- Academic Editor
Background: Hypertrophic cardiomyopathy is the most frequent autosomal dominant disease, yet due to genetic heterogeneity, incomplete penetrance, and phenotype variability, the prognosis of the disease course in pathogenic variant carriers remains an issue. Identifying common patterns among the effects of different genetic variants is important. Methods: We investigated the cause of familial hypertrophic cardiomyopathy (HCM) in a family with two patients suffering from a particularly severe disease. Searching for the genetic variants in HCM genes was performed using different sequencing methods. Results: A new missense variant, p.Leu714Arg, was identified in exon 19 of the beta-myosin heavy chain gene (MYH7). The mutation was found in a region that encodes the ‘converter domain’ in the globular myosin head. This domain is essential for the conformational change of myosin during ATP cleavage and contraction cycle. Most reports on different mutations in this region describe severe phenotypic consequences. The two patients with the p.Leu714Arg mutation had heart failure early in life and died from HCM complications. Conclusions: This case presents a new likely pathogenic variant in MYH7 and supports the hypothesis that myosin converter mutations constitute a subclass of HCM mutations with a poor prognosis for the patient.
Hypertrophic cardiomyopathy (HCM) is a primary myocardium disease characterized by an asymmetric increase in heart muscle mass, predominantly in the interventricular septum, hypertrophied cardiocytes, cellular and sarcomeric disarray, and fibrosis [1]. Major functional impairments are compromised relaxation and hypercontractility [2]. In most cases, the disease has a genetic cause that leads to an autosomal dominant trait being transmitted throughout families. Occasionally, a de novo variant may be responsible for the phenotype in previously unaffected families. Significant contributors to the disease are mutations in the beta-myosin heavy chain (MYH7) and the cardiac isoforms of myosin binding protein-C (MYBPC3). These two genes may account for about two-thirds of all genetically confirmed cases [3]. Most genes that possess pathogenic variants code for proteins constituting the sarcomere or contributing to controlling contraction.
The heterogeneity of inherited causes of HCM corresponds to a broad spectrum of highly variable phenotypes. Mild cases with onset of symptoms in the fourth decade of life or even later contrast with early onset, severe symptoms, and a high risk of sudden cardiac death. The underlying genetic defect can partly explain the variability in the phenotype; however, genetic modifiers and polymorphisms may modulate the functional consequences of the mutation [4]. Thus, it is frequently difficult to predict the clinical outcome if a diagnosis has been established early or before the symptomatic stage. Therefore, familial case studies are frequently useful to improve our understanding of the biological consequences of a genetic alteration. Here, we document the new likely pathogenic variant together with its clinical phenotype and discuss the functional implications that this variant could have.
The initial (tentative) diagnosis of HCM in the proband was performed in an army hospital during military service. A son was first diagnosed as having a congenital atrial septal defect following surgical correction of that defect. Treatment and follow-up investigations of both patients were conducted in the Cardiology Research Institute of Tomsk National Research Medical Center. The diagnosis of hypertrophic cardiomyopathy was based on the evaluation of symptoms, 12-lead ECG monitoring at rest and during exercise, X-ray studies, 2D- and Doppler echocardiography, and quantitative coronary angiography with intracardiac blood pressure measurement. Informed consent for the genetic analysis was obtained from the patients and unaffected relatives.
For three family members (two affected, one unaffected), genomic DNA was extracted from venous EDTA blood samples using phenol–chloroform extraction. The genes coding for beta-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) were investigated as candidate genes by analyzing single-strand conformation polymorphism (SSCP) using gel migration behavior of the amplified exons of the two genes. For PCR amplification, PCR primers were designed based on reference gene sequences using Primer3 v 4.1.0 software [5]. For SSCP analysis, non-denaturing polyacrylamide gradient gels (5 to 17% acrylamide and 0 to 0.5% sucrose) were used. Electrophoresis was performed in horizontal gel chambers at two temperatures (12 °C and 24 °C). DNA was visualized by silver staining. Sanger sequencing of PCR products with altered SSCP patterns was performed in both directions by the ABI3730 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) using PCR primers. Additional screening was conducted for the proband using NGS (TruSight™ Cardiomyopathy Sequencing Panel, Illumina, San Diego, CA, USA) to prevent additional clinically relevant genetic variants.
Initial SSCP screening and sequencing were performed at the Max Planck Institute
for Physiological and Clinical Research in Bad Nauheim, Germany. Sample
preparation and confirmation sequencing were conducted in the Institute of
Medical Genetics using the Core Facility
The Local Biomedical Ethics Committee approved the study at the Research Institute of Medical Genetics and the Cardiology Research Institute, Tomsk National Research Medical Center. The DNA samples were stored in the collection of samples “Biobank of the North Eurasia population” in the Research Institute of Medical Genetics, Tomsk NRMC.
A small Tomsk (Russia) family with two seriously affected patients (father and son) was studied. The pedigree of the family is shown in Fig. 1. The proband (father, II-2) had one unaffected brother (II-1) and one affected child (III-1). The parents of the proband died from unidentified causes (no further information available). Cardiac disease was first recognized in the proband upon hospitalization at the age of 22 years. Reduced exercise tolerance, dyspnea, extrasystoles, myocardial hypercontractility, and an ECG suggestive of a cardiomyopathy in the absence of systemic conditions able to explain these features (e.g., arterial hypertension) led tentatively to diagnosing hypertrophic cardiomyopathy.
Pedigree of the family. Squares and circles indicate male and female members of the family, respectively. Slashed symbols designate deceased members. Filled and open symbols refer to affected and unaffected members, respectively. The clinical status of the grandparents (generation I) was unassessed. The arrow points to the proband.
The clinical status worsened gradually in subsequent years. At 30 years, thickening of the interventricular septum was apparent (see Fig. 2A; documented at 37 years), together with mild mitral regurgitation. Severe arrhythmia (atrial flutter and frequent extrasystoles), X-ray evidence of pulmonary hypertension, impaired ventricular contractility and relaxation, and dilatation of the left atrium and ventricle were indicative of advanced cardiac failure (New York Heart Association (NYHA) stage III). The absence of other vascular or systemic malfunctions supported HCM as the primary diagnosis. The disease was complicated by the development of exudative pericarditis (Fig. 2A). Medication (angiotensin converting enzyme (ACE) inhibitors, cardiac glycosides, diuretics) was of limited use. At the age of 42 years, the patient had dyspnea at rest, severely impaired systolic and diastolic function, an ejection fraction of 40%, atrial fibrillation, and severe complications by pulmonary hypertension and pericarditis, indicating that the patient’s condition was close to end-stage heart failure (NYHA stage III-IV). The patient died soon after.
Echocardiographic investigation of the two patients in the family. (A) Father. (B) Son. IVS, interventricular septum; PW, left ventricular posterior wall; LV and RV, left and right ventricle; LA, left atrium; Ao, aorta. The arrow indicates pericardial space filled with exsudative fluid in panel A.
The son suffered from early childhood onwards. His case was confounded by an
atrial septal defect diagnosed at the age of 2.5 years and corrected by surgery
at the age of 6. Even before surgery, conspicuous interventricular septum
hypertrophy became apparent. At the age of 17, his condition was characterized by
grossly reduced exercise tolerance, dyspnea at rest, hypertrophy of the posterior
wall (14 mm) in addition to the septum (23 mm, see Fig. 2B), impaired cardiac
performance (ejection fraction at rest 61%) and cardiomegaly (based on chest
X-ray and an index of LV myocardial mass of 205.6 g/m
Two genes (MYH7 and MYBPC3), whose mutations account for about
50% of all familial cases of HCM, were screened in the patients using the SSCP
method. PCR products containing exon sequences in the myosin binding protein C
gene did not show differences in SSCP patterns obtained from the DNA of the
patients, while some SNPs were observed (not shown). Among 40 MYH7 exons
tested, a unique altered SSCP pattern was identified for exon 19. Sanger
sequencing of this exon revealed T to G transversion (A to C transversion in the
genomic sequence) in the second position of codon 714 leading to a Leu to Arg
substitution: MYH7(NM_000257.4):c.2141T
The mutation introduces a positive charge in a normally hydrophobic position
located in the region of the so-called converter domain of the protein that
contributes to the movement of the myosin head during contraction [9]. According
to the ACMG/AMP guidelines for the interpretation of sequence variants [10], this
new variant meets criteria PM1, PM2, PP2, PP3, and PP4 and can be classified as
likely pathogenic, according to the rule “2 Moderate (PM1–PM6) and
The severe course of the disease suggested a contribution by additional genetic variants because patients with two pathogenic variants usually have a “malignant” phenotype [3]. To identify/exclude additional mutations in the patient, we performed sequencing coding regions of 46 cardiomyopathy genes with TruSight™ Cardiomyopathy Sequencing Panel, Illumina, for the DNA of the proband (Fig. 1, II-2). The results confirmed a p.Leu714Arg variant in MYH7. No additional pathogenic/likely pathogenic/uncertain significance variants were identified. No additional genetic tests were conducted for the son (Fig. 1, III-1).
We have identified a novel MYH7 variant in two patients from a nuclear
family, a father and son, who were both affected by hypertrophic cardiomyopathy
and cardiac failure. The mutation was a c.2141T
However, the contribution of the mutation to the phenotype cannot easily be assessed because both patients had additional conditions not generally associated with HCM. The father was suffering from recurrent pericarditis for many years with apparently serious consequences for cardiac performance. Volume overload caused by or associated with HCM and pulmonary hypertension presumably contributed to a vicious cycle resulting in end-stage failure. The son was born with a congenital defect, which led to hemodynamic consequences. To summarize the clinical evaluation, these patients were exposed not only to HCM but to confounding conditions, adding to the myocardial dysfunction caused by the myosin heavy chain mutation. However, the uncommonly severe phenotype in both patients is a solid reason to argue for a severe characterization of the 714 mutation. Sometimes, the severe disease course may be explained by more than one pathogenic variant in the family or additional variants in other genes that are not pathogenic but have some modifying effect [3, 12]. However, sequencing of the coding regions for 46 cardiomyopathy-associated genes in the proband did not find candidate variants with likely functional effects. It should be noted that the possible genetic cause for the congenital heart defect in the son was not investigated.
Exon 19 MYH7 contributes to the part of the C-terminal end of the myosin head known as the “converter domain” [13]. This region consists of a stretch of 68 amino acids (in the human beta-myosin chain residues 709 to 777) thought to serve as a socket for the adjacent (C-terminal) alpha-helical extension (or “neck”) stabilized by two light chains. It has been concluded from the model building as well as from functional assays that the “neck” is in the correct orientation and position to act as a lever (for review and references, see [14]). The converter contributes to the “power stroke” of the contractile cycle by, presumably, transducing a conformational change resulting from ATP cleavage in the globular myosin head to the alpha-helical “lever arm” extending from the C-terminus of the head. The position between the myosin head and the lever arm may function as a “relay station” between the ATP cleavage center and the lever arm. Internal elastic distortions of the converter domain may be how the converter mediates a reorientation (“swinging”) of the lever arm. That swinging contributes to the translocation of actin along myosin filaments. The function of the lever in terms of speed of actin transport promoted by myosin heads depends on the length of the neck. The addition of light chain binding sites increased the speed of actin movement in vitro, and truncations led to a decrease, as was shown by exposing actin filaments to myosin heads of Dictyostelium discoideum immobilized on a solid surface [15]. Altered functions and pleiotropic effects, including changes in ATPase activity patterns, have also been demonstrated with truncated converter regions of myosin II from Dictyostelium discoideum [16]. These experiments strongly suggest that the converter region and the adjacent lever are essential for the motor system. Recently, it has been shown that conformational change in the converter is integral to the mechanochemical coupling of myosin, particularly for the ADP release step [17].
A query to the ClinVar database [6] (accessed September 2023) provided information on more than 90 different amino acid variants in 48 positions within the converter region (71% of the converter amino acids), classified from uncertain significance to pathogenic. In the first 16 amino acids (709–726), at least one missense variant was described in each position (except 709 and 711), and altogether with p.Leu714Arg, there are 33 different amino acid variations in patients for these 18 positions (Fig. 3A, Ref. [6, 18, 19, 20]). It means that this converter region is frequently mutated in cardiomyopathies. Indeed, analysis of 2913 patients with HCM allowed us to identify significant enrichment of disease-associated variants in the converter, with earlier disease onset in the carriers of these variants [18]. According to the summary of published and own data provided in [18], among 526 patients with 25 different mutations in the converter, 407 had HCM (77%), the mean age of diagnosis was 8 years, and 97% of the variant carriers were symptomatic at the age of 35; in addition, 184 patients (37%) had either sudden cardiac death or cardiac transplantation [19]. A recent study showed that variants in the converter domain have significantly higher penetrance than variants in other MYH regions [20].
Missense substitutions in the MYH7 converter domain and
localization of Leu714 in the myosin molecular structure. (A) A summary of known
missense variants in amino acids 709-777 in MYH7, according to the ClinVar
database [6]. The amino acid variants are classified as pathogenic (red), likely
pathogenic (dark red), “conflicting interpretations of pathogenicity” (lilac),
or uncertain significance (black). Leu714 (L) and the novel Arg714 variant (R)
are highlighted in green. (B) Secondary structure of part of MYH7 motor domain:
(1) myosin active center bound with Mg
Some experimental studies analyzed the consequences of mutations in the converter. For instance, using isolated muscle fibers (obtained from m. soleus of an HCM patient suffering from a p.Arg719Trp mutation), increased generation of force, and fiber stiffness were demonstrated as a consequence of the Arg to Trp exchange [23]. M. soleus expresses the same beta-myosin isoform as the myocardium. Stiffness under rigorous conditions and during relaxation and force development increased by similar amounts (about 50%). Increased fiber stiffness would imply a reduced ability by the myosin molecule to switch back to the original conformation at the end of one power stroke. The authors suggested that the converter is a sub-domain in myosin within which the transient structural change is needed to drive the action of the lever arm [23]. Further studies demonstrated that the p.Arg723Gly variant has a similar effect on the fiber stiffness, whereas p.Ile736Thr has essentially no effect [24, 25]. Other investigators showed slightly decreased contractile force but 15% faster velocity for the Arg719Trp and Arg723Gly variants; however, no change was noted in these characteristics for the Gly741Arg variant. In the transgenic Drosophila model carrying the p.Arg713Glu mutation, the mutants showed no actin motility, and it was demonstrated that the mutation disrupts interdomain interaction, namely with the Glu497 residue in the myosin “relay” domain [26]. The Leu714 is close to the 712 and 719 positions, at which the former participates in the myosin interdomain interactions, and the latter leads to the reported change in fiber stiffness. It may be hypothesized that the functional consequences of the p.Leu714Arg transition are similar to the substitutions of neighboring amino acids.
Molecular modeling shows that p.Leu714 constitutes the last part of the
A new likely pathogenic variant in MYH7 (NM_000257.4):c.2141T
Data sharing is not applicable to this article, as no datasets were generated or analyzed during the current study.
MVG designed and performed the research, analysed the data, wrote the manuscript. ENP and KVP peformed diagnostics and observation of the patients, collected clinical data, prepared and discussed clinical section of the manuscript. OAM participated conducting the experiments, and discussing the results. RRS and OSG performed NGS experiments and interpreted the results. VPP and MSN planned and designed the research, discussed and edited the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
Informed consent was obtained from the participants of the study. The study was conducted in accordance with the Declaration of Helsinki and approved by the Local Biomedical Ethics Committee at the Research Institute of Medical Genetics, Tomsk National Research Medical Center (protocol # 10, February 15, 2021) and the Local Biomedical Ethics Committee at the Cardiology Research Institute Tomsk National Research Medical Center (protocol # 151, December 22, 2016).
The authors dedicate this article to the memory of Professor Hans-Peter Vosberg, who made a significant contribution to the genetic studies of hypertrophic cardiomyopathy and who participated in writing the first version of the manuscript.
The study was performed as a part of basic research program of the Institute of Medical Genetics, Tomsk NRMC. M.V.G. received a postdoc fellowship of Max-Planck Society (Germany).
The authors declare no conflict of interest.
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