Loss-of-function variants in ciliary genes confer high risk for Tetralogy of Fallot

Tetralogy of Fallot (TOF), the most common severe cyanotic congenital heart disease, has poorly understood genetic causes. Through next-generation sequencing in 131 nonsyndromic TOF patients, we identified an increased burden of rare deleterious variants in ciliary genes and cilium pathway, and observed a multigenic inheritance pattern, with an OR of 1.672 for more than two deleterious variants and a cumulative OR of 3.158 for six variants. Functional validation in single- and double-heterozygous mouse models carrying these variants recapitulated TOF-like phenotypes and impaired normal cilia structure and function, particularly disrupting Hedgehog signaling in cardiomyocytes, downregulating key transcription factors Gata4 and Nkx2-5. scRNA-Seq analysis of mice E10.5 cardiac OFTs from wild-type and ciliary gene knockout/mutant littermates of each strain.