Decoding genetic determinants of heart development
Congenital heart defects (CHDs) are the most common human birth defects, affecting nearly 2% of newborns and remaining the leading cause of fetal mortality. Although surgical outcomes have greatly improved, our understanding of how genetic variation causes CHD is still limited. Roughly half of all patients carry potentially damaging variants, yet in most cases the causal mutation, affected cell type, developmental stage, and molecular mechanism remain unclear.
Genomic studies underscore the central role of genetics in CHD pathogenesis. Large chromosomal aberrations account for about 25% of cases, while smaller copy-number variants, indels, and loss-of-function mutations are found in another ~20%. Advances in whole-exome and whole-genome sequencing have revealed over 400 candidate genes, yet strong statistical evidence supports only a small subset (~14%, or 18 of 132 genes in a recent meta-analysis). Strikingly, many of these de novo variants cluster in genes encoding chromatin regulators—especially in severe syndromic CHDs associated with neurodevelopmental delay.
This enrichment of chromatin-modifying and transcriptional regulators among CHD-linked mutations points to a fundamental mechanism: that disruptions in genome organization and gene-expression control drive cardiac malformation. These same processes form the foundation of our broader research paradigm, Genome Architecting, which seeks to understand how the three-dimensional structure of the genome governs cell identity. CHD therefore offers a powerful and medically relevant model for decoding how 3D genome architecture shapes human development and disease.
Genomic studies underscore the central role of genetics in CHD pathogenesis. Large chromosomal aberrations account for about 25% of cases, while smaller copy-number variants, indels, and loss-of-function mutations are found in another ~20%. Advances in whole-exome and whole-genome sequencing have revealed over 400 candidate genes, yet strong statistical evidence supports only a small subset (~14%, or 18 of 132 genes in a recent meta-analysis). Strikingly, many of these de novo variants cluster in genes encoding chromatin regulators—especially in severe syndromic CHDs associated with neurodevelopmental delay.
This enrichment of chromatin-modifying and transcriptional regulators among CHD-linked mutations points to a fundamental mechanism: that disruptions in genome organization and gene-expression control drive cardiac malformation. These same processes form the foundation of our broader research paradigm, Genome Architecting, which seeks to understand how the three-dimensional structure of the genome governs cell identity. CHD therefore offers a powerful and medically relevant model for decoding how 3D genome architecture shapes human development and disease.
Functional genomics with iPS2-seq
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To functionally annotate the expanding list of candidate CHD genes, our lab developed iPS2-seq — an inducible, clone-aware screening platform for human induced pluripotent stem cells (hiPSCs). iPS2-seq integrates pooled shRNA perturbations with single-cell multi-omics to capture transcriptomic, chromatin-accessibility, and protein-level effects (RNA, ATAC, and CITE-seq modalities).
iPS2-seq supports both pooled discovery assays and arrayed, polyclonal validation studies, allowing rapid follow-up across specific developmental stages. Its clone-tracking architecture distinguishes genuine gene-specific phenotypes from epigenetic or clonal bias, increasing sensitivity and reproducibility. For instance, the system uncovered an unexpected ZIC1-dependent epigenetic priming toward neuroectodermal fates that can confound differentiation outcomes — highlighting the importance of controlling for intrinsic lineage bias in hiPSC models. We apply iPS2-seq to systematically interrogate DNA- and RNA-binding proteins recurrently mutated in de novo CHD patients. In proof-of-principle studies, we identified SMAD2 as a key determinant of early cardiac fate specification: its knockdown diverts mesodermal progenitors toward fibroblast and epicardial lineages. |
Graphical abstract of Balmas E. et al., SSRN 2024
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Modeling human heart development with cardioids
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To capture these mechanisms in a physiologically relevant setting, we combine iPS2-seq with human cardiac organoids (“cardioids”), developed by our close collaborator Sasha Mendjan (IMBA, Vienna). These self-organizing 3D structures recapitulate early human heart morphogenesis, including the formation of chamber-like cavities, endothelial layers, and epicardial spreading. They offer a unique opportunity to study how specific genetic perturbations alter tissue architecture, signaling, and cell–cell interactions — features not accessible in 2D cultures.
By integrating iPS2-seq functional genomics with 3D cardioid models, we aim to build a comprehensive functional gene atlas of human cardiogenesis. This atlas will systematically link CHD-associated mutations to their developmental consequences, reveal the “genome architects” that shape cardiac differentiation and maturation, and provide an experimental foundation for predictive diagnostics and targeted therapies. |
Section of a day 7.5 left ventricular cardioid stained with alpha actinin (magenta), ki67 (yellow), and DAPI (cyan). Credit: Silvia Becca
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Dissecting the role of established genome architects in cardiomyogenesis
Besides screening for novel regulators of cardiogenesis, we also mechanistically investigate established genome architects that orchestrate chromatin topology during heart development. In recent work, we focused on the interplay between the pioneer factor GATA4 and the architectural protein CTCF. We found that GATA4 is essential for the B-to-A compartment transitions that activate key cardiac genes, while CTCF mediates intergenic chromatin loops whose partial loss facilitates this remodeling. At the cellular level, GATA4 depletion delays differentiation and sustains cardiomyocyte proliferation, whereas premature CTCF depletion accelerates but disrupts maturation. Together, these findings indicate that GATA4 and CTCF act antagonistically to fine-tune the tempo of chromatin remodeling during cardiomyogenesis, and that imbalance in this rheostat may contribute to congenital heart defects.
Working model for the antagonistic function of GATA4 and CTCF during cardiomyogenesis, from Becca S. et al., bioRxiv 2025