Tetralogy of Fallot, the most common form of cyanotic congenital heart disease, may be influenced by genetic changes hidden outside protein-coding genes. A study led by researchers at Nanjing Medical University has identified disease-associated variants in a regulatory region that controls the activity of TBX1, a gene essential for early cardiovascular development. The findings suggest that disruption of this noncoding DNA can interfere with the formation and stability of blood vessels involved in the developing heart, offering a new explanation for a subset of cases that cannot be fully accounted for by conventional genetic testing.
Tetralogy of Fallot is characterized by four structural abnormalities of the heart, including a ventricular septal defect, narrowing of the pulmonary outflow tract, overriding of the aorta, and thickening of the right ventricle. Although the condition is treatable with surgery, its biological origins are complex. One of the strongest known genetic contributors is deletion of a segment on chromosome 22 known as 22q11.2. This region contains TBX1, a gene that helps coordinate the development of the cardiac outflow tract and nearby tissues. However, not every patient carries a large chromosomal deletion, leaving the contribution of smaller regulatory changes largely unresolved.
In the new study, published in Science China Life Sciences, investigators used whole-genome sequencing to examine 428 patients diagnosed with tetralogy of Fallot. The analysis identified 31 patients carrying 22q11.2 deletions involving the TBX1 region. The researchers also found seven functional noncoding variants located within an enhancer associated with TBX1. Enhancers are stretches of DNA that do not encode proteins but act as molecular control panels, helping determine when, where, and how strongly a gene is expressed. Together, the deletions and enhancer variants were found in 42 patients, representing 9.81 percent of the cohort.
The discovery is important because noncoding regions make up most of the human genome, yet they remain more difficult to interpret than mutations within protein-coding genes. A variant in an enhancer may not alter the structure of a protein directly. Instead, it can weaken the instructions that activate a gene during a specific developmental window or in a particular cell type. In the case of TBX1, the researchers proposed that enhancer disruption may reduce gene activity in endothelial progenitor cells, which contribute to the formation and specialization of blood vessels during embryonic development.
To test this possibility, the team used human embryonic stem cells and removed the TBX1 enhancer before directing the cells to form blood vessel organoids. These three-dimensional laboratory models reproduce several features of vascular development, including the organization of endothelial cells and their interaction with supporting pericytes. Compared with control organoids, those lacking the enhancer produced substantially less TBX1 messenger RNA. They also displayed impaired angiogenesis, a process in which new blood vessels grow from existing ones, as well as vessel regression and reduced structural stability.
The altered organoids showed additional abnormalities at the microscopic level. Pericytes, cells that wrap around small blood vessels and help maintain their integrity, covered a smaller proportion of the vessel networks. The endothelial cells also developed abnormal tight junctions. These junctions act as seals between neighboring cells, controlling the movement of molecules across the vessel wall and preserving vascular barrier function. Disorganized tight junction formation may indicate that the vessels are not maturing correctly, potentially compromising the circulation required by developing cardiac tissues.
Further molecular analysis pointed to a regulatory network connecting TBX1 with genes involved in vascular signaling and cardiac morphogenesis. Among the downstream targets identified were DLL4 and TGFBR2. DLL4 participates in the Notch signaling pathway, which helps regulate endothelial cell specialization and the patterning of growing blood vessels. TGFBR2 is part of the transforming growth factor beta pathway, a signaling system involved in cell differentiation, tissue remodeling, and heart development. Both genes have previously been associated with processes relevant to formation of the cardiac outflow tract.
The researchers then restored TBX1 expression in organoids carrying the enhancer deletion. This intervention recovered the activity of downstream target genes and improved several of the abnormal vascular features. Pericyte coverage increased, and tight junction formation became more organized, supporting the conclusion that reduced TBX1 activity was not merely associated with the defects but contributed directly to them. The rescue experiments provide functional evidence that the enhancer operates as an essential developmental control element and that its disruption can alter vascular behavior through a TBX1-dependent mechanism.
The study does not establish that every identified variant independently causes tetralogy of Fallot, nor does it show that the organoid findings reproduce the entire complexity of a developing human heart. Nevertheless, it provides a mechanistic link between noncoding variation, reduced gene expression, vascular dysfunction, and congenital heart malformation. The results also highlight why whole-genome sequencing may reveal clinically relevant information missed by approaches focused primarily on exons, the protein-coding portions of genes. By combining genomic analysis with human stem cell-derived organoids, the researchers have shown how regulatory DNA can be tested experimentally rather than treated as genetic background noise. Their findings could eventually improve risk assessment and deepen understanding of how early vascular defects contribute to congenital heart disease.
Subject of Research: Functional noncoding variants in a TBX1 enhancer and their role in vascular development and tetralogy of Fallot.
Article Title: Noncoding Variants in a TBX1 Enhancer Disrupt Early Vascular Development in Tetralogy of Fallot
Web References: https://doi.org/10.1007/s11427-025-3303-x
References: Science China Life Sciences, DOI: 10.1007/s11427-025-3303-x
Image Credits: © Science China Press
Keywords: Tetralogy of Fallot, TBX1, noncoding variants, genetic enhancers, congenital heart disease, vascular development, angiogenesis, blood vessel organoids, 22q11.2 deletion, whole-genome sequencing
Tags: gene regulation in cardiovascular developmentgenetic basis of Tetralogy of FallotGenetic regulation of TBX1 in congenital heart defectsgenetic testing limitations in heart defect diagnosisimpact of noncoding mutations on heart congenital anomalieslong-range DNA regulatory elements in heart diseasemolecular mechanisms underlying cyanotic congenital heart diseasenoncoding DNA variants and heart developmentregulatory regions controlling cardiac gene expressionrole of 22q11.2 deletions in heart defectssilent DNA switches and blood vessel formation
