NOTCH1 missense alleles associated with left ventricular outflow tract defects exhibit impaired receptor processing and defective EMT

Abstract

Notch signaling is essential for proper cardiac development. We recently identified missense variants in the NOTCH1 receptor in patients with diverse left ventricular outflow tract (LVOT) malformations (NOTCH1G661S and NOTCH1A683T) that reduce ligand-induced Notch signaling. Here, we examine the molecular mechanisms that contribute to reduced signaling and perturbed development. We find that NOTCH1A683T exhibits reduced S1 cleavage due to impaired trafficking through the endoplasmic reticulum (ER). This observation is consistent with improper localization of the variant receptor to the ER and decreased presentation at the cell surface. In contrast, the nearby mutation NOTCH1G661S exhibits reduced cell-surface presentation in the absence of overt folding or trafficking defects. To examine the implications of these variants in disease pathogenesis, we investigated their effect on epithelial-to-mesenchymal transition (EMT), a critical process for development of the outflow tract. We find that these LVOT-associated NOTCH1 alleles can contribute to defective EMT in endothelial cell lines through impaired induction of Snail and Hes family members. These data represent the first description of a molecular mechanism underlying NOTCH1 mutations in individuals with LVOT malformations, and have important implications regarding the functional contribution of these alleles to a complex set of developmental defects.

Compound heterozygous NOTCH1 mutations underlie impaired cardiogenesis in a patient with hypoplastic left heart syndrome

“…In contrast, three were unreported loss of function mutations, including an inherited nonsense mutation in two family members with BAV (Foffa et al 2014) and de novo frameshift and splice site mutations in two unrelated probands with HLHS (Iascone et al 2012). Functional characterization of two of the missense mutations, each linked to diverse CHDs and reported in ExAC (A683T = 0.0028 and G661S = 0.038), demonstrated impaired EMT due to perturbed downstream Notch1 signaling (Riley et al 2011). To our knowledge, genetic or environmental factors that modify phenotypic expression of NOTCH1 mutations and account for the severe HLHS phenotype have not been identified previously.…”

“…Heterozygous NOTCH1 mutations have in fact been identified in patients with a spectrum of left-sided cardiac malformations, including HLHS (Garg et al 2005;Mohamed et al 2006;McKellar et al 2007;McBride et al 2008;Iascone et al 2012;Foffa et al 2014). Functional characterization of mutant Notch1 (G661S and A683T) in mouse fibroblasts did not demonstrate altered protein expression, but both mutations reduced ligandinduced signaling (McBride et al 2008) and ultimately impaired the efficiency of epithelial to mesenchymal cell transition (EMT) (Riley et al 2011). Incomplete penetrance of NOTCH1 missense mutations with extreme variability of intra-and inter-familial phenotypic expression implicates unknown genetic or environmental modifiers (Garg et al 2005;McBride et al 2008).…”

Copy Number Variation in Congenital Heart Defects

“…While there is substantial evidence for the underlying genetic contribution to HLHS, and left ventricular outflow tract obstructive defects (LVOTO) [53][54][55][56][57], only a few genes have been identified as important contributors [58,59]. Several studies have investigated the potential role of CNVs in the pathogenicity of HLHS.…”

Notch signaling in cardiac valve development and disease

“…In two families, truncation mutations leading to an unstable mRNA or the expression of a truncated polypeptide lacking the intracellular domain were predicted to occur, implying that the mechanism of disease in these patients was haploinsufficiency. Missense variants in the NOTCH1 receptor, located mainly within the epidermal growth factor (EGF)-like repeats (EGFR) of NOTCH1, were later identified in patients with sporadic BAV or LVOT malformations (aortic valve stenosis, coarctation of the aorta, and hypoplastic left heart syndrome; McKellar et al, 2007;McBride et al, 2008;Riley et al, 2010). The A683T mutant exhibited reduced S1 cleavage because of impaired trafficking through the endoplasmic reticulum.…”

“…In contrast, the nearby G661S mutation exhibited reduced cellsurface presentation in the absence of obvious folding or trafficking defects. Disease variant receptor-driven EMT was found to be defective in endothelial cell lines through impaired induction of Snail and Hes family members, providing a molecular mechanism underlying NOTCH1 mutations in individuals with left ventricular outflow tract obstruction (LVOTO) malformations (Riley et al, 2010).…”