Basic science methods for the characterization of variants of uncertain significance in hypertrophic cardiomyopathy

Figures

• 

  Figure 1. (A) Structural models of cardiac myosin folded into the interacting heads motif (left) and cardiac myosin binding protein-C (right). Individual domains are labelled accordingly. RLC, regulatory light chain; ELC, essential light chain; MHC, myosin heavy chain; P/A, proline/alanine-rich linker. (B) High throughput in vitro screening process for VUS in genes associated with HCM. Gene synthesis and expression allows an initial screen for protein folding and production of purified proteins for characterization. A variety of biophysical techniques such as differential scanning fluorimetry, circular dichroism spectroscopy, and size exclusion multi-angle scattering can aid in characterizing the folding pathway, protein stability, and aggregation potential. More advanced techniques such as microscale thermophoresis or NADH-coupled ATPase assays can also be performed in high throughput micro-well formats. By utilizing this high throughput in vitro screening pipeline, it will be possible to obtain mechanistically and clinically meaningful information for VUS in HCM.

• 

  Figure 2. Mathematical modeling and simulation framework for HCM-associated VUS in cardiac MyBPC. The left-hand panels illustrate a multiscale system in which a sarcomere-level model of myofilament function is embedded inside a ventricle and pumps blood around a closed circulation. Growth can be added to this framework by allowing myocytes to add myofibrils in parallel (concentric growth, wall thickening) or to add sarcomeres in series (eccentric growth, chamber dilation). If the approach is to be useful for testing the potential impact of VUS, the sarcomere-level model must reproduce the different potential biophysical actions of cardiac MyBPC.