Thermodynamic destabilization informs pathogenicity assessment of a variant of uncertain significance in cardiac myosin binding protein C

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 significance (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 studied a novel VUS (c.1809T>G-p.I603M) in the most frequently mutated gene in HCM, MYBPC3, which codes for cardiac myosin-binding protein C (cMyBPC). Our determinations of pathogenicity integrate bioinformatics evaluation and functional studies of RNA splicing and protein thermodynamic stability. In silico prediction and mRNA analysis indicated no alteration of RNA splicing induced by the variant. At the protein level, the p.I603M mutation maps to the C4 domain of cMyBPC. Although the mutation does not perturb much the overall structure of the C4 domain, the stability of C4 I603M is severely compromised as detected by circular dichroism and differential scanning calorimetry experiments. Taking into account the highly destabilizing effect of the mutation in the structure of C4, we propose reclassification of variant p.I603M as likely pathogenic. Looking into the future, the workflow described here can be used to refine the assignment of pathogenicity of variants of uncertain significance in MYBPC3.

Protein haploinsufficiency drivers identifyMYBPC3mutations that cause hypertrophic cardiomyopathy

“…defects in RNA splicing that result in the appearance of premature stop codons 21 , and reduction of protein stability 22 . Both haploinsufficiency drivers have been reported before in MYBPC3 variants linked to HCM 7,8,16,18,23–26 . However, the association of these molecular features to disease remains unknown due to the absence of systematic comparison with non-pathogenic variants.…”

Protein haploinsufficiency drivers identify MYBPC3 variants that cause hypertrophic cardiomyopathy

“…The two most frequent mechanisms of protein haploinsufficiency induced by putative nontruncating variants in monogenic diseases are RNA splicing defects that result in the appearance of premature stop codons (25) and protein destabilization (26). Both of these haploinsufficiency drivers have been reported in MYBPC3 variants linked to HCM (7,8,19,20,(27)(28)(29)(30). However, how these molecular features cause disease remains unknown due to the lack of systematic comparison with non-pathogenic variants.…”

Does protein unfolding play a functional role in vivo?

“…The elastic part of titin (in the nonoverlapping myosin-actin region) has up to~100 immunoglobulin (Ig)-like and fibronectin III (FNIII)like domains, in series with unstructured regions, and this number varies among muscle types [44,45]. Other bridging molecules, such as titin kinase [46,47], obscurin [48], and myosin binding protein C [49,50] may act as mechanical force sensors during muscle contractions. A long-standing question has been if in vivo titin domains unfold regularly or only due to some catastrophic events [51][52][53].…”