-Myosin Heavy Chain A Sarcomeric Gene Associated With Dilated and Hypertrophic Phenotypes of Cardiomyopathy

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1 -Myosin Heavy Chain A Sarcomeric Gene Associated With Dilated and Hypertrophic Phenotypes of Cardiomyopathy Elisa Carniel, MD; Matthew R.G. Taylor, MD, PhD; Gianfranco Sinagra, MD; Andrea Di Lenarda, MD; Lisa Ku, MS; Pamela R. Fain, PhD; Mark M. Boucek, MD; Jean Cavanaugh, MS; Snjezana Miocic, MD; Dobromir Slavov, PhD; Sharon L. Graw, PhD; Jennie Feiger, MS, MA; Xiao Zhong Zhu, BS; Dmi Dao, BA; Debra A. Ferguson, MS; Michael R. Bristow, MD, PhD; Luisa Mestroni, MD Background Mutations in the -myosin heavy-chain ( MyHC) gene cause hypertrophic (HCM) and dilated (DCM) forms of cardiomyopathy. In failing human hearts, downregulation of MyHC mrna or protein has been correlated with systolic dysfunction. We hypothesized that mutations in MyHC could also lead to pleiotropic cardiac phenotypes, including HCM and DCM. Methods and Results A cohort of 434 subjects, 374 (134 affected, 214 unaffected, 26 unknown) belonging to 69 DCM families and 60 (29 affected, 30 unaffected, 1 unknown) in 21 HCM families, was screened for MyHC gene (MYH6) mutations. Three heterozygous MYH6 missense mutations were identified in DCM probands (P830L, A1004S, and E1457K; 4.3% of probands). A Q1065H mutation was detected in 1 of 21 HCM probands and was absent in 2 unaffected offspring. All MYH6 mutations were distributed in highly conserved residues, were predicted to change the structure or chemical bonds of MyHC, and were absent in at least 300 control chromosomes from an ethnically similar population. The DCM carrier phenotype was characterized by late onset, whereas the HCM phenotype was characterized by progression toward dilation, left ventricular dysfunction, and refractory heart failure. Conclusions This study suggests that mutations in MYH6 may cause a spectrum of phenotypes ranging from DCM to HCM. (Circulation. 2005;112:54-59.) Key Words: genetics myosin cardiomyopathy, hypertrophic cardiomyopathy, dilated Seventeen genes encoding cytoskeletal, sarcomeric, and nuclear proteins, including -myosin heavy chain ( MyHC), have been associated with dilated cardiomyopathy (DCM). 1 Hypertrophic cardiomyopathy (HCM) is caused by mutations in 9 genes encoding sarcomeric proteins; among them, mutations in the MyHC gene (MYH7) account for the majority of cases. 2 Two cardiac MyHC isoforms 3 have been identified in humans, with the genes tandemly located on chromosome 14. MYH6 encodes MyHC and MYH7 encodes MyHC. 3 MyHC and MyHC are present in different amounts in mammalian hearts 4 ; human hearts express predominantly MyHC. 4 6 In nonfailing human hearts, MyHC mrna represents 20% to 30% of the total myosin mrna, whereas MyHC protein represents 7% of the total MyHC. These are downregulated to 10% and 1%, respectively, in failing hearts, whereas MyHC is upregulated. 5,6 Few data exist on the role of MYH6 mutations in mammals. 7 In humans, an MYH6 mutation has been found in one case of elderly-onset sporadic HCM. 8 On the basis of its behavior in human myocardial failure, we hypothesized that MyHC may be relevant for myocardial function and that mutations could cause a spectrum of cardiac phenotypes ranging from HCM to DCM, as observed in the case of MyHC. Methods Patient Population Ninety families, 69 with DCM (48 familial, 21 sporadic) and 21 with HCM, for a total of 434 subjects, 163 of whom were affected, were studied in the Cardiology Divisions of the University of Colorado Hospital and the University Hospital of Trieste, Italy, and were enrolled in the Familial Cardiomyopathy Registry. 1 Informed consent was obtained from all subjects enrolled in the study, according to the institutional review committee. Accurate family history was obtained from each individual, and family screening was performed. All of the subjects underwent physical examination, ECG, and laboratory analysis. Echocardiography was performed in 407 of 434 individuals (echocardiograms were not obtained for 22 relatives classified as healthy by history, physical examination, and ECG). Received January 30, 2004; de novo received September 17, 2004; revision received February 24, 2005; accepted March 2, From the Familial Cardiomyopathy Registry Research Group. Reprint requests to Dr Luisa Mestroni, University of Colorado Cardiovascular Institute, Bioscience Park Center, E Montview Blvd, Suite 150, Aurora, CO Luisa.Mestroni@UCHSC.edu 2005 American Heart Association, Inc. Circulation is available at DOI: /CIRCULATIONAHA

2 Carniel et al MyHC Mutations in DCM and HCM 55 TABLE 1. Major and Minor Criteria for the Diagnosis of Familial DCM Major Criteria LV systolic dysfunction: EF 45% (2SD) and/or FS 25% ( 2SD) LV dilation: LVEDD 117% of predicted value Minor Criteria Unexplained SV arrhythmias (AF or sustained arrhythmias) Frequent ( 1000/24 h) or repetitive (3 or more ectopic beats with a heart rate 120 bpm) Ventricular arrhythmias 50 years old LVEDD 112% of predicted value LVEF 50% or FS 28% Unexplained conduction abnormalities (grade II or III AV blocks, complete LBBB, or sinus nodal dysfunction) Unexplained SD or stroke 50 years old Segmental wall-motion abnormalities in 1 segment, or 1 if not previously present, in the absence of ischemic heart disease LV indicates left ventricular; FS, fractional shortening; EF, ejection fraction; LVEDD, LV end-diastolic diameter; SV, supraventricular; AF, atrial fibrillation; AV, atrioventricular; LBBB, left bundle-branch block; and SD, sudden death. When clinically indicated, additional studies were performed, including right and left heart catheterization, ventriculography, coronary angiography, endomyocardial biopsy, and neuromuscular evaluation. TABLE 2. Diagnostic Criteria of HCM Major Criteria Echocardiography LV wall thickness 13 mm in the anterior septum or posterior wall or 15 mm in the posterior septum or free wall. Severe SAM (septal-leaflet contact) Electrocardiography LVH repolarization changes TWI in leads I and avl ( 3mm) (with QRS T-wave axis difference 30 ), V 3 V 6 ( 3 mm) or II and III and avf ( 5 mm) Abnormal Q wave ( 40 ms or 25% R wave) in at least 2 leads from II, III, avf (in absence of LAFB, V 1 V 4 ; or I, avl, V 5 V 6 ) Diagnostic Criteria of DCM and HCM Criteria for the diagnosis of DCM were the presence of left ventricular fractional shortening 25% ( 2SD) and/or an ejection fraction 45% ( 2SD) and left ventricular end-diastolic diameter 117% of the predicted value by the Henry formula, corrected for age and body surface area. 9 Exclusion criteria included any of the following conditions: blood pressure 160/110 mm Hg, obstruction 50% of a major coronary artery branch, alcohol intake 100 g/d, persistent high-rate supraventricular arrhythmia, systemic diseases, pericardial diseases, congenital heart diseases, cor pulmonale, and myocarditis. Familial DCM was defined by the presence of 2 or more affected subjects in the same family with DCM meeting the published criteria. 9 Family members were classified as affected, unaffected, or unknown on the base of major and minor criteria (Table 1). The affected status was defined by the presence of 2 major criteria, 1 major criterion and 1 minor criterion, or 3 minor criteria. The unknown status was defined by the presence of 1 or 2 minor criteria and the unaffected status by the presence of a normal heart or the determination of other causes of myocardial dysfunction. HCM was diagnosed in the presence of unexplained left ventricular hypertrophy, 10 excluding secondary causes of cardiac hypertrophy, such as hypertension and valvular disease. The diagnostic criteria of HCM are summarized in Table 2. The affected status was defined by the presence of 1 major criterion, 2 minor echocardiographic criteria, or 1 minor echocardiographic criterion and 1 electrocardiographic criterion. Molecular Genetic Screening Blood samples were collected from 427 of 434 subjects for DNA analysis. MYH6 was screened for mutations by denaturing highperformance liquid chromatography and sequence analysis. In the families in which we found either a putative disease-causing mutation or a polymorphism, all available relatives were screened for mutations. Criteria for classifying variants as putative disease-causing mutations 11 included changes in predicted amino acid sequences, segregation within the family (when available), conservation across different species ( absence in a control population of at least 150 healthy ethnically similar subjects, and changes in protein secondary structure, with the use of GOR 4 12 and NNPREDICT software. 13 The control panels were screened by denaturing high-performance liquid chromatography, and profiles different from the wild type were sequenced. Results Four putative disease-causing mutations were detected (Figures 1 and 2; Table 3) in 3 of 69 probands with DCM (4.3%) and 1 of 21 probands with HCM (4.8%). In exon 21, a P830L substitution was found in a sporadic DCM case. The mutation affects a highly conserved residue of the globular head of MyHC and is predicted to alter the secondary structure of the light-chain binding domain. 3,12 14 Minor Criteria LV wall thickness of 12 mm in the anterior septum or posterior wall or 14 mm in the posterior septum or free wall Moderate SAM (no leaflet-septal contact) Redundant MV leaflets Complete BBB or (minor) interventricular conduction defect (in LV leads) Minor repolarization changes in LV leads Deep S wave in V 2 ( 25 mm) Clinical features None Unexplained chest pain, dyspnea, or syncope SAM indicates systolic anterior motion of the mitral valve; LVH, left ventricular hypertrophy; TWI, T-wave inversion; LAFB, left anterior fascicular block; and BBB, bundle-branch block. See text and the footnote to Table 1 for explanation of other abbreviations.

3 56 Circulation July 5, 2005 Figure 1. MYH6 mutations detected in 4 families with DCM and HCM. Top, Pedigrees of families. Filled symbols indicate affected subjects; open symbols with N (normal) indicate tested, unaffected subjects; open empty symbols indicate subjects without history of cardiomyopathy. In genotyped individuals, and indicate presence of mutant allele and wildtype allele, respectively. Middle, Elution profiles for subjects carrying wild type (red) and mutation (blue). Bottom, Sequence analysis showing heterozygous nucleotide substitution. In exon 23, an A1004S substitution was found in a different sporadic DCM case. The change leads to an alteration in polarity: An alanine (hydrophobic/nonpolar) is replaced by a serine (hydrophilic/polar) in a highly conserved region of the rod domain. In exon 24, a glutamine (neutral) to histidine (basic) substitution (Q1065H) was found in a family with familial HCM. This mutation occurred in a highly conserved residue of the rod domain and was absent in 2 unaffected relatives. The A1004S and Q1065H mutations occurred in the fourth (g) and second (e) position, respectively, of the heptad repeat motif of the -helical coiled-coil 15 (Figure 2B). In exon 31, a glutamic acid (acidic) to a lysine (basic) (E1457K) Figure 2. A and B, Human MyHC protein structure (Swiss-Prot P13533). Amino acid sequence, functional domains, 3,14,15,24 28 and putative disease-causing mutations are shown. Gray shaded areas represent completely conserved sequences among human MyHC proteins. 3 Mutated residues are highlighted in red. NBP indicates nucleotide-binding protein. B, S2 and LMM domains with heptad motif. 15,25 28 ACD indicates assembly-competent domain.

4 Carniel et al MyHC Mutations in DCM and HCM 57 TABLE 3. Genotypes and Phenotypes of 4 Patients With a Putative Mutation of MYH6 and Their Relatives Family Mutation Codon ID Phenotype DNA Genotype Age at Diagnosis, y NYHA* LVEDD, mm* LVFS* (%) LVEF, %* MWT, mm* Arrhythmias and Conduction Outcome (Age, y) TSSDC C3T P830L II:2 DCM / 56 I SVT, VF, LBBB, CHF (75) 1 AVB II:4 Unaffected NA 43 I NA NA NA NA NS (53) III:2 Unaffected NA 17 I NA 7 NS (27) TSSDC G3T A1004S II:2 DCM / 51 I CHF (59) III:1 Unaffected NA 16 I NS (24) II:1 Unaffected NA 47 I NS (55) TSSDC G3A E1457K II:2 DCM / 44 II LBBB, 1 AVB, PM CHF, heart transplant (57) III:2 Unaffected / 22 I NS (24) III:1 Unaffected NA 23 I NS (31) DNHOCM G3C Q1065H II:2 HCM / 27 III NSVT, RBBB Refractory CHF, death (45) III:1 Unaffected / 22 I NS (23) III:2 Unaffected / 15 I NS (17) ID indicates identification in pedigree; NYHA, New York Heart Association functional class; MWT, maximal wall thickness; /, presence of the mutant allele; NA, not available; /, absence of the mutant allele; SVT, supraventricular tachycardia; VF, ventricular fibrillation; 1 AVB, first-degree atrioventricular block; PM, pacemaker; NSVT, nonsustained ventricular tachycardia; RBBB, right bundle-branch block; CHF, congestive heart failure; and NS, no symptoms. Other abbreviations are as defined in text and in the footnotes to Tables 1 and 2. *At enrollment. Age at assessment. substitution was found in a sporadic DCM case. This mutation is predicted 12 to alter the -helix of the rod domain, changing the conformation of a 4 amino acid region from an organized -helix to a random-coil pattern. 12 All putative mutations were absent in at least 300 normal control chromosomes (and absent in an overall number of 500 chromosomes tested) and conserved across different species. Mutations in lamin A/C, actin, MyHC, troponin T, desmin, -sarcoglycan, and lamina-associated polypeptide-2 genes were excluded in the DCM MYH6 mutation carriers. Mutations in the MyHC, troponin T, and myosin-binding protein C genes were excluded in the HCM Q1065H carrier. In addition to the putative disease-associated mutations, 7 nonsynonymous single-nucleotide polymorphisms were identified: 6 new (G56R, I275N, A1130T, E1295Q, R1502Q, and G1826N) and 1 (A1101V) already reported ( ncbi.nlm.nih.gov/entrez/query.fcgi?db snp). 16 Of these new variants, G56R was present in 8 different families in the studied population, with no segregation with the disease within the families, and was found in 10 of 150 healthy controls. A1130T and E1295Q were found in 3 and 2 different families, respectively, without cosegregation with the disease. The polymorphisms I275N, R1502Q, and G1826S were present in the same Italian DCM family in healthy relatives of affected subjects. The reported variants did not meet the criteria for consideration as a diseasecausing mutation and therefore, were classified as polymorphisms. Disease Characteristics Both patients and controls were white. DCM mutation carriers had a late onset of the disease (mean age, 50 6 years), mild symptoms, and mild to moderate left ventricular dysfunction (Table 3). All had slow progression of the disease (follow-up, 8 to 19 years). The proband with HCM had an early onset of the disease and evolution toward dilation and dysfunction, with death due to refractory heart failure while awaiting heart transplantation. The family history was significant for sudden death at the age of 47 years in the proband s affected mother. The proband s offspring were clinically unaffected and did not carry the mutation (Figure 1, Table 3). Discussion This study provides genetic evidence of putative MYH6 mutations in patients that are associated with a spectrum of phenotypes ranging from ventricular hypertrophy to dilation (Figure 1). At enrollment, DCM mutation carriers (4.3% of DCM probands) had mild to moderate ventricular dysfunction at diagnosis and a slow progression of the disease. The Q1065H mutation found in familial HCM was associated with a severe phenotype, characterized by early onset, severe hypertrophy, and evolution toward myocardial dilation, severe dysfunction and death in the fifth decade due to refractory heart failure in the proband, or sudden death in the proband s mother. Overall, the currently available data suggest that MyHC may represent a rare cause of HCM, 8 and consequently, the small number of carriers limits genotypephenotype correlations.

5 58 Circulation July 5, 2005 The MYH6 mutation previously reported in 1 HCM case 8 and the 4 novel putative mutations identified in our study are located in both the head and rod domains of MyHC and affect highly conserved residues of the protein (Figure 2) P830L occurs in a sharp bend (amino acids 829 to 832), which connects a long -helix region, 3 the light-chain binding site, with a short C-terminal -helix. 14 The proline-to-lysine substitution could alter the binding of myosin light chain, compromise the movement of the light chain on MyHC, and interfere with force generation. The E1457K missense mutation is predicted to alter the structure of the rod domain, its assembly, and its interactions with surrounding molecules. The A1004S and the Q1065H mutations are expected to interrupt the heptad repeat motif of the -helical coiled-coil and alter the hydrogen bonds that stabilize the structure of the rod domain. 15 The evidence that MyHC, characterized by higher ATPase activity and faster contraction, 22 is downregulated in failing hearts 5 supports the hypothesis that MyHC is critical for normal myocardial function. In human left ventricles, MyHC mrna represents 20% to 30% of the total MyHC RNA. However, the abundance of MyHC protein is low, 7% of total MyHC in nonfailing hearts, decreasing to 1% in failing left ventricles. 6 The relatively small amount of MyHC protein present in nonfailing left ventricles has called into question the physiological significance of MyHC isoform changes in failing human ventricles. 23 Interesting observations come from studies of myocardial gene expression in patients with heart failure Lowes et al 24 studied 53 subjects (45 with DCM, 8 normal controls) assigned to treatment with -blockers or placebo. Before treatment, patients with DCM had downregulation of MyHC mrna and upregulation of MyHC mrna expression compared with controls. Responders to -blocker therapy had a significant improvement in ejection fraction and functional capacity, as well as a significant increase in the amount of MyHC mrna and a decrease in the level of MyHC mrna. The authors concluded that -blocker therapy could reverse a pathological fetal gene program, leading to restoration of the fast-contracting MyHC fibers and to a consequent improvement of myocardial function. Interestingly, similar changes were also observed in placebo-treated patients who improved spontaneously. Ladenson et al 26 reported the case of a patient with DCM and hypothyroidism: Treatment with levothyroxine led to an improvement in the patient s clinical and echocardiographic parameters, associated with an 11-fold increase in MyHC mrna level. Similar data were obtained by Sabbah et al, 27 who studied the effect of passive mechanical ventricular restraint with the Acorn cardiac support device in a dog model of chronic heart failure. At baseline, heart failure dogs had a decreased level of MyHC mrna and an increased amount of MyHC mrna compared with normal dogs. Therapy with the cardiac support device was associated with improved contractility and normalization of MyHC mrna levels. Finally, recent data from a study in rat myocardium 28 demonstrated that even a small amount of MyHC may have physiological or biological significance. All of these studies support the hypothesis that decreased expression of the fast-contracting MyHC can cause a loss of contractile function as observed in DCM. In summary, we have provided genetic data suggesting that MYH6 mutations may lead to a spectrum of dilated and hypertrophic phenotypes, including myocardial hypertrophy with evolution toward dilation and systolic dysfunction. Functional studies are currently in progress to clarify the role of MYH6 mutations, to determine the mechanisms by which these mutations can lead to the development of a cardiomyopathy phenotype, and to exclude whether any of the mutations reported here are rare polymorphisms. Familial Dilated Cardiomyopathy Registry Research Group University of Colorado Cardiovascular Institute: Brian D. Lowes, MD; Human Medical Genetics Program: Katherine Gowan, MS; Hospital and University of Trieste, Italy: Mauro Driussi, MD, Giulio Scherl. Acknowledgments The authors are supported by grants from the NIH/NHLBI (1RO1 HL , 5K23 HL ), the Muscular Dystrophy Association USA (PN ), and the American Heart Association ( N). We thank the family members for their participation in this study and Stanislav Miertus, PhD, for his critical reading and suggestions. References 1. Taylor MRG, Carniel E, Mestroni L. Familial dilated cardiomyopathy. Orphanet Databases, Available at: patho/gb/uk-fdcardiomyopathy.pdf. Accessed June 8, Richard P, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C, Benaiche A, Isnard R, Dubourg O, Burban M, Gueffet JP, Millaire A, Desnos M, Schwartz K, Hainque B, Komajda M; EUROGENE Heart Failure Project. 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