A New Leu714Arg Variant in the Converter Domain of MYH7 is Associated with a Severe Form of Familial Hypertrophic Cardiomyopathy

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


Introduction
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 symp-toms, 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.

Clinical Evaluation
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.

Genetic Analysis
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 4.1.0software [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

Clinical Features
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.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.
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 2 .The son also exhibited signs of pulmonary hypertension.Therapy using calcium antagonists and beta-blockers was ineffective or even worsened the condition.At the age of 18, the son died of sudden cardiac death provoked by physical efforts.In summary, both patients severely suffered from cardiac disease, which started early in life.The brother of the proband (see Fig. 1, II-1) had typical echocardiographic characteristics and no clinical symptoms.The proband's wife (Fig. 1, II-3) had no symptoms and was not clinically evaluated.

Genetic Analysis
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>G,m.14:23895194-A-C (GRCh37/hg19), or m.14:23425985-A-C (GRCh38/hg38).The substitution was identified in both patients but not in the unaffected brother of the proband.This variant is novel and is absent in population and mutation genomic databases (ClinVar, GnomAD, RuSeq) [6][7][8].
The mutation introduces a positive charge in a normally hydrophobic position located in the region of the socalled 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 ≥2 Supporting (PP1-PP5) criteria" (see Tables 4, 5 in [10]).Online resource VarSome [11] also designates this variant as likely pathogenic.Hence, we suppose that this new variant is the cause of the observed hypertrophic cardiomyopathy in this family.
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).

Discussion
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>G transversion, resulting in the replacement of leucine by arginine in codon 714.The variant was found in the two patients but not in the unaffected brother of the proband.Since both patients had early onset variations of the disease, relatively fast progression of symptoms, and premature cardiac death, the cardiomyopathy course in this family can be described as "malignant".
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 Cterminal 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].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 (https://doi.org/10.2210/pdb8EFE/pdb)[17], created using Mol* Viewer [22].
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 β-sheet before α-spiral in the converter domain (Fig. 3B,C), and the variant increases the positive charge in this region by replacing the non-charged leucine with a positive-charged arginine.The closest known variant with a similar effect is p.Gly716Arg, which has been reported in several publications.Some publications provide clinical information about patients with this variant.Indeed, the first report was in 1994, when one of three affected individuals required cardiac transplantation [27].In one family from Korea, four cases of premature sudden death and two children with full expression of the disease were seen in a pedigree with 13 affected members, all carrying this variant with complete penetrance [28].The variant was also associated with sudden cardiac death in an American family, and the proband received an internal cardioverter defibrillator [29].Then, the variant was identified as a de novo mutation in an Indian family, with sudden cardiac death of the proband at the age of 21 years [30].Another de novo mutation, p.Gly716Arg, was reported in a Chinese family, and analysis of available published data on this variant (altogether 43 patients) showed that p.Gly716Arg is characterized by complete penetrance and extremely poor prognosis, with a median survival age of 42 years and 25 cases of sudden cardiac death [31].Therefore, it could be proposed that a charge change in this part of the converter region seems to cause "malignant" HCM.Alternatively, according to the ClinVar database, two variations in the codon 714 were recently reported, p.Leu714Ile and p.Leu714Pro [32], and both were classified as variants of uncertain significance.However, it was noted that they do not change the molecular charge.It might be proposed that clinically severe phenotypes mark the majority of converter mutations in the region at or near 714, with a high risk of life-threatening complications.Using epidemiological inference, published evidence suggests a common or at least related mechanism for these mutations.Based on this suggestion, we propose that the p.Leu714Arg mutation is responsible for a similarly severe dysfunction in the converter domain as the p.Gly716Arg mutation.

Conclusions
A new likely pathogenic variant in MYH7 (NM_000257.4):c.2141T>Gleading to amino acid replacement p.Leu714Arg in the cardiac beta-myosin heavy chain has been identified in two related patients suffering from "malignant" hypertrophic cardiomyopathy.Analysis of the mutation spectrum in this gene showed that the substitution is located in the critical functional converter domain, marked by a high rate of functionally relevant variants.In most cases, mutations in the converter myosin domain lead to early-onset hypertrophic cardiomyopathy with a high risk of life-threatening complications.
(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 «Medical Genomics» equipment of Tomsk NRMC.In particular, DNA sequencing was performed on the ABI3730 Genetic Analyzer.NGS was performed on a MiSeq instrument (Illumina, Inc., San Diego, CA, USA) according to the TruSight™ Cardiomyopathy Sequencing Panel protocol in the Clinical Genetic Research department of the City Hospital No. 40, St. Petersburg, Russia.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.

Fig. 1 .
Fig. 1.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.

Fig. 2 .
Fig. 2. 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 ven-tricle; LA, left atrium; Ao, aorta.The arrow indicates pericardial space filled with exsudative fluid in panel A.

Fig. 3 .
Fig. 3. 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 2+ • ADP; (2) converter domain with highlighted Leu714; (3) essential light myosin chain.(C) Enlarged part of the converter region.Images B and C are from the RCSB PDB (RCSB.org)[21]; PDB ID 8EFE