RESUMO
Pre-existing coronary artery disease (CAD), and thrombotic, inflammatory, or virus infectivity response phenomena have been associated with COVID-19 disease severity. However, the association of candidate single nucleotide variants (SNVs) related to mechanisms of COVID-19 complications has been seldom analysed. Our aim was to test and validate the effect of candidate SNVs on COVID-19 severity. CARGENCORS (CARdiovascular GENetic risk score for Risk Stratification of patients positive for SARS-CoV-2 [COVID-19] virus) is an age- and sex-matched case-control study with 818 COVID-19 cases hospitalized with hypoxemia, and 1636 controls with COVID-19 treated at home. The association between severity and SNVs related to CAD (n = 32), inflammation (n = 19), thrombosis (n = 14), virus infectivity (n = 11), and two published to be related to COVID-19 severity was tested with adjusted logistic regression models. Two external independent cohorts were used for meta-analysis (SCOURGE and UK Biobank). After adjustment for potential confounders, 14 new SNVs were associated with COVID-19 severity in the CARGENCORS Study. These SNVs were related to CAD (n = 10), thrombosis (n = 2), and inflammation (n = 2). We also confirmed eight SNVs previously related to severe COVID-19 and virus infectivity. The meta-analysis showed five SNVs associated with severe COVID-19 in adjusted analyses (rs11385942, rs1561198, rs6632704, rs6629110, and rs12329760). We identified 14 novel SNVs and confirmed eight previously related to COVID-19 severity in the CARGENCORS data. In the meta-analysis, five SNVs were significantly associated to COVID-19 severity, one of them previously related to CAD.
Assuntos
COVID-19 , Doença da Artéria Coronariana , Trombose , Humanos , Estudos de Casos e Controles , SARS-CoV-2/genética , InflamaçãoRESUMO
The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (NaV1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles.
Assuntos
Epigênese Genética , Denervação Muscular , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/biossíntese , Elementos de Resposta , Transcriptoma , Animais , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Masculino , Músculo Esquelético/inervação , Músculo Esquelético/patologia , RNA-Seq , Ratos , Ratos Sprague-DawleyRESUMO
Brugada syndrome (BrS) is an inherited electrical heart disease associated with a high risk of sudden cardiac death (SCD). The genetic characterization of BrS has always been challenging. Although several cardiac ion channel genes have been associated with BrS, SCN5A is the only gene that presents definitive evidence for causality to be used for clinical diagnosis of BrS. However, more than 65% of diagnosed cases cannot be explained by variants in SCN5A or other genes. Therefore, in an important number of BrS cases, the underlying mechanisms are still elusive. Common variants, mostly located in non-coding regions, have emerged as potential modulators of the disease by affecting different regulatory mechanisms, including transcription factors (TFs), three-dimensional organization of the genome, or non-coding RNAs (ncRNAs). These common variants have been hypothesized to modulate the interindividual susceptibility of the disease, which could explain incomplete penetrance of BrS observed within families. Altogether, the study of both common and rare variants in parallel is becoming increasingly important to better understand the genetic basis underlying BrS. In this review, we aim to describe the challenges of studying non-coding variants associated with disease, re-examine the studies that have linked non-coding variants with BrS, and provide further evidence for the relevance of regulatory elements in understanding this cardiac disorder.
Assuntos
Síndrome de Brugada , Genoma Humano , RNA não Traduzido , Elementos Reguladores de Transcrição , Síndrome de Brugada/genética , Síndrome de Brugada/metabolismo , Morte Súbita Cardíaca , Feminino , Humanos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
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 disease.
Assuntos
Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Transcrição Gênica , Animais , Sítios de Ligação , Linhagem Celular , Fator de Transcrição GATA5/metabolismo , Perfilação da Expressão Gênica , Humanos , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RatosRESUMO
Human induced pluripotent and embryonic stem cell cultures (hiPSC/hESC) are phenotypically heterogeneous and prone to clonal deviations during subculturing and differentiation. Clonal deviations often emerge unnoticed, but they can change the biology of the cell culture with a negative impact on experimental reproducibility. Here, we describe a computational workflow to profile the bulk clonal composition in a hiPSC/hESC culture that can also be used to infer clonal deviations. This workflow processes data obtained with two versions of the same method. The two versions-epigenetic and transcriptomic-rely on a mechanism of stochastic H3K4me3 deposition during hiPSC/hESC derivation. This mechanism generates a signature of ten or more H3K4me3-enriched clustered protocadherin (PCDH) promoters distinct in every single cell. The aggregate of single-cell signatures provides an identificatory feature in every hiPSC/hESC line. This feature is stably transmitted to the cell progeny of the culture even after differentiation unless there is a clonal deviation event that changes the internal balance of single-cell signatures. H3K4me3 signatures can be profiled by chromatin immunoprecipitation and next-generation sequencing (ChIP-seq). Alternatively, an equivalent PCDH-expression version can be profiled by RNA-seq in PCDH-expressing hiPSC/hESC-derived cells (such as neurons, astrocytes, and cardiomyocytes; and, in long-term cultures, such as cerebral organoids). Notably, our workflow can also distinguish genetically identical hiPSC/hESC lines derived from the same patient or generated in the same editing process. Together, we propose a method to improve data sharing and reproducibility in the hiPSC and hESC fields.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias , Humanos , Reprodutibilidade dos TestesRESUMO
The global SARS-CoV-2 pandemic requires a rapid, reliable, and user-friendly diagnostic test to help control the spread of the virus. Reverse transcription and quantitative PCR (RT-qPCR) is currently the gold standard method for SARS-CoV-2 detection. Here, we develop a protocol based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and demonstrate increased sensitivity of this technique using fresh RNA extracts compared to RNA samples subjected to freezing/thawing cycles. We further compare RT-LAMP to RT-qPCR and demonstrate that the RT-LAMP approach has high sensitivity in fresh RNA extracts and can detect positive samples with Ct values between 8 and 35.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , Colorimetria/métodos , Primers do DNA , Humanos , Nasofaringe/virologia , Transcrição Reversa , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
Genome-wide association studies (GWASs) are instrumental in identifying loci harboring common single-nucleotide variants (SNVs) that affect human traits and diseases. GWAS hits emerge in clusters, but the focus is often on the most significant hit in each trait- or disease-associated locus. The remaining hits represent SNVs in linkage disequilibrium (LD) and are considered redundant and thus frequently marginally reported or exploited. Here, we interrogate the value of integrating the full set of GWAS hits in a locus repeatedly associated with cardiac conduction traits and arrhythmia, SCN5A-SCN10A. Our analysis reveals 5 common 7-SNV haplotypes (Hap1-5) with 2 combinations associated with life-threatening arrhythmia-Brugada syndrome (the risk Hap1/1 and protective Hap2/3 genotypes). Hap1 and Hap2 share 3 SNVs; thus, this analysis suggests that assuming redundancy among clustered GWAS hits can lead to confounding disease-risk associations and supports the need to deconstruct GWAS data in the context of haplotype composition.
Assuntos
Síndrome de Brugada/genética , Predisposição Genética para Doença/genética , Desequilíbrio de Ligação/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Síndrome de Brugada/diagnóstico , Testes Genéticos/métodos , Estudo de Associação Genômica Ampla/métodos , Genótipo , Haplótipos/genética , Humanos , Pessoa de Meia-Idade , Fenótipo , Locos de Características Quantitativas/genéticaRESUMO
The cardiac voltage-gated sodium channel (gene: SCN5A, protein: NaV1.5) 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 (LDI-DII) of NaV1.5 contains a nuclear localization signal (residues 474-481) that is necessary to localize LDI-DII into the nucleus, and 2) nuclear LDI-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 NaV1.5 degradation is signaled to the cell transcriptional machinery via nuclear localization of LDI-DII and subsequent stimulation of the SCN5A promoter.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Potenciais de Ação , Linhagem Celular , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Ativação do Canal Iônico , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , ProteóliseRESUMO
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.
RESUMO
BACKGROUND: 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. CONCLUSIONS: In summary, the present study is the first comprehensive genetic evaluation of 14 BrS-susceptibility 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 range.