Novel SMAD3 variant identified in a patient with familial aortopathy modeled using a zebrafish embryo assay

Abstract

In human, pathogenic variants in smad3 are one cause of familial aortopathy. We describe a novel SMAD3 variant of unknown significance (VUS), V244F, in a patient who presented with aortic root dilation, right coronary artery ectasia, abdominal aortic aneurysm, right vertebral artery atresia, and cavernoma. Determination of variant pathogenicity impacted multiple aspects of the patient’s care, including the most appropriate surgical threshold for which to recommend a valve-sparing aortic root replacement. To determine whether the newly identified SMAD3 variant, and whether SMAD3 induced aortopathy in general, can be assayed in a zebrafish embryo model, we injected smad3a mRNA into Tg[kdrl:mCherry] zebrafish embryos. By measuring the size of the dorsal aorta at 48hpf we found a correlation between pathogenic SMAD3 variants and increased dorsal aortic diameter. The newly identified V244F variant increased dorsal aortic diameter (p < 0.0001) similar to that of the pathogenic control variant T261I (p < 0.0084). In addition, we examined several previously identified variants of uncertain significance and found P124T (p < 0.0467), L296P (p < 0.0025) and A349P (p < 0.0056) to behave like T261I. These results demonstrate that the zebrafish embryo assay was successful in validating known pathogenic variants, classifying our newly identified variant V244F as likely pathogenic, and classifying previously identified variants P124T, L296P, and A349P as likely pathogenic. Overall, our findings identify a novel SMAD3 variant that is likely pathogenic as well as offer a new mechanism to model SMAD3 VUSs in vivo.

Inducing aortic aneurysm/dissection in zebrafish: evaluating the efficacy of β-Aminopropionic Nitrile as a model

“…These results are similar to the toaortic phenotype sought by the gene mutation model constructed in Prendergast's laboratory (Andrew Prendergast et al 2022 ). A 28% widening of the dorsal aorta after induction (statistically significant P < 0.05, as shown in Figure 1 A and B) as well as the development of aortic arch malformations with a rate of 75% (6/8) ( Figure 1 C), is significantly correlated with the development of AAD (Shao-Qing Kuang et al 2016 ; Mary B Sheppard et al 2023 ). In the adult models, we found zebrafish treated with 1 mM BAPN for 22 days developed symptoms replicating the AAD pathological changes according to the Pathological Evaluation of AAD-like Alterations Table (Table S1; Cosmin Marian Banceu et al 2023 ).…”

“…To determine whether BAPN could be used as a modeling method for AAD in zebrafish, larval zebrafish of the Tg[flk:eGFP] strain were chosen for the intervention. The widening and deformity of the dorsal aorta and the aortic arch were used as evaluation indicators (Marine Abrial et al 2022 ; Shao Qing Kuang et al 2016 ; Mary B Sheppard et al 2023 ). We administered various concentrations of BAPN to zebrafish at different developmental stages (24 hpf-7 dpf), and observed the diameter of dorsal aortic within the first five internodal vessels at the ejaculatory orifice by fluorescence microscopy ( Figure 1 B).…”

“…Kuang et al demonstrated that knocking down FOXE3 in zebrafish larvae can lead to aortic arch malformation, which can reflect ascending aortic aneurysm in mice (Shao Qing Kuang et al 2016 ). Transient overexpression of SMAD3a in zebrafish embryos induced aneurysms, indicated by an enlarged dorsal aorta diameter (Mary B Sheppard et al 2023 ).…”