Functional validation reveals the novel missense V419L variant in associated with Loeys-Dietz syndrome (LDS) impairs canonical TGF-β signaling.

TGF-β-related heritable connective tissue disorders are characterized by a similar pattern of cardiovascular defects, including aortic root dilatation, mitral valve prolapse, vascular aneurysms, and vascular dissections and exhibit incomplete penetrance and variable expressivity. Because of the phenotypic overlap of these disorders, panel-based genetic testing is frequently used to confirm the clinical findings. Unfortunately in many cases, variants of uncertain significance (VUSs) obscure the genetic diagnosis until more information becomes available. Here, we describe and characterize the functional impact of a novel VUS in the kinase domain (c.1255G>T; p.Val419Leu), in a patient with the clinical diagnosis of Marfan syndrome spectrum. We assessed the structural and functional consequence of this VUS using molecular modeling, molecular dynamic simulations, and in vitro cell-based assays. A high-quality homology-based model of TGFBR2 was generated and computational mutagenesis followed by refinement and molecular dynamics simulations were used to assess structural and dynamic changes. Relative to wild type, the V419L induced conformational and dynamic changes that may affect ATP binding, increasing the likelihood of adopting an inactive state, and, we hypothesize, alter canonical signaling. Experimentally, we tested this by measuring the canonical TGF-β signaling pathway activation at two points; V419L significantly delayed SMAD2 phosphorylation by western blot and significantly decreased TGF-β-induced gene transcription by reporter assays consistent with known pathogenic variants in this gene. Thus, our results establish that the V419L variant leads to aberrant TGF-β signaling and confirm the diagnosis of Loeys-Dietz syndrome in this patient.