Computational and biophysical determination of pathogenicity of variants of unknown significance in cardiac thin filament

Abstract

Point mutations within sarcomeric proteins have been associated with altered function and cardiomyopathy development. Difficulties remain, however, in establishing the pathogenic potential of individual mutations, often limiting the use of genotype in management of affected families. To directly address this challenge, we utilized our all-atom computational model of the human full cardiac thin filament (CTF) to predict how sequence substitutions in CTF proteins might affect structure and dynamics on an atomistic level.Utilizing molecular dynamics (MD) calculations, we simulated 21 well-defined genetic pathogenic cardiac troponin T and tropomyosin variants to establish a baseline of pathogenic changes induced in computational observables. Computational results were verified via differential scanning calorimetry on a subset of variants to develop an experimental correlation.Calculations were performed on 9 independent variants of unknown significance (VUS) and results were compared to pathogenic variants to identify high resolution pathogenic signatures.Results for VUS were compared to the baseline set to determine induced structural and dynamic changes and potential variant reclassifications were proposed. This unbiased, highresolution computational methodology can provide unique structural and dynamic information that can be incorporated into existing analyses to facilitate classification both for de novo variants and those where established approaches have provided conflicting information.

Thin filament cardiomyopathies: A review of genetics, disease mechanisms, and emerging therapeutics

“…However, with increasing use of high-sensitivity genetic testing there is a constantly expanding list of variants of unknown significance, often contributing more to patient confusion and undue distress rather than clinical intervention. As a potential solution, Mason et al have demonstrated the use of a high-fidelity computational model of the cardiac thin filament to predict point mutation behavior at the atomic level, suggesting the future possibility of characterization of a given VUS without necessitating animal models or familial studies ( 158 ). But until mutation characterization matches available screening diagnostics, clinicians should be encouraged to limit any interpretation of genetic testing to only well-understood variants.…”

Free-Energy Surfaces of Two Cardiac Thin Filament Conformational Changes during Muscle Contraction

“…Structures utilized in this study were created starting from the cryo-EM structures published by Yamada, Namba, and Fujii, as previously described, , which provided structures for both Ca 2+ -saturated and -unsaturated conformations of the CTF (PDB 6KN8 and 6KN7, respectively). In the context of saturation of the CTF, a fully saturated system refers to Ca 2+ bound to site II of cTnC, while unsaturated refers to no Ca 2+ bound to site II of cTnC.…”

“…All structured fragments were aligned and the proteins were subjected to minimization until all bonds and angles were acceptable values. Any clashing of proteins was resolved by rotating dihedral angles in the unstructured regions, as previously described. , The helical pitch of Tm in the Yamada structure was corrected to agree with the protein docking study by Pavadai et al, allowing Tm to span 7 actin monomers instead of the 6 monomers in the original pdb. The structure for the cTnI C-terminal domain of the Ca 2+ -unsaturated state was also corrected to agree with the docking study by Lehman et al, which found a more energetically favorable location compared to the originally published structure.…”

“…Following this, studies by both Pavadai et al and Lehman et al have since refined certain segments in the models: Tm helical pitch and location and cTnI placement on actin, respectively. Our group has previously published results using these updated structures while also providing initial locations for unstructured segments not included in the cryo-EM maps to include all of the complete proteins of the CTF. , …”

“…Our group has previously published results using these updated structures while also providing initial locations for unstructured segments not included in the cryo-EM maps to include all of the complete proteins of the CTF. 15,16 Understanding the interactions between proteins in the cTn complex is crucial for understanding contraction at a molecular level. Previous work analyzing the opening of cTnC has shown that binding of Ca 2+ to site II creates a strain on the protein.…”