SCN5A Research Articles

P.100 Early recognition of unique conventional and amplitude-integrated EEG patterns and clinical semiology of neonatal seizures caused by SCN2A and KCNQ3 mutations

Background: Early recognition of neonatal seizures secondary to pathogenic variants in potassium or sodium channel coding genes is crucial, as these seizures are often resistant to commonly used anti-seizure medications, but respond well to sodium-channel blockers. We report a unique aEEG pattern in neonatal seizures caused by SCN2A and KCNQ3 pathogenic variants, as well as adding regular EEG description. Methods: International multicentre descriptive study, reporting clinical characteristics, aEEG and conventional EEG findings of 10 newborns with seizures due to pathogenic SCN2A and KCNQ3 gene variants. Results: Seizures started in the first postnatal week. Seizure semiology typically included tonic posturing with apnea and desaturation. The aEEG showed a characteristic sequence of brief onset with a decrease, followed by a quick rise, and then postictal amplitude attenuation. This pattern correlated with bilateral attenuation in the EEG at onset, followed by rhythmic discharges ending in several seconds of post-ictal amplitude suppression. The majority of patients became seizure free upon initiation of a sodium-channel blocker. Conclusions: Neonatal seizures caused by SCN2A and KCNQ3 mutations can be recognized by a characteristic ictal aEEG pattern and clinical semiology. Awareness of this pattern facilitates the prompt initiation of precision treatment with sodium-channel blockers even before genetic test results are available.

Electrophysiological characterization of schizophrenia-associated variants in NaV1.2 sodium channel

IntroductionA major pathophysiological hypothesis of schizophrenia states an increased activity of glutamatergic neurons leading to an imbalance of neural excitation and inhibition (E/I-imbalance). One potential molecular mechanism of E/I-imbalance is a dysfunction of voltage-gated sodium channels, which are crucial for the generation of action potentials, the fundamental event of neuronal excitation. Indeed, patients with schizophrenia exhibit an increased burden of rare exonic variants of sodium channel genes, but the literature describing their electrophysiological effect is scarce.ObjectivesThe aim of this project is to assess the functional impact of three mutations of the Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A) gene / NaV1.2 channel which were identified in four patients with schizophrenia, using a heterologous expression system.MethodsThree variants of the human SCN2A gene (R850P, V1282F and S1656P) were created using site-directed mutagenesis. HEK293T cells transfected with either the mutant or wild type constructs are being investigated by voltage-clamp technique, applying activation, steady-state fast inactivation, use dependency and ramp protocols.ResultsAll three mutated constructs were successfully created. Preliminary recordings from the V1282F mutant indicate a shift of both the activation and steady-state fast inactivation to the hyperpolarized direction.ConclusionsIn a subgroup of patients, E/I imbalance may be a consequence of Nav1.2 mutations leading to increased excitability of glutamatergic neurons. By integrating insights from different mutations we aim to identify traits of a potentially shared disease pathway which may provide a basis for the development of novel therapeutics.DisclosureNo significant relationships.

Customised targeted massively parallel sequencing enables more precise diagnosis of patients with epilepsy.

Advancement in genetic technology has led to the identification of an increasing number of genes in epilepsy. This will provide a lot of information in clinical practice and improve the diagnosis and treatment of epilepsy. To show the importance of genes in the next-generation sequencing (NGS) panel during the evaluation of epilepsy and to emphasise the importance of genetic studies in different populations for the evaluation of genes that cause disease. This was a single-centre retrospective cohort study of 80 patients who underwent NGS testing with a customised epilepsy panel. In a total of 54 (67.5%) out of 80 patients, pathogenic or likely pathogenic variants and variants of uncertain significance (VOUS) were identified according to the American College of Medical Genetics and Genomics criteria. Pathogenic or likely pathogenic variants (n = 35) were identified in 29 (36.25%) out of 80 individuals. VOUS (n = 34) were identified in 28 (35%) out of 80 patients. Pathogenic, likely pathogenic and VOUS were most frequently identified in TSC2 (n = 11), SCN1A (n = 6) and TSC1 (n = 5) genes. Other common genes were KCNQ2 (n = 3), AMT (n = 3), CACNA1H (n = 3), CLCN2 (n = 3), MECP2 (n = 2), ASAH1 (n = 2) and SLC2A1 (n = 2). NGS-based testing panels contribute to the diagnosis of epilepsy and might change the clinical management by preventing unnecessary and potentially harmful diagnostic procedures and management in patients. Thus, our results highlight the benefit of genetic testing in children suffering with epilepsy.

Concurrent sodium channel myotonia and amyotrophic lateral sclerosis supports shared pathogenesis

Amyotrophic lateral sclerosis (ALS) is an invariably fatal adult-onset neurodegenerative disorder; 10% of ALS is autosomal dominant but even sporadic ALS exhibits significant heritability. Dysfunctional voltage- gated sodium channels (NaVs) have been implicated in both sporadic and familial forms of the disease, but it is unclear whether these are an upstream cause or a downstream consequence. Rare missense mutations in the SCN4A gene, encoding NaV1.4, disrupt membrane excitability and cause autosomal dominant muscle channelopathies such as myotonia and hypo/hyperkalaemic periodic paralysis. We report two patients with congenital SCN4A channelopathy due to p.S1159P and p.R672H mutations who developed ALS in later life. Based on our finding we hypothesised that ALS may be linked to genetic mutations in other voltage gated ion channels. Rare-variant burden testing using whole genome sequenc- ing data from 4,495 ALS patients and 1,925 controls within the superfamily of voltage-gated ion channels (Accession: ssf81324) identified one gene, SCN7A, which passed multiple testing correction (p=0.0002, Firth logistic regression) consistent with an enrichment of ALS-associated mutations. We propose that genetic mutations within NaVs leading to neuromuscular excitotoxicity are an upstream cause of ALS. Early identification of ALS patients carrying NaV mutations might lead to personalised therapy with a sodium channel-blocking agent such as riluzole.j.franklin@doctors.org.uk

The Gain-of-Function R222S Variant in Scn11a Contributes to Visceral Hyperalgesia and Intestinal Dysmotility in Scn11a R222S/R222S Mice.

BackgroundThe SCN11A gene encodes the α-subunit of the Nav1. 9 channel, which is a regulator of primary sensory neuron excitability. Nav1.9 channels play a key role in somatalgia. Humans with the gain-of-function mutation R222S in SCN11A exhibit familial episodic pain. As already known, R222S knock-in mice carrying a mutation orthologous to the human R222S variant demonstrate somatic hyperalgesia. This study investigated whether Scn11aR222S/R222S mice developed visceral hyperalgesia and intestinal dysmotility.MethodsWe generated Scn11aR222S/R222S mice using the CRISPR/Cas9 system. The somatic pain threshold in Scn11aR222S/R222S mice was assessed by Hargreaves' test and formalin test. The excitability of dorsal root ganglia (DRG) neurons was assessed by whole-cell patch-clamp recording. Visceralgia was tested using the abdominal withdrawal reflex (AWR), acetic acid-induced writhing, and formalin-induced visceral nociception tests. Intestinal motility was detected by a mechanical recording of the intestinal segment and a carbon powder propelling test. The excitability of the enteric nervous system (ENS) could influence gut neurotransmitters. Gut neurotransmitters participate in regulating intestinal motility and secretory function. Therefore, vasoactive intestinal peptide (VIP) and substance P (SP) were measured in intestinal tissues.ResultsThe R222S mutation induced hyperexcitability of dorsal root ganglion neurons in Scn11aR222S/R222S mice. Scn11aR222S/R222S mice exhibited somatic hyperalgesia. In addition, Scn11aR222S/R222S mice showed lower visceralgia thresholds and slowed intestinal movements when compared with wild-type controls. Moreover, Scn11aR222S/R222S mice had lower SP and VIP concentrations in intestinal tissues.ConclusionsThese results indicated that Scn11aR222S/R222S mice showed visceral hyperalgesia and intestinal dysmotility.

Plasma MicroRNAs as noninvasive diagnostic biomarkers in patients with Brugada syndrome.

BackgroundBrugada syndrome (BrS) can be diagnosed by a type 1 BrS tracing in a 12-lead electrocardiogram (ECG). However, there are daily variations in the ECGs of BrS patients, which presents a challenge when diagnosing BrS. Although many susceptibility genes have been identified, the SCN5A gene is reportedly the main causative gene of BrS. However, most patients do not have an evidence of genetic predisposition to develop BrS. In addition, the diagnosis and risk stratification for ventricular fibrillation (VF) in patients with BrS presents some problems. Meanwhile, circulating micro RNAs (miRNAs) have drawn increased attention as potential biomarkers of various diseases. We hypothesize that circulating miRNAs may be potential diagnostic biomarkers for BrS.MethodsWe enrolled 70 Japanese BrS patients and 34 controls for the screening cohort. A total of 2,555 miRNA sequences were detected using the 3D-Gene miRNAs labeling kit and 3D-Gene Human miRNAs Oligo Chip. We compared the expression of the miRNAs between the BrS patients and the controls. We validated whether the miRNA were significantly up- or downregulated in the screening cohort using RT-PCR. We also enrolled 72 Japanese BrS patients and 56 controls to replicate these miRNAs.ResultsEight miRNAs (hsa-miR-223-3p, hsa-miR-22-3p, hsa-miR-221-3p, hsa-miR-4485-5p, hsa-miR-550a-5p, hsa-miR-423-3p, hsa-miR-23a-3p, and hsa-miR-30d-5p) were downregulated, and one miRNA (hsa-miR-873-3p) was upregulated by more than 3-fold in BrS patients. The multivariate logistic regression analysis determined that hsa-miR-423-3p, hsa-miR-223-3p, and hsa-miR-23a-3p were independently associated with BrS (P < 0.0001). The AUC based on cross validation was 0.871 with a sensitivity and specificity of 83.5% and 81.1%, respectively.ConclusionsThe plasma miRNAs are potential noninvasive biomarkers of BrS, and the constructed logistic model was useful for discriminating BrS.

SCN5A mutation is associated with severity of abnormalities detected by ECG imaging in Brugada syndrome

Abstract Funding Acknowledgements Type of funding sources: None. Background Brugada syndrome (BrS) is caused by mutations in SCN5A gene in 15%-20% of cases. Previous studies showed a worse prognosis in SCN5A mutation carriers (SCN5A+). Purpose To evaluate genotype-phenotype correlation with ECG imaging (ECGI). Methods All consecutive patients who underwent ECGI with CardioInsight for BrS were retrospectively analyzed. ECGI parameters analyzed before and after ajmaline administration were: 1) Right ventricular outflow tract (RVOT) activation time (RVOT-AT) and 2) RVOT recovery time (RVOT-RT). Genetic analysis was performed in all patients with SeqCap EZ Human Exome Probes v3.0 for BrS. Patients were defined as SCN5A+ if they had a pathogenic/likely pathogenic mutation in SCN5A following ACMG guidelines. Results Thirty-six BrS patients were included and 8 patients (22%) were SCN5A+. At baseline ECGI map, mean RVOT-RT was higher in SCN5A+ [384.8 ms vs 362 ms, p=0.037], with no difference in RVOT-AT (p=0.65). After ajmaline administration SCN5A+ patients showed higher RVOT-AT [125.6 ms vs 102.4 ms, p=0.045] (Figure) and higher RVOT-RT [426.4 ms vs 400 ms, p=0.038]. At univariate logistic regression, baseline RVOT-RT was a predictor of SCN5A mutation (specificity: 0.64, sensitivity 0.88, AUC 0.75). Conclusion In BrS syndrome SCN5A+ patients exhibit marked depolarization and repolarization abnormalities as assessed by ECGI.

P379 CARDIAC CONDUCTION DISEASE IN YOUNG PEOPLE: DON‘T FORGET THE GENES

Abstract Introduction Heart rhythm disorders in young individuals are often associated with mutations in genes encoding ion channels. A frequently encountered mutation is in the SCN5A gene, which encodes the alpha subunit of the sodium channel. Mutations in this gene can be expressed through a wide spectrum of phenotypic manifestations including rhythm disturbances, structural cardiac changes and/or overlap syndromes (long/short QT syndrome, Brugada syndrome, sinus node syndrome, progressive cardiac conduction disorder, dilated cardiomyopathy and arrhythmogenic cardiomyopathy). Case Report 23–year–old boy with no relevant history. During the induction of anaesthesia for a surgical intervention of nasal polyposis, episodes of asystole with a maximum duration of about 6 seconds were documented. During the hospital stay, a syncopal episode followed, with electrocardiographic monitoring showing a sinus arrest of approximately 16 seconds (Fig. 1). Echocardiography and cardiac resonance ruled out the presence of structural alterations. In view of the documented bradyarrhythmia, the clinical findings and the absence of secondary causes, a dual–chamber pacemaker implant was performed. The patient was subsequently admitted for ectopic atrial tachycardia (Fig. 2) symptomatic for heart palpitations, treated with medical therapy. After careful reconstruction of the family tree (Fig. 3), a positive family history of bradyarrhythmias emerged (grandfather and sister of the paternal grandfather were pacemaker carriers). A blood sample was taken for genetic analysis and a mutation in the SCN5A gene (ex28 c5207 A&amp;gt;G) was detected. The genetic study was extended to first–degree relatives (still in progress). During follow–up visits the patient reported subjective well–being and at pacemaker interrogation evidence of 29% atrial pacing, in the absence of ventricular pacing. Conclusions When faced with a young patient with a conduction disorder, once possible secondary causes have been excluded, genetic analysis should be included in routine examinations because it helps to define an adequate clinical–instrumental follow–up and to identify, at an early stage, the possible progression of the disease and/or the onset of concomitant structural heart disease. Family genetic screening is also essential in order to identify patients at risk of developing the disease.

P175 ELECTROCARDIOGRAPHIC ELEMENTS ASSOCIATED WITH ARRHYTHMIC RISK IN BRUGADA SYNDROME

Abstract Introduction Brugada syndrome is an inherited disease characterized by an increased arrhythmic risk and sudden death. The type 1 ECG pattern is the only standard required to make a diagnosis but the stratification of arrhythmic risk remains a controversial element. Some electrocardiographic signs have been described as associated with an increased arrhythmic risk. We present a case of Brugada Syndrome in which different electrocardiographic elements of incremental risk could have predicted the later evolution of the clinical case. Clinical Case A 68–year–old male patient with family history of sudden cardiac death (brother 51aa) and occasional finding of Brugada type 1 ECG pattern (2009), implanted with Medtronic bicameral ICD device in primary prevention. The electrocardiogram shows type 1 Brugada pattern in the right precordials with characteristic ST–elevation, followed by a concave ST segment, one of the signs of association with an increased arrhythmic risk; first–degree AV block with a long PR (323 msec) associated with the presence of the SCN5A mutation and an increased risk of arrhythmic events and sudden cardiac death; in the end a fragmented QRS in V2 with extended duration (153 ms). At the next follow–up the patient had some episodes of arrhythmic storm, effectively treated by the device; in consideration of the arrhythmic burden, he was subjected to epicardial ablation and was also subjected to genetic analysis that was positive for pathological mutation on the SCN5A gene (SCN5A ex 22: c.3929C&amp;gt;T;p.Pro1310Leu). Conclusions Brugada Syndrome can be diagnosed from the type 1 ECG pattern, but the current risk stratification score remains a controversial element. Electrocardiographic signs of malignancy can contribute to create a multiparametric evaluation of the patient in order to predict future arrhythmic events. Fig 1. ECG: RS 74 bpm, first–degree AV block (PR 323 msec), BFA, Brugada type 1 pattern (QRS 153 ms), fragmented QRS in V2. Fig. 2 Family tree.

P377 CHALLENGING RISK STRATIFICATION IN BRUGADA SYNDROME: A CASE OF VENTRICULAR TACHYCARDIA IN A LOW–RISK PATIENT

Abstract A 22–year–old healthy athlete was referred to our hospital for electrocardiographic findings consistent with intermittent type–1 Brugada pattern. A routine electrocardiogram (ECG) performed during a check–up showed sinus rhythm, normal PR interval and QRS duration, rSR’ with a coved type ST–segment elevation in V2 lead (Figure 1).The patients denied history of syncope or palpitations and his family history did not show sudden cardiac death or Brugada syndrome (BrS). Transthoracic echocardiography was normal and a 12–lead Holter monitoring revealed the intermittence of the type–1 Brugada pattern. The patient was hospitalized for further investigation. On admission, ECG showed a type–2 Brugada pattern; therefore, Ajmaline challenge test (1 mg/kg injection in 10 minutes) was performed and resulted positive. Subsequently, the patient underwent an electrophysiological study (EPS) which was negative for induction of sustained arrhythmias. The protocol adopted consisted of double extrastimuli programmed electrical stimulation until refractoriness (drive 600/220 ms and 400/210 ms), at double sites (right ventricle apex and right ventricular outflow tract). Furthermore a genetic test was performed using next–generation sequencing, showing a heterozygous mutation in the SCN5A gene, encoding for sodium channel alpha subunit (variant c.5363delA), not previously described in the literature. Since the low arrhythmic risk, the patient was implanted with a loop recorder (ILR, Medtronic Reveal LINQ), entering a program of home monitoring. After a 15–months follow–up, an episode of ventricular polymorphic tachycardia lasting 2 minutes and 11 seconds, associated with lightheadedness and palpitations, was detected by the remote monitoring (Figure 2). The patient was therefore hospitalized. During the in–hospital stay a cardiac magnetic resonance was performed to exclude other underlying diseases, identifying an enlarged right ventricle (EDVi 114 ml/m2) and abnormal free wall motion (Figure 3), thus reaching one major criteria of arrhythmogenic cardiomyopathy (however not sufficient for the diagnosis). The patient was then implanted with a subcutaneous implantable cardiac defibrillator (Boston Scientific EMBLEM MRI S–ICD). We presented a case of complex arrhythmic risk stratification in a possible overlap of an arrhythmogenic cardiomyopathy and a Brugada Syndrome in a young athlete who experienced a sustained ventricular tachycardia during loop–recorder monitoring.

P152 R222Q MUTATION OF THE SCN5A GENE IN PEDIATRIC AGE: PHENOTYPE AND RESPONSE TO DRUG THERAPY

Abstract Some studies have shown the association between ventricular and atrial arrhythmias, dilated cardiomyopathy and the R222Q mutation of the SCN5A gene. The mutation causes impaired activation of the sodium channel, the genesis of extrasystoles and the development of dilated cardiomyopathy; both respond better to antiarrhythmic therapy with flecainide and amiodarone than therapy for heart failure. Other studies have shown the efficacy of hydroquinidine. Purpose of the Study To observe the clinical characteristics and the response to drug therapy of pediatric subjects carrying the R222Q mutation. Materials and methods we observed subjects received at our centre for arrhythmias, carriers of the R222Q variant, subjected to ECG, Echocardiogram, Holter, stress test and follow up. Results 4 patients were observed, 3 males and 1 female, with ages ranging from 0 to 12 years. The first patient was familiar with dilated cardiomyopathy and rhythm disturbances; he had arrhythmia already in foetal age, he then developed first and second–degree BAV, junctional rhythm and polyform TVNS. Echocardiography and exercise testing were normal. Arrhythmia control was achieved with flecainide. The second patient, with negative familiarity, from the age of 8 is under observation for polyform ventricular ectopic beats, repetitive, unresponsive to propafenone, flecainide, nadolol and sotalol. Echocardiography showed minimal impairment of the contractile function of the left ventricle. Ventricular extrasystoles did not disappear during exertion. At 15, a defibrillator was implanted and hydroquinidine was associated. The third patient presented with foetal tachyarrhythmias; at the age of 6, atrial tachycardia, well–controlled by sotalol, and ventricular ectopic beats were detected. The fourth patient, a cousin of the previous one, developed repetitive ventricular ectopic beats and periods of atrial and junctional rhythm at 12 years; he responded to flecainide. Conclusions The R222Q mutation of the SCN5A gene is responsible for arrhythmias already from the pediatric age and is expressed with a variable phenotype. Only one patient with therapy–resistant arrhythmia had contractile dysfunction. Dilated cardiomyopathy could be a consequence of a high arrhythmic burden and occur at a later age if the arrhythmia is not treated. An early and genetic diagnosis is essential.

Pathological changes of the sural nerve in patients with familial episodic pain syndrome.

Familial episodic pain syndrome type 3 (FEPS3) is an inherited disorder characterized by the early-childhood onset of severe episodic pain that primarily affects the distal extremities. As skin biopsy has revealed a reduction in intraepidermal nerve fiber density and degeneration of the unmyelinated axons, it remains unclear whether FEPS3 patients have pathological changes in the peripheral nerve. The clinical features of patients with FEPS3 were summarized in a large autosomal dominant family. Sural nerve biopsies were conducted in two patients. Whole exome sequencing (WES) was performed in the index patient. Sanger sequencing was used to analyze family co-segregation. Fourteen members exhibited typical and uniform clinical phenotypes characterized by length-dependent and age-dependent severe episodic pain affecting the distal extremities, which can be relieved with anti-inflammatory medicine. The WES revealed a heterozygous mutation c.665G > A (p.R222H) in the SCN11A gene, which was co-segregated with the clinical phenotype in this family. A sural biopsy in patient V:1, who was experiencing episodic pain at 16years old, showed normal structure, while the sural nerve in patient IV:1, whose pain attack had completely diminished at 42years old, displayed a decrease of the density of unmyelinated axons with the axonal degeneration. The clinical phenotype of FEPS3 showed distinctive characteristics that likely arise from dysfunctional nociceptive neurons that lack detectable pathological alterations in the nerve fibers. Nevertheless, long-term dysfunction of the Nav1.9 channel may cause degeneration of the unmyelinated fibers in FEPS3 patient with pain remission.

Human iPSC Modeling of Genetic Febrile Seizure Reveals Aberrant Molecular and Physiological Features Underlying an Impaired Neuronal Activity.

Mutations in SCN1A gene, encoding the voltage-gated sodium channel (VGSC) NaV1.1, are widely recognized as a leading cause of genetic febrile seizures (FS), due to the decrease in the Na+ current density, mainly affecting the inhibitory neuronal transmission. Here, we generated induced pluripotent stem cells (iPSCs)-derived neurons (idNs) from a patient belonging to a genetically well-characterized Italian family, carrying the c.434T > C mutation in SCN1A gene (hereafter SCN1AM145T). A side-by-side comparison of diseased and healthy idNs revealed an overall maturation delay of SCN1AM145T cells. Membranes isolated from both diseased and control idNs were injected into Xenopus oocytes and both GABA and AMPA currents were successfully recorded. Patch-clamp measurements on idNs revealed depolarized action potential for SCN1AM145T, suggesting a reduced excitability. Expression analyses of VGSCs and chloride co-transporters NKCC1 and KCC2 showed a cellular “dysmaturity” of mutated idNs, strengthened by the high expression of SCN3A, a more fetal-like VGSC isoform, and a high NKCC1/KCC2 ratio, in mutated cells. Overall, we provide strong evidence for an intrinsic cellular immaturity, underscoring the role of mutant NaV1.1 in the development of FS. Furthermore, our data are strengthening previous findings obtained using transfected cells and recordings on human slices, demonstrating that diseased idNs represent a powerful tool for personalized therapy and ex vivo drug screening for human epileptic disorders.

PO-676-01 A VERSATILE CHEMICAL-GENETIC APPROACH TO DETERMINE BASES FOR ARRHYTHMOGENESIS AND SODIUM CHANNELOPATHIES

The voltage gated sodium channel NaV1.5 controls cardiac excitability and is an established therapeutic target; mutations in the Scn5a gene, which encodes NaV1.5, are associated with inherited arrhythmia syndromes such as Brugada and Long-QT. Other NaV isoforms, especially NaV1.1 and 1.8, have been strongly implicated in cardiac rhythmogenesis, however their presence in cardiomyocytes and functional roles are unresolved and contentious. Direct observation of non-NaV1.5 isoform currents would serve to address this outstanding question on a fundamental level, however the inability to selectively and completely silence NaV1.5 has prevented such a study.

PO-646-03 REDUCED EPHAPTIC COUPLING EXACERBATES CONDUCTION SLOWING IN THE SCN5A HETEROZYGOUS MOUSE

Conduction dependence on ephaptic coupling (EpC) is mediated by voltage gated sodium channels (Nav1.5) localized to the gap junction adjacent extracellular nanodomain: the perinexus. Pharmacologic Nav1.5 inhibition exacerbates conduction slowing due to reduced EpC. Yet, a 50% reduction of the SCN5a gene encoding Nav1.5 either modestly slows or does not change conduction velocity (CV) in genetically modified mice. Thus, the mechanism by which predicted dramatic conduction slowing during Nav1.5 LOF is attenuated in hearts is unknown.

PO-629-03 IN-DEPTH ANALYSIS OF THE SCN5A LOCUS HIGHLIGHTS DISTINCT GENETIC ARCHITECTURES FOR BRUGADA SYNDROME IN DIFFERENT ANCESTRIES AND IDENTIFIES A NOVEL RARE ENHANCER VARIANT ASSOCIATED WITH DISEASE IN SOUTHEAST ASIAN PATIENTS

Brugada syndrome (BrS) is a cardiac arrhythmia disorder that can cause sudden death in young adults. Genetic variation in the Nav1.5 cardiac sodium channel encoding SCN5A gene is robustly associated with BrS, across both rare coding and common non-coding variants. Despite a markedly higher prevalence of BrS, the diagnostic yield from SCN5A genetic testing in east and southeast Asia has been reported to be much lower than other regions. To compare the contribution of different classes of SCN5A variants (by variant type and rarity) to BrS between northwest European and Thai populations in order to investigate the apparent paradox between high disease prevalence and low diagnostic yield in southeast Asia. Genome sequencing data from European (412 cases, 769 controls) and Thai (202 cases, 410 controls) samples was analysed. SCN5A coding variants were binned by gnomAD exomes (v2.1) filtering allele frequency (FAF) and case-control odds ratios (OR) were calculated for each bin and ancestry. Rare non-coding variants in promoter and enhancer regions of SCN5A and SCN10A were assessed for enrichment or depletion in BrS cohorts. Ultra-rare (FAF<0.00001) SCN5A coding variants are more prevalent and enriched in European (22.6% of cases, OR=22.1, 11.4-43.0) compared to Thai (5.9% of cases, OR=4.3, 1.6-11.5) BrS patients. In contrast, low frequency variants (FAF 0.00005-0.001) that are expected to have intermediate effect sizes are uniquely enriched in Thai samples, occurring in 9.9% of patients. 3/6 of these variants (p.R965C, p.A1428S, p.V2016M) are known to affect Nav1.5 function. A rare non-coding variant in the SCN5A RE5 enhancer region (GRCh37:3-38621871-A-C), affecting a base conserved across species, was highly and uniquely enriched in Thai BrS cases (4.5% vs 0.2%, OR=19.1, p=3e-04). Distinct SCN5A genetic architectures are observed in European and southeast Asian BrS patients. The increased prevalence of BrS in southeast Asia is not due to an increased population burden of ultra-rare and highly deleterious SCN5A coding variants (which are relatively depleted in case cohorts). Our data suggest that other SCN5A variant classes, including coding variants of intermediate effect size and rare non-coding enhancer variants, are genetic risk factors in Thai BrS cases.

RNA Interference Via shRNA Decreases the Number of Transcripts of Scn8a in Glutamatergic Interneurons in the preBötC

Rhythmic neural activity that drives the inspiratory phase of the breathing cycle in mammals emanates from glutamatergic interneurons of the pre‐Bötzinger Complex (preBötC). A longstanding question in neuroscience is: what are the underlying mechanisms for inspiratory rhythmogenesis? One proposed mechanism involves the persistent sodium current (INaP), which is the ionic mechanism that underlies the voltage‐dependent bursting‐pacemaker activity of neurons whose rhythmic burst discharges are putatively obligatory for inspiratory rhythm generation. Specifically in brain tissues, the Scn8a gene codes for Nav1.6 ion channels, and those ion channels give rise to INaP.Different genetic tools can help us to understand the role of specific molecules in the generation of motor behaviors, such as breathing. The short hairpin RNA (shRNA) is an artificial RNA molecule, delivered through adeno‐associated virus (AAV), that can inhibit gene expression and promote long‐term knock‐down of the targeted gene.Here we administered Cre‐dependent shRNA to knock‐down Scn8a (thus Nav1.6) in Cre‐expressing glutamatergic neurons in preBötC and quantified mRNA per glutamatergic neuron. We used adult Vglut2Cre mice (10‐14 weeks old) and injected 50 nL of AAVs carrying the shRNA. We used in situ hybridization (RNAscope® Fluorescent Assay) to label mRNA of Scn8a and to identify transduced glutamatergic neurons in the preBötC for RNA quantification at 2 and 6 weeks after injection. Samples were visualized under the Four‐Channel, Upright Confocal Microscope Body Based on Cerna® System (ThorLabs), and images were acquired using the ThorImage®LS Data Acquisition Software, at 40x magnification. The amount of RNA per cell was quantified using the open‐source software Quantitative Pathology(QuPath). We obtained the mean of the number of transcripts per cell ± the standard deviation (SD), and unpaired t‐test was used to evaluate the statistical difference between the shRNA injected mice and their control group (mice whose viral vector contained a non‐targeting shRNA sequence).Our results show that at 2 weeks, the control group had 27.5±9.5 RNA/neuron, whereas the shRNA‐injected had 12.8±3.5, which represents a 53% of decrease in the number of transcripts (P&lt;0.0001). A similar pattern was observed after 6 weeks of the injection in a different cohort, where control animals showed 35.4±6.1 RNA/cells, and the shRNA group only had 24.1±5.2, representing 32% drop in the transcripts for that group (P&lt;0.0001). This initial protocol allowed us to verify that the gene expression has been knocked down at critical time points, encouraging further studies using shRNA to evaluate whether attenuation of Nav1.6 expression in rhythmogenic preBötC neurons impairs breathing, which could resolve the longstanding question regarding the putatively rhythmogenic role of INaP.

Analysis of a Family with Brugada Syndrome and Sudden Cardiac Death Caused by a Novel Mutation of SCN5A.

Background Brugada syndrome is a hereditary cardiac disease associated with mutations in ion channel genes. The clinical features include ventricular fibrillation, syncope, and sudden cardiac death. A family with Brugada syndrome with sudden cardiac death was analyzed to locate the associated mutation in the SCN5A gene. Methods and Results Three generations of a Han Chinese family with Brugada syndrome were recruited in the study; their clinical phenotype data were collected and DNA samples extracted from the peripheral blood. Next-generation sequencing was carried out in the proband, and candidate genes and mutations were screened using the full exon capture technique. The family members who participated in the survey were tested for possible mutations using Sanger sequencing. Six family members were diagnosed with Brugada syndrome, including four asymptomatic patients. A newly discovered heterozygous mutation in the proband was located in exon 25 of SCN5A (NM_000335.5) at c.4313dup(p.Trp1439ValfsTer32). Among the surviving family members, only those with a Brugada wave on their electrocardiogram carried the c.4313dup(p.Trp1439ValfsTer32) variant. Bioinformatics prediction revealed that the frameshift of the c.4313dup (p.Trp1439ValfsTer32) mutant led to a coding change of 32 amino acids, followed by a stop codon, resulting in a truncated protein product. Conclusion The newly discovered mutation site c.4313dup(p.Trp1439ValfsTer32) in exon 25 of SCN5A may be the molecular genetic basis of the family with Brugada syndrome.

Non-canonical endogenous expression of voltage-gated sodium channel NaV 1.7subtype by the TE671 rhabdomyosarcoma cell line.

The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage‐sensitive sodium currents in these cells, the specific voltage‐gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription–PCR (qRT‐PCR), western blot and whole‐cell patch clamp electrophysiology were performed. qRT‐PCR profiling revealed that expression of the SCN9A gene was ∼215‐fold greater than the SCN4A gene and over 400‐fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V 50 values of −31.9 ± 1.1 and −69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV1.7 specific blockers Huwentoxin‐IV and Protoxin‐II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV1.7. Combined, the results revealed the non‐canonical and highly dominant expression of NaV1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost‐effective model for the study of NaV1.7, a major target for the development of analgesic drugs and more generally for the study of pain.Key points Undifferentiated TE671 cells produce a voltage‐sensitive sodium current when depolarised.The voltage‐gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT‐PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost‐effective model to study NaV1.7‐targeting drugs.

Life-span characterization of epilepsy and comorbidities in Dravet syndrome mice carrying a targeted deletion of exon 1 of the Scn1a gene

ObjectiveDravet Syndrome (DS) is a catastrophic form of paediatric epilepsy associated with multiple comorbidities mainly caused by mutations in the SCN1A gene. DS progresses in three different phases termed febrile, worsening and stabilization stage. Mice that are haploinsufficient for Scn1a faithfully model each stage of DS, although various aspects have not been fully described, including the temporal appearance and sex differences of the epilepsy and comorbidities. The aim of the present study was to investigate the epilepsy landscape according to the progression of DS and the long-term co-morbidities in the Scn1a(+/−)tm1Kea DS mouse line that are not fully understood yet. MethodsMale and female F1.Scn1a(+/+) and F1.Scn1a(+/−)tm1Kea mice were assessed in the hyperthermia model or monitored by video electroencephalogram (vEEG) and wireless video-EEG according to the respective stage of DS. Long-term comorbidities were investigated through a battery of behaviour assessments in ~6 month-old mice. ResultsAt P18, F1.Scn1a(+/−)tm1Kea mice showed the expected sensitivity to hyperthermia-induced seizures. Between P21 and P28, EEG recordings in F1.Scn1a(+/−)tm1Kea mice combined with video monitoring revealed a high frequency of SRS and SUDEP (sudden unexpected death in epilepsy). Power spectral analyses of background EEG activity also revealed that low EEG power in multiple frequency bands was associated with SUDEP risk in F1.Scn1a(+/−)tm1Kea mice during the worsening stage of DS. Later, SRS and SUDEP rates stabilized and then declined in F1.Scn1a(+/−)tm1kea mice. Incidence of SRS ending with death in F1.Scn1a(+/−)tm1kea mice displayed variations with the time of day and sex, with female mice displaying higher numbers of severe seizures resulting in greater SUDEP risk. F1.Scn1a(+/−)tm1kea mice ~6 month-old displayed fewer behavioural impairments than expected including hyperactivity, impaired exploratory behaviour and poor nest building performance. SignificanceThese results reveal new features of this model that will optimize use and selection of phenotype assays for future studies on the mechanisms, diagnosis, and treatment of DS.