Protein haploinsufficiency drivers identifyMYBPC3mutations that cause hypertrophic cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Mutations in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are a leading cause of HCM. However, it remains challenging to define whether specific gene variants found in patients are pathogenic or not, limiting the reach of cardiovascular genetics in the management of HCM. Here, we have examined cMyBP-C haploinsufficiency drivers in 68 clinically annotated non-truncating variants of MYBPC3. We find that 45% of the pathogenic variants show alterations in RNA splicing or protein stability, which can be linked to pathogenicity with 100% and 94% specificity, respectively. Relevant for variant annotation, we uncover that 9% of non-truncating variants of MYBPC3 currently classified as of uncertain significance induce one of these molecular phenotypes. We propose that alteration of RNA splicing or protein stability caused by MYBPC3 variants provide strong evidence of their pathogenicity, leading to improved clinical management of HCM patients and their families

Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy

“…The variants are located in exon 17 of the MYBPC3 gene, which together with exon 16 codifies for the C3 domain of cMyBP-C (Supplementary Text S1; Figure ). Many HCM missense mutations cluster in C3, , but no protein interactors have been described for this domain, as expected from its location far from the anchoring points of cMyBP-C to actomyosin filaments. , Hence, mutations targeting C3 are arguably not predicted to affect cMyBP-C interactions, which is in agreement with the normal sarcomere localization of missense mutant cMyBP-C proteins in HCM myocardium. , In addition, the high-resolution structure of C3 is known, which can help interpret the effects of the mutations (Figure ). We first verified that the pathogenic mutations do not induce RNA splicing defects or extensive protein structural destabilization, two classical protein haploinsufficiency drivers linked to pathogenicity in 45% of cMyBP-C missense mutations …”

“…All mutations retain close-to-WT thermal stability (Supplementary File S1; Supplementary Figure S4b). The maximum drop in T m was 5.3 °C for mutant R502Q; however, this limited decrease in thermal stability can also be found in nonpathogenic missense variants targeting C3 and therefore cannot explain the pathogenicity of the mutation …”

“…Many HCM-causing mutations in myosin affect directly the mechanochemical cycle of the protein, resulting in enhanced force generation. , In addition, the proportion of low-activity SRX myosin heads is decreased in HCM myocytes devoid of cMyBP-C but is normalized upon treatment with mavacamten. ,, The emerging unifying view is that, regardless of the specific mutation, HCM is primarily a mechanical disease at the molecular level; however, it remains unknown how missense mutations in the central region of cMyBP-C can lead to hypercontractility . In this work we have studied several pathogenic mutations targeting the central C3 domain of cMyBP-C, the region of the protein with the highest number of HCM-causing missense mutations. ,, We verified that our variants do not induce classical protein haploinsufficiency drivers specifically associated with other HCM mutations (Supplementary Figures S1, S4). Hence, these variants trigger HCM by yet-to-identify molecular mechanisms.…”

“…The molecular deficits, or phenotypes, of these missense mutations remain largely unexplored. Some of them have been proposed to disrupt cMyBP-C interaction with actomyosin filaments − or to induce extensive protein destabilization; , however, many HCM-causing missense mutants appear to operate via alternative, unidentified mechanisms . Prompted by the ability of cMyBP-C to establish mechanical tethers that modulate sarcomere contraction (Figure b), we hypothesized that HCM-causing mutations may perturb the nanomechanics of cMyBP-C, leading to altered sarcomere activity.…”

Molecular Epidemiology of Mitochondrial Cardiomyopathy: A Search Among Mitochondrial and Nuclear Genes

“…Moreover, the correct classification of novel variants needs functional evidence, conferred by studies often complicated to perform and requiring specific laboratory competences [ 148 , 149 ]. Conversely, in silico evaluations, using several bioinformatics tools, are used to predict the likelihood of pathogenicity of missense and splicing variants.…”