Endothelial defects unveil cardiovascular phenotype in iPSC-based disease modelling across three generations of a DiGeorge syndrome family

Aims DiGeorge syndrome (DGS) due to 22q11.2 microdeletion is characterized by a high degree of phenotypic variability. This study aimed to elucidate the molecular and cellular mechanisms underlying this variability and exacerbation of cardiovascular manifestations by developing a human induced pluripotent stem cell (hiPSC)-based model using a three-generation family. Methods and results We established hiPSC lines and their cardiovascular derivatives from a family carrying the identical 22q11.2 deletion but displaying a wide spectrum of cardiovascular involvement, allowing for a direct comparison of genetics and phenotypes at the cellular level. Genetic analyses revealed no de novo mutation in the DG region genes in patients, and no correlation between the cumulative number of genetic variants and the severity of DGS. However, we observed the presence of gene variants that may modulate predisposition to the disease with possible role in cardiovascular development. Cardiomyocytes from DGS-hiPSC with prominent cardiac symptoms exhibited altered expression of connexin-43, suggesting disruptions in early cardiac morphogenesis compared to asymptomatic relatives. Endothelial cells differentiated from symptomatic DGS-hiPSC showed impaired migration and disrupted tubular morphology with dysregulation of angiogenesis and vascular integrity pathways at the transcriptomic level. To bridge in vitro and transcriptomics findings with in vivo relevance, we performed deep clinical phenotyping for each participant and applied gene-to-phenotype correlation algorithms using large-scale clinical registry data. Conclusion Our results demonstrate that hiPSC-derived cardiovascular cells from DGS patients recapitulate disease-specific morphological and functional abnormalities, enabling identification of genotype-phenotype relationships that underlie clinical heterogeneity. Translational Perspective By modelling DGS with patient-derived hiPSC from a uniquely multigenerational family, our study reveals how shared genetic microdeletions can result in divergent pathophysiological outcomes at the cellular level. This work establishes that individualised hiPSC models faithfully reflect cardiac and vascular anomalies observed in patients and delineate the cellular and transcriptional mechanisms underlying these differences. These results facilitate early identification of pathogenic mechanisms, potentially enabling more refined risk assessment and personalised monitoring strategies for DGS patients - both for cardiovascular manifestations and potentially for extra-cardiac features.