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An unusual clinical history of a 23-year-old male proband with obstructive hypertrophic cardiomyopathy associated with a rare genotype is presented. Genetic analysis of the proband found evidence for two distinct mutations of the MYH7 gene (the gene coding for the beta-myosin heavy chain): 403Arg→ Trp in exon 13 and a novel mutation, 453Arg→ His, in exon 14. A heterozygous site mutation was identified in exon 13 in the proband’s father but no mutation site was found in his mother. Thus, the novel mutation in exon 14 is a de novo mutation.
Hypertrophic cardiomyopathy (HCM) is a fascinating primary heart disease characterized by left or right ventricular hypertrophy, or both. Typical microscopic changes, arrhythmias and sudden cardiac death are also common. The prevalence of HCM is about 0.2% (1,2).
Left ventricular hypertrophy is the most typical morphological sign of HCM. The hypertrophy is usually asymmetrical and involves mainly the interventricular septum. In about a quarter of patients, hypertrophy of the subvalvular part of the interventricular septum leads to dynamic obstruction of the left ventricular outflow tract. Both ventricular hypertrophy and dynamic obstruction may change substantially during adolescence, in later stages of the disease or as a result of therapy (1,2). The first signs of the disease usually occur in adolescence, but the diagnosis is most often made between the ages of 30 and 50 years. The annual mortality rate is believed to be around 1% (3). Death is often sudden and may occur in previously asymptomatic or stable patients. Young age, positive family history, specific genetic defects, sustained ventricular tachycardia and a history of recurrent syncopes or aborted sudden death are the most important risk factors. Diastolic dysfunction with relaxation abnormalities is responsible for dyspnea, myocardial hypertrophy for angina pectoris, and arrhythmias for palpitations and syncope. On physical examination, systolic murmur at the left sternal border, increasing with the Valsalva manoeuvre, is typical of the obstructive form. The diagnosis of HCM is based on an echocardiographic or magnetic resonance image finding of left ventricular hypertrophy that cannot be explained by arterial hypertension or aortal stenosis. The systolic anterior movement (SAM) is a specific feature of dynamic outflow tract obstruction. In therapy, beta-blockers and verapamil are the drugs used most often. Patients with a high outflow gradient may benefit from interventional methods, such as dual-chamber pacing, transcoronary ablation or surgical myotomy. Unfortunately, none of these methods has been proved to lower the overall mortality rate.
Over the past two decades, substantial progress has been achieved in defining the molecular basis of the HCM. A mendelian trait with an autosomal dominant pattern and incomplete penetrance was proved in a vast majority of cases, and more than 100 different mutations in eight distinct genes have been identified (4,5). All of these genes code for structural or regular proteins of cardiac sarcomere (Table 1).
Experimental studies on isolated myofibrils or mice models proved that these mutations lead to a decreased contractility of cardiac sarcomeres. Based on these conclusions, a hypothesis of compensational hypertrophy was suggested (4). Unfortunately, no direct association between genotype and phenotype of HCM was found; however, it is very likely that genetic testing can identify patients with a high risk of sudden death (6).
Mutations on the MYH7 gene (7), which codes for the beta-myosin heavy chain, are the most frequent cause of HCM. Myosin is a primary contractile protein present in hex-amer form consisting of two heavy chains and two pairs of regulatory and essential subunits of light chains. Thick myofibrillar filaments are composed of a few hundred molecules of myosin that interact with thin filaments, enabling a muscle to contract and relax. More than 50 mutations of the MYH7 gene have been identified. Almost all of them are single base substitutions and affect the globular region of myosin, where most functional regions of a protein are concentrated. Some of them are associated with a relatively benign clinical picture of the disease, and an almost normal length of life is assumed (606Val→ Met, 908Leu→ Val) (8), whereas others confer a higher probability of sudden cardiac death or failure (403Arg→ Gln, 453Arg→ Cys, 719Arg→ Trp) (8–10).
Denaturing gradient gel electrophoresis (DGGE) in DNA analysis is useful since it resolves mutant DNA from wild type DNA (11,12). After DNA amplification (13), DGGE allows for screening of point mutation because DGGE techniques are based on the ability of DNA to denature and melt, depending on its primary structure (14). When a mutation is present in the heterozygous status, after denaturation and subsequent reannealing, polymerase chain reaction (PCR) products create heteroduplexes that form separate bands on DGGE because of their abnormal migration.
The proband is now a 23-year-old man with no known family history of heart disease. When he was nine years old, a benign mediastinal teratoma was surgically removed. At that time, the boy had a history of at least one documented syncope while exercising. The physical examination was normal except for a moderate systolic murmur and third heart sound. On chest x-ray, enlargement of the left atrium was found with no signs of congestion; the electrocardiogram was clearly abnormal with signs of left ventricular hypertrophy, right bundle branch block, left atrial enlargement and abnormal Q waves in the precordial leads. On echocardiography, the left ventricle was hypertrophic, involving mainly the interventricular septum (29 mm), and the left atrium was enlarged (39 mm). There was a typical SAM of the mitral valve. Left ventricular end-diastolic diameter (LVEDD) was 41 mm and ejection fraction (EF) 65%. The right ventricle was normal in size and in the thickness of its wall. The 24 h electrocardiographic monitoring showed no abnormalities. Obstructive HCM was diagnosed and therapy with 120 mg of verapamil was started.
When he was 17 years old, the patient had another syncope on exercise and started to complain of dyspnea. On the chest x-ray, signs of mild congestion and cardiomegaly (cardiac index 16/29) were detected. Only a slight progression of the size of the left atrium and ventricle (left atrium 43 mm, LVEDD 42 mm with EF 55%) was found by ultrasound examination. The outflow gradient due to the SAM was markedly increased. A dual-chamber pacemaker in DDD mode was implanted and atrioventricular delay optimized at 120 ms. The dose of verapamil was increased to 240 mg/day with good clinical effect. On outpatient checkups a progression of the left side chambers was detected; however, the clinical status remained stable. At 21 years of age, the patient developed paroxysmal atrial fibrillation, which was successfully cardioverted, and the patient was put on amiodarone. Echocardiography detected further left atrial dilation (left atrium 51 mm) and marked right ventricular hypertrophy.
At the age of 23, the young man was hospitalized again, this time due to symptoms of congestive heart failure, classified as New York Heart Association functional class (NYHA) III. The chest x-ray revealed cardiomegaly (cardiac index 19/30) and signs of congestion. On echocardiography, further dilation of the left atrium (56 mm) and the left ventricle (LVEDD 55 mm) with severe systolic dysfunction (EF 25%) was found. Hypertrophy of the right ventricle and right atrial dilation were confirmed, and no SAM was found. Treatment was therefore changed from verapamil to angiotensin-converting enzyme inhibitors and diuretics, with a beneficial clinical effect. At present, the patient is complaining of dyspnea classified as NYHA I to II. Echocardiographic findings seem to be stable.
Primers were designed to be complementary to both introns adjacent to exon 13 or exon 14, respectively. One primer from each pair comprised a 45-nucleotide long GC clamp at the 5′ end to ensure appropriate melting behaviour of amplified DNA subjected to DGGE.
For amplification of the exon 13 region, two primers were made: ex13A-GC (5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC GCC CGC GTC ATC CCA CCA TGC CAG TCT CC-3′) and ex13B (5′-TCA TCT CTT TAC CAA CTT TGC TAC TTG CC-3′).
For amplification of the exon 14 region, two other primers were made: ex14A-GC (5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC GCC CGC GCC GGG TCT CTC CTC CAC CTT GCA GG-3′) and ex14B (5′-CCA GGG GTC CCA ACT CAC ATC G-3′).
Genomic DNA was isolated from the peripheral blood according to standard protocols. The amplification reactions were performed using 0.8 U Taq DNA polymerase (MBI Fermentas, Lithuania) in a 40 μl volume according to the manufacturer’s protocol.
The samples were first denatured at 96°C for 3.5 min, followed by 38 thermal cycles, each consisting of heat denaturation at 96°C, annealing at 66°C (for amplification of the exon 13 region), or 64°C (for amplification of the exon 14 region) and extension at 72°C.
To search for point mutations in the amplified exon regions, parallel DGGE was performed. The gels were prepared with a vertical denaturing gradient from 50% to 80% (for the exon 13 region) or from 25% to 80% (for the exon 14 region) from top to bottom. This scale of gradient was first found by perpendicular DGGE. The 80% denaturant contained 7.5% acrylamide, 40% formamide and 7 M urea in Tris-acetate EDTA buffer. The 0% denaturant contained 7.5% acrylamide in Tris-acetate EDTA buffer. For preparation of the gradient gel, 50 ml of each denaturing solution was mixed with 95 μl of 20% ammonium persulphate and 9 μl N,N,N′,N′-tetramethylenediamine and mixed in a gradient chamber (Model 385 Gradient Former, Bio-Rad Laboratories, USA).
Following amplification, PCR products were subjected to heat denaturation and subsequent reannealing. Electrophoresis was performed at 95 W at a strictly constant temperature of 60°C in a custom apparatus based on Protean II xi (Bio-Rad).
Electrophoretic separation was performed on 7.5% poly-acrylamide gels (in a ratio of 37.5 acrylamide to 1 bisacrylamide) for 4.5 h. The gels were stained with ethidium bromide and photographed under ultraviolet transillumination.
Direct sequencing was performed using an automated CEQ2000 Beckman sequencer (Beckman Instruments, USA) and dye-terminator chemistry. Forward and reverse strands of amplified DNA were sequenced.
On parallel DGGE, migration patterns typical of heterozygous mutation (two bands or more) were found in exon 13 in the proband and his father. No mutations were detected in either the proband’s mother or his sister (Figure 1).
On parallel DGGE, a migration pattern typical of heterozygous mutation was found in exon 14 only in the proband. No mutations were detected in the proband’s mother, father or sister (Figure 2).
Automated sequencing found in the proband and in his father a 403Arg→Trp mutation in exon 13, which has already been described (15). However, a novel mutation, 453Arg→His, was also found in exon 14 (Figure 3).
We have described the case of a young man with an unusual clinical history of obstructive HCM, including early manifestation. Furthermore, in the proband we observed right ventricular involvement and a rapid shift from diastolic dysfunction with obstructed left ventricular outflow tract toward systolic dysfunction, with marked congestive failure.
The genetic analysis of this patient revealed two distinct mutations of different types in the MYH7 gene. Although a double heterozygote for mutations in the MYH7 gene has been reported (16), such a case is quite unusual.
Whereas the mutation in exon 13 was inherited from the proband’s father, who carries the same mutation, the mutation in exon 14 was not detected in either his father or his mother. Thus, we conclude that the mutation in exon 14 is the proband’s de novo mutation. However, it remains unclear whether the mutations are in the cis or trans position (in the cis position, both mutations are in the same chromosome; in the trans position, one mutation is located on one chromosome and the other is located on the other chromosome).
The novel 453Arg→ His mutation has been recorded in the Familial Hypertrophic Cardiomyopathy Database (Australian National Genomic Information Service, www.angis.org.au) under code FHC0196. This mutation does not influence the restriction map of the locus; therefore, it is not detectable by restriction analysis, only by sequencing.
This work was supported by grant IGA MZ CR 4966-3. We are also grateful to Shawn McNamara for correction of the manuscript.