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

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          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.

          Most cited references27

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          T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension

          This article introduces a new interface for T-Coffee, a consistency-based multiple sequence alignment program. This interface provides an easy and intuitive access to the most popular functionality of the package. These include the default T-Coffee mode for protein and nucleic acid sequences, the M-Coffee mode that allows combining the output of any other aligners, and template-based modes of T-Coffee that deliver high accuracy alignments while using structural or homology derived templates. These three available template modes are Expresso for the alignment of protein with a known 3D-Structure, R-Coffee to align RNA sequences with conserved secondary structures and PSI-Coffee to accurately align distantly related sequences using homology extension. The new server benefits from recent improvements of the T-Coffee algorithm and can align up to 150 sequences as long as 10 000 residues and is available from both http://www.tcoffee.org and its main mirror http://tcoffee.crg.cat.
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            Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism.

            The surface comparison of different serine-threonine and tyrosine kinases reveals a set of 30 residues whose spatial positions are highly conserved. The comparison between active and inactive conformations identified the residues whose positions are the most sensitive to activation. Based on these results, we propose a model of protein kinase activation. This model explains how the presence of a phosphate group in the activation loop determines the position of the catalytically important aspartate in the Asp-Phe-Gly motif. According to the model, the most important feature of the activation is a "spine" formation that is dynamically assembled in all active kinases. The spine is comprised of four hydrophobic residues that we detected in a set of 23 eukaryotic and prokaryotic kinases. It spans the molecule and plays a coordinating role in activated kinases. The spine is disordered in the inactive kinases and can explain how stabilization of the whole molecule is achieved upon phosphorylation.
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              VADAR: a web server for quantitative evaluation of protein structure quality.

              VADAR (Volume Area Dihedral Angle Reporter) is a comprehensive web server for quantitative protein structure evaluation. It accepts Protein Data Bank (PDB) formatted files or PDB accession numbers as input and calculates, identifies, graphs, reports and/or evaluates a large number (>30) of key structural parameters both for individual residues and for the entire protein. These include excluded volume, accessible surface area, backbone and side chain dihedral angles, secondary structure, hydrogen bonding partners, hydrogen bond energies, steric quality, solvation free energy as well as local and overall fold quality. These derived parameters can be used to rapidly identify both general and residue-specific problems within newly determined protein structures. The VADAR web server is freely accessible at http://redpoll.pharmacy.ualberta.ca/vadar.
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                Author and article information

                Journal
                Cold Spring Harb Mol Case Stud
                Cold Spring Harb Mol Case Stud
                cshmcs
                cshmcs
                cshmcs
                Cold Spring Harbor Molecular Case Studies
                Cold Spring Harbor Laboratory Press
                2373-2873
                July 2017
                : 3
                : 4
                : a001727
                Affiliations
                [1 ]Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota 55905, USA;
                [2 ]Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA;
                [3 ]Laboratory of Epigenetics and Chromatin Dynamics, Gastroenterology Research Unit, Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA;
                [4 ]Department of Health Sciences Research, Mayo Clinic, Jacksonville, Florida 32224, USA;
                [5 ]Center for Individualized Medicine, Mayo Clinic, Jacksonville, Florida 32224, USA;
                [6 ]Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, USA;
                [7 ]Department of Clinic Genomics, Mayo Clinic, Rochester, Minnesota 55905, USA
                Author notes
                Article
                CousinMCS001727
                10.1101/mcs.a001727
                5495030
                28679693
                89c70e8f-2526-4ca2-9f04-65fbb4aaffda
                © 2017 Cousin et al.; Published by Cold Spring Harbor Laboratory Press

                This article is distributed under the terms of the Creative Commons Attribution-NonCommercial License, which permits reuse and redistribution, except for commercial purposes, provided that the original author and source are credited.

                History
                : 6 January 2017
                : 12 April 2017
                Page count
                Pages: 16
                Funding
                Funded by: Center for Individualized Medicine at Mayo Clinic , open-funder-registry 10.13039/100000871;
                Categories
                Research Article

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

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