Protein Thermodynamic Destabilization in the Assessment of Pathogenicity of a Variant of Uncertain Significance in Cardi
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ORIGINAL ARTICLE
Protein Thermodynamic Destabilization in the Assessment of Pathogenicity of a Variant of Uncertain Significance in Cardiac Myosin Binding Protein C Maria Rosaria Pricolo 1,2 & Elías Herrero-Galán 1 & Cristina Mazzaccara 2,3 & Maria Angela Losi 4 & Jorge Alegre-Cebollada 1 & Giulia Frisso 2,3 Received: 18 October 2019 / Accepted: 20 January 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords VUS . HCM . cMyBC
Associate Editor Paul J. R. Barton oversaw the review of this article Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12265-020-09959-6) contains supplementary material, which is available to authorized users. * Maria Rosaria Pricolo [email protected] * Jorge Alegre-Cebollada [email protected]
Giulia Frisso [email protected] 1
Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
Elías Herrero-Galán [email protected]
2
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Naples, Italy
Cristina Mazzaccara [email protected]
3
CEINGE Biotecnologie Avanzate s.c. a r.l., Naples, Italy
4
Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, Italy
Maria Angela Losi [email protected]
J. of Cardiovasc. Trans. Res.
Introduction Hypertrophic cardiomyopathy (HCM) constitutes the most common inherited disease of the myocardium with an estimated prevalence ranging from 1:500 to 1:200 [1, 2]. Although the clinical manifestations of HCM are highly variable, the disease is chara
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