P5722Thermodynamic phenotypes guide the pathogenicity assessment of a variant of uncertain significance in cardiac myosin binding protein C

Abstract

Abstract In the era of Next Generation Sequencing (NGS), genetic testing for inherited disorders identifies an ever-increasing number of variants, whose pathogenicity remains unclear. These variants of uncertain significant (VUS) limit the reach of genetic testing in clinical practice. The VUS for Hypertrophic Cardiomyopathy (HCM), the most common familial heart disease, constitute over 60% of entries for missense variants shown in ClinVar database. We have proposed a workflow to determine pathogenicity of VUS, which integrates bioinformatics evaluation and functional studies of RNA splicing and protein thermodynamic stability. This workflow was applied on a novel VUS in MYBPC3 gene, coding for cardiac myosin-binding protein C (cMyBP-C), which is the most mutated gene in HCM. The c.1809T>G-p.I603M variant was identified during genetic screening of patient clinically affected by HCM and it was classified as VUS applying ACMG criteria. In silico prediction and mRNA analysis of c.1809T>G-p.I603M variant indicated no alteration of RNA splicing (Figure 1). At protein level, the p.I603M mutation maps to the C4 domain of cMyBP-C. The homology modelling of domain C4 shows I603 to be buried in the protein structure, suggesting a potential destabilising role of this mutant. The Circular Dichroism (CD) confirms that the I603M variant does not perturb much the structure of domain, according to the far-UV and near-UV CD spectra. However, the thermodynamic stability of domain C4 is severely compromised, as shown by lower Tm and van't Hoff enthalpy (ΔHv) in thermal denaturation experiments by CD and Differential Scanning Calorimetry (DSC) (Figure 1). Furthermore, the Gibbs free energy change (ΔG) at 25°C of the unfolding process was also calculated from the DSC thermograms. The resulting ΔΔG of C4 I603M (4.5 kcal/mol) is comparable to other pathogenetic variants in other MYBPC3 domains. Taking into account our results, we propose reclassification of variant p.I603M as likely pathogenic. Figure 1. Workflow In conclusion, our strategy is useful to refine the assignment of pathogenicity in the absence of enough genetic support, improving the clinical management of HCM patients and their families. Acknowledgement/Funding MCNU: BIO2014-54768-P, BIO2017-83640-P (AEI/FEDER, UE), RYC-2014-16604, SEV-2015-0505- ISCIII: CB16/11/00425. Comunidad de Madrid: PEJ16/MED/TL-1593.