Do sodium channel proteolytic fragments regulate sodium channel expression?

© 2017 Taylor & Francis The cardiac voltage-gated sodium channel (gene: SCN5A, protein: Na V 1.5) is responsible for the sodium current that initiates the cardiomyocyte action potential. Research into the mechanisms of SCN5A gene expression has gained momentum over the last few years. We have recently described the transcriptional regulation of SCN5A by GATA4 transcription factor. In this addendum to our study, we report our observations that 1) the linker between domains I and II (L DI-DII ) of Na V 1.5 contains a nuclear localization signal (residues 474–481) that is necessary to localize L DI-DII into the nucleus, and 2) nuclear L DI-DII activates the SCN5A promoter in gene reporter assays using cardiac-like H9c2 cells. Given that voltage-gated sodium channels are known targets of proteases such as calpain, we speculate that Na V 1.5 degradation is signaled to the cell transcriptional machinery via nuclear localization of L DI-DII and subsequent stimulation of the SCN5A promoter

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in freshfrozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease

Large Genomic Imbalances in Brugada Syndrome

Purpose Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field. Methods 220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS). Results The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes. Conclusion CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related genes

Regulome-seq: a novel approach for the identification of non-coding variants associated with human disease. Assessment of its applicability in 89 Brugada syndrome individuals

Brugada syndrome (BrS) is a cardiac electrical disease with high susceptibility to sudden cardiac death. Approximately 25-30% of BrS patients are explained by pathogenic variants in coding sequences of cardiac ion channels, especially in the cardiac sodium channel gene SCN5A. However, the role of genetic variants in regulatory elements affecting cardiac ion channels remains largely unknown. We integrated ENCODE information of topological organization, chromatin accessibility, histone marks, and transcription factor binding in human cardiac cells to define 1,293 putative regulatory regions of six BrS-associated genes (SCN5A, SCN2B, SCN3B, CACNA1C, CACNB2 and CACNA2D). We selectively captured and sequenced these regions in 89 BrS patients and compared the genetic variation identified to that present in a cohort of 200 healthy-aging individuals. Finally, we scored the variants based on the tolerance to variation and other parameters, allowing us to propose candidate regulatory variants that may explain the molecular basis of some BrS casesLa síndrome de Brugada (SBr) és una malaltia elèctrica cardíaca associada a mort sobtada cardíaca. Aproximadament un 25-30% dels pacients amb SBr s’expliquen per variants patogèniques en les seqüències codificants dels canals iònics cardíacs, especialment en el gen del canal de sodi cardíac SCN5A. Tot i així, els paper de les variants genètiques en els elements reguladors dels canals iònics cardíacs és encara desconegut. Utilitzant informació sobre l’organització topològica, accessibilitat de la cromatina i unió de factors de transcripció en cèl·lules cardíaques humanes, hem definit 1.293 regions reguladores de sis gens associats a SBr (SCN5A, SCN2B, SCN3B, CACNA1C, CACNB2 i CACNA2D). Hem seqüenciat aquestes regions en 89 pacients amb SBr i hem comparat les variants identificades amb les variants presents en 200 individus sans. Finalment, hem anotat les variants segons la tolerància a la variació i altres paràmetres, permetent-nos proposar variants reguladores candidates que podrien explicar alguns casos amb SB

Regulome-seq: a novel approach for the identification of non-coding variants associated with human disease. Assessment of its applicability in 89 Brugada syndrome individuals

Brugada syndrome (BrS) is a cardiac electrical disease with high susceptibility to sudden cardiac death. Approximately 25-30% of BrS patients are explained by pathogenic variants in coding sequences of cardiac ion channels, especially in the cardiac sodium channel gene SCN5A. However, the role of genetic variants in regulatory elements affecting cardiac ion channels remains largely unknown. We integrated ENCODE information of topological organization, chromatin accessibility, histone marks, and transcription factor binding in human cardiac cells to define 1,293 putative regulatory regions of six BrS-associated genes (SCN5A, SCN2B, SCN3B, CACNA1C, CACNB2 and CACNA2D). We selectively captured and sequenced these regions in 89 BrS patients and compared the genetic variation identified to that present in a cohort of 200 healthy-aging individuals. Finally, we scored the variants based on the tolerance to variation and other parameters, allowing us to propose candidate regulatory variants that may explain the molecular basis of some BrS casesLa síndrome de Brugada (SBr) és una malaltia elèctrica cardíaca associada a mort sobtada cardíaca. Aproximadament un 25-30% dels pacients amb SBr s’expliquen per variants patogèniques en les seqüències codificants dels canals iònics cardíacs, especialment en el gen del canal de sodi cardíac SCN5A. Tot i així, els paper de les variants genètiques en els elements reguladors dels canals iònics cardíacs és encara desconegut. Utilitzant informació sobre l’organització topològica, accessibilitat de la cromatina i unió de factors de transcripció en cèl·lules cardíaques humanes, hem definit 1.293 regions reguladores de sis gens associats a SBr (SCN5A, SCN2B, SCN3B, CACNA1C, CACNB2 i CACNA2D). Hem seqüenciat aquestes regions en 89 pacients amb SBr i hem comparat les variants identificades amb les variants presents en 200 individus sans. Finalment, hem anotat les variants segons la tolerància a la variació i altres paràmetres, permetent-nos proposar variants reguladores candidates que podrien explicar alguns casos amb SB

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac diseas

Large Genomic Imbalances in Brugada Syndrome

PURPOSE: Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field. METHODS: 220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS). RESULTS: The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes. CONCLUSION: CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related gene

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort

Brugada syndrome (BrS) is a rare genetic cardiac arrhythmia that can lead to sudden cardiac death in patients with a structurally normal heart. Genetic variations in SCN5A can be identified in approximately 20-25% of BrS cases. The aim of our work was to determine the spectrum and prevalence of genetic variations in a Spanish cohort diagnosed with BrS. Methodology/Principal Findings: We directly sequenced fourteen genes reported to be associated with BrS in 55 unrelated patients clinically diagnosed. Our genetic screening allowed the identification of 61 genetic variants. Of them, 20 potentially pathogenic variations were found in 18 of the 55 patients (32.7% of the patients, 83.3% males). Nineteen of them were located in SCN5A, and had either been previously reported as pathogenic variations or had a potentially pathogenic effect. Regarding the sequencing of the minority genes, we discovered a potentially pathogenic variation in SCN2B that was described to alter sodium current, and one nonsense variant of unknown significance in RANGRF. In addition, we also identified 40 single nucleotide variations which were either synonymous variants (four of them had not been reported yet) or common genetic variants. We next performed MLPA analysis of SCN5A for the 37 patients without an identified genetic variation, and no major rearrangements were detected. Additionally, we show that being at the 30-50 years range or exhibiting symptoms are factors for an increased potentially pathogenic variation discovery yield. In summary, the present study is the first comprehensive genetic evaluation of 14 BrSsusceptibility genes and MLPA of SCN5A in a Spanish BrS cohort. The mean pathogenic variation discovery yield is higher than that described for other European BrS cohorts (32.7% vs 20-25%, respectively), and is even higher for patients in the 30-50 years age rang

Large Genomic Imbalances in Brugada Syndrome

Purpose Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field. Methods 220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS). Results The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes. Conclusion CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related genes