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.

Keywords: aneurysm of an abdominal artery; aortic root dilatation; arterial tortuosity; arthralgia/arthritis; dental crowding; dolichocephaly; hip subluxation; inguinal hernia; joint laxity; malar flattening; pes planus; shoulder subluxation.

Figure 1.

Canonical TGF-β signaling pathway.

Figure 2.

Family history and vascular imaging. (A) Pedigree (arrow indicates proband). (B) Aortic root dilatation and (C) aneurysms of the common iliac arteries of the proband visualized using computer tomography angiography.

Figure 3.

Protein domains and ortholog and paralog analyses. (A) V419 is located in the kinase domain and (B) is highly conserved in orthologs. (C) Paralog analysis reveals high conservation and disease-associated variants in nearby residues. V419E was reported as likely pathogenic in ClinVar. For a variant reported in more than one source, shading prioritization follows HGMD>ClinVar>UMD. Only variants reported as pathogenic or likely pathogenic in ClinVar were included. HGMD, Human Gene Mutation Database; UMD, Universal Mutation Database.

Figure 4.

TGFBR2 structure and dynamics. (A) V419 is within the activation loop in close proximity to R254 in the ATP binding domain. Locations of pathogenic and likely pathogenic variants reported in ClinVar are marked by spheres. (B) Probability density of root-mean-square deviation (RMSD) from simulations demonstrated that V419L affects conformation. (C) Principal component analysis (PCA) reveals that V419L exhibits significant dynamic departures from wild type (WT) and many other pathogenic variants. Snapshots from each simulation are shown as points in the first two PCs. (D) The motion indicated by the first PC is shown, projected onto the initial WT structure. The direction and relative magnitude of each residue's motion is indicated—a twisting of the N- and C-lobes with respect to one another. V419L samples conformations that are further activated in the +PC1 direction than WT and most pathogenic variants. (E) We quantified the changes in reference distances along PC1. The median PC1 coordinates for WT and V419L simulations are indicated by dashed vertical lines and the full range of sampling by shaded regions. The angle of the ATP binding site is measured using N332–C396–R254. (F) We show the structure with the N-lobe colored blue, C-lobe orange, and αC-helix pink. Two conformations along PC1 are shown: a 2 Å RMSD deformation in the negative direction (left) and a 2 Å deformation in the positive direction (right). (G) Zooming in on the top of the hydrophobic spine reveals that PC1 also leads to misalignment of these residues. Thus, V419L may lead to enhanced sampling of an inactive state.

Figure 5.

TGFBR2 V419L has decreased canonical TGF-β signaling in vitro. Heterologous expression of TGFBR2 V419L in TGFBR2-deficient HCT116 cells leads to (A) decreased TGF-β-associated gene transcription using a luciferase reporter, p3TP-Lux, and (B) decreased phosphorylated SMAD2 following TGF-β1 treatment compared with wild type. The bar graph represents the quantification of the western blot density. Each TGFBR2 WT and TGFBR2 mutant was normalized to its respective unstimulated (0 h) control.