Patient-Specific Ipsc Model for Tbx5 Reveals Transcriptomic and Metabolic Disturbances Causing Holt–Oram Syndrome

Congenital heart defects are the most common congenital malformations, yet their mechanisms remain poorly understood. We generated the first patient-specific induced pluripotent stem cell model of Holt-Oram syndrome carrying the TBX5 Pro85Thr mutation, along with a CRISPR-corrected isogenic control, to investigate developmental mechanisms underlying ventricular septal defects and tricuspid valve dysplasia. Using 2D cardiac differentiation and 3D human heart organoids, we identified impaired induction of key cardiac transcription factors ( TBX5 , NKX2-5 , GATA4 ), consistent with disrupted first heart field lineage specification. Temporal and single-cell transcriptomic profiling further revealed altered developmental, contractile, and metabolic programs, across cardiac progenitors, cardiomyocytes, and endocardial-, endothelial-, and fibroblast-like cells, including myogenesis, sarcomere organization, oxidative phosphorylation, and lipid metabolism. At the functional level, mutant cardiomyocytes showed delayed structural maturation, reduced ATP production and glucose uptake, and increased cholesterol biosynthesis. Together, these transcriptional and metabolic alterations reveal developmental and maturation defects that provide a mechanistic framework for the patient’s ventricular septal defects and dysplastic tricuspid valve with leaflet prolapse. Importantly, maturation defects and metabolic dysregulation were independently reproduced in cardiomyocytes derived from a second CRISPR-engineered isogenic TBX5 Pro85Thr pair from a healthy male donor. Our findings identify disrupted cardiac metabolic maturation, characterized by impaired energy metabolism and cholesterol homeostasis, as a key downstream consequence of TBX5 haploinsufficiency and provide broader mechanistic insight into TBX5 -associated congenital heart defects.