Calcification of the aortic valve is the third leading cause of heart disease in adults. The incidence increases with age, and it is often associated with a bicuspid aortic valve present in 1-2% of the population. Despite the frequency, neither the mechanisms of valve calcification nor the developmental origin of a two, rather than three, leaflet aortic valve is known. Here, we show that mutations in the signalling and transcriptional regulator NOTCH1 cause a spectrum of developmental aortic valve anomalies and severe valve calcification in non-syndromic autosomal-dominant human pedigrees. Consistent with the valve calcification phenotype, Notch1 transcripts were most abundant in the developing aortic valve of mice, and Notch1 repressed the activity of Runx2, a central transcriptional regulator of osteoblast cell fate. The hairy-related family of transcriptional repressors (Hrt), which are activated by Notch1 signalling, physically interacted with Runx2 and repressed Runx2 transcriptional activity independent of histone deacetylase activity. These results suggest that NOTCH1 mutations cause an early developmental defect in the aortic valve and a later de-repression of calcium deposition that causes progressive aortic valve disease.

Congenital heart defects (CHDs) are the most common developmental anomaly and are the leading non-infectious cause of mortality in newborns. Only one causative gene, NKX2-5, has been identified through genetic linkage analysis of pedigrees with non-syndromic CHDs. Here, we show that isolated cardiac septal defects in a large pedigree were linked to chromosome 8p22-23. A heterozygous G296S missense mutation of GATA4, a transcription factor essential for heart formation, was found in all available affected family members but not in any control individuals. This mutation resulted in diminished DNA-binding affinity and transcriptional activity of Gata4. Furthermore, the Gata4 mutation abrogated a physical interaction between Gata4 and TBX5, a T-box protein responsible for a subset of syndromic cardiac septal defects. Conversely, interaction of Gata4 and TBX5 was disrupted by specific human TBX5 missense mutations that cause similar cardiac septal defects. In a second family, we identified a frame-shift mutation of GATA4 (E359del) that was transcriptionally inactive and segregated with cardiac septal defects. These results implicate GATA4 as a genetic cause of human cardiac septal defects, perhaps through its interaction with TBX5.