakefulness and highly activated at sleep onset and in NREM sleep, which could show nearly continuous spike activity. This activity dramatically decreased in REM sleep and after awakening. This pattern of “dents de scie” (sawtooth) spikes maintained the same morphology throughout the entire EEG recording. In both patients, the spikes were favored by passive eye closure. During wakefulness, the spikes could evolve into atypical absences while keeping the same “dents de scie” pattern. Neither patient had tonic or myoclonic seizures at the time of the EEG assessment. Both were moderately retarded, and neither one had a typical DS gait disorder. Previous EEG recordings of case 1 performed at 9.5 and 18.5 years showed spike-waves, but the morphology did not correspond to the EEG recording observed at 22 years. ConclusionsBoth patients have a similar electro-clinical phenotype. This “dents de scie” pattern appears to be very specific and could be pathognomonic in a subgroup of young adults with DS. Results of sleep EEG recording could be added to the diagnostic criteria for this syndrome.

Modern treatment of epileptic encephalopathies in young children: improvement of precision medicine

Background. Treatment of epileptic seizures in young children, especially with epileptic encephalopathies (EE), is a difficult task, which is impossible in modern conditions without the use of a personified (precision) therapy. The diagnostic algorithm for EE must include genetic examination by the next-generation sequencing, which makes it possible to prescribe targeted therapy depending on the genetic etiology of the disorder. The article presents the results of own research on the effectiveness and approaches to targeted therapy of genetic epileptic encephalopathies in young children. Materials and methods. Fifty-eight children aged 0–3 years with clinical manifestations of epileptic encephalopathies, onset of seizures in the first year of life and diagnosed genetic etiology were included in the study. Pathogenic variants in genes associated with the development of epileptic seizures were identified in all children using the next-generation sequencing. The study included assessment of neurological status, history collection, evaluation of semiology and seizure type, development and screening for autism spectrum disorders at the age of 18 and 24 months, video-electroencephalography during night sleep, magnetic resonance imaging of the brain, assessment of antiepileptic treatment received by the child. Results. Of 58 children with EE who were prescribed antie­pileptic drugs, 10 (17.2 %) received monotherapy, 40 patients (69 %) received combined therapy with two anticonvulsants, and 8 children (13.8 %) — combined therapy with three or more anticonvulsants. Levetiracetam (31 patients), valproic acid salt preparations (20 cases), topiramate (11 children) and vigabatrin (10 cases) were most used antiepileptic drugs. In all examined patients with EE, we used schemes of targeted (persona­lized) antiepileptic therapy focused on the genetic etiology of the disorder. In children with tuberous sclerosis caused by mutations in the TSC1 and TSC2 genes, vigabatrin (50–150 mg/kg per day) was included in the antiepileptic therapy and showed efficacy in 75.0 % (9/12) of children with infantile spasms. Corticosteroids (adrenocorticotropic hormone or prednisone) were additionally included in the treatment regimen and showed effectiveness in 66.7 % of cases (4/6). In children with mutations in SCN1A gene, combined therapy including valproic acid, topiramate and clobazam, or valproic acid with levetiracetam and corticosteroids was used, which showed effectiveness in reducing the frequency of seizures in 100 % of cases. Conclusions. Epileptic encephalopathies are a heterogeneous group of genetic disorders in young children that are difficult to treat and often have a malignant course. Since standard antiepileptic drugs are often insufficiently effective in epileptic encephalopathies, the use of targeted therapy drugs and alternative treatments such as hormone therapy are extremely important. The goal of treatment for epileptic encephalopathies is not only to control seizures, but also to prevent the development of neurological and cognitive deficits and restore lost functions.

SCN1A Pathogenic Variants in a Turkish Cohort with GEFS+ Spectrum and Dravet Syndrome.

The α subunit of voltage-gated sodium channels in mammals is encoded by nine different genes, and the mutations in the SCN1A, SCN2A, SCN3A and SCN8A genes highly expressed in the CNS have been associated with epilepsy phenotypes. This study aimed at investigating the frequency of SCN1A gene mutations in DS and GEFS+ spectrum phenotype cases and discussing the molecular results in the context of genotype-phenotype correlation. 15 patients diagnosed with DS and 54 patients meeting the GEFS+ spectrum criteria were included in this study. All patients were evaluated by next-generation sequencing (NGS) method using a SCN1A gene commercial kit. A total of 17 different variants were detected in 18 index cases (26%) as a result of sequencing of the SCN1A gene. Note that 7 of the total 17 different variations (p.M1R, p.M147T, p.I767L, p.N1391Ifs *5, p.R1886G, p.E1915G, p.R1933Q) were novel variations. Of the 18 cases with variation in the SCN1A gene, 12 had DS and 6 had GEFS+ phenotype. The variations were de novo in all DS cases and in one case with a GEFS+ phenotype; in 5 GEFS+ cases, it was inherited from the affected parent. This study contributed to the mutation spectrum with the novel variations detected in cases with DS and GEFS+ phenotype. SCN1A genetic analysis can help in determining antiepileptic drugs to be selected or avoided in cases with mutations. The elucidation of the molecular etiology makes it possible to provide the family with effective genetic counseling for future pregnancies.

Morphometry and network-based atrophy patterns in SCN1A-related Dravet syndrome.

Mutations of the voltage-gated sodium channel SCN1A gene (MIM#182389) are among the most clinically relevant epilepsy-related genetic mutations and present variable phenotypes, from the milder genetic epilepsy with febrile seizures plus to Dravet syndrome, a severe developmental and epileptic encephalopathy. Qualitative neuroimaging studies have identified malformations of cortical development in some patients and mild atrophic changes, partially confirmed by quantitative studies. Precise correlations between MRI findings and clinical variables have not been addressed. We used morphometric methods and network-based models to detect abnormal brain structural patterns in 34 patients with SCN1A-related epilepsy, including 22 with Dravet syndrome. By measuring the morphometric characteristics of the cortical mantle and volume of subcortical structures, we found bilateral atrophic changes in the hippocampus, amygdala, and the temporo-limbic cortex (P-value < 0.05). By correlating atrophic patterns with brain connectivity profiles, we found the region of the hippocampal formation as the epicenter of the structural changes. We also observed that Dravet syndrome was associated with more severe atrophy patterns with respect to the genetic epilepsy with febrile seizures plus phenotype (r = -0.0613, P-value = 0.03), thus suggesting that both the underlying mutation and seizure severity contribute to determine atrophic changes.

Single coronary artery presenting dilated cardiomyopathy and hyperlipidemia with the SCN5A and APOA5 gene mutation: A case report and review of the literature.

We present a 55-year-old man with chest tightness and dyspnoea after activity lasting for 2 months who was diagnosed with single coronary artery (SCA) and presented with dilated cardiomyopathy (DCM) with the c.1858C > T mutation in the SCN5A gene. The computed tomography coronary angiogram (CTCA) showed congenital absence of the right coronary artery (RCA), and the right heart was nourished by the left coronary artery branch with no apparent stenosis. Transthoracic echocardiography (TTE) revealed enlargement of the left heart and cardiomyopathy. Cardiac magnetic resonance imaging (CMR) revealed DCM. Genetic testing showed that the c.1858C > T variant of the SCN5A gene could lead to Brugada syndrome and DCM. SCA is a rare congenital anomaly of the coronary anatomy, and this case reported as SCA accompanied by DCM is even rarer. We present a rare case of a 55-year-old man with DCM with the c.1858C > T (p. Arg620Cys)/c.1008G > A (p.(Pro336=) variant of the SCN5A gene, congenital absence of RCA, and c.990_993delAACA (p. Asp332Valfs*5) variant of the APOA5 gene. To our knowledge, this is the first report of DCM combined with the SCN5A gene mutation in SCA after searching the PubMed, CNKI and Wanfang databases.

Molecular mechanisms of Brugada syndrome related to p.R211H variant in RRAD gene

Brugada syndrome (BrS) is a rare inherited arrhythmic disorder with increased risk of sudden cardiac death. More than 20% of BrS cases have been linked to mutations in SCN5A gene, which encodes the voltage-gated Na+ channel Nav1.5. We have previously identified a variant (p.R211H) in RRAD gene that co-segregates with a familial form of BrS. RRAD encodes the Rad (Ras associated with diabetes) monomeric GTPase. RRAD p.R211H variant induces a reduction of the Na+ current (INa) amplitude and morphological defects in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs). To determine the mechanisms linking RRAD variant to INa reduction and the cardiac phenotype of a knock-in (KI) mouse model carrying the equivalent p.R210H variant. Mouse phenotyping was performed using ECG recordings, and patch-clamp technique on ventricular cardiomyocytes. Western blot and immunostaining were performed on mouse ventricular tissue and hiPSC-CMs. Co-immunoprecipitation experiments were performed on mouse ventricle and on HEK cells transfected with plasmids encoding Nav1.5 and either wild type (WT) or p.R211H-Rad. Twelve-week-old homozygous KI mice showed longer QRS complexes (12.3 ± 0.2 ms; n = 22) than WT (11.1 ± 0.2 ms; n = 13; P < 0.01) and heterozygous KI (11.7 ± 0.2 ms; n = 22) mice, a defect persisting in older mice. In homozygous KI mice INa density was decreased by 38% compared to WT mice (P < 0.01; n = 22 & 19). This was linked to a lower ventricular expression of Nav1.5 (by 49% in left ventricle; P < 0.01), Connexin 43 (Cx43; by 34%; P = 0.06) and Rad (by 33%; P < 0.01) in homozygous KI mice versus WT mice. Similar results were obtained in the hiPS-CMs of the BrS index case, in which an altered location of Rad (more nuclear than cytoplasmic) and Cx43 (lower clusters, but of larger size) was also observed, compared to control hiPS-CMs. Coimmunoprecipitation studies on left ventricular tissue of 30-week-old WT mice showed an interaction of Rad with Nav1.5, a result that was confirmed in HEKs, and of Rad with Cx43. Our results suggest that BrS-related RRAD p.R211H variant affects Rad expression and leads to a decrease of Nav1.5 and Cx43 expression, explaining both sodium current lower amplitude and conduction defects. Our results also show that Rad interacts with Nav1.5, and possibly Cx43.

PO-04-164 MOLECULAR MECHANISMS OF BRUGADA SYNDROME RELATED TO P.R211H MUTATION IN RRAD GENE

Brugada syndrome (BrS) is a rare inherited arrhythmic disorder with increased risk of sudden cardiac death. More than 20% of BrS cases have been linked to mutations in SCN5A gene, which encodes the voltage-gated Na+ channel Nav1.5. We have previously identified a novel variant (p.R211H) in RRAD gene that co-segregates with a familial form of BrS. RRAD encodes the Rad (Ras associated with diabetes) monomeric GTPase. We have shown that p.R211H RRAD variant induces a reduction of the Na+ current (INa) amplitude and morphological defects in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs). To characterize the cardiac phenotype of a knock-in (KI) mouse model carrying the equivalent (p.R210H) mutation and identify the mechanisms linking RRAD mutation to INa decrease and conduction defects. Mouse phenotype was determined using ECG recordings, and patch-clamp technique on ventricular cardiomyocytes. Western blot and immunostaining were performed on mouse ventricular tissue and iPSC-CMs. Co-immunoprecipitation experiments were performed on mouse ventricle and on HEK cells transfected with plasmids encoding Nav1.5 and either wild type or p.R211H-Rad. Homozygous KI mice (12 weeks old) showed longer QRS complexes (12.3±0.2 ms; n=22) than wildtype (WT; 11.1±0.2 ms; n=13; P<0.01) and heterozygous KI (11.7±0.2 ms; n=22) mice, a defect persisting in older mice. Homozygous KI mice showed a 38% decrease in INa density versus WT mice (P<0.01; n=22 & 19). This was associated with a lower ventricular expression of Nav1.5 (by 49% in left ventricle; P < 0.01) and Connexin 43 (Cx43; by 34%; P = 0.06) in homozygous KI mice versus WT mice. Rad expression was also decreased by 33% (P < 0.05). Similar results were obtained in the BrS index case hiPS-CMs, in which an altered location of Rad (more nuclear than cytoplasmic) and Cx43 (lower clusters, but of larger size) was also observed, compared to control hiPS-CMs. Coimmunoprecipitation studies on left ventricular tissue of 30-week-old WT mice showed an interaction of Rad with Nav1.5, and of Rad with Cx43. Nav1.5 interaction with Rad has been confirmed in HEK cells, which are used to determine the effect of the mutation on this interaction. To conclude, BrS-related RRAD p.R211H mutation affects Rad expression and leads to a decrease of Nav1.5 and Cx43 cardiac expression, explaining both sodium current lower amplitude and conduction defects.

Rare Compound Heterozygous Missense Mutation of the SCN5A Gene with Childhood-Onset Sick Sinus Syndrome in Two Chinese Sisters.

Sick sinus syndrome (SSS) is a group of syndromes characterized by pathological changes in the sinoatrial node and its adjacent tissues. Although several mutations in the SCN5A gene have been associated with early-onset SSS, pediatric patients are still less common. Here, we report a rare compound missense mutation in the SCN5A gene [c.2893C>T (p. R965C) and c.2431C>T (p. R811C) ] in two sisters with childhood-onset SSS in Chinese population. The proband (5 years and 5 months old) was the second child of a clinically normal and nonconsanguineous couple. Her elder sister was 12 years old and had been implanted with a pacemaker because of the diagnosis of SSS at another hospital one year ago. The proband was presented to the hospital with a slowed heart rate and reduced endurance exercise capacity for more than three months. After a comprehensive clinical examination, she was diagnosed with SSS and underwent pacemaker implantation. Exome and Sanger sequencing were used to determine the compound heterozygous missense mutation of [c.2893C>T (p. R965C) and c.2431C>T (p. R811C) ] in the SCN5A in the patient and her elder sister. Each healthy parent carried a different heterozygous missense mutation. The compound heterozygous mutation of c.2893C>T (p. R965C) and c.2431C>T (p. R811C) rather than the single mutation might be the primary cause of familial early-onset SSS in Chinese population. Our current findings expanded the current understanding of the SCN5A gene mutations. We further confirmed the essential role of the SCN5A gene on the diagnosis, family cascade screening, early intervention, and prognostic evaluation of SSS.

Patient profile, management, and quality of life associated with Dravet syndrome: a cross-sectional, multicentre study of 80 patients in Spain

The aim of this study was to describe the profile of patients diagnosed with Dravet syndrome (DS), their clinical management, and the impact of DS on their quality of life (QoL) and family. Data of 80 patients from 11 centres in Spain was collected. Patients (47.5% female) were 12.7 (9.6) years on average (SD, standard deviation). Despite the first episode occurred when patients were a mean (SD) of 0.4 (0.2) years, DS was not diagnosed until they were 6.9 (10.1) years old. The majority (86.7%) had SCN1A gene mutations and 73.4% had seizures during the last year (mostly generalized motor seizures [47.8%]). The mean (SD) number of status epilepticus episodes was 3.6 (8.0) since diagnosis and 0.1 (0.5) in the last year. On the Health Utilities Index Mark (HUI) multi-attribute scale, the mean global score (SD) was 0.56 (0.24) in HUI2 and 0.32 (0.37) in HUI3. The impact of the disease was severe in most patients (HUI2, 81%; HUI3, 83.5%). In the Care-related QoL (CarerQol) the mean (SD) well-being score was 7.2 (2.1). Most caregivers (90%) were satisfied with their caregiving tasks, although 75% had difficulties combining these tasks with daily activities, 68.8% reported mental health problems and 61.2% physical problems.

Coexistence of SCN4A and CLCN1 mutations in a family with atypical myotonic features: A clinical and functional study

Non-dystrophic myotonias include several entities with possible clinical overlap, i.e. myotonia congenita caused by CLCN1 gene mutations, as well as paramyotonia congenita and sodium channel myotonia caused by SCN4A gene mutations. Herein, we describe the clinical features of five relatives affected by clinical and neurophysiological myotonia, with an aspecific and mixed phenotype. Next-generation sequencing identified the novel p.K1302R variant in SCN4A and the p.H838P variant in CLCN1. Segregation of the two mutations with the disease was confirmed by genotyping affected and non-affected family members. Patch-clamp experiments showed that sodium currents generated by p.K1302R and WT hNav1.4 were very similar. Mutant channel showed a small negative shift (5 mV) in the voltage-dependence of activation, which increased the likelihood of the channel to open at more negative voltages. The p.H838P mutation caused a reduction in chloride current density and a small voltage-dependence shift towards less negative potentials, in agreement with its position into the CBS2 domain of the C-terminus. Our results demonstrated that the mild functional alterations induced by p.K1302R and p.H838P in combination may be responsible for the mixed myotonic phenotypes. The K1302R mutant was sensitive to mexiletine and lamotrigine, suggesting that both drugs might be useful for the K1302R carriers.

The Generation of Human iPSC Lines from Three Individuals with Dravet Syndrome and Characterization of Neural Differentiation Markers in iPSC-Derived Ventral Forebrain Organoid Model.

Dravet syndrome (DRVT) is a rare form of neurodevelopmental disorder with a high risk of sudden unexpected death in epilepsy (SUDEP), caused mainly (&gt;80% cases) by mutations in the SCN1A gene, coding the Nav1.1 protein (alfa-subunit of voltage-sensitive sodium channel). Mutations in SCN1A are linked to heterogenous epileptic phenotypes of various types, severity, and patient prognosis. Here we generated iPSC lines from fibroblasts obtained from three individuals affected with DRVT carrying distinct mutations in the SCN1A gene (nonsense mutation p.Ser1516*, missense mutation p.Arg1596His, and splicing mutation c.2589+2dupT). The iPSC lines, generated with the non-integrative approach, retained the distinct SCN1A gene mutation of the donor fibroblasts and were characterized by confirming the expression of the pluripotency markers, the three-germ layer differentiation potential, the absence of exogenous vector expression, and a normal karyotype. The generated iPSC lines were used to establish ventral forebrain organoids, the most affected type of neurons in the pathology of DRVT. The DRVT organoid model will provide an additional resource for deciphering the pathology behind Nav1.1 haploinsufficiency and drug screening to remediate the functional deficits associated with the disease.

820 VARIABLE EXPRESSIVITY OF SCN5A MUTATION IN A FAMILY WITH CARDIAC DYSFUNCTION AND SUSPECTED BRUGADA SYNDROME

Abstract Background Inherited arrhythmogenic diseases (IADs) represent a group of life-threatening genetic cardiomyopathies that predispose young individuals to sudden cardiac death (SCD), often in the absence of structural cardiac alterations. Some IADs share mutations in genes encoding for surface membrane ion channels, and the gene for the alpha subunit of the voltage-dependent channel Nav1.5 (SCN5A) is among the ones mostly involved. Brugada syndrome represents one of the possible spectrum deriving from SCN5A gene mutations, and it is characterized by “loss-of-function” genetic alterations. Interestingly, both loss-of and gain-of-function mutations have been associated with familial dilated cardiomyopathy (DCM). Materials and Methods In the context of an Italian Ministry of Health-funded research project devoted to identify genetic isolates of IADs in the Calabria region, we found a family affected by different cardiovascular manifestations including cardiac dysfunction, ventricular tachyarrhythmias, atrioventricular block and suspected Brugada syndrome pattern in first-degree relatives in association with a newly identified SCN5A mutation. Results The proband came to our attention with a diagnosis of DCM, echocardiographic assessment of reduced left ventricular ejection fraction (EF) to 38%, high occurrence of premature ventricular complexes (PVCs) and first-degree atrioventricular block (PR = 240ms) at electrocardiogram. In spite of negative electrophysiologic study, he underwent implantable cardioverter defibrillator implantation (ICD), hypothesizing lamin A/C mutation; genetic analysis turned out negative for lamin A/C and CACNA1C, while a SCN5A gene mutation (c.4554 G&amp;lt;T p.Gln1518His) was detected. Proband's sons and his brother underwent clinical evaluations as well as genetic testing: the second son's ECG showed first-degree AV block (PR = 220ms), QTc interval 426ms, and coved type ST-segment elevation of 1.5mm, in the context of normal cardiac function; the remaining male (see Figure 1) was asymptomatic, while the daughter had a hypokinetic non-dilated cardiomyopathy. The proband's brother had first-degree AV block and very frequent monomorphic ventricular couplets. Conclusions These phenotypic differences in first degree-relatives, can be explained by the concept of variable expressivity of genes, according to which individuals carrying the same mutation can have different clinical features. Therefore, the search for genetic mutations assumes a preponderant role for stratifying the risk of SCN5A-related diseases and to adopt appropriate diagnostic and therapeutic strategies.