Novel mutations in sarcomeric protein genes in dilated cardiomyopathy

doi:10.1016/S0006-291X(02)02374-4

Biochemical and Biophysical Research Communications

Volume 298, Issue 1, 18 October 2002, Pages 116-120

https://doi.org/10.1016/S0006-291X(02)02374-4 Get rights and content

Abstract

Mutations in sarcomeric protein genes have been reported to cause dilated cardiomyopathy (DCM). In order to detect novel mutations we screened the sarcomeric protein genes β-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), troponin T (TNNT2), and α-tropomyosin (TPM1) in 46 young patients with DCM. Mutation screening was done using single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The mutations in MYH7 were projected onto the protein data bank-structure (pdb) of myosin of striated muscle. In MYH7 two mutations (Ala223Thr and Ser642Leu) were found in two patients. Ser642Leu is part of the actin–myosin interface. Ala223Thr affects a buried residue near the ATP binding site. In MYBPC3 we found one missense mutation (Asn948Thr) in a male patient. None of the mutations were found in 88 healthy controls and in 136 patients with hypertrophic cardiomyopathy (HCM). Thus mutations in HCM causing genes are not rare in DCM and have potential for functional relevance.

Section snippets

Materials and methods

Clinical evaluation. This study is based on 46 unrelated patients with a diagnosis of DCM, who gave written informed consent for genotyping and detailed prospective phenotyping. The study was approved by the Ethic Committee of Charité, Humboldt University Berlin. Thirty eight of 46 patients were male, eight female. The patients’ age at study was 35±8 years; the age at diagnosis was 29±8 years. Diagnosis was based on echocardiography, coronary angiography, and in selected cases, endomyocardiac

Results

By systematic mutation screening of 46 DCM patients using SSCP analysis we identified a total of three mutations, two in MYH7 and one in MYBPC3. All three mutations lead to the substitution of an amino acid. In addition, we found a number of silent variants and polymorphisms, which were partially already detected in HCM (Table 1, Table 2).

In exon 8 of MYH7 a G to A transition occurs in one male patient at nucleotide 7799 (GenBank No. M57965). This mutation is predicted to result in the

Discussion

The novel findings of our study consist in the detection of three novel rare mutations, leading to amino acid exchanges in the HCM related genes MYH7 and MYBPC3 in patients with DCM.

Inspection of the protein structure of β-MHC predicts effects on thermostability and protein folding for the mutation Ala223Thr and severe conformational changes for Ser642Leu located close to the actin–myosin interaction site. Both mutations did not occur in healthy controls or HCM patients. Since both occurred in

Acknowledgements

The study was Supported by BMBF, Grant NBL III, TP3.1.4.

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Cited by (131)

Novel Mutations in β-MYH7 Gene in Indian Patients With Dilated Cardiomyopathy
2022, CJC Open
Heart failure is a hallmark of severe hypertrophic cardiomyopathy and dilated cardiomyopathy (DCM). Several mutations in the β-MYH7 gene lead to hypertrophic cardiomyopathy. Recently, causative mutations in the β-MYH7 gene have also been detected in DCM from different populations.
Here, we sequenced the β-MYH7 gene in 137 Indian DCM patients and 167 ethnically matched healthy controls to detect the frequency of mutations and their association.
Our study revealed 27 variations, of which 7 mutations (8.0%) were detected exclusively in Indian DCM patients for the first time. These included 4 missense mutations—Arg723His, Phe510Leu, His358Leu, and Ser384Tyr (2.9%); a frameshift mutation—Asn676_T-del (1.5%); and 2 splice-site mutations (IVS17+2T) T>G and (IVS19-1G) G>A (3.6%). Remarkably, all 4 missense mutations altered evolutionarily conserved amino acids. All 4 missense mutations were predicted to be pathogenic by 2 bioinformatics tools—polymorphism phenotyping v2 (PolyPhen-2) and sorting intolerant from tolerant (SIFT). In addition, the 4 homology models of β-MYH7—p.Leu358, p.Tyr384, p.Leu510, and p.His723—displayed root-mean-square deviations of ∼2.55 Å, ∼1.24 Å, ∼3.36 Å, and ∼3.86 Å, respectively.
In the present study, we detected numerous novel, unique, and rare mutations in the β-MYH7 gene exclusively in Indian DCM patients (8.0%). Here, we demonstrated how each mutant (missense) uniquely disrupts a critical network of non-bonding interactions at the mutation site (molecular level) and may contribute to development of dilated cardiomyopathy (DCM). Therefore, our findings may provide insight into the understanding of the molecular bases of disease and into diagnosis along with promoting novel therapeutic strategies (through personalized medicine).
L’insuffisance cardiaque est une caractéristique de la cardiomyopathie hypertrophique grave et de la cardiomyopathie dilatée (CMD). Plusieurs mutations dans le gène β-MYH7 conduisent à la cardiomyopathie hypertrophique. Récemment, les mutations causales dans le gène β-MYH7 ont également été détectées au sein de différentes populations atteintes de CMD.
Ici, nous avons séquencé le gène β-MYH7 de 137 patients indiens atteints de CMD et de 167 témoins sains appariés selon l’origine ethnique pour détecter la fréquence des mutations et leur association.
L’étude nous a permis de révéler 27 variations, dont sept mutations (8,0 %) étaient exclusivement détectées chez les patients indiens atteints de CMD pour la première fois. Parmi ces mutations, nous avons observé quatre mutations faux-sens—Arg723His, Phe510Leu, His358Leu et Ser384Tyr (2,9 %), une mutation par déphasage—Asn676_T-del (1,5 %) et deux mutations des sites d’épissage (IVS17+2T) T>G et (IVS19-1G) G>A (3,6 %). Étonnamment, les quatre mutations faux-sens changeaient les acides aminés évolutivement conservés. Selon deux outils bioinformatiques—PolyPhen-2 (de l’anglais, polymorphism phenotyping v2) et SIFT (de l’anglais, sorting intolerant from tolerant), les quatre mutations faux-sens devaient être pathogènes. De plus, les quatre modélisations de β-MYH7 par homologie—p.Leu358, p.Tyr384, p.Leu510 et p.His723—affichaient de façon respective des écarts quadratiques moyens de ∼2,55 Å, ∼1,24 Å, ∼3,36 Å et ∼3,86 Å.
Dans la présente étude, nous avons détecté de nombreuses nouvelles mutations, uniques et rares, dans le gène β-MYH7, exclusivement chez les patients indiens atteints de CMD (8,0 %). Ici, nous avons démontré comment chaque mutant (faux-sens) perturbe de manière unique un réseau essentiel d’interactions non liantes au site de mutation (moléculaire) et peut contribuer à la survenue de la CMD. Par conséquent, les conclusions de notre étude peuvent donner un aperçu des bases moléculaires de la maladie et du diagnostic tout en favorisant la promotion de nouvelles stratégies thérapeutiques (par la médecine personnalisée).
High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex
2021, Structure
Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.
Acute NelfA knockdown restricts compensatory gene expression and precipitates ventricular dysfunction during cardiac hypertrophy
2020, Journal of Molecular and Cellular Cardiology
Citation Excerpt :
We did not observe significant difference in ubiquitously expressed essential genes, which could be due to contaminating transcripts from non-cardiomyocyte population in these hearts. Mutations in several sarcomeric genes, like Mybpc3, Myh7, Tnnt2, mostly involved in coordinated muscle contraction and Ca+2 handling have been linked to the development of hypertrophic and dilated cardiomyopathy [34,35,36]. Our ChIP-Seq data shows high pol II occupancy and active promoter at these genes in quiescent hearts, which is further increased with pressure overload (Group 2a, Fig. 2A and B).
Coordinated functional balance of negative and positive transcription complexes maintain and accommodate gene expression in hearts during quiescent and hypertrophic conditions, respectively. Negative elongation factor (Nelf) complex has been implicated in RNA polymerase II (pol II) pausing, a widespread regulatory transcriptional phenomenon observed across the cardiac genome. Here, we examine the role of NelfA aka, Wolf-Hirschhorn syndrome candidate 2 (Whsc2), a critical component of the negative elongation complex in hearts undergoing pressure-overload induced hypertrophy. Alignment of high-resolution genome-wide occupancy data of NelfA, Pol II, TFIIB and H3k9ac from control and hypertrophied hearts reveal that NelfA associates with active gene promoters. High NelfA occupancy is seen at promoters of essential and cardiac-enriched genes, expressed under both quiescent and hypertrophic conditions. Conversely, de novo NelfA recruitment is observed at inducible gene promoters with pressure overload, accompanied by significant increase in expression of these genes with hypertrophy. Interestingly, change in promoter NelfA levels correlates with the transcript output in hypertrophied hearts compared to Sham, suggesting NelfA might be playing a critical role in the regulation of gene transcription during cardiac hypertrophy. In vivo knockdown of NelfA (siNelfA) in hearts subjected to pressure-overload results in early ventricular dilatation and dysfunction, associated with decrease in expression of inducible and cardiac-enriched genes in siNelfA hypertrophied compared to control hypertrophied hearts. In accordance, in vitro knockdown of NelfA in cardiomyocytes showed no change in promoter pol II, however significant decrease in in-gene and downstream pol II occupancy was observed. These data suggest an inhibited pol II progression in transcribing and inducible genes, which reflects as a decrease in transcript abundance of these genes. These results indicate that promoter NelfA occupancy is essential for pol II –dependent transcription. Therefore, we conclude that NelfA is required for active transcription and gene expression during cardiac hypertrophy.
Cardiomyocyte SMAD4-Dependent TGF-β Signaling is Essential to Maintain Adult Heart Homeostasis
2019, JACC: Basic to Translational Science
Citation Excerpt :
MyBP-C is a crucial sarcomeric protein that has structural as well as regulatory functions in muscles. Genetic studies have identified MYBPC3 (cardiac isoform) as one of the major mutated genes that results in the development of hypertrophic and dilated cardiomyopathies (33,34). Several mouse models bearing such mutations showed haploinsufficiency and reduction in cMyBP-C levels (35–38).
The role of the transforming growth factor (TGF)-β pathway in myocardial fibrosis is well recognized. However, the precise role of this signaling axis in cardiomyocyte (CM) biology is not defined. In TGF-β signaling, SMAD4 acts as the central intracellular mediator. To investigate the role of TGF-β signaling in CM biology, the authors deleted SMAD4 in adult mouse CMs. We demonstrate that CM-SMAD4–dependent TGF-β signaling is critical for maintaining cardiac function, sarcomere kinetics, ion-channel gene expression, and cardiomyocyte survival. Thus, our findings raise a significant concern regarding the therapeutic approaches that rely on systemic inhibition of the TGF-β pathway for the management of myocardial fibrosis.
Dilated cardiomyopathy myosin mutants have reduced force-generating capacity
2018, Journal of Biological Chemistry
Citation Excerpt :
Affected individuals presented with early-onset ventricular dilatation without antecedent symptoms or signs of hypertrophy and often progressed to heart failure. A223T was identified in a 35-year-old male DCM patient (10). R237W, a possible disease-causing mutation, was identified in a patient with familial DCM (11).
Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human β-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M·D complex in the steady state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force-holding capacity due to the reduced occupancy of the force-holding state.
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2023, Nature Communications

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Novel mutations in sarcomeric protein genes in dilated cardiomyopathy

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

J. Am. Coll. Cardiol.

J. Am. Coll. Cardiol.

Cell

J. Mol. Cell. Cardiol.

J. Mol. Cell. Cardiol.

Am. J. Hum. Genet.

J. Biol. Chem.

Epidemiology of idiopathic dilated and hypertrophic cardiomyopathy. A population-based study in Olmsted County, Minnesota, 1975–1984

Circulation

Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies

Circulation

The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy

N. Engl. J. Med.

Genetics of dilated cardiomyopathy

Z. Kardiol.

First mutation in cardiac troponin C, L29Q, in a patient with hypertrophic cardiomyopathy

Hum. Mutat.