We identified eight missense mutations, six of them novel, in patients with sporadic LVOT malformations. Of these, five were either completely absent or were under-represented in over 200 ethnically matched controls, suggesting they may be disease predisposition alleles. The frequency of mutations in our cohort (15.4%) exceeds that reported previously in subjects with BAV and thoracic aortic aneurysms (2/48, 4.2%) or BAV alone (2/48, 4.2%). If we consider only mutations found in cases at highly conserved sites that have functional alterations (A683T), our frequency is 2/91 (2.2%). G661S, which is present in one control but over-represented among the cases, also involves a conserved site and demonstrates functional change. The addition of that variant would increase the frequency to 6/91 (6.6%). The reason for the higher rate of missense changes in our group is unknown, and may be due to chance. Screening of a larger cohort will be required to better define the frequency of NOTCH1 mutations among individuals with LVOT defects.
A possible limitation of this study is the absence of echocardiography screening of the control group. While all controls are healthy without evidence of heart defects, there is a 1–2% possibility of asymptomatic BAV in this group (19
). This could result in mutation ascertainment bias in the controls, causing a false negative result for specific mutations. We would estimate this rate to be very low, i.e. [rate of BAV in controls (0.02)] × [rate of mutation among individuals with BAV (0.04)]=rate of mutations in control group (0.0008).
We show in this study that mutations in NOTCH1
that alter function of the signaling pathway are found in individuals with AVS, COA and HLHS. The presumed common pathogenetic cause for LVOT defects is underscored by the finding that one variant (G661S) was found in all three LVOT defects (AVS, COA and HLHS), as well as in a patient with BAV. Interestingly, the previously reported R1108X mutation also caused a spectrum of LVOT phenotypes, including aortic valve disease and an individual with mitral valve atresia, double outlet right ventricle and hypoplastic left ventricle (a possible variation of HLHS) (12
). These findings support the hypothesis that a wide spectrum of LVOT defects are developmentally related, and a single genetic defect can underlie diverse phenotypic outcomes. This is reminiscent of findings that mutations in NKX2–5
are observed in rare patients with non-syndromic LVOT defects, although functional studies of the identified mutation demonstrated minor effects (23
). Taken together, these results support the suggestion that LVOT defects have a common developmental cause, and are governed by complex genetic inheritance.
We note that while G661S significantly decreases ligand induced signaling compared to wild-type, and R1279H does not have a statistically significant effect, the comparison between G661S and R1279 does not suggest a large difference in activity between these two mutants. The experiments reported here were specifically designed to allow comparison of the variant proteins to the wild-type control, thus future analyses will be required to determine whether G661S and R1279H actually have significantly different activities or whether there may be a subtle alteration in ligand-induced signaling through R1279H.
Our results indicate that LVOT-associated mutations reduce ligand-activated NOTCH1 signaling. This is consistent with two NOTCH1
mutations found previously to cause familial calcific aortic valve disease (R1108X and H1505del) which are proposed to be null mutations, as they truncate the protein in the extracellular domain (12
). These findings suggest that familial aortic valve disease can be caused by haploinsufficiency for the NOTCH1 receptor. The reduction of ligand-induced signaling observed through the NOTCH1G661S
variants would support a similar model for the spectrum of non-syndromic LVOT defects, namely that Notch signaling levels must be tightly regulated and that relatively minor alterations in Notch1 activity levels may promote LVOT defects.
The molecular mechanisms by which these mutations affect NOTCH1 activation are, as yet, unclear. NOTCH1
mutations observed in leukemia have been shown to increase ligand-independent NOTCH1 activation by promoting S2 and S3 cleavage (26
). In contrast, in this system where ligand-dependent activation is reduced, we observe a reduction in S1 cleavage of the A683T variant. These findings are typical of developmentally important genes, where gain-of-function mutations are associated with cancers and loss-of-function mutations cause congenital defects. This alteration in processing may change the cellular localization of the receptor, or may simply reduce the amount of S1 cleaved heterodimeric NOTCH1 available for ligand-induced signaling. This may be similar in some respects to mutations identified in Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), in which mutations in the NOTCH3
receptor have been variously reported to affect NOTCH3 signaling, protein processing and cellular localization (27
). Further experimentation will be required to assess the functional implications of the LVOT-associated NOTCH1
mutants identified here.
The mechanism(s) by which NOTCH1
mutations and the subsequent presumed reduction of NOTCH1 signaling promote LVOT defects remains unclear. Notch signaling has been proposed to play multiple functions during cardiac development, including cardiomyocyte differentiation, valve formation and outflow tract remodeling (30
). In mice, Notch1 activation promotes epithelial to mesenchymal transisition (EMT) during valve development through the activation of Snail
, and this may represent a developmental stage where NOTCH1 dosage is critically important (31
). Interestingly, some NOTCH1
mutations may play additional roles in cardiac disease. Garg et al
) showed that RUNX2 levels are repressed by Notch1, suggesting that the de-repression of RUNX2 may contribute to valve calcification in some BAV patients. None of our subjects showed evidence of aortic valve calcification, but all are young and may not have had time to develop calcification. It is not yet known whether the variants identified here could additionally contribute to calcification phenotypes.
The findings of an increased proportion of NOTCH1 variants among LVOT malformation cases, compared to controls, suggest that these mutations are indeed LVOT disease susceptibility alleles. This is further supported by the findings that two variants perturb ligand-induced signaling. Clearly, as LVOT-associated variants are found in unaffected parents of our patients, the NOTCH1 mutations with their relatively subtle effects on signaling are not sufficient in and of themselves to perturb cardiac development. The variants presumably act in concert with genetic background, other specific gene mutations and/or developmental insults in order to cause phenotypes. The presence of a mutation in a normal parent, indicating reduced penetrance, is typical of many complex genetic diseases. This requirement for additional genetic or environmental causes may also partially explain why no cardiac defects have been reported in Notch1+/− mice, while NOTCH1 haploinsufficiency has been suggested as a cause of familial BAV.
In summary, we note for the first time a common molecular mechanism for AVS, BAV, COA and HLHS. This substantiates the observations from familial studies demonstrating a higher concordance for these LVOT defects in families with multiple individuals affected with a CVM. Notch signaling is involved in endothelial–mesenchymal transformation in the ventricular chamber (32
), atrioventricular valves (31
) and vasculature (33
). We hypothesize that altered Notch1 signaling affecting this transformation is the unifying event in the pathogenesis of the LVOT disorders. Future studies will focus effort on candidate genes important in endothelial–mesenchymal transformation and on further defining the functional mechanisms of these NOTCH1