Mutations in the DES gene, which encodes the intermediate filament desmin, lead to desminopathy, a rare disease characterized by skeletal muscle weakness and different forms of cardiomyopathies ...associated with cardiac conduction defects and arrhythmias. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying the DES p.R406W mutation, and employed CRISPR/Cas9 to rectify the mutation in the patient's hiPSC line and introduced the mutation in an hiPSC line from a control individual unrelated to the patient. These hiPSC lines represent useful models for delving into the mechanisms of desminopathy and developing new therapeutic approaches.
This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 ...technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time.
For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).
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•Generation of knockout and knockin edits in hiPSCs using the CRISPR-Cas9 RNP system•FACS-assisted genome editing of hiPSCs•An optimized approach for culturing and genotyping hiPSC clones
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time.
Abstract
Aims
Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The ...limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD.
Methods and results
Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-β and inflammation in the disease.
Conclusion
The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.
Graphical Abstract
Graphical Abstract
While 3D chromatin organization in topologically associating domains (TADs) and loops mediating regulatory element-promoter interactions is crucial for tissue-specific gene regulation, the extent of ...their involvement in human Mendelian disease is largely unknown. Here, we identify 7 families presenting a new cardiac entity associated with a heterozygous deletion of 2 CTCF binding sites on 4q25, inducing TAD fusion and chromatin conformation remodeling. The CTCF binding sites are located in a gene desert at 1 Mb from the Paired-like homeodomain transcription factor 2 gene (PITX2). By introducing the ortholog of the human deletion in the mouse genome, we recapitulate the patient phenotype and characterize an opposite dysregulation of PITX2 expression in the sinoatrial node (ectopic activation) and ventricle (reduction), respectively. Chromatin conformation assay performed in human induced pluripotent stem cell-derived cardiomyocytes harboring the minimal deletion identified in family#1 reveals a conformation remodeling and fusion of TADs. We conclude that TAD remodeling mediated by deletion of CTCF binding sites causes a new autosomal dominant Mendelian cardiac disorder.
Brugada syndrome is a significant cause of sudden cardiac death (SCD), but the underlying mechanisms remain hypothetical.
This study aimed to elucidate this knowledge gap through detailed ex vivo ...human heart studies.
A heart was obtained from a 15-year-old adolescent boy with normal electrocardiogram who experienced SCD. Postmortem genotyping was performed, and clinical examinations were done on first-degree relatives. The right ventricle was optically mapped, followed by high-field magnetic resonance imaging and histology. Connexin-43 and NaV1.5 were localized by immunofluorescence, and RNA and protein expression levels were studied. HEK-293 cell surface biotinylation assays were performed to examine NaV1.5 trafficking.
A Brugada-related SCD diagnosis was established for the donor because of a SCN5A Brugada-related variant (p.D356N) inherited from his mother, together with a concomitant NKX2.5 variant of unknown significance. Optical mapping demonstrated a localized epicardial region of impaired conduction near the outflow tract, in the absence of repolarization alterations and microstructural defects, leading to conduction blocks and figure-of-8 patterns. NaV1.5 and connexin-43 localizations were normal in this region, consistent with the finding that the p.D356N variant does not affect the trafficking, nor the expression of NaV1.5. Trends of decreased NaV1.5, connexin-43, and desmoglein-2 protein levels were noted; however, the RT-qPCR results suggested that the NKX2-5 variant was unlikely to be involved.
This study demonstrates for the first time that SCD associated with a Brugada-SCN5A variant can be caused by localized functionally, not structurally, impaired conduction.
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In a first family (family#1), we identified 53 members of whom 17 present a new syndromic cardiac disorder characterized by electrical disorders (sinus node dysfunction, atrial fibrillation..) and ...developmental defects (atrial septal defect, valvopathy, left ventricle non-compaction..) following an autosomal dominant model. Six are implanted with a pacemaker and one experienced a sudden death at 43yo. Interestingly, 6 additional non-related families presenting the same phenotype have been also identified.
Our aims are to identity the causal mutation and the molecular mechanism underlying this complex cardiac syndrome.
Genetic study has been performed using whole genome sequencing (WGS). Based on transgenic mouse strains, we assessed the impact of Family#1 mutation on the phenotype and on gene expression. Then, we generated human cardiomyocytes derived iPS cells (CM-iPS) isogenic models to evaluate the epigenome (CUT&RUN and ATAC-seq), transcriptome (RNA-seq) and topological associated domain (TAD) remodelling (Hi-C).
By WGS we uncovered a deletion of 15kb in a gene desert area on 4q25, segregating in all affected relatives of Family#1. The 6 other families present overlapping deletions. Mouse model recapitulates the cardiac phenotype and exhibit an increase of Pitx2 expression in right atria, unchanged in left atria and a decrease in ventricle. Based on human CM-iPS models, epigenetic data highlight among the 15kb deletion a unique open region containing a CTCF binding site, crucial for delimiting TAD boundaries. Hi-C assay reveals the fusion of 2 TADs and highlights new interactions between PITX2 and atrial specific regulatory elements.
We identified a deletion located within a gene desert area associated with a new complex cardiac disorder. The CTCF binding site contained in the deletion seems key for the TAD border. The TAD remodelling leads to new (regulatory) interactions and expression dysregulation of PITX2. We describe a new molecular mechanism implying a yet unidentified non-coding regulatory element of PITX2 and responsible for a complex electrical and developmental cardiac syndrome.
