Utilization of Whole Exome Sequencing to Identify Causative Mutations in Familial Congenital Heart Disease.

BACKGROUND: Congenital heart disease (CHD) is the most common type of birth defect with family- and population-based studies supporting a strong genetic cause for CHD. The goal of this study was to determine whether a whole exome sequencing (WES) approach could identify pathogenic-segregating variants in multiplex CHD families. METHODS AND RESULTS: WES was performed on 9 kindreds with familial CHD, 4 with atrial septal defects, 2 with patent ductus arteriosus, 2 with tetralogy of Fallot, and 1 with pulmonary valve dysplasia. Rare variants (<1% minor allele frequency) that segregated with disease were identified by WES, and variants in 69 CHD candidate genes were further analyzed. These selected variants were subjected to in silico analysis to predict pathogenicity and resulted in the discovery of likely pathogenic mutations in 3 of 9 (33%) families. A GATA4 mutation in the transactivation domain, p.G115W, was identified in familial atrial septal defects and demonstrated decreased transactivation ability in vitro. A p.I263V mutation in TLL1 was identified in an atrial septal defects kindred and is predicted to affect the enzymatic functionality of TLL1. A disease-segregating splice donor site mutation in MYH11 (c.4599+1delG) was identified in familial patent ductus arteriosus and found to disrupt normal splicing of MYH11 mRNA in the affected individual. CONCLUSIONS: Our findings demonstrate the clinical utility of WES to identify causative mutations in familial CHD and demonstrate the successful use of a CHD candidate gene list to allow for a more streamlined approach enabling rapid prioritization and identification of likely pathogenic variants from large WES data sets. CLINICAL TRIAL REGISTRATION: URL: https://clinicaltrials.gov; Unique Identifier: NCT0112048.

Endothelial Notch1 Is Required for Proper Development of the Semilunar Valves and Cardiac Outflow Tract.

BACKGROUND: Congenital heart disease is the most common type of birth defect, affecting ≈2% of the population. Malformations involving the cardiac outflow tract and semilunar valves account for >50% of these cases predominantly because of a bicuspid aortic valve, which has an estimated prevalence of 1% in the population. We previously reported that mutations in NOTCH1 were a cause of bicuspid aortic valve in nonsyndromic autosomal-dominant human pedigrees. Subsequently, we described a highly penetrant mouse model of aortic valve disease, consisting of a bicuspid aortic valve with thickened cusps and associated stenosis and regurgitation, in Notch1-haploinsufficient adult mice backcrossed into a Nos3-null background. METHODS AND RESULTS: Here, we described the congenital cardiac abnormalities in Notch1(+/-);Nos3(-/-) embryos that led to ≈65% lethality by postnatal day 10. Although expected Mendelian ratios of Notch1(+/-);Nos3(-/-) embryos were found at embryonic day 18.5, histological examination revealed thickened, malformed semilunar valve leaflets accompanied by additional anomalies of the cardiac outflow tract including ventricular septal defects and overriding aorta. The aortic valve leaflets of Notch1(+/-);Nos3(-/-) embryos at embryonic day 15.5 were significantly thicker than controls, consistent with a defect in remodeling of the semilunar valve cushions. In addition, we generated mice haploinsufficient for Notch1 specifically in endothelial and endothelial-derived cells in a Nos3-null background and found that Notch1(fl/+);Tie2-Cre(+/-);Nos3(-/-) mice recapitulate the congenital cardiac phenotype of Notch1(+/-);Nos3(-/-) embryos. CONCLUSIONS: Our data demonstrate the role of endothelial Notch1 in the proper development of the semilunar valves and cardiac outflow tract.

Evidence of Aortopathy in Mice with Haploinsufficiency of Notch1 in Nos3-Null Background.

Thoracic aortic aneurysms (TAA) are a significant cause of morbidity and mortality in humans. While the exact etiology is unknown, genetic factors play an important role. Mutations in NOTCH1 have been linked to bicuspid aortic valve (BAV) and aortopathy in humans. The aim of this study was to determine if haploinsufficiency of Notch1 contributes to aortopathy using Notch1+/-; Nos3-/- mice. Echocardiographic analysis of Notch1+/-; Nos3-/- mice reveals effacement of the sinotubular junction and a trend toward dilation of the aortic sinus. Furthermore, examination of the proximal aorta of Notch1+/-; Nos3-/- mice reveals elastic fiber degradation, a trend toward increased matrix metalloproteinase 2 expression, and increased smooth muscle cell apoptosis, features characteristic of aneurysmal disease. Although at a lower penetrance, we also found features consistent with aortopathic changes in Notch1 heterozygote mice and in Nos3-null mice. Our findings implicate a novel role for Notch1 in aortopathy of the proximal aorta.

Endothelial nitric oxide signaling regulates Notch1 in aortic valve disease.

The mature aortic valve is composed of a structured trilaminar extracellular matrix that is interspersed with aortic valve interstitial cells (AVICs) and covered by endothelium. Dysfunction of the valvular endothelium initiates calcification of neighboring AVICs leading to calcific aortic valve disease (CAVD). The molecular mechanism by which endothelial cells communicate with AVICs and cause disease is not well understood. Using a co-culture assay, we show that endothelial cells secrete a signal to inhibit calcification of AVICs. Gain or loss of nitric oxide (NO) prevents or accelerates calcification of AVICs, respectively, suggesting that the endothelial cell-derived signal is NO. Overexpression of Notch1, which is genetically linked to human CAVD, retards the calcification of AVICs that occurs with NO inhibition. In AVICs, NO regulates the expression of Hey1, a downstream target of Notch1, and alters nuclear localization of Notch1 intracellular domain. Finally, Notch1 and NOS3 (endothelial NO synthase) display an in vivo genetic interaction critical for proper valve morphogenesis and the development of aortic valve disease. Our data suggests that endothelial cell-derived NO is a regulator of Notch1 signaling in AVICs in the development of the aortic valve and adult aortic valve disease.

Genetic ablation of neural crest cell diversification.

The neural crest generates multiple cell types during embryogenesis but the mechanisms regulating neural crest cell diversification are incompletely understood. Previous studies using mutant zebrafish indicated that foxd3 and tfap2a function early and differentially in the development of neural crest sublineages. Here, we show that the simultaneous loss of foxd3 and tfap2a function in zebrafish foxd3(zdf10);tfap2a(low) double mutant embryos globally prevents the specification of developmentally distinct neural crest sublineages. By contrast, neural crest induction occurs independently of foxd3 and tfap2a function. We show that the failure of neural crest cell diversification in double mutants is accompanied by the absence of neural crest sox10 and sox9a/b gene expression, and that forced expression of sox10 and sox9a/b differentially rescues neural crest sublineage specification and derivative differentiation. These results demonstrate the functional necessity for foxd3 and tfap2a for neural crest sublineage specification and that this requirement is mediated by the synergistic regulation of the expression of SoxE family genes. Our results identify a genetic regulatory pathway functionally discrete from the process of neural crest induction that is required for the initiation of neural crest cell diversification during embryonic development.