Functional noncoding variants within the TBX1 enhancer contribute to tetralogy of Fallot

The TBX1 deletion is frequently observed in patients with tetralogy of Fallot (TOF), the most prevalent cyanotic congenital heart defect (CHD); however, the role of noncoding variants in its regulatory region remains unclear. We performed whole-genome sequencing (WGS) on a cohort of 428 patients diagnosed with TOF, which identified the presence of 22q11.2 deletion in 31 cases, including the TBX1 genomic region, as well as 7 functional noncoding variants in its enhancer (Enh_TBX1). These findings accounted for a total of 9.81% (42/428) of TOF cases. We then knocked out Enh_TBX1 (Enh_TBX1^-/-) in ES cells and utilized blood vessel organoids (BVOs) as a model to elucidate the mechanisms underlying outflow tract (OFT) vessel dysfunction. The Enh_TBX1^-/- BVOs exhibited significantly reduced TBX1 mRNA expression, leading to impaired angiogenesis, vessel regression with decreased pericyte coverage, and alterations in tight junction morphology. Mechanistically, we have identified TBX1 as a specific transcription factor for endothelial progenitor cells that influences their differentiation. The main targets of TBX1 are genes involved in angiogenesis-related pathways, such as DLL4 and TGFBR2, which have been reported to play crucial roles during OFT formation. Finally, rescue experiments with TBX1 overexpression not only reinstated the expression of aforementioned target genes but also recovered tight junction formation and enhanced pericyte coverage rate of Enh_TBX1^-/- BVOs. In conclusion, this study demonstrates the involvement of noncoding variants in the TBX1 enhancer in TOF pathogenesis, highlighting the feasibility of using organoids to decipher the underlying mechanisms of unconserved noncoding regions in developmental malformations.