Voltage-gated Sodium Channel Alpha Subunit Research Articles

Whole genome sequencing in adults with clinical hallmarks of hypophosphatasia negative for ALPL variants.

Hypophosphatasia (HPP) is a rare disease caused by deficient activity of tissue-nonspecific alkaline phosphatase (ALP), encoded by the ALPL gene. The primary objective was to explore novel ALPL variants by whole genome sequencing (WGS) in patients with HPP who previously tested negative by standard methods for ALPL variants. The secondary objective was to search for genes beyond ALPL that may reduce ALP activity or contribute to HPP symptoms. WGS was performed in 16 patients clinically diagnosed with HPP who had ALP activity below the normal range and tested negative for ALPL variants. Genetic variants in ALPL and genes possibly associated with low ALP activity or phenotypic overlap with HPP were assessed. All 16 patients had ALP activity below the normal range. WGS did not identify any novel disease-causing ALPL variants. Positive findings for other gene variants were identified in 4 patients: 1 patient presented with variants in COL1A1, NLRP12, and SCN9A, coding for collagen, type, I alpha-1 chain, nod-like receptor pyrin domain containing 12, and sodium voltage-gated channel alpha subunit 9, respectively; 1 presented with a heterozygous, likely pathogenic variant in P3H1 coding for prolyl 3 hydroxylase 1; 1 presented with a heterozygous pathogenic variant in SGCE, coding for sarcoglycan epsilon; and 1 presented with a heterozygous variant of uncertain significance in VDR, encoding vitamin D receptor. Genomic analysis did not identify novel ALPL variants or a pattern of disease-causing variants in genes other than ALPL to explain the HPP phenotype in these patients. Clinicaltrials.gov identifier: NCT04925804.

NaV1.1 contributes to the cell cycle of human mesenchymal stem cells by regulating AKT and CDK2.

Non-excitable cells express sodium voltage-gated channel alpha subunit 1 gene and protein (SCN1A/NaV1.1); however, the functions of NaV1.1 are unclear. SCN1A was expressed in human mesenchymal stem cells (MSCs). Nav1.1 was abundantly expressed in the endoplasmic reticulum of MSCs; however, its expression was not found to be related to sodium currents. SCN1A-silencing reduced MSC proliferation and delayed the cell cycle in the S phase. SCN1A-silencing also suppressed the protein levels of CDK2 and AKT, despite similar mRNA expression, and inhibited AKT phosphorylation in MSCs. Cycloheximide-chase assay showed that SCN1A-silencing induced CDK2 but not AKT protein degradation in MSCs. Proteolysis inhibition assay using epoxomicin, bafilomycin A1, and NH4Cl, revealed that the ubiquitin-proteasome and autophagy/endo-lysosome systems were irrelevant to CDK2 and AKT protein reduction in SCN1A-silenced MSCs. AKT inhibitor LY294002 did not affect the degradation and nuclear localization of CDK2 in MSCs. Likewise, AKT activator SC79 did not attenuate the SCN1A-silencing effects on CDK2 in MSCs. These results suggest that NaV1.1 contributes to the cell cycle of MSCs by regulating the post-translational control of AKT and CDK2.

Single-Base Gene Variants in MIR-146A and SCN1A Genes Related to the Epileptogenic Process in Drug-Responsive and Drug-Resistant Temporal Lobe Epilepsy-A Preliminary Study in a Brazilian Cohort Sample.

The drug-resistant temporal lobe epilepsy (TLE) has recently been associated with single nucleotide variants (SNVs) in microRNA(miR)-146a (MIR-146A) (rs2910164) and Sodium Voltage-Gated Channel Alpha Subunit 1 (SCN1A) (rs2298771 and rs3812718) genes. Moreover, no studies have shown an association between these SNVs and susceptibility to drug-resistant and drug-responsive TLE in Brazil. Thus, deoxyribonucleic acid (DNA) samples from 120 patients with TLE (55 drug-responsive and 65 drug-resistant) were evaluated by real-time polymerase chain reaction (RT-PCR). A total of 1171 healthy blood donor individuals from the Online Archive of Brazilian Mutations (ABraOM, from Portuguese Arquivo Brasileiro On-line de Mutações), a repository containing genomic variants of the Brazilian population, were added as a control population for the studied SNVs. MIR-146A and SCN1A relative expression was performed by quantitative RT-PCR (qRT-PCR). The statistical analysis protocol was performed using an alpha error of 0.05. TLE patient samples and ABraOM control samples were in Hardy-Weinberg equilibrium for all studied SNVs. For rs2910164, the frequencies of the homozygous genotype (CC) (15.00% vs. 9.65%) and C allele (37.80% vs. 29.97%) were superior in patients with TLE compared to controls with a higher risk for TLE disease [odds ratio (OR) = 1.89 (95% confidence interval (95%CI) = 1.06-3.37); OR = 1.38 (95%CI = 1.04-1.82), respectively]. Drug-responsive patients also presented higher frequencies of the CC genotype [21.81% vs. 9.65%; OR = 2.58 (95%CI = 1.25-5.30)] and C allele [39.09% vs. 29.97%; OR = 1.50 (95%CI = 1.01-2.22)] compared to controls. For rs2298771, the frequency of the heterozygous genotype (AG) (51.67% vs. 40.40%) was superior in patients with TLE compared to controls with a higher risk for TLE disease [OR = 2.42 (95%CI = 1.08-5.41)]. Drug-resistant patients presented a higher AG frequency [56.92% vs. 40.40%; OR = 3.36 (95%CI = 1.04-17.30)] compared to the control group. For rs3812718, the prevalence of genotypes and alleles were similar in both studied groups. The MIR-146A relative expression level was lower in drug-resistant compared to drug-responsive patients for GC (1.6 vs. 0.1, p-value = 0.049) and CC (1.8 vs. 0.6, p-value = 0.039). Also, the SCN1A relative expression levels in samples from TLE patients were significantly higher in AG [2.09 vs. 1.10, p-value = 0.038] and GG (3.19 vs. 1.10, p-value < 0.001) compared to the AA genotype. In conclusion, the rs2910164-CC and rs2298771-AG genotypes are exerting significant risk influence, respectively, on responsive disease and resistant disease, probably due to an upregulated nuclear factor kappa B (NF-kB) and SCN1A loss of function.

Genomic analysis of severe COVID-19 considering or not asthma comorbidity: GWAS insights from the BQC19 cohort

BackgroundThe severity of COVID-19 is influenced by various factors including the presence of respiratory diseases. Studies have indicated a potential relationship between asthma and COVID-19 severity.ObjectiveThis study aimed to conduct a genome-wide association study (GWAS) to identify genetic and clinical variants associated with the severity of COVID-19, both among patients with and without asthma.MethodsWe analyzed data from 2131 samples sourced from the Biobanque québécoise de la COVID-19 (BQC19), with 1499 samples from patients who tested positive for COVID-19. Among these, 1110 exhibited mild-to-moderate symptoms, 389 had severe symptoms, and 58 had asthma. We conducted a comparative analysis of clinical data from individuals in these three groups and GWAS using a logistic regression model. Phenotypic data analysis resulted in the refined covariates integrated into logistic models for genetic studies.ResultsConsidering a significance threshold of 1 × 10−6, seven genetic variants were associated with severe COVID-19. These variants were located proximal to five genes: sodium voltage-gated channel alpha subunit 1 (SCN10A), desmoplakin (DSP), RP1 axonemal microtubule associated (RP1), IGF like family member 1 (IGFL1), and docking protein 5 (DOK5). The GWAS comparing individuals with severe COVID-19 with asthma to those without asthma revealed four genetic variants in transmembrane protein with EGF like and two follistatin like domains 2 (TMEFF2) and huntingtin interacting protein-1 (HIP1) genes.ConclusionThis study provides significant insights into the genetic profiles of patients with severe forms of the disease, whether accompanied by asthma or not. These findings enhance our comprehension of the genetic factors that affect COVID-19 severity.Key messagesSeven genetic variants were associated with the severe form of COVID-19;Four genetic variants were associated with the severe form of COVID-19 in individuals with comorbid asthma;These findings help define the genetic component of the severe form of COVID-19 in relation to asthma as a comorbidity.

Elevated SCN11A concentrations associated with lower serum lipid levels in patients with major depressive disorder

The pathogenesis of major depressive disorder (MDD) involves lipid metabolism. Our earlier research also revealed that MDD patients had much lower total cholesterol (TC) concentrations than healthy controls (HCs). However, it is still unclear why TC decreased in MDD. Here, based on the Ingenuity Knowledge Base’s ingenuity pathway analysis, we found that sodium voltage-gated channel alpha subunit 11A (SCN11A) might serve as a link between low lipid levels and MDD. We analyzed the TC levels and used ELISA kits to measure the levels of SCN11A in the serum from 139 MDD patients, and 65 HCs to confirm this theory and explore the potential involvement of SCN11A in MDD. The findings revealed that TC levels were considerably lower and SCN11A levels were remarkably increased in MDD patients than those in HCs, while they were significantly reversed in drug-treatment MDD patients than in drug-naïve MDD patients. There was no significant difference in SCN11A levels among MDD patients who used single or multiple antidepressants, and selective serotonin reuptake inhibitors or other antidepressants. Pearson correlation analysis showed that the levels of TC and SCN11A were linked with the Hamilton Depression Rating Scales score. A substantial association was also found between TC and SCN11A. Moreover, a discriminative model made up of SCN11A was discovered, which produced an area under a curve of 0.9571 in the training set and 0.9357 in the testing set. Taken together, our findings indicated that SCN11A may serve as a link between low lipid levels and MDD, and showed promise as a candidate biomarker for MDD.

Molecular characterization of positively selected genes contributing aquatic adaptation in marine mammals.

Marine mammals, which have evolved independently into three distinct lineages, share common physiological features that contribute to their adaptation to the marine environment. To identify positively selected genes (PSGs) for adaptation to the marine environment using available genomic data from three taxonomic orders: cetaceans, pinnipeds, and sirenians. Based on the genomes within each group of Artiodactyla, Carnivora and Afrotheria, we performed selection analysis using the branch-site model in CODEML. Based on the branch-site model, 460, 614, and 359 PSGs were predicted for the cetaceans, pinnipeds, and sirenians, respectively. Functional enrichment analysis indicated that genes associated with hemostasis were positively selected across all lineages of marine mammals. We observed positive selection signals for the hemostasis and coagulation-related genes plasminogen activator, urokinase (PLAU), multimerin 1 (MMRN1), gamma-glutamyl carboxylase (GGCX), and platelet endothelial aggregation receptor 1 (PEAR1). Additionally, we found out that the sodium voltage-gated channel alpha subunit 9 (SCN9A), serine/arginine repetitive matrix 4 (SRRM4), and Ki-ras-induced actin-interacting protein (KRAP) are under positive selection pressure and are associated with cognition, neurite outgrowth, and IP3-mediated Ca2 + release, respectively. This study will contribute to our understanding of the adaptive evolution of marine mammals by providing information on a group of candidate genes that are predicted to influence adaptation to aquatic environments, as well as their functional characteristics.

Effect of NaV1.7 blockers on post-ganglionic sympathetic nerve function

NaV1.7, also known as the sodium voltage-gated channel alpha subunit 9 (SCN9A), plays a critical role in action potential induction and conduction in pain-causing nociceptors. Accordingly, NaV1.7 blockers may be effective non-opioids analgesics. SCN9A is also expressed in autonomic neurons, but its functional role in the autonomic system is less established. We employed three highly selective NaV1.7 inhibitors to investigate the role of NaV1.7 in action potential conduction in postganglionic sympathetic nerves and in sympathetic adrenergic contractions of blood vessels. Our single neuron rt-PCR analysis revealed that nearly all neurons (32/39) isolated from guinea pig stellate ganglia expressed NaV1.7 mRNA. Genentech Pharmaceuticals provided a highly selective NaV1.7 blocker, GNE8493, which exhibits a selectivity ranging from 500 to 5000-fold for NaV1.7 over all other NaV subtypes. SiteOne Pharmaceuticals contributed another mechanistically distinct and highly selective NaV1.7 blocker, ST2262, with a selectivity of 1000-fold for human NaV1.7 over other NaV subtypes (as reported in Sci Rep. 10:14791, 2020). Unfortunately, ST2262 has little affnity for rodent and guinea pig NaV1.7. We quantified the effect of GNE8493 on postganglionic compound actional potentials (CAP) within the sympathetic trunk of superior cervical ganglia (SCG). GNE8493 inhibited the CAP in the postganglionic neurons of SCG by ~ 70% (n=5, p&lt;0.01). With such a significant impact of the NaV1.7 blocker on post-ganglionic actional potential conduction, we were prompted to explore its potential physiological relevance. Our study involved the isolated pulmonary arteries from anonymous human lung donors (obtained from IIAM) and isolated pulmonary arteries and the abdominal aorta from guinea pigs. We measured smooth muscle tension using standard tissue bath techniques. Stimulation of intrinsic nerves was achieved through electrical field stimulation (EFS) at 12V, 1msec, and 10Hz for 30 seconds, resulting in rapid contractions in all tissues. These contractions were completely prevented by the nonselective NaV1 blocker tetrodotoxin and by the alpha adrenoceptor antagonist prazosin. The contractions were quantified as a percentage of the maximum adrenergic contraction achievable, induced by the addition of 100 μM phenylephrine at the end of the experiment. In guinea pigs, GNE8493 completely blocked sympathetic contractions of the isolated pulmonary arteries (IC50 of -log (M) 5.8) and abdominal aorta (IC50 of -log (M) 6.4 ± 0.3). In human pulmonary arteries, GNE8493 (1 μM) and ST2262 inhibited the sympathetic contractions 94% and 87%, respectively (p&lt;0.01, n=6). These findings support the hypothesis that pharmacological inhibition of NaV1.7 using selective inhibitors has the potential to reduce sympathetic function in specific vascular beds. R35HL155671 - BJU F32HL170490 - JSK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Clinical and Genetic Features of Dravet Syndrome: A Prime Example of the Role of Precision Medicine in Genetic Epilepsy.

Dravet syndrome (DS), also known as severe myoclonic epilepsy of infancy, is a rare and drug-resistant form of developmental and epileptic encephalopathies, which is both debilitating and challenging to manage, typically arising during the first year of life, with seizures often triggered by fever, infections, or vaccinations. It is characterized by frequent and prolonged seizures, developmental delays, and various other neurological and behavioral impairments. Most cases result from pathogenic mutations in the sodium voltage-gated channel alpha subunit 1 (SCN1A) gene, which encodes a critical voltage-gated sodium channel subunit involved in neuronal excitability. Precision medicine offers significant potential for improving DS diagnosis and treatment. Early genetic testing enables timely and accurate diagnosis. Advances in our understanding of DS's underlying genetic mechanisms and neurobiology have enabled the development of targeted therapies, such as gene therapy, offering more effective and less invasive treatment options for patients with DS. Targeted and gene therapies provide hope for more effective and personalized treatments. However, research into novel approaches remains in its early stages, and their clinical application remains to be seen. This review addresses the current understanding of clinical DS features, genetic involvement in DS development, and outcomes of novel DS therapies.

Genetic testing in inflammatory cardiomyopathy/myocarditis: a red flag for cardiomyopathy-like histologic findings

Abstract Background Myocarditis/inflammatory cardiomyopathy is characterized by the presence of an inflammatory infiltrate in the myocardium. Its etiology is heterogeneous, predominantly mediated by viral infection, as well as a wide variety of toxic substances and drugs and systemic immune-mediated diseases. Outcome could vary from a complete recovery to a residual myocardial injury with the subsequent development of dilated cardiomyopathy (DCM). The factors that play a role in the disease progression are not clear, but it has been shown that inflammatory cardiomyopathy complicated by left ventricular dysfunction, heart failure or arrhythmia is associated with a poor prognosis. Purpose To characterize the genetic background of inflammatory cardiomyopathy affected patients to determine the prevalence of pathogenic/likely pathogenic (P/LP) variants in cardiomyopathy-related genes and correlate to histopathologic findings in endomyocardial biopsies (EMB). Methods Eighty-nine myocarditis-affected patients (mean age 43±14, 55 males) underwent genetic screening of 200 genes related to inherited cardiomyopathies. Definite/moderate gene association with DCM and Arrhythmogenic Cardiomyopathy was based on the ClinGen framework. Variant prioritization was carried out using American College of Medical Genetics and Genomics rules. Correlation with histopathologic findings of the endomyocardial biopsy and family history was appraised. Results Twenty of the 89 suspected myocarditis patients (22%) carried a P/LP variant in cardiac-related genes: 8 in Titin, 3 in Myosin Heavy Chain 7, 2 in Desmoplakin, 2 in Lamin A/C; and Troponin T2, Filamin-C, Myosing Binding Protein C3, Desmin and Sodium Voltage-Gated Channel Alpha Subunit 5 carried one variant each. A re-analysis of the EMBs revealed that 82% of patients showed cardiomyopathy-like features at first histologic findings, with fibrosis in the myocardium and DCM-like phenotype at the second EMB. Further, 13 of the genotype-positive patients referred positive family history for cardiomyopathy and/or sudden cardiac death, whereas only 7 genotype-negative patients declared family history, showing a significant difference among both groups (p-value&amp;lt;0,0001). Conclusion The prevalence of P/LP variants in inflammatory cardiomyopathy/myocarditis patients is 22%, most of them associated with DCM. Cardiomyopathy-like features were already observed at the first EMB during the histologic re-evaluation in the majority of genotype-positive patients, and among them, high frequency of positive family history was observed. These findings suggest that genotype-positive inflammatory cardiomyopathy/myocarditis affected patients should be deeply clinically evaluated in order to guarantee an appropriate management for them and their families.

Identification of five novel SCN1A variants.

Epilepsy is characterized by recurrent unprovoked seizures. Mutations in the voltage-gated sodium channel alpha subunit 1 (SCN1A) gene are the main monogenic cause of epilepsy. Type and location of variants make a huge difference in the severity of SCN1A disorder, ranging from the mild phenotype (genetic epilepsy with febrile seizures plus, GEFS+) to the severe phenotype (developmental and epileptic encephalopathies, DEEs). Dravet Syndrome (DS) is an infantile-onset DEE, characterized by drug-resistant epilepsy and temperature sensitivity or febrile seizures. Genetic test results reveal SCN1A variants are positive in 80% DS patients and DS is mainly caused by de novo variants. Trio-whole exome sequencing (WES) was used to detect variants which were associated with clinical phenotype of five probands with epilepsy or twitching. Then, Sanger sequencing was performed to validate the five novel SCN1A variants and segregation analysis. After analyzing the location of five SCN1A variants, the pathogenic potential was assessed. In this study, we identified five novel SCN1A variants (c.4224G > C, c.3744_3752del, c.209del, c.5727_5734delTTTAAAACinsCTTAAAAAG and c.5776delT) as the causative variants. In the five novel SCN1A variants, four were de novo and the remaining one was inherited. All novel variants would be classified as "pathogenic" or "likely pathogenic." The five novel SCN1A variants will enrich the SCN1A mutations database and provide the corresponding reference data for the further genetic counseling.

Twelve-Lead ECG, Holter Monitoring Parameters, and Genetic Testing in Brugada Syndrome: Insights from Analysis of Multigenerational Family with a History of Sudden Cardiac Arrest during Physical Activity.

Brugada syndrome (BrS) is an arrhythmogenic disorder increasing the risk of syncopal episodes and sudden cardiac death. BrS usually runs through families with reduced penetrance and variable expression. We analyzed the multigenerational family of a patient who died after sudden cardiac arrest with post-mortem diagnosis of BrS. We analyzed clinical history, comprehensive arrhythmic risk, genetic findings, and additional tests, including electrocardiogram (ECG), detailed 24-hour Holter ECG results, and standard echocardiography findings, and followed up the patients in the ambulatory clinic. We analyzed a pedigree of 33 members of four generations of the family (19 male and 14 female patients). In this family, we identified 7 patients with BrS (median Modified Shanghai Score and Sieira model: 4.5 (4-6) and 1 (0-4) points, respectively), including both parents of the deceased patient, and 8 relatives with negative sodium channel blocker drug challenge test. Genetic testing revealed a novel mutation in sodium voltage-gated channel alpha subunit 5 (SCN5A) c.941A>G, (p.Tyr314Cys) inherited from the father of the proband. Patients with BrS were characterized by longer P-wave duration (120 (102-155) vs. 92.5 (88-110) ms, p = 0.013) and longer PR intervals (211.3 ±26.3 vs. 161.6 ± 18.9 ms, p = 0.001), along with more frequent positive aVR sign, but did not differ in terms of QRS duration or T-wave characteristics in resting ECGs. BrS patients were characterized by lower mean, minimal, and maximal (for all p ≤ 0.01) heart rates obtained from Holter ECG monitoring, while there was no difference in arrhythmias among investigated patients. Moreover, visual diurnal variability of ST segment changes and fragmented QRS complexes were observed in patients with BrS in Holter ECG monitoring. There were no major arrhythmic events during median follow-up of 68.7 months of alive BrS patients. These results suggest ECG features which may be associated with a diagnosis of BrS and indicate a novel SCN5A variant in BrS patients. Twelve-lead Holter ECG monitoring, with modified precordial leads placement, may be useful in BrS diagnostics and risk stratification in personalized medicine.

Long non-coding RNA Snhg16 Lessens Ozone Curative Effect on Chronic Constriction Injury mice via microRNA-719/SCN1A axis.

We investigated the function and molecular mechanism of long non-coding RNA (lncRNA) small nucleolar RNA host gene 16 (Snhg16) in modifying ozone treatment for neuropathic pain (NP) in a mouse model of chronic constriction injury (CCI). Pain-related behavioral responses were evaluated using paw withdrawal threshold (PWT), paw lifting number (PLN), and paw withdrawal latency (PWL) tests. Interleukin (IL)-1β, IL-10, IL-6, and tumor necrosis factor-alpha (TNF-α) were measured by ELISA and qRT-PCR to evaluate neuroinflammation. qRT-PCR was performed to detect expressions of Snhg16, microRNA (miR)-719, sodium voltage-gated channel alpha subunit 1 (SCN1A), and inflammatory factors. Bioinformatics, dual-luciferase reporter assay, and RNA pull-down verified the underlying molecular mechanisms. Snhg16 expression increased in CCI mice. Snhg16 overexpression retarded the curative effect of ozone and induced NP. miR-719 was sponged by Snhg16. SCN1A was a target of miR-719. Inhibition of miR-719 markedly reversed the effects of Snhg16 on pain-related behavioral responses and neuroinflammation. Upregulation of SCN1A partly abrogated the effects of elevated miR-719 levels on the occurrence of NP. The findings demonstrate that lncRNA Snhg16 promotes NP progression in CCI mice by binding to miR-719 to increase SCN1A expression. The Snhg16/miR-719/SCN1A axis may influence the curative effects of ozone therapy in treating NP.

An SCN1A gene missense variant in a Chinese Tujia ethnic family with genetic epilepsy with febrile seizures plus.

Genetic epilepsy with febrile seizures plus (GEFSP) is a familial epileptic syndrome that is genetically heterogeneous and inherited in an autosomal dominant form in most cases. To date, at least seven genes have been reported to associate with GEFSP. This study aimed to identify the disease-causing variant in a Chinese Tujia ethnic family with GEFSP by using whole exome sequencing, Sanger sequencing, and in silico prediction. A heterozygous missense variant c.5725A>G (p.T1909A) was identified in the sodium voltage-gated channel alpha subunit 1 gene (SCN1A) coding region. The variant co-segregated with the GEFSP phenotype in this family, and it was predicted as disease-causing by multiple in silico programs, which was proposed as the genetic cause of GEFSP, further genetically diagnosed as GEFSP2. These findings expand the genetic and phenotypic spectrum of GEFSP and should contribute to genetic diagnoses, personalized therapies, and prognoses.

Generation of an induced pluripotent stem cell line from a patient with conduction disease and recurrent ventricular fibrillation with a sodium voltage-gated channel alpha subunit 5 (SCN5A) gene c.392 + 3A > G splice-site variant

Variants in the sodium voltage-gated channel alpha subunit 5 gene (SCN5A) produce variable cardiac phenotypes including Brugada syndrome, conduction disease and cardiomyopathy. These phenotypes can lead to life-threatening arrhythmias, heart failure, and sudden cardiac death. Novel variants in splice-site regions of SCN5A require functional studies to characterise their pathogenicity as they are poorly understood. The generation of an induced pluripotent stem cell line provides a valuable resource to investigate the functional effects of potential splice-disrupting variants in SCN5A.

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing

PurposeNeurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains below 50%, suggesting that clinically relevant variants may be missed by standard analyses. Here, we analyze “poison exons” (PEs), which are evolutionarily conserved alternative exons often absent from standard gene annotations. Variants that alter PE inclusion can lead to loss of function and may be highly penetrant contributors to disease. MethodsWe curated published RNA sequencing data from developing mouse cortex to define 1937 conserved PE regions potentially relevant to NDDs, and we analyzed variants found by genome sequencing in multiple NDD cohorts. ResultsAcross 2999 probands, we found 6 novel clinically relevant variants in PE regions. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family (SCN1A, SCN2A, and SCN8A), which is associated with epilepsies. One variant is in SNRPB, associated with cerebrocostomandibular syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and in genes with gene-phenotype associations consistent with each probands reported features. ConclusionWith a very minimal increase in variant analysis burden (average of 0.77 variants per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

A Novel Approach to Assess the Impact of Disease in Patients with SCN8A-Related Developmental and Epileptic Encephalopathy (P3-9.008)

<h3>Objective:</h3> Clinical data generated using Invitae’s Ciitizen patient-consented, real-world data platform were used to explore disease burden in <i>SCN8A</i>-DEE. <h3>Background:</h3> <i>SCN8A</i>-related DEE is a rare disease associated with pathogenic variants in the <i>SCN8A</i> gene encoding the voltage-gated sodium channel alpha subunit Na_v1.6. Due to significant phenotypic heterogeneity, comprehensive understanding of disease impact and progression is limited. <h3>Design/Methods:</h3> Real-world clinical data were extracted from patients with an <i>SCN8A</i> variant using the Ciitizen platform, which employs a proprietary approach combining machine learning and expert human review to streamline generation of research-grade data from unstructured health records spanning ~10 years. Extracted data including seizure history, comorbidities, and therapeutic interventions were analyzed. <h3>Results:</h3> 79 patients were enrolled and contributed data for analyses. Mean patient age was 8.2 years (1–23), with similar percentages of males and females. Patients were categorized based on age at seizure onset; ≤6 months (75.6%) or &gt;6 months (34.2%). A further group included patients without seizures (5%). Mean age at seizure onset was 70.4 days (1–175) for patients with early (≤6 months) seizures and 749.3 days (224–4409) for those with later (&gt;6 months) seizures. While seizure frequency was variable, most patients continued to experience seizures over time. Procedural interventions and hospitalizations were high across the lifespan; nearly a third occurring in the first year of life. Mean number of medications prescribed over a patient’s lifetime was 17.6 (3–44). Patients experienced multiple comorbidities; global developmental delay was the most common (81%), with sleep disorders, hypotonia, feeding difficulties and GERD reported in &gt;50%. <h3>Conclusions:</h3> This work represents a new generation of natural history study providing the most robust real-world dataset to date on disease burden and progression in <i>SCN8A</i>-DEE. Knowledge from this study is anticipated to accelerate drug development efforts by providing new insights into the broad, longitudinal impact of disease, therefore informing trial endpoints beyond seizure symptomatology. <b>Disclosure:</b> Kelley Dalby has received personal compensation for serving as an employee of Praxis Precision Medicines. Kelley Dalby has stock in Praxis Precision Medicines. Kelley Dalby has stock in RogCon. Mr. Snyder has received personal compensation for serving as an employee of Praxis Precision Medicines. Mr. Snyder has received stock or an ownership interest from Praxis Precision Medicines. Dr. G Matthews has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Praxis Precision Medicines. Dr. G Matthews has stock in Praxis Precision Medicines. Dr. Oldham has received personal compensation for serving as an employee of Praxis Precision Medicines. Steven Petrou has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Praxis Precision Medicines. Steven Petrou has stock in Praxis Precision Medicines. The institution of Steven Petrou has received research support from Praxis Precision Medicines. The institution of Steven Petrou has received research support from Medical Research Future Fund. Steven Petrou has received intellectual property interests from a discovery or technology relating to health care. Steven Petrou has received intellectual property interests from a discovery or technology relating to health care. Dr. Souza has received personal compensation for serving as an employee of Praxis Precision Medicines. Dr. Souza has stock in Praxis Precision Medicines.

HiPSC-derived cardiomyocyte to model Brugada syndrome: both asymptomatic and symptomatic mutation carriers reveal increased arrhythmogenicity

Brugada syndrome is an inherited cardiac arrhythmia disorder that is mainly associated with mutations of the cardiac voltage-gated sodium channel alpha subunit 5 (SCN5A) gene. The clinical symptoms include ventricular fibrillation and an increased risk of sudden cardiac death. Human-induced pluripotent stem cell (hiPSC) lines were derived from symptomatic and asymptomatic individuals carrying the R1913C mutation in the SCN5A gene. The present work aimed to observe the phenotype-specific differences in hiPSC-derived cardiomyocytes (CMs) obtained from symptomatic and asymptomatic mutation carriers. In this study, CM electrophysiological properties, beating abilities and calcium parameters were measured. Mutant CMs exhibited higher average sodium current densities than healthy CMs, but the differences were not statistically significant. Action potential durations were significantly shorter in CMs from the symptomatic individual, and a spike-and-dome morphology of action potential was exclusively observed in CMs from the symptomatic individual. More arrhythmias occurred in mutant CMs at single cell and cell aggregate levels compared with those observed in wild-type CMs. Moreover, there were no major differences in ionic currents or intracellular calcium dynamics between the CMs of asymptomatic and symptomatic individuals after the administration of adrenaline and flecainide.In conclusion, mutant CMs were more prone to arrhythmia than healthy CMs but did not explain why only one of the mutation carriers was symptomatic.

Abstract 2057: Pan-cancer analysis of ion channel genes reveals patterns of large-scale transcriptional dysregulation and novel oncogenes with functions in cellular connectivity

Abstract Ion channels (IC) are one of the largest families of highly druggable proteins targeted in therapies for many human diseases; however, their role in cancer remains understudied. IC drug repurposing for cancer therapies is an appealing avenue; however, prioritizing ICs for pre-clinical research, mechanistic elucidation, and target development remains a challenge. Here we performed a systematic, pan-cancer analysis to prioritize IC genes as putative targets for therapeutic and biomarker development. Using machine learning, we studied ~10,000 human transcriptomes from 32 cancer types of The Cancer Genome Atlas (TCGA) to identify ICs whose expression significantly associated with patient survival. IC genes showed dysregulation in all cancer types: elevated IC expression was characteristic of a subset of tumors often associated with poor patient prognosis. Excessive IC expression was unexpectedly frequent and high-magnitude compared to control sets of protein-coding genes and genes of major druggable families. We focused on ICs in glioblastoma (GBM), the most prevalent and lethal form of brain cancer in adults, prioritizing GJB2 (gap junction protein beta 2) and SCN9A (sodium voltage-gated channel alpha subunit 9) as potential GBM oncogenes. High expression of these ICs associated with poor GBM prognosis in TCGA and independent transcriptomics datasets. GJB2 and SCN9A expression was higher in GBMs than normal brain tissues and associated with GBM subtypes, histological features, and immune cell infiltration. At the single-cell level, GJB2 and SCN9A were predominantly expressed in malignant cell types in GBMs. We validated GJB2 and SCN9A with shRNA-mediated knockdowns (KDs) in patient-derived GBM cell lines and mouse xenograft models. First, GJB2 and SCN9A KDs significantly impaired cell viability and spheroid formation. We then validated survival associations, showing that GJB2 and SCN9A KDs prolonged survival of mice with GBM xenografts. Finally, we showed GBJ2 KD impaired tunneling nanotube length in GBM cells. Transcriptome-wide profiling of IC KD cells revealed hundreds of differentially expressed genes. Integrative pathway enrichment across IC KDs in GBM cells and IC-stratified TCGA samples identified dysregulated pathways with functional themes of neuronal development, cancer hallmarks, and regulation of glial cells, including tumor cell connectivity pathways in GBM, corroborating the phenotypes observed in our functional validations. Our pan-cancer analysis prioritized ICs for further pre-clinical development and mechanistic study. The functional validation experiments of GJB2 and SCN9A highlight their possible roles in GBM pathology and lends confidence to our analysis. This study demonstrates the utility of investigating ICs as avenues for drug repurposing and the development of novel cancer therapeutics. Citation Format: Alexander Bahcheli, Hyun Min, Xi Huang, Jüri Reimand. Pan-cancer analysis of ion channel genes reveals patterns of large-scale transcriptional dysregulation and novel oncogenes with functions in cellular connectivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2057.

Underexpression of SCN7A is associated with poor prognosis in lung adenocarcinoma

Lung cancer is one of the most common malignancies and the leading cause of cancer-related deaths worldwide. Elucidating the mechanism behind the development of lung cancer at a molecular level could reveal new biomarkers and clinical therapeutic targets. A growing body of evidence has shown an association between ion channel-related genes and the progression of various diseases, including cancer. However, the association is not well-understood. In this study, we identified ion channel-related genes differentially expressed between cancer and normal lung tissue cells. Data were extracted from GSE31552, GSE33532, and GSE103512 lung adenocarcinoma (LUAD) datasets, and the prognostic value of the differentially expressed genes between LUAD and normal lung tissue was evaluated using data in The Cancer Genome Atlas. Only sodium voltage-gated channel alpha subunit 7 (SCN7A) was found to be significantly correlated to prognosis. The expression of SCN7A in LUAD was assessed further by immunological analysis. We also constructed a competing endogenous RNA network of SCN7A. Further analyses revealed that the underexpression of SCN7A predicted a poor prognosis in LUAD, with a strong correlation between SCN7A expression and immune cell infiltration as well as immune checkpoint expression. We found that SCN7A is potentially regulated via the OTUD6B-AS1-miR-21-5p-SCN7A axis in LUAD cells and proved that SCN7A inhibits the proliferation and migration of lung cancer cells in vitro. Taken together, SCN7A, as an independent prognostic factor, is a promising diagnostic biomarker for LUAD, and the OTUD6B-AS1-miR-21-5p-SCN7A axis is a potential regulatory network of SCN7A expression.

Developmental changes in brain activity of heterozygous Scn1a knockout rats.

Dravet syndrome (DS) is an infantile-onset developmental and epileptic encephalopathy characterized by an age-dependent evolution of drug-resistant seizures and poor developmental outcomes. Functional impairment of gamma-aminobutyric acid (GABA)ergic interneurons due to loss-of-function mutation of SCN1A is currently considered the main pathogenesis. In this study, to better understand the age-dependent changes in the pathogenesis of DS, we characterized the activity of different brain regions in Scn1a knockout rats at each developmental stage. We established an Scn1a knockout rat model and examined brain activity from postnatal day (P) 15 to 38 using a manganese-enhanced magnetic resonance imaging technique (MEMRI). Scn1a heterozygous knockout (Scn1a +/-) rats showed a reduced expression of voltage-gated sodium channel alpha subunit 1 protein in the brain and heat-induced seizures. Neural activity was significantly higher in widespread brain regions of Scn1a +/- rats than in wild-type rats from P19 to P22, but this difference did not persist thereafter. Bumetanide, a Na+-K+-2Cl- cotransporter 1 inhibitor, mitigated hyperactivity to the wild-type level, although no change was observed in the fourth postnatal week. Bumetanide also increased heat-induced seizure thresholds of Scn1a +/- rats at P21. In Scn1a +/- rats, neural activity in widespread brain regions increased during the third postnatal week, corresponding to approximately 6 months of age in humans, when seizures most commonly develop in DS. In addition to impairment of GABAergic interneurons, the effects of bumetanide suggest a possible contribution of immature type A gamma-aminobutyric acid receptor signaling to transient hyperactivity and seizure susceptibility during the early stage of DS. This hypothesis should be addressed in the future. MEMRI is a potential technique for visualizing changes in basal brain activity in developmental and epileptic encephalopathies.