Ontology highlight
ABSTRACT:
PROVIDER: PRJNA985079 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24957189.fastq.gz | Fastqsanger.gz | |||
| SRR24957190.fastq.gz | Fastqsanger.gz | |||
| SRR24957191.fastq.gz | Fastqsanger.gz | |||
| SRR24957192.fastq.gz | Fastqsanger.gz | |||
| SRR24957193.fastq.gz | Fastqsanger.gz |
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Project description:BACKGROUND: 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. METHODS: 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). RESULTS: 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 wildtype. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A downregulation 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 and increased arrhythmia risk with disruption of PDGF sigaling were both 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
2022-12-31 | GSE221945 | GEO
Project description:Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used to examine in vitro the effect of mutations in the cardiac sodium channel gene SCN5A, associated with cardiac arrhythmias. Postnatally SCN5A undergoes a fetal-to-adult isoform switch, but hiPSC-CMs in conventional 2-dimensional cultures are fetal-like. This impedes evaluation of mutations in the adult isoform. Here, we derived hiPSC-CMs from a patient carrying compound mutations in the adult SCN5A exon 6B and in exon 4 and generated isogenic corrected lines. In hiPSC-CM 2-dimensional culture, exon 6B mutation did not affect single-cell electrophysiology because of its limited expression. CRISPR/Cas9-mediated excision of the fetal exon 6A with did not promote adult SCN5A expression, rather it impaired the splicing. By maturing hiPSC-CMs in three-dimensional tri-cell type cardiac microtissues, SCN5A underwent isoform switch and revealed the functional effect of exon 6B mutation. Upregulation of the splicing factor MBNL1 in hiPSC-CMs either by culture in microtissues or by overexpression was sufficient to promote exon 6B inclusion. Our results support the ability to study developmentally regulated cardiac genes and postnatal cardiac arrhythmias using hiPSC cardiac cells.
2023-05-03 | GSE180290 | GEO
Project description:Brugada syndrome (BrS) is a cardiac arrhythmia disorder that causes sudden death in young adults. Rare genetic variants in the SCN5A gene, encoding the Nav1.5 sodium channel, and common non-coding variants at this locus, are robustly associated with the condition. BrS is particularly prevalent in Southeast Asia but the underlying ancestry-specific factors remain largely unknown. Methods: Genome sequencing of BrS probands and population-matched controls from Thailand was performed to identify rare non-coding variants at the SCN5A-SCN10A locus that were enriched in BrS cases. A likely causal variant was prioritised by computational methods and introduced into human pluripotent stem cell (hiPSC) lines using CRISPR-Cas9. The effect of the variant on SCN5A expression and Nav1.5 sodium channel current was then assessed in hiPSC-derived cardiomyocytes (hiPSC-CMs). Results: A rare non-coding variant in an SCN5A intronic enhancer region was highly enriched in BrS cases (detected in 3.9% of cases with a case-control odds ratio of 45.2). The variant affects a nucleotide conserved across all mammalian species and predicted to disrupt a Mef2 transcription factor binding site. Heterozygous introduction of the enhancer variant in hiPSC-CMs caused significantly reduced SCN5A expression from the variant-containing allele and a 30% reduction in Nav1.5-mediated sodium current density compared to isogenic controls, confirming its pathogenicity. Patients with the variant had severe phenotypes, with 89% experiencing cardiac arrest. Conclusions: This is the first example of a functionally validated rare non-coding variant at the SCN5A locus and highlights how genome sequencing in understudied populations can identify novel disease mechanisms. The variant partly explains the increased prevalence of BrS in this region and enables the identification of at-risk variant carriers to reduce the burden of sudden cardiac death in Thailand.
2025-01-02 | GSE264359 | GEO
Project description:This study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs. BrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression.
2017-01-12 | GSE93530 | GEO
Project description:CACNA1A encodes the pore-forming α1A subunit of CaV2.1 calcium channel, whose altered function is associated with different neurological disorders including variable forms of ataxia, epilepsy, and migraine. In this study, we generated isogenic iPSC-derived neural cultures carrying two CACNA1A loss-of-function variants differently affecting CaV2.1 isoforms. Morphological, molecular, and functional assays highlighted distinct neurodevelopmental defects. F1491S mutation, which is located in a constitutive domain of the channel, impaired neural induction at very early stages, as demonstrated by changes in migratory capacity and single-cell transcriptomic signatures in neural progenitors, and by defective polarization in neurons. Instead, cells carrying Y1854X mutation, which selectively affects the [EFa] isoform, behaved normally in terms of neural induction but showed altered neuronal network composition and lack of synchronized activity. Our findings highlighted important roles of CACNA1A in mechanisms underlying early neural induction, neuronal maturation, and neural network dynamics.
2025-02-03 | GSE276494 | GEO
Project description:We investigated the ALDH2*2 genetic polymorphism and its underlying mechanisms for the first time in a human model system of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation confers elevated levels of reactive oxygen species (ROS) and toxic aldehydes such as 4HNE, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. ALDH2 exerts control of cell survival decisions via modulation of oxidative stress levels. This regulatory circuitry was found to be dysfunctional in the loss-of-function ALDH2*2 genotype, causing upregulation of apoptosis in cardiomyocytes following ischemic insult. These results reveal a novel function of the metabolic enzyme ALDH2 in modulation of cell survival decisions. Molecular mechanism of increased ischemic damage in cardiomyocytes of ALDH2*2 genotype.
2014-07-07 | E-GEOD-59100 | biostudies-arrayexpress
Project description:Background: The phospholamban (PLN) p.Arg14del mutation belongs to the established causes of dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, CRISPR/Cas9 gene editing and patient heart samples to investigate the molecular pathomechanisms of PLN p.Arg14del cardiomyopathy. Methods and results: Patient dermal fibroblasts carrying the PLN p.Arg14del mutation were reprogrammed into hiPSC, isogenic controls were established by CRISPR/Cas9. Cells were differentiated into cardiomyocytes and characterized in 2D and 3D-engineered heart tissue (EHT) format. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern and lower peak force compared to isogenic controls. While this data support the reported super-inhibitory effect of PLN p.Arg14del on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, an unchanged SR Ca2+ content argued against disturbed SR Ca2+ cycling as the sole cause of contractile dysfunction. Label-free proteomic analysis of p.Arg14del EHTs revealed less endoplasmic reticulum (ER), ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the rough ER, large lipid droplets in close association with mitochondria and reduced mitochondrial number. Follow-up experiments confirmed impairment of the rough ER/mitochondria compartment and enhanced oxidative stress in PLN p.Arg14del. Relevance for human disease was demonstrated by immunohistochemistry of human heart samples. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison to ischemic heart failure - and non-failing donor heart samples. Surprisingly, transduction of PLN p.Arg14del EHTs with the Ca2+ binding protein GCaMP6f reversed the contractile, molecular and morphological disease phenotype. Conclusion: This study identified impairment of the rough ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.
2021-04-12 | PXD020175 | Pride
Project description:Phospholamban R14del mutazion (PLN-R14del) has been identified in a large family pedigree in which heterozygous carriers exhibited inherited dilated cardiomyopathy (DCM) and death by middle age. To better understand the causal link between the mutations in PLN and DCM pathology, we derived induced pluripotent stem cells from a DCM patient carrying the PLN R14del mutation. We showed that iPSC-derived cardiomyocytes recapitulated the DCM-specific phenotype and demonstrated that either TALEN-mediated genetic correction or combinatorial gene therapy resulted in phenotypic rescue. Our findings offer novel insights into the pathogenesis caused by mutant PLN and point to the development of potential new therapeutics of pathogenic genetic variants associated with inherited cardiomyopathies. iPSCs were derived from a female patient carrying a heterozygous mutation (R14del) in the PLN gene. Tree samples were analyzed: Cardiomyocytes derived from PLN-R41del iPSC cells (R14del-CM); R14del-CMs infected with AAV6-EGFP-miR-PLN and R14del-CMs infected with AAV6-EGFP-miR-luc used as a negative control
2015-02-09 | E-GEOD-65761 | biostudies-arrayexpress
Project description:Mutations in the lamin A (LMNA) gene, which encodes the lamin A and C proteins, cause severe human diseases collectively known as laminopathies. These conditions are often devastating and lack effective therapies. In this study, we developed precise base editing (BE) strategies targeting the human LMNA gene variants L35P and R249Q, which cause Congenital Muscular Dystrophy (CMD) and Dilated Cardiomyopathy with Conduction Defects (DCM-CD), respectively. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying the R249Q mutation displayed nuclear aberrations, DNA damage, and abnormal Ca2+ transients. Similarly, L35P iPSC-CMs exhibited abnormal contraction, DNA damage, and reduced lamin A/C protein expression. We also generated novel “humanized” mouse models carrying these pathogenic human mutations. R249Q homozygous mice exhibited cardiac conduction abnormalities, cardiac arrhythmias, and premature death. Mice with the homozygous L35P mutation displayed severe muscle-wasting and reduced lifespan, while heterozygous L35P mice displayed dilated cardiomyopathy (DCM). We developed an adenine base editing (ABE) approach for correcting the R249Q mutation and a cytosine base editing (CBE) strategy for the L35P variant. Precise correction of these mutations in iPSC-CMs successfully rescued all of the in vitro abnormalities. Furthermore, delivery of the BE components using adeno-associated virus (AAV) prevented the pathological phenotypes and extended longevity of mice carrying the LMNA L35P and the R249Q mutations. These results demonstrate the efficacy of ABE and CBE in correcting pathogenic LMNA mutations that cause cardiac disease, highlighting BE as a promising therapeutic approach for human laminopathies.
2025-09-23 | GSE308777 | GEO
Project description:Sustained Ca2+ entry into CD4+CD8+ double-positive thymocytes is required for positive selection. We identified a voltage-gated Na+ channel (VGSC), essential for positive selection of CD4+ T cells. Pharmacological inhibition of VGSC activity inhibited sustained Ca2+ influx induced by positive-selecting ligands and in vitro positive selection of CD4+ but not CD8+ T cells. In vivo shRNA knockdown of Scn5a specifically inhibited positive selection of CD4+ T cells. Ectopic expression of VGSC in peripheral AND CD4+ T cells bestowed the ability to respond to a positively selecting ligand, directly demonstrating VGSC expression was responsible for increased sensitivity. Thus active VGSCs in thymocytes provides a mechanism by which a weak positive selecting signal can induce sustained Ca2+ signals required for CD4+ T cell development. Pre-selected AND DP thymocytes were stimulated with plate bound peptide-loaded I-Ek Ig dimers. Genes were differentially regulated by positive and negative selection signals. The goal of this study is to identify ion-channel related genes critical for thymic positive selection, given the sustained Ca2+ entry into double positive thymocytes is required for positive selection. Total RNA of pre-selected AND thymocytes stimulated with I-Ek Ig dimers loaded with the positively-selecting peptide gp250, the negatively-selecting agonist peptide MCC, or the non-selecting control peptide Hb were used for the transcriptional profiling analysis. We found 28 genes were differentially down-regulated by MCC stimulation but upregulated by gp250 stimulation. One of them, scn4b, was ion-channel related.
2012-06-30 | E-GEOD-38909 | biostudies-arrayexpress