Functional validation reveals the novel missense V419L variant in TGFBR2 associated with Loeys–Dietz syndrome (LDS) impairs canonical TGF-β signaling

Abstract

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 TGFBR2 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.

Molecular mechanics and dynamic simulations of well-known Kabuki syndrome-associated KDM6A variants reveal putative mechanisms of dysfunction

“…To test the utility of our biophysically parametrized structural bioinformatic methods [ 1 – 4 ], we selected KDM6A variants within the catalytic domain found in patients with a Kabuki syndrome diagnosis. These have previously predicted to be damaging in most cases (with a few exceptions) by available impact predictors but for which more studies remain necessary to determine if their classification is valid and to identify the mechanisms underlying their dysfunction (Table 1 ).…”

“…Our studies are focused on the use and optimization of structural bioinformatics methods for improving the interpretation of data derived from next generation sequencing in cases presenting with rare diseases [ 1 – 4 ]. Extensive studies in our laboratories have previously demonstrated that these approaches yield not only information on the damaging effects of likely pathogenic variants but also yield useful mechanistic information on their pathophysiological impact on human health.…”

Identification of a Pathogenic TGFBR2 Variant in a Patient With Loeys–Dietz Syndrome

“…Previous variants about TGFBR2 mutant reported in ClinVar showed that 86 of the 87 plausible pathogenic missense variants are located in the kinase domain. Study showed that p.Val419Leu variant in the TGFBR2 affected the receptor function through alteration of its structure and inactivation of kinase conformations (Cousin et al, 2017). In this study, we discovered that the c.1613T > C/p.Val538Ala variant also located in the kinase domain of TGFBR2 protein, and the overall scaffold of the mutated structure is similar to the native one by MD stimulation.…”

“…); ACMG, American College of Medical Genetics and Genomics; AMP, Association for Molecular Pathology. Cousin et al, 2017). Since the c.1613T > C/p.Val538Ala variant of TGFBR2 was reported as a likely pathogenic variant in 2016 (Luo et al, 2016), these cellular results confirmed the pathogenicity of this variant.…”

“…Intuitively, gene mutation-associated diseases are caused by loss of function of the encoded protein (Doyle et al, 2012;Franken et al, 2013). Surprisingly, as shown in many clinical case reports, the TGF-β signaling pathway was activated in LDS patient tissue samples (Cousin et al, 2017;Hara et al, 2019). Although the underlying detailed pathogenic mechanisms for this phenomenon remain largely unknown, it might be related to compensatory activation of a non-canonical pathway (Sorrentino et al, 2008;Iwata et al, 2012) and maladaptation of negative feedback mechanism (Lindsay and Dietz, 2011).…”