NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling

Abstract

Congenital aortic valve stenosis (AVS), coarctation of the aorta (COA) and hypoplastic left heart syndrome (HLHS) are congenital cardiovascular malformations that all involve the left ventricular outflow tract (LVOT). They are presumably caused by a similar developmental mechanism involving the developing endothelium. The exact etiology for most LVOT malformations is unknown, but a strong genetic component has been established. We demonstrate here that mutations in the gene NOTCH1, coding for a receptor in a developmentally important signaling pathway, are found across the spectrum of LVOT defects. We identify two specific mutations that reduce ligand (JAGGED1) induced NOTCH1 signaling. One of these mutations perturbs the S1 cleavage of the receptor in the Golgi. These findings suggest that the levels of NOTCH1 signaling are tightly regulated during cardiovascular development, and that relatively minor alterations may promote LVOT defects. These results also establish for the first time that AVS, COA and HLHS can share a common pathogenetic mechanism at the molecular level, explaining observations of these defects co-occurring within families.

Figure 1.

NOTCH1 mutations in LVOT subjects. (A) Molecular characterization of NOTCH1 missense mutations identified in patients. DNA sequence analysis of the eight missense mutations is shown. (B) Schematic representation of the NOTCH1 protein indicating the localization of protein variants identified in this study. LNR=Lin12/Notch repeats. TM, transmembrane domain. (B) Cross species sequence comparison for the highly conserved non-synonymous changes G661S, A683T, R1279H and A1343V.

Figure 2.

Mutations in the extracellular domain alter ligand-induced activation in variant NOTCH1 proteins. (A) Luciferase assays of Notch1 pathway activity in the absence of Jagged1 ligand. Low levels of NOTCH activity are seen in the wild-type and most variants, similar to that seen in the vector control. Results are presented as normalized luciferase values with wild-type Notch1 arbitrarily set to 1, and no significant differences are observed between the wild-type and any mutant (one way ANOVA, Dunnett’s post hoc, P > 0.05). (B) Luciferase assays of Notch1 pathway activity in the presence of Jagged1 ligand. Results are expressed as the fold Notch1 activation seen after culture with JAGGED1 cells compared to control L-cells, with wild-type NOTCH1 set to 100% (actual fold activation = 5.5 ± 1.4). Fold activation was significantly lower than wild-type NOTCH1 for NOTCH1^G661S (66.72 ± 0.87) and NOTCH1^A683T (52.2 ± 10.6). No significant change in activation was observed for NOTCH1^R1279H (79.7 ± 22.2) or NOTCH1^A1343V (110.6 ± 23.8). Error bars indicate the standard deviation from the mean. All experiments were performed at least three times in triplicate. Statistical significance was calculated by one-way ANOVA followed by Dunnett’s post hoc test. *P <.05, **P <.01.

Figure 3.

The A683T mutation affects S1 cleavage of the NOTCH1 receptor. (A) Western blot analysis of N-terminally HA-tagged NOTCH1 protein. NIH3T3 cells transfected with Notch1 expression vectors were lysed and the expression of NOTCH1 protein was assessed with an anti-HA antibody. HA-NOTCH1 bands are observed at 300 and 180 kDa, representing full-length (p300) and S1 cleaved (p180) NOTCH1 receptor. All proteins are expressed robustly, and to similar levels as seen in wild-type. (B) The levels of p300 and p180 Notch1 proteins were quantitated in western blots, demonstrating that the NOTCH1^A683T variant undergoes S1 cleavage at reduced levels. Results are expressed as the percent of total protein found in the p180 band. Error bars represent the standard deviation from the mean of at least four experiments. ** indicates P < 0.01 by ANOVA followed by Dunnett’s post hoc.