Channel Alpha Research Articles

Method Development of Nested Allele-Specific Multiplex Polymerase Chain Reaction (NASM-PCR) for the Irritable Bowel Syndrome (IBS)-related Gene Polymorphisms

Polymorphisms in the genes of G-protein subunit beta 3 (GNB3); rs5443, tryptophan hydroxylase 1 (TPH1); rs211105 and rs4537731, tryptophan hydroxylase 2 (TPH2); rs4570625 and sodium voltage-gated channel alpha subunit 5 (SCN5A); rs1805124, have known to cause the abnormalities in the gastrointestinal tract that are implicated to irritable bowel syndrome (IBS) predisposition. Upfront genetic polymorphism genotyping in IBS-related gene polymorphisms will help to intervene and guide the decision-making in the management of IBS patients. This study aimed to develop a genotyping method to detect the respective polymorphisms using nested allele-specific multiplex polymerase chain reaction (NASM-PCR). A combination of nested and allele-specific multiplex PCR method was developed to determine the five single nucleotide polymorphisms (SNPs) mentioned. Annealing temperature, annealing and extension times, and the concentrations of MgCl2, primers, and DNA samples were optimized in the PCR. Sanger sequencing was performed to validate the genotyping results. NASM-PCR for IBS-related gene polymorphisms were successfully developed. DNA bands which correspond to the genes and SNPs have shown 100% homologous with the gene database. The developed method of NASM-PCR was reproducible and specific to be used for determining the respective polymorphisms of IBS. Notably, the described method can easily be integrated into other laboratories for population study or clinical use.

Brugada Syndrome: From Molecular Mechanisms and Genetics to Risk Stratification

Brugada syndrome (BrS) is a rare hereditary arrhythmia disorder, with a distinctive ECG pattern, correlated with an increased risk of ventricular arrhythmias and sudden cardiac death (SCD) in young adults. BrS is a complex entity in terms of mechanisms, genetics, diagnosis, arrhythmia risk stratification, and management. The main electrophysiological mechanism of BrS requires further research, with prevailing theories centered on aberrant repolarization, depolarization, and current-load match. Computational modelling, pre-clinical, and clinical research show that BrS molecular anomalies result in excitation wavelength (k) modifications, which eventually increase the risk of arrhythmia. Although a mutation in the SCN5A (Sodium Voltage-Gated Channel Alpha Subunit 5) gene was first reported almost two decades ago, BrS is still currently regarded as a Mendelian condition inherited in an autosomal dominant manner with incomplete penetrance, despite the recent developments in the field of genetics and the latest hypothesis of additional inheritance pathways proposing a more complex mode of inheritance. In spite of the extensive use of the next-generation sequencing (NGS) technique with high coverage, genetics remains unexplained in a number of clinically confirmed cases. Except for the SCN5A which encodes the cardiac sodium channel NaV1.5, susceptibility genes remain mostly unidentified. The predominance of cardiac transcription factor loci suggests that transcriptional regulation is essential to the Brugada syndrome's pathogenesis. It appears that BrS is a multifactorial disease, which is influenced by several loci, each of which is affected by the environment. The primary challenge in individuals with a BrS type 1 ECG is to identify those who are at risk for sudden death, researchers propose the use of a multiparametric clinical and instrumental strategy for risk stratification. The aim of this review is to summarize the latest findings addressing the genetic architecture of BrS and to provide novel perspectives into its molecular underpinnings and novel models of risk stratification.

Gene therapy as an emerging treatment for Scn2a mutation-induced autism spectrum disorders

Autism spectrum disorder (ASD) is a complex neurological and developmental disorder that affects how a person acts, communicates, learns, and interacts with others. It affects the structure and function of the brain and nervous system. How ASD is caused remains uncertain and there is no effective treatment for this disorder. Searching for new technologies for treatment of this disorder becomes a priority. Genetic alterations have been implicated in the generation of this disorder. One of the most promising genes for potential treatment of ASD is sodium voltage-gated channel alpha subunit 2 gene (SCN2A). SCN2A, encoding the neuronal voltage-gated Na+ channel NaV1.2, is one of the most commonly affected loci linked to ASD. Here, we discuss the implications of loss of function (LOF) mutations in SCN2A and the potential efficacy of gene therapy by highlighting the usage of CRISPR restoration of various Scn2a-insufficiencies. Various therapeutics exist that can be extrapolated to address the needs of Scn2a LOF induced ASD. The current treatment that exists for ASD can be seen as outdated in comparison to the advent of new technologies that build upon CRISPR. Due to complications in treatment of ASD, genetic therapies may induce alterations such as insertion–deletion mutations, which may lead to further complications along with a negative public outlook on CRISPR technologies. Gene therapy can be applied to ASD but much work is yet to be done in order to address both biochemical and ethical considerations.

Classical trigeminal neuralgia is associated with gephyrin and sodium voltage-gated channel alpha subunit 8

Trigeminal neuralgia is highly debilitating, and its etiology is still undefined. The goal of this work was to define associations between well-characterized trigeminal neuralgia cases and common genetic variants in the population. two hundred and fifty-seven individuals diagnosed with classical trigeminal neuralgia were compared to 865 individuals without classical trigeminal neuralgia and with an assessment for lower or higher pain threshold based on the amount of anesthetic required for routine dental treatment. Genotypes of 24 variants marking genes in the VGSC (voltage-gated sodium channels) or GABA (gamma-aminobutyric acid) pathways were obtained using TaqMan chemistry end end-point analysis. Chi-square was used for all comparisons with an alpha of 0.002. An association between classical trigeminal neuralgia and individuals requiring less or more anesthetic for routine dental treatments showed associations with SCN8A rs1601012 and GPHN rs723432 (p = 0.0009 and p = 0.0002, respectively). In conclusion, classical trigeminal neuralgia is associated with SCN8A and GPHN and markers rs1601012 rs723432 may be useful to determine individual risks for the condition.

SCN1A: bioinformatically informed revised boundaries for promoter and enhancer regions.

Pathogenic variations in the sodium voltage-gated channel alpha subunit 1 (SCN1A) gene are responsible for multiple epilepsy phenotypes, including Dravet syndrome, febrile seizures (FS) and genetic epilepsy with FS plus. Phenotypic heterogeneity is a hallmark of SCN1A-related epilepsies, the causes of which are yet to be clarified. Genetic variation in the non-coding regulatory regions of SCN1A could be one potential causal factor. However, a comprehensive understanding of the SCN1A regulatory landscape is currently lacking. Here, we summarized the current state of knowledge of SCN1A regulation, providing details on its promoter and enhancer regions. We then integrated currently available data on SCN1A promoters by extracting information related to the SCN1A locus from genome-wide repositories and clearly defined the promoter and enhancer regions of SCN1A. Further, we explored the cellular specificity of differential SCN1A promoter usage. We also reviewed and integrated the available human brain-derived enhancer databases and mouse-derived data to provide a comprehensive computationally developed summary of SCN1A brain-active enhancers. By querying genome-wide data repositories, extracting SCN1A-specific data and integrating the different types of independent evidence, we created a comprehensive catalogue that better defines the regulatory landscape of SCN1A, which could be used to explore the role of SCN1A regulatory regions in disease.

Parallel changes in the promoter methylation of voltage-gated T-type calcium channel alpha 1 subunit G and histone deacetylase activity in the WAG/Rij model of absence epilepsy

Objective: In the last two decades, research on epigenetic mechanisms has expanded dramatically. Recent studies demonstrated that epigenetic mechanisms regulate epilepsy and epileptogenic pathologies. In this study, we aimed to investigate changes in the promoter methylation status of the voltage-gated T-type calcium channel alpha 1 subunit G (CACNA1G) gene and total histone deacetylase activity in Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats which is one of the commonly used genetic absence rat models of epilepsy in the three different age groups (3, 6, and 9 months old) on both sexes. 
 Material and Method: Evaluation of changes in the spike-wave discharges (SWDs) was performed with electrocorticography (ECoG). The promoter methylation status of the CACNA1G gene was determined by methylation-specific PCR (MSP), and histone deacetylase (HDAC) activity was determined spectrophotometrically. 
 Results: Our results demonstrated that the number of SWDs increased time-dependent in WAG/Rij. Additionally, it was observed that CACNA1G promoter methylation decreased, and total HDAC activity increased with age in both sexes. 
 Conclusion: Our results provide further support for epigenetic regulation in the absence epilepsy phenotype and suggest that the underlying mechanism behind the increase in the number of SWDs with age in the WAG/Rij animals might be regulated by CACNA1G promoter methylation or HDAC activity.

Upregulation of α-ENaC induces pancreatic β-cell dysfunction, ER stress, and SIRT2 degradation.

Islet beta cells (β-cells) produce insulin in response to high blood glucose levels, which is essential for preserving glucose homeostasis. Voltage-gated ion channels in β-cells, including Na +, K +, and Ca 2+ channels, aid in the release of insulin. The epithelial sodium channel alpha subunit (α-ENaC), a voltage-independent sodium ion channel, is also expressed in human pancreatic endocrine cells. However, there is no reported study on the function of ENaC in the β-cells. In the current study, we found that α-ENaC was expressed in human pancreatic glandule and pancreatic islet β-cells. In the pancreas of db/db mice and high-fat diet-induced mice, and in mouse islet β-cells (MIN6 cells) treated with palmitate, α-ENaC expression was increased. When α-ENaC was overexpressed in MIN6 cells, insulin content and glucose-induced insulin secretion were significantly reduced. On the other hand, palmitate injured islet β-cells and suppressed insulin synthesis and secretion, but increased α-ENaC expression in MIN6 cells. However, α-ENaC knockout ( Scnn1a -/-) in MIN6 cells attenuated β-cell disorder induced by palmitate. Furthermore, α-ENaC regulated the ubiquitylation and degradation of sirtuin 2 in β-cells. α-ENaC also modulated β-cell function in correlation with the inositol-requiring enzyme 1 alpha/X-box binding protein 1 (IRE1α/XBP1) and protein kinase RNA-like endoplasmic reticulum kinase/C/EBP homologous protein (PERK/CHOP) endoplasmic reticulum stress pathways. These results suggest that α-ENaC may play a novel role in insulin synthesis and secretion in the β-cells, and the upregulation of α-ENaC promotes islet β-cell dysfunction. In conclusion, α-ENaC may be a key regulator involved in islet β-cell damage and a potential therapeutic target for type 2 diabetes mellitus.

Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization.

Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001). These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

Exploring the neurogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins βIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.

SCN1A polymorphisms influence the antiepileptic drugs responsiveness in Jordanian epileptic patients.

The aim of this study was to evaluate whether the voltage-gated sodium channel alpha subunit 1 (SCN1A) gene polymorphisms influence the responsiveness of Jordanian epileptic patients to antiepileptic drugs (AEDs). A total of 72 AEDs-treated epileptics were polymerase chain reaction (PCR)-genotyped for six single nucleotide polymorphisms (SNPs), including SCN1A rs2298771, rs3812718, rs3812719, rs2217199, rs2195144 and rs1972445. Genotype and allele distributions in drug-responsive and drug-resistant patients were compared. The six SNPs haplotypes were examined, and the linkage disequilibrium (LD) was assessed. The genotypes of drug-resistant and drug-responsive groups were in Hardy-Weinberg equilibrium. Three genetic polymorphisms of the SCN1A gene seemed to influence the resistance to AEDs, on the level of alleles and genotypes. Data revealed that rs2298771 G allele, rs3812719 C allele, and rs2195144 T allele increased the risk of developing AEDs-resistance (OR=2.9; 95%CI= 1.4-5.9, p=0.003; OR=2.4; 95%CI=1.2-4.7, p=0.01; OR=2.3; 95%CI=1.2-4.7, p=0.01), respectively. Haplo type analysis of SCN1A polymorphisms revealed high-degree LD associated with resistance to AEDs. A synergetic effect appears with highly significant association in GCCATG haplotype of rs2298771, rs3812718, rs3812719, rs2217199, rs2195144, and rs1972445 respectively (OR=2.8; 95%CI=1.5-6.2, p=0.002). Data suggests that SCN1A polymorphisms could influence the resistance to AEDs in Jordanian epileptics at three SNPs (rs2298771; rs3812719; rs2195144). Additionally, haplotype analysis indicated a substantial degree of LD between the six SCN1A polymorphisms. Further investigation with larger sample size is needed to confirm the results of the current study.

Effects of chronic intermittent hypoxia on atrial electrical remodeling in rats

The aim of this study was to investigate the effects of chronic intermittent hypoxia (CIH) on atrial electrical remodeling in Sprague-Dawley (SD) rats, which provide the explication for the mechanisms of CIH promoting atrial fibrillation (AF). Eighty SD rats were randomly divided into 2 groups: control group and CIH group (n=40). CIH rats were subjected to CIH 8 h/d for 30 days. After the echocardiography and hemodynamics examination, cardiac electrophysiological experiments, histological experiments, and molecular biological experiments were executed. AF susceptibility was measured by isolated heart electrophysiological experiments. Masson's trichrome stain was used to assess the degree of atrial fibrosis. The protein expression levels of sodium voltage-gated channel alpha subunit 5 (SCN5A/Nav1.5), calcium voltage-gated channel subunit alpha1 C (CACNA1C/Cav1.2) and potassium voltage-gated channel subfamily D member 3 (KCND3/Kv4.3) were measured by Western blot. In whole-cell patch clamp experiments, current clamp mode was used to record AP, and APD90 and APD50 were analyzed and compared between the two groups. In voltage clamp mode, INa, ICa-L, Ito and their kinetic parameters were recorded and compared between the two groups. Compared to the control rats, atrial interstitial collagen deposition (P＜0.01) and AF inducibility (P＜0.05) were increased in CIH rats, whereas the expression levels of Nav1.5, Cav1.2 and Kv4.3 were decreased (P＜0.05). APD90 and APD50 in CIH rats' atrial myocytes were longer than those of control rats, and CIH rats showed decreased current density of INa, ICa-L(P＜0.01) and Ito(P＜0.01). CIH-induced changes in the protein expression levels of ion channel subunits, current intensity, APD, and AF susceptibility, which may be the mechanisms of CIH promoting AF.

Complex interactions between p.His558Arg and linked variants in the sodium voltage-gated channel alpha subunit 5 (Na V 1.5).

Common genetic polymorphisms may modify the phenotypic outcome when co-occurring with a disease-causing variant, and therefore understanding their modulating role in health and disease is of great importance. The polymorphic p.His558Arg variant of the sodium voltage-gated channel alpha subunit 5 (NaV1.5) encoded by the SCN5A gene is a case in point, as several studies have shown it can modify the clinical phenotype in a number of cardiac diseases. To evaluate the genetic backgrounds associated with this modulating effect, we reanalysed previous electrophysiological findings regarding the p.His558Arg variant and further assessed its patterns of genetic diversity in human populations. The NaV1.5 p.His558Arg variant was found to be in linkage disequilibrium with six other polymorphic variants that previously were also associated with cardiac traits in GWAS analyses. On account of this, incongruent reports that Arg558 allele can compensate, aggravate or have no effect on NaV1.5, likely might have arose due to a role of p.His558Arg depending on the additional linked variants. Altogether, these results indicate a major influence of the epistatic interactions between SCN5A variants, revealing also that phenotypic severity may depend on the polymorphic background associated to each individual genome.

Primary erythromelalgia mainly manifested by hypertensive crisis: A case report and literature review.

BackgroundPrimary erythrocytic (PEM) is a rare autosomal dominant single gene disease. Most of the changes of gene loci can be found by whole exon gene sequencing, and the clinical symptoms and patient survival can be improved by specific site-to-site drug treatment. The other manifestations of this patient population are not remarkable. After the application of common drugs, the toxicity and side effects can be limiting. In addition to other common clinical manifestations, we found that the only unique manifestation of this patient was hypertensive crisis. Following multidisciplinary diagnosis and treatment (MDT), we decided to first control hypertension to alleviate the acute and critical patients. However, after controlling the hypertensive crisis, we unexpectedly found that the clinical symptoms of the patients had been significantly improved. Therefore, we concluded that the use of antihypertensive drugs can treat erythematous limb pain with the clinical manifestation of hypertensive crisis. Here, we describe a typical PEM disease, primary clinical features, diagnosis and treatment.MethodsMedical records of an 8-year-old boy with PEM were analyzed retrospectively, which included clinical characteristics, follow-up information, and SCN9A (Sodium Voltage-Gated Channel Alpha Subunit 9) gene analysis.ResultsThe 8-year-old boy had complained of abnormal paresthesia in his feet and ankles with burning sensation and pain for 2 years. The skin of both lower legs was red and underwent ichthyosis and lichenification. Genetic analysis confirmed the existence of a SCN9A gene mutation. The symptoms were gradually improved by treating with intravenous drip and oral administration of nitroglycerin to slow his heart rhythm.ConclusionPrimary erythrocytic is characterized by skin ulceration, redness, elevated temperature, and severe burning pain primarily in both lower extremities. PEM can be diagnosed by genetic analysis. As this case demonstrates, treating with nitroglycerin as the drug of choice to control the hypertensive crisis significantly improved the symptoms of PEM and hypertension in this patient.

Expression of voltage-gated Ca2+ channels, Insp3Rs, and RyRs in the immature mouse ovary

BackgroundThe postnatal mammalian ovary undergoes a series of changes to ensure the maturation of sufficient follicles to support ovulation and fecundation over the reproductive life. It is well known that intracellular [Ca2+]i signals are necessary for ovulation, fertilization, and egg activation. However, we lack detailed knowledge of the molecular identity, cellular distribution, and functional role of Ca2+ channels expressed during folliculogenesis. In the neonatal period, ovarian maturation is controlled by protein growth factors released from the oocyte and granulosa cells. Conversely, during the early infantile period, maturation becomes gonadotropin-dependent and is controlled by granulosa and theca cells. The significance of intracellular Ca2+ signaling in folliculogenesis is supported by the observation that mice lacking the expression of Ca2+/calmodulin-dependent kinase IV in granulosa cells suffer abnormal follicular development and impaired fertility.ResultsUsing immunofluorescence in frozen ovarian sections and confocal microscopy, we assessed the expression of high-voltage activated Ca2+ channel alpha subunits and InsP3 and ryanodine receptors in the postnatal period from 3 to 16 days. During the neonatal stage, oocytes from primordial and primary follicles show high expression of various Ca2+-selective channels, with granulosa and stroma cells expressing significantly less. These channels are likely involved in supporting Ca2+-dependent secretion of peptide growth factors. In contrast, during the early and late infantile periods, Ca2+ channel expression in the oocyte diminishes, increasing significantly in the granulosa and particularly in immature theca cells surrounding secondary follicles.ConclusionsThe developmental switch of Ca2+ channel expression from the oocytes to the perifollicular cells likely reflects the vanishing role of the oocytes once granulosa and theca cells take control of folliculogenesis in response to gonadotropins acting on their receptors.

Migraine with Brainstem Aura Associated with SCN2A Mutation: A Case Report

Abstract Background Migraine with brainstem aura (MBA) is a rare subtype of migraine with aura that associates headache with paroxysmal episodes of ataxia, dysarthria, and diplopia among other symptoms attributed to brainstem involvement. The manifestation of this disease in the absence of typical aura symptoms (i.e., fully reversible visual, sensory and/or speech/language symptoms) is even more infrequent. Case Report In this article, we reported a case of a 16-year-old male patient with MBA associated with a sodium voltage-gated channel alpha subunit 2 (SCN2A) de novo mutation. The patient did not have typical aura symptoms. Ataxia was the most prominent clinical feature, leading to initial diagnosis of episodic ataxia. Patient responded partially to treatment with onabotulinum toxin A. Conclusion MBA needs to be taken into consideration in the differential diagnosis of recurrent ataxia, even in the absence of typical aura symptoms. Finding a de novo SCN2A mutation in our patient helps in further understanding of MBA genetics.

Whole-Exome Sequencing Identifies Novel SCN1A and CACNB4 Genes Mutations in the Cohort of Saudi Patients With Epilepsy.

Epilepsy is a neurological disorder described as recurrent seizures mild to severe convulsions along with conscious loss. There are many different genetic anomalies or non-genetic conditions that affect the brain and cause epilepsy. The exact cause of epilepsy is unknown so far. In this study, whole-exome sequencing showed a family having novel missense variant c.1603C>T, p. Arg535Cys in exon 10 of Sodium Voltage-Gated Channel Alpha Subunit 1 (SCN1A) gene. Moreover, targeted Sanger sequencing analysis showed c.1212A>G p.Val404Ile in SCN1A gene in 10 unrelated patients and a mutation in Calcium Voltage-Gated Channel Auxiliary Subunit Beta 4 gene where one base pair insertion of “G” c.78_79insG, p.Asp27Glyfs*26 in the exon 3 in three different patients were observed from the cohort of 25 epileptic sporadic cases. The insertion changes the amino acid sequence leading to a frameshift mutation. Here, we have described, for the first time, three novel mutations that may be associated with epilepsy in the Saudi population. The study not only help us to identify the exact cause of genetic variations causing epilepsy whereas but it would also eventually enable us to establish a database to provide a foundation for understanding the critical genomic regions to control epilepsy in Saudi patients.

Exogenous H2S promotes ion channel reconstruction to regulate colonic motility in rats with dinitrobenzene sulfonic acid-induced colitis.

BackgroundHydrogen sulfide (H2S) is a newly recognized endogenously generated gasotransmitter. Endogenous H2S has been shown to be related to several gastrointestinal diseases, including irritable bowel syndrome, Crohn’s disease, and ulcerative colitis. We explored the functional role of H2S in colitis and investigated the underlying mechanism.MethodsRats were randomly divided into control, model, and sodium hydrosulfide (NaHS) groups. Rat colitis was evaluated using the disease activity index and hematoxylin and eosin (HE) staining. The collected rat feces and intestinal charcoal transit were examined to determine intestinal motility. Enzyme-linked immunoassay kits were used to determine inflammatory factors in the rat blood. Calcium ion distribution and the protein expression of Potassium voltage gated channel subfamily D member 3 (KCND3), Maxi Potassium channel alpha (KCNMA1) and potassium inwardly rectifying channel J8 (KCNJ8) in rat smooth muscle were determined using an intracellular calcium detection kit, quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and Western blot, respectively.ResultsThe results showed that H2S donor NaHS improved the intestinal mucosa of colitis rats by alleviating the tissue edema and congestion and repairing the gland structure of the intestinal mucosa. NaHS also reduced the levels of inflammatory factors containing interleukin (IL)-1β, tumor necrosis factor alpha, and IL-6 in the rat blood. Additionally, the NaHS intervention partially restored rat intestinal motility by downregulating the proteins and mRNA expression of KCND3, KCNMA1, and KCNJ8 to reconstruct the ion channels.ConclusionsOur findings provide novel insights into the effect of H2S regulation on the anti-inflammatory action.

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.

SCN1A-Related Epilepsy: Novel Mutations and Rare Phenotypes.

ObjectivesTo expand the genotypes and phenotypes of sodium voltage-gated channel alpha subunit 1 (SCN1A)-related epilepsy.MethodsWe retrospectively collected the clinical and genetic information of 22 epilepsy patients (10 males, 12 females; mean: 9.2 ± 3.9 years; 3.9–20.3 years) carrying 22 variants of SCN1A. SCN1A mutations were identified by next-generation sequencing.ResultsTwenty-two variants were identified, among which 12 have not yet been reported. The median age at seizure onset was 6 months. Sixteen patients were diagnosed with Dravet syndrome (DS), two with genetic epilepsy with febrile seizures plus [one evolved into benign epilepsy with centrotemporal spikes (BECTS)], one with focal epilepsy, one with atypical childhood epilepsy with centrotemporal spikes (ABECTS) and two with unclassified epilepsy. Fourteen patients showed a global developmental delay/intellectual disability (GDD/ID). Slow background activities were observed in one patient and epileptiform discharges were observed in 11 patients during the interictal phase.SignificanceThis study enriches the genotypes and phenotypes of SCN1A-related epilepsy. The clinical characteristics of patients with 12 previously unreported variants were described.

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.