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Patient-derived induced pluripotent stem cells to study non-canonical splicing variants associated with Hypertrophic
Joanna Jager1, Marta Ribeiro2, Marta Furtado3
1University College London Institute of Cardiovascular Science, Rayne Institute, 5 University Street, London WC1E 6JF, United Kingdom.
Insights
Genetic variants in intronic regions of MYBPC3 cause hypertrophic cardiomyopathy (HCM) by affecting mRNA splicing. Induced pluripotent stem cell-derived cardiomyocytes reveal differential responses to these splicing defects, aiding disease research.
Area of Science:
- Cardiovascular Genetics
- Molecular Cardiology
- Stem Cell Biology
Background:
- Hypertrophic cardiomyopathy (HCM) is a primary inherited heart muscle disease and a significant cause of sudden cardiac death.
- Current genetic testing for HCM often misses intronic splicing variants, leaving some cases genetically undiagnosed.
- Intronic variants in MYBPC3 are implicated in HCM pathogenesis but require further functional investigation.
Purpose of the Study:
- To investigate the functional impact of intronic MYBPC3 variants on mRNA splicing and cardiomyocyte function.
- To generate and characterize induced pluripotent stem cell (iPSC) lines carrying specific intronic HCM-associated variants.
- To explore the differential mRNA processing and quality control responses to distinct intronic variants.
Main Methods:
- Generation of patient-derived iPSC lines using a non-integrative reprogramming strategy.
- Differentiation of iPSCs into iPSC-derived cardiomyocytes (iPSC-CMs).
- Analysis of mRNA splicing patterns and assessment of nonsense-mediated decay (NMD) activation in iPSC-CMs.
Main Results:
- Identified mis-spliced mRNAs in iPSC-CMs with intronic MYBPC3 variants (c.1224-52G>A and c.1898-23A>G).
- Both variants led to premature termination codons (PTCs), but only c.1224-52G>A transcripts triggered NMD-mediated degradation.
- The c.1898-23A>G variant resulted in transcripts that escaped NMD, potentially leading to truncated protein production.
Conclusions:
- Intronic MYBPC3 variants can cause HCM through aberrant mRNA splicing and differential NMD pathway engagement.
- Generated iPSC lines are crucial tools for studying the functional consequences of intronic variants in HCM.
- These iPSC models offer a platform for developing therapeutic strategies targeting splicing abnormalities in inherited cardiomyopathies.
Abstract:
Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiomyopathy and a leading cause of sudden death. Genetic testing and familial cascade screening play a pivotal role in the clinical management of HCM patients. However, conventional genetic tests primarily focus on the detection of exonic and canonical splice site variation. Oversighting intronic non-canonical splicing variants potentially contributes to a proportion of HCM patients remaining genetically undiagnosed. Here, using a non-integrative reprogramming strategy, we generated induced pluripotent stem cell (iPSC) lines from four individuals carrying one of two variants within intronic regions of MYBPC3: c.1224-52G > A and c.1898-23A > G. Upon differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), mis-spliced mRNAs were identified in cells harbouring these variants. Both abnormal mRNAs contained a premature termination codon (PTC), fitting the criteria for activation of nonsense mediated decay (NMD). However, the c.1898-23A > G transcripts escaped this mRNA quality control mechanism, while the c.1224-52G > A transcripts were degraded. The newly generated iPSC lines represent valuable tools for studying the functional consequences of intronic variation and for translational research aimed at reversing splicing abnormalities to prevent disease progression.
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