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(Circulation. 1995;92:1336-1347.)
© 1995 American Heart Association, Inc.

Articles

Recent Advances in the Molecular Genetics of Hypertrophic Cardiomyopathy

A.J. Marian, MD; R. Roberts, MD

From the Department of Medicine, Section of Cardiology, Baylor College of Medicine, Houston, Tex.

Correspondence to Robert Roberts, MD, Section of Cardiology, Baylor College of Medicine, 6535 Fannin, MS F905, Houston, TX 77030.

Key Words: genetics • molecular biology • hypertrophy • cardiomyopathy

	Introduction

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
The recent evolution of molecular genetic techniques and their application in deciphering the molecular genetic basis of inherited diseases have facilitated the dawning of molecular medicine. A complete genetic map of the human genome, based on easily identifiable highly polymorphic DNA markers, has been developed.^{1 2 3} This achievement is a milestone in that it provides the foundation for genetic linkage analysis, a technique essential to the mapping of the chromosomal locus responsible for a disease. Before the development of multiple informative markers, identification of a disease-related gene required a priori knowledge of the defective protein, which was known for only a few diseases. The previous approach of "from a defective protein to a defective gene" has been replaced with the approach of "from a defective gene to a defective protein." The recent availability of the highly informative STRP markers compared with previous markers based on RFLP has greatly accelerated chromosomal mapping by linkage analysis.⁴ Furthermore, the RFLP markers detected by Southern blotting required 5 to 7 days, whereas STRP markers are detected by PCR and require as few as 1 to 2 days.^{4 5} The loci for more than 400 disease-related genes have been mapped, and the responsible genes have been identified for more than 40 of these diseases.¹ Theoretically, it is possible to map the chromosomal locus of any disease-related gene if a family with 10 or more living affected individuals spanning two or more generations is available.^{4 5} Subsequent to the chromosomal mapping of the locus by genetic linkage analysis, several techniques, such as positional cloning, are used to identify the responsible gene.^{6 7} Positional cloning refers to cloning of a segment of DNA with only its chromosomal position in relation to a marker known. This process of identifying the genes, which may require years, has been accelerated recently through the development of two techniques: YAC and PFGE.⁸ Before the availability of YAC, one could clone fragments of DNA only as large as 45 000 bp, whereas with YAC, it is possible to clone fragments as large as 1 to 2 million bp. Separation of DNA fragments by agarose gel electrophoresis was limited to those of 10 000 bp, whereas with PFGE, fragments as large as 2 million bp can be separated.

HCM was the first primary cardiomyopathy that was subjected to these techniques. During the short period of 4 years, three genes and a fourth locus responsible for this disease have been identified.^{9 10 11 12} In addition, structure-function analysis has shed significant light on the molecular basis of this disease. It is hoped that within the next few years the application of molecular genetic tools will not only facilitate the ability to diagnose HCM but also help to stratify and develop more definitive therapy.

	Clinical Epidemiology

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
Familial HCM is inherited as an autosomal dominant disease that is characterized by hypertrophy, often of the left ventricle, with predominant involvement of the interventricular septum in the absence of other causes of hypertrophy, such as hypertension or valvular heart disease.¹³ The predominant cardiac pathology is myocyte hypertrophy and sarcomere disarray; the former is found in most cardiac diseases, whereas the latter is the hallmark of HCM.¹⁴ The ventricular systolic function is usually normal or supernormal, with an average left ventricular ejection fraction of 65% to 70%. However, ventricular diastolic function often is impaired in patients with HCM. The clinical manifestations of HCM are diverse, ranging from a benign asymptomatic course to severe heart failure and SCD.¹³ SCD is a well-recognized outcome of HCM. The annual incidence of SCD is higher in younger patients with HCM (6%) than in the elderly (1%).^{15 16 17} In young, apparently healthy athletes with HCM, SCD often is the first manifestation of the disease.^{15 16 17} In a study of 29 highly conditioned young (<35 years old) athletes who died suddenly, HCM was present at autopsy in 48% of the cases, and idiopathic left ventricular hypertrophy (probably HCM) was present in an additional 18% of the cases.¹⁷

The true prevalence of HCM remains unknown. Clinical diagnostic criteria probably underestimate the prevalence of the disease as the phenotypic expression of the disease (ie, development of hypertrophy) is age dependent.¹⁸ The presence of concomitant diseases such as hypertension and valvular heart disease also confounds the diagnosis of HCM.¹⁹ Several investigators have estimated the prevalence of the disease to be approximately 0.1 to 1 per 1000 population.^{20 21 22 23 24} The prevalence of the disease is higher in older individuals and in those with an abnormal ECG.^{23 24} In a study of 3607 men (452 men with ECG abnormalities and 3155 men with no ECG abnormalities), the overall prevalence of HCM was 1.1% in the study group, 0.8% in those with no ECG abnormalities, and 3.6% in those with ECG abnormalities.²³ This probably reflects the fact that HCM is often associated with ECG abnormalities.

	Genetic Basis of HCM

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
The disease was first described in the 19th century,²⁵ but it was not until 1958 that Teare described the familial inheritance of HCM.²⁶ Braunwald et al in 1964²⁷ and Frank et al in 1968²⁸ described several families with HCM, and they further delineated its familial nature. Clark et al²⁹ and van Dorp et al³⁰ performed routine echocardiography on all family members of patients with HCM and showed that the pattern of inheritance of HCM was autosomal dominant with a high but variable degree of penetrance. Echocardiographic screening of the families in their studies showed that cardiac hypertrophy was present in many asymptomatic relatives of the affected individuals.²⁹ The data, indicating that 55% of the cases of HCM are familial and the remainder are sporadic, may have to be reevaluated once genetic screening becomes routinely available.^{31 32} Essentially, HCM is always a genetic disorder. When de novo mutations occur, the disease may not be transmitted to offspring in a small family as each offspring has only a 50% chance of inheriting the mutation. Thus, despite being an underlying genetic cause of HCM, the disease may not exhibit familial inheritance. It is clear that many ß-MHC mutations occur spontaneously and have an independent origin.³³

In 1989, Jarcho et al³⁴ applied linkage analysis to a large French Canadian family and showed linkage of the disease to the chromosomal locus of 14q1. Through similar studies, Hejtmancik et al³⁵ showed that the chromosome 14 locus was linked to HCM in several families from North America. The ß-MHC gene was identified as the responsible gene, and several mutations were shown to co-segregate with inheritance of the disease, suggesting a causal role for ß-MHC in these families as well as confirming an autosomal dominant pattern of inheritance.^{36 37} The first mutation identified was that of a missense mutation in which A substituted for G in exon 13, resulting in a change in the coding of arginine for glutamine.³⁶ Subsequently, at least 36 mutations in the ß-MHC gene have been shown to be responsible for HCM, and these mutations have been reported worldwide in families with HCM^{5 36 37 38 39 40 41} (Table 1).

View this table: [in this window] [in a new window]   Table 1. Mutations in Patients With HCM

Several families, however, have been identified who did not show linkage to chromosome 14q1.^{42 43 44} Two new chromosomal loci responsible for HCM have been mapped^{9 10} to 1q3 and 15q2, and the responsible genes are cardiac troponin T and -tropomyosin,¹² respectively (Table 2). A fourth locus has been mapped, 11q11, but the gene has not been identified.¹¹ Mutations in the cardiac troponin T and the -tropomyosin genes have been identified that co-segregate with inheritance of the disease¹² (Table 1). It appears that the mutations in the ß-MHC gene occur in approximately 20% to 30% of the families with HCM.⁴⁵ However, the true incidence of ß-MHC mutations and of mutations in cardiac troponin T, -tropomyosin, and additional genes remains unknown. Furthermore, linkage to a fragile site on chromosome 16 and linkage to the prealbumin gene on chromosome 18 have been reported without identification of the responsible genes.^{46 47} Thus, HCM exhibits genetic heterogeneity with regard to both the number of responsible genes and the number of mutations. It is highly likely that many genes responsible for HCM remain to be identified.

View this table: [in this window] [in a new window]   Table 2. HCM Genes, mRNA, and Proteins

	ß-MHC Protein and Its Role in the Sarcomere

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
The ß-MHC gene on chromosome 14 is separated from the -MHC gene by only 3.5 kb DNA.⁴⁸ It is composed of 40 exons that span approximately 23 kb of the genome.^{49 50} The mature ß-MHC mRNA is 6008 bp and encodes for a protein with a molecular weight of 220 kD. The ß-MHC is the contractile protein of the heart with enzymatic activity that in the presence of actin hydrolyzes ATP to ADP and P_i. The ß-MHC protein is divided into three functional segments referred to as the globular head, hinge region, and rod segment. The first 23 exons of the ß-MHC gene encode for the globular head and hinge regions of the protein, whereas the remaining exons encode for the rod portion of the ß-MHC protein. The globular head of the myosin molecule contains the major domains of the protein, such as actin and ATP-binding sites. The hinge region flexes during cardiac contraction, contracting from a 90° angle to a 45° angle, which, in turn, pulls the actin filaments toward the center, resulting in shortening (contraction). The rod is essential for tail-to-tail interbinding of the myosin molecules that form the thick filament. The rod, a coiled helix, also coils around the rod of another ß-MHC molecule, with the two heads separated and folded backward onto each other to form a globular shape. Attached to each ß-MHC head are one regulatory and one essential myosin light chain molecule, which together form a hexameric protein. Each thick filament of the sarcomere is formed from more than 400 ß-MHC molecules bound together by their tails (Fig 1).

View larger version (29K): [in this window] [in a new window]   Figure 1. Structure of sarcomeres. The sarcomere is the contractile unit of cardiac muscle, composed of thin and thick filaments encompassed by Z bands. More than 10 different sarcomeric proteins have been identified. Thick filaments are formed by tail-to-tail binding of several hundred myosin molecules, whereas thin filaments are composed of actin, troponin T, I and C complex, and -tropomyosin.

The ß-MHC protein is the major contractile protein of the sarcomere and makes up approximately 30% of the myocardial protein.^{51 52} It is the predominant protein in the cardiac ventricles of large mammals as it is in the adult human heart, comprising more than 95% of the myosin in the human ventricles.^{51 52} The three-dimensional structure of the S1 fragment of the myosin molecule (globular head and myosin light chains) has been determined from atomic resolution after the molecule was crystallized.⁵³ ATP binds to a small groove in the globular head of the myosin molecule, which results in detachment of the myosin molecule from actin.⁵⁴ Enzymatic activity of the myosin ATPase results in hydrolysis of ATP to ADP and P_i, and with removal of the latter end products, flexion of the hinge region of the myosin molecule occurs that displaces the globular head over the actin filaments (contraction and cardiac systole). Subsequently, ATP is again taken up into the groove, and myosin is again released from the actin filaments (Fig 2). New techniques have been developed that can accurately quantify the movement of a single myosin head over an actin filament during contraction.^{54 55 56} These data indicate that each myosin molecule is capable of generating a force of 10 pN, which results in approximately 12-nm displacement of the globular head over the actin filament during each contraction.^{55 56}

View larger version (46K): [in this window] [in a new window]   Figure 2. Diagram illustrating the actin-myosin interaction of cardiac contractility through one cycle of systole and diastole. Globular head of the myosin molecule contains a nucleotide-binding cleft, where an ATP molecule binds to a myosin. The latter results in conformational changes in the ATP binding site, which is transmitted to a stiff "level arm." The level arm of the myosin is composed of a long helix surrounded by regulatory and essential myosin light chains. Enzymatic activity of the myosin hydrolyzes ATP to ADP and Pi, which results in rebounding of the globular head and tight binding to actin filament. Strong binding of the myosin to actin results in reopening of the ATP binding site and release of ADP, which induces conformational changes in the level arm and displaces the globular head over actin filament (systole). A new ATP molecule occupies the now reopened ATP binding site and detaches the myosin molecule from the actin filament (diastole).

	ß-MHC Gene Mutations

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
A large number of mutations in the ß-MHC gene have been identified and shown to co-segregate with inheritance of the disease.^{5 36 37 38 39 40 41} All of the mutations of the ß-MHC gene except one are missense mutations located within the first 23 exons that encode for the globular head of the ß-MHC protein. Although Arg⁴⁰³Gln (G1208A in exon 13) is the most commonly described mutation in families with HCM and the 403 codon is considered a hot spot for mutations,⁴⁰ no single mutation appears to be predominant in HCM. The majority of the ß-MHC mutations involve replacement of guanine or cytosine nucleotides, as expected, and involve an evolutionary conserved amino acid. Only one mutation encoding for the rod region has been described³⁹ ; this was a deletion mutation that deletes part of intron 39, exon 40, and the entire 3' untranslated region.³⁹ In the ß-MHC protein, the deletion mutation results in deletion of the last five amino acids in the C-terminus. The C-terminus of the ß-MHC protein is essential for tail-to-tail interbinding of the myosin molecules to form the thick filament of the sarcomere. Haplotype analysis of families with HCM who have identical mutations has indicated an independent origin of the ß-MHC mutations in these families³³ rather than arising from a single founder. This indicates that the ß-MHC gene is highly vulnerable to mutagenesis, and one would expect to find many new mutations arising sporadically.

	Cardiac Troponin T and -Tropomyosin Genes and Proteins

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
The cardiac troponin T gene, which is located on chromosome 1q3,¹² is composed of 15 exons and transcribes a mature mRNA of approximately 1.2 kb.⁵⁷ The cardiac troponin T gene undergoes multiple alternative splicing, resulting in several isoforms of cardiac troponin T.⁵⁸ Expression of cardiac troponin T is developmentally regulated.⁵⁸ Cardiac troponin T comprises approximately 5% of the total myofibrillar protein and, along with troponin I and troponin C, is involved in Ca²⁺ regulation.⁵⁹ Troponin T binds to tropomyosin and is responsible for positioning of the troponin complex on the thin filaments.

Thus far, seven missense mutations and a deletion mutation in the cardiac troponin T gene have been identified in patients with HCM.^{12 60} The missense mutations are located in exons 8, 9, 11, 14, and 16. The deletion mutation is a 5' splice donor site GA transition in residue 1 of intron 15, which is expected to produce a truncated cardiac troponin T with loss of the terminal 28 amino acid residues. The 5' splice donor mutation is likely to function as a null allele and result in synthesis of a truncated troponin T that degrades rapidly. A decreased quantity of stable cardiac troponin T is likely to alter the stoichiometry of the sarcomeric proteins, which could be responsible for the phenotype of HCM. The truncated cardiac troponin T mutation is the second deletion mutation described in families with HCM.^{12 39} The first deletion mutation described in a family with HCM was a deletion mutation in the 3' region of the ß-MHC gene, which is also postulated to result in expression of a truncated unstable message.³⁹ These mutations in the cardiac troponin T gene appear to be uncommon mutations in families with HCM. In addition, screening for cardiac troponin T gene mutations in families with HCM indicates that such mutations account for HCM in 15% of affected families.⁶⁰

-Tropomyosin protein is a rod-shaped sarcomeric protein that comprises approximately 5% of the total myofibrillar protein.⁵⁹ Each -tropomyosin binds to another molecule in an helix coil, forming a dimer, which is the functional form of the protein. The function of-tropomyosin is to bridge the binding of the troponin protein complex to thin actin filament.⁵⁹ The gene for-tropomyosin is located on chromosome 15q2 and is composed of 15 exons with the corresponding mature mRNA of 1 kb.⁵⁹ Two missense mutations have been described in exon 5 of the -tropomyosin gene in affected individuals from families with HCM. The first mutation is due to substitution of adenine for guanine at position 579, which alters the amino acid sequence from aspartic acid to asparagine at position 175 (Asp¹⁷⁵Asn).¹² The second missense mutation is an AG substitution at position 595, which changes the glutamic acid residue at amino acid 180 to a glycine residue. Fewer than 5% of cases of HCM are caused by mutations in -tropomyosin gene.⁶⁰

	Evidence That ß-MHC Mutations Are Responsible for HCM

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
The evidence that mutations in the ß-MHC gene are responsible for the disease in families with the mutation is conclusive: (1) multiple families have been shown to be genetically linked to the ß-MHC locus on chromosome 14; (2) DNA analysis shows that each affected individual within a single family has the same mutation, which is not present in the unaffected individuals; (3) ß-MHC mutations are absent in the general population; and (4) ß-MHC mutations are expressed in the cardiac mRNA and protein of individuals affected with HCM. Perryman et al⁶¹ showed that the Arg⁴⁰³Gln mutation was expressed in the mRNA extracted from the myocardium of a patient with HCM, and Greve et al⁶² showed expression of Arg⁷⁴¹Lys in the mRNA and protein in the explanted heart of a patient with sporadic HCM. Yu et al⁶³ showed expression of the ß-MHC mutation Arg⁴⁰³Gln in mRNA extracted from skeletal muscle, and Cuda et al^{64 65} isolated several different mutant ß-MHC proteins from skeletal muscle of patients with HCM. (5) De novo mutations occurring in the ß-MHC gene have been shown to result in transmittal of the disease to offspring. Greve et al⁶² showed that the mutation Arg⁷⁴¹Lys, which was proved by haplotyping to occur de novo, was transmitted to two subsequent generations and induced the disease in an autosomal dominant manner. Furthermore, the explanted heart from the proband showed expression of the mutation in the cardiac mRNA and protein. These studies provide conclusive evidence that ß-MHC mutations are responsible for HCM in these families. In addition, a de novo mutation in -tropomyosin that causes HCM has also been reported.⁶⁶ (6) In vitro motility studies (discussed later) show the mutant ß-MHC protein to have impaired contractile function and decreased actin-dependent ATPase activity.⁶⁷ (7) Expression of the mutant human ß-MHC gene with the Arg⁴⁰³Gln mutation in feline adult cardiac myocytes was associated with disarray of the sarcomeres⁶⁸ (discussed later).

	Genotype-Phenotype Correlation in HCM

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
The consistent phenotype of patients with HCM is cardiac hypertrophy, which predominantly affects the interventricular septum. However, the degree of hypertrophy, its distribution, patient age at onset, and type and severity of clinical manifestations vary markedly. The natural course of HCM in certain families is riddled with SCD, whereas in others SCD is almost absent, and the life span is essentially normal.⁶⁹ Identification of the underlying genetic defect provides the opportunity to relate phenotype to specific genotypes. Results of these studies indicate⁷⁰ that in the majority of families described, the ß-MHC mutations Arg⁴⁰³Gln, Arg⁴⁵³Cys, and Arg⁷¹⁹Trp are associated with a poor prognosis and a high incidence of SCD.^{38 71 72 73 74 75} In contrast, the ß-MHC mutation Leu⁹⁰⁸Val is associated with a near-normal life expectancy.⁷⁴ In addition, the mutations Glu⁹³⁰Lys and Arg²⁴⁹Gln are associated with an intermediary risk of SCD.^{38 75}

The phenotypes associated with Arg⁴⁰³Gln have been described in several families with HCM and are characterized by high penetrance, a high incidence of SCD, and severe hypertrophy.^{38 72 73} In the majority of families described, the incidence of premature death in affected individuals with Arg⁴⁰³Gln mutations is approximately 50%.⁷⁵ We identified two families with the Arg⁴⁰³Gln mutation in whom 11 of the 20 affected individuals died prematurely—9 from SCD.⁷⁵ The mean age at the time of SCD was 33 years. There also was an early onset of symptoms, a high penetrance, and a high prevalence of ECG and echocardiographic abnormalities. Watkins et al³⁸ observed a high incidence of SCD in two families with the Arg⁴⁰³Gln mutation in whom 21 of 44 affected individuals died prematurely—9 from SCD, at a mean age of 33±15 years. A third group of investigators reported two families with HCM who had the Arg⁴⁰³Gln mutation.^{73 74} In both families, there was a high penetrance and a high incidence of myocardial ischemia, but the survival rates of affected individuals in these two families were different. In the white family, SCD occurred in 6 of the 15 affected individuals between the ages of 19 and 45 years, resulting in a cumulative SCD rate at 40 years of age of 45%. In contrast, in the Korean family with the Arg⁴⁰³Gln mutation, none of the 6 affected individuals had died. The variability in the incidence of SCD in two families with the identical mutation may in part reflect the different genetic background of these families or the small size of the Korean family, which precludes meaningful assessment of survival rate.

A second ß-MHC mutation associated with a high incidence of SCD is Arg⁷¹⁹Gln, which has been described in four families composed of 61 individuals.⁷⁶ Thirty-five of the 61 affected individuals with Arg⁷¹⁹Gln mutation have died—22 from SCD.⁷¹ The life expectancy of the affected individuals in these four families was 38 years. The Arg⁴⁵³Cys mutation is another malignant mutation that has been described in one family in whom 9 of the 13 affected individuals have died—6 from SCD.³⁸ The mean age at the time of death of the affected individuals was 30±12 years.

The Leu⁹⁰⁸Val mutation has been associated with a low penetrance, a benign course, and a low incidence of SCD.⁷³ The genotype-phenotype correlation was described in a large family with HCM composed of 46 genotype-positive individuals, only 2 of whom have died.⁷³ The cumulative survival rate at 60 years of age was 92%. Similarly, the Gly²⁵⁶Glu mutation has been associated with a benign prognosis in a large family with HCM composed of 39 affected individuals.⁷⁴ The cumulative SCD rate at age 50 in the affected individuals with the Gly²⁵⁶Glu mutation was only 2%. The Val⁶⁰⁶Met mutation has also been associated with a benign prognosis in four families with HCM.^{38 72} Watkins et al³⁸ reported the Val⁶⁰⁶Met mutation in three small families composed of 18 affected individuals, 1 of whom has died. We identified the Val⁶⁰⁶Met mutation in a family with HCM composed of 12 affected individuals, only 1 of whom has died.⁷² However, Fananapazir et al⁷⁴ reported a family with the Val⁶⁰⁶Met mutation in whom there is a severe form of the disease and a high incidence of SCD. These correlations must be interpreted with caution as the number of families studied remains too small for definitive conclusions to be made.

Two mutations have been characterized as being associated with an intermediary prognosis: the Glu⁹³⁰Lys and Arg²⁴⁹Gln mutations.^{38 75} We described a family composed of 16 affected individuals with Glu⁹³⁰Lys mutation 2 of whom have died at ages 14 and 16 and 1 had undergone cardiac transplantation due to progressive heart failure at age 58. The ß-MHC mutation Arg²⁴⁹Gln has been described in a family composed of 26 affected individuals, 10 of whom have died—4 from SCD.³⁸ The average age of the affected individuals at the time of death was 49 years.³⁸

Echocardiography is the most sensitive and specific clinical method of diagnosing HCM. The diagnosis is based on echocardiographic detection of cardiac hypertrophy in the absence of other causes for hypertrophy, such as hypertension or valvular disease.³⁵ However, hypertrophy is not usually evident before puberty and, when the penetrance is low, may not develop until middle age.^{18 20 39} In individuals with other causes for hypertrophy or in the elderly, the echocardiographic detection of hypertrophy is inconclusive, and only a genetic diagnosis is definitive. Mutations with low penetrance will characteristically provide families with many individuals who have the defective genotype but no disease that can be detected with the use of echocardiography. In a study by Solomon et al,³² a considerable overlap was present in the left ventricular wall thickness between genotype-positive individuals and genotype-negative individuals. Thus, in these situations, echocardiography is inadequate for the diagnosis of HCM.³² Individuals with the same HCM genotype have a broad spectrum of echocardiographic findings, but the presence and extent of hypertrophy on the echocardiogram are reflective of clinical severity. Mutations that are associated with high penetrance and poor prognosis are more likely to show a greater degree of left ventricular hypertrophy or septal thickness than are those associated with low penetrance and good prognosis.^{32 72 73 74 75} In our preliminary research, the mean septal thickness as measured with echocardiography for 14 patients with HCM due to the Arg⁴⁰³Gln mutation (associated with a poor prognosis) was 18.1±6.4 mm, whereas in 9 patients with the benign Val⁶⁰⁶Met mutation,⁷² the mean septal thickness was 13.3±3.9 mm (P=.04). Similarly, we found that the left ventricular mass index was greater in patients with the Arg⁷¹⁹Trp mutation than in those with the Val⁶⁰⁶Met mutation (unpublished data). Large-scale analysis is required to verify these preliminary findings and to characterize the diagnostic and prognostic implications of echocardiographic findings in the context of each mutation.

Two additional genes for HCM have been described—cardiac troponin T and -tropomyosin, as well as a fourth locus, to be discussed. The spectrum of clinical manifestations observed in these families is similar to that caused by the ß-MHC gene.^{9 10 11} Genotype-phenotype correlations of cardiac troponin T mutations show the presence of relatively mild and sometimes subclinical hypertrophy but a high incidence of SCD.

	Influence of Environmental Factors and Genetic Background on Phenotypic Expression of HCM Mutations

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
Even within the same family, the phenotypic expression of HCM among affected individuals sharing the same mutation varies markedly, indicating a role for environmental factors and possibly other genetic factors. The most striking example to indicate an environmental effect is the observation that despite ß-MHC being expressed similarly in the right and left ventricles, HCM is primarily a disease of the left ventricle. Hypertrophy presumably occurs in response to the higher systolic pressure in the left ventricle.

There also is evidence suggesting that other genetic factors play a role in the expression of cardiac hypertrophy associated with HCM. ACE genotype DD (rather than ID or II) was found to be more common in patients from families with HCM, showing a malignant phenotype characterized by a high incidence of SCD.⁷⁷ We have shown in a study of 120 patients with HCM that a left ventricular mass index of 100 g/m² (defined as an increased mass index) was six times more likely to occur in patients with the ACE genotype DD than in those with the ACE genotype II.⁷⁸ Furthermore, the ACE genotype DD was associated with more extensive left ventricular hypertrophy characterized by involvement of the entire interventricular septum, apex, and lateral wall.⁷⁸ The genotypes of the ACE gene DD, II, and ID, are independent of the genes responsible for HCM, but in an individual with HCM genotype who also has the ACE genotype DD, hypertrophy is more likely to be manifested and to be more extensive than if the genotype is II. It appears that genotypes such as DD may have a significant role in the degree of penetrance and the variation in phenotype (expressivity) observed in patients with HCM. Hypertrophy requires the coordination of a large number of genes, and thus, it is highly likely that many other genes such as the ACE genotypes influence expressivity of the primary genetic defect. It should not be concluded that the DD allele is necessarily causative of the hypertrophy as it may simply reflect a more important causative gene that is located in close proximity. However, the finding that ACE genotype DD influences the phenotypic expression of HCM, probably through the mitogenic effect of angiotensin II,⁷⁹ raises the intriguing question of the role of ACE inhibitors in HCM. It is now well established that ACE inhibitors induce regression of hypertrophy due to pressure overload, independent of afterload.⁸⁰ It is intriguing whether ACE inhibitors could also induce regression of hypertrophy in patients with HCM. The data are inadequate to recommend such therapy for HCM at this time, and should such therapy be used in the future, it might be contraindicated in patients with outflow tract obstruction. Nevertheless, ACE inhibitors have recently been shown to improve diastolic function in patients with aortic stenosis.⁸¹ However, the knowledge that angiotensin II is a mitogen that induces cardiac hypertrophy and that ACE inhibitors have been shown to induce regression of hypertrophy makes this observation worth pursuing, since ACE inhibitors may be beneficial.

	Structure-Function Analysis of ß-MHC Mutations to Identify the Primary Defect

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
Studies are under way to determine the primary defect initiated by the mutations that lead to the phenotype of HCM. It is most appropriate to concentrate on the myocardial lesion of hypertrophy since the heart is the only organ responsible for the phenotype. Hypertrophy, along with myocyte and sarcomere disarray, is associated with normal systolic but impaired diastolic function. Three of these studies have used a recently developed in vitro motility assay to assess the effect of the mutation on the contractile properties of the ß-MHC protein. In this assay, microscopic beads are coated with actin, and the myosin globular head is fixed to a nitrocellulose membrane. The extent to which myosin moves the beads on binding with actin is detected with a laser beam. The displacement of the bead for each individual myosin molecule averages 10 nm with an average force of 10 pN.^{54 55 56} Cuda et al⁶⁴ isolated the ß-MHC protein from skeletal muscle of patients with HCM and showed that the velocity of contraction was significantly less than that of ß-MHC isolated from skeletal muscle of unaffected individuals. Furthermore, Cuda et al⁶⁵ demonstrated that the degree of impairment of velocity of the contraction varied with the site of mutation. The results of these studies were corroborated by Sweeney et al⁶⁷ with in vitro expression of rat cardiac -myosin that was mutated in a region homologous to that of the human mutation (Arg⁴⁰³Gln) in the ß-MHC gene. A normal and a mutant (Arg⁴⁰³Gln) full-length rat -cardiac myosin were expressed in Sf9 cells, and the heavy meromyosin fragment was isolated for in vitro motility studies. The mutant fragment showed an approximately fivefold reduction in the rate of displacement of actin filaments compared with the normal fragment.⁶⁷ Furthermore, by mixing different ratios of normal and mutant fragments, they showed that the mutant myosin disproportionately reduced the velocity of actin displacement. In these experiments, an increase in the ratio of mutant to normal myosin to 50% reduced the rate of actin filament displacement to 20% of control. The actin-activated ATPase activity of the mutant (Arg⁴⁰³Gln) fragment was also significantly reduced compared with control.

Lankford et al⁸² compared the force-velocity relationship and power output of single slow-twitch muscle fibers isolated from the soleus muscle of patients with three distinct ß-MHC gene mutations. They showed that fibers with Arg⁴⁰³Gln and Gly⁷⁴¹Arg exhibited a significantly reduced maximum velocity of shortening and isometric force generation. In contrast, fibers containing the Gly²⁵⁶Gln mutation displayed essentially normal contractile properties.

Straceski et al⁸³ studied the formation of microfilaments in COS cells after expression of normal and mutant rat -MHC constructs. COS cells normally do not form filamentous structure; however, expression of the normal rat cardiac -myosin in COS cells resulted in formation of structures similar to thick filaments⁸⁴ in 25% of the transfected cells, whereas the mutant -MHC showed filament-like structures in only 2% of the transfected cells.⁸³

To determine whether the sarcomere disarray observed in the myocardium of patients affected with HCM was the primary lesion resulting from ß-MHC mutations, we selected feline cardiac myocytes because they normally form sarcomeres as their contractile unit and have ß-MHC as their adult cardiac myosin, as in humans. Furthermore, HCM is a common disease in cats.⁶⁸ A full-length human ß-MHC cDNA was incorporated into an adenoviral vector with a cytomegalovirus promoter and was expressed in adult feline cardiac myocytes. Electron microscopic examination of myocytes 48 hours after infection showed only minor changes in the structure of sarcomeres in cardiac myocytes. However, 120 hours after infection, approximately 50% of the myocytes infected with the mutant ß-MHC construct showed severe disruption of the sarcomere. In contrast, the structure of the sarcomere remained largely intact in myocytes transfected with normal ß-MHC construct.⁶⁸

These studies have important implications for understanding the primary defect and the pathogenesis of the phenotype. The results of the in vitro studies are very compelling in that the mutant protein is inherently defective as a contractile molecule. This is in accord with our postulated hypothesis that the hypertrophy is compensatory.^{61 63} This suggests that the hypertrophy is similar to that occurring in response to pressure or volume overload or, more appropriately, that of myocardial infarction. The studies in COS cells and, more specifically, in feline adult cardiac myocytes suggest sarcomere disarray to be an early lesion. To further understand and confirm the significance of these studies will require confirmation by other investigators and, more important, documentation in the intact animal, as with overexpression in transgenic animals, or with targeted mutations, such as with replacement of genes by homologous recombination. A major obstacle delaying both studies is the presence of -MHC in the mouse adult heart compared with ß-MHC in human myocardium. Nevertheless, if one assumes the mutant ß-MHC protein exhibits impaired contractility and disrupts formation of the normal sarcomeres, this would lead to increased stress encountered by the normal contractile units and fibers, a stimulus known to induce cardiac hypertrophy.

	Evidence That Hypertrophy Is Compensatory in Familial HCM

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
Hypertrophy is the common response of the heart to pressure overload. The mutant ß-MHC protein, with impaired contractility, may result in increased fiber stress that leads to compensatory hypertrophy. Evidence favoring the hypothesis that the hypertrophy is compensatory⁶¹ in response to the primary defect is as follows. (1) The phenotypes of HCM resulting from several distinct genes and a variety of mutations are similar, suggesting a common mechanism for the hypertrophy. (2) The disease occurs almost exclusively in the left ventricle, despite ß-MHC accounting for 35% of the myosin in the right and left ventricles. This suggests that the hypertrophy is secondary to an environmental stimulus such as the higher pressure and work load of the left ventricle. (3) Skeletal muscle, which also expresses the mutant ß-MHC, appears to have normal function in patients with HCM. Although this may reflect the differences in the quantity of the mutant myosin present in the heart and the skeletal muscles, it may also reflect the different physiological properties of these two organs. (4) The age-dependent manifestation of the cardiac hypertrophy of HCM indicates that the phenotype is secondary to environmental or other genetic factors. (5) The genes upregulated in pressure-overload–induced cardiac hypertrophy are also upregulated in the hypertrophied myocardium of patients with HCM, indicating a common hypertrophic response. Oncogenes, such as c-fos, c-jun, and c-myc, are upregulated, as are ANP and BNP in the hypertrophied myocardium of patients with HCM.^{85 86 87} (6) The phenotypic expression of hypertrophy in patients with HCM is influenced by the underlying genetic background in which mutations occur. Patients with HCM and ACE genotype DD are more likely to develop severe hypertrophy and involvement of the entire interventricular septum, apex, and lateral wall than are patients with ACE genotype II. These data indicate that the mutant protein harbinger of an impaired function triggers a cascade that induces the final phenotype of hypertrophy in patients with HCM.

	Response of the Heart to Injury Is Limited to Hypertrophy, Dilatation, or a Combination

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
It is an important caveat to appreciate that the heart responds to injury, whether it is pressure overload, volume overload, loss of contractile mass (myocardial infarction), or inherited defects, by one of two mechanisms: hypertrophy, dilation (or a combination).⁴¹ This is why familial HCM, being a disease in which the heart responds only through hypertrophy, is important to an ultimate understanding of the hypertrophic response of the myocardium. The reciprocal disease is familial DCM, in which the heart responds predominantly by dilatation.⁸⁸ Many inherited defects induce hypertrophy. Several mutations in the mitochondrial genome have been associated with cardiac hypertrophy.^{89 90} In mitochondrial DNA mutations, HCM is part of a general phenotypic expression of a systemic disease that is characterized by metabolic disorders and usually involves the central nervous and skeletal muscle systems. A characteristic feature of mitochondrial DNA mutation is the maternal inheritance of the disease, due to the inheritance of mitochondrial DNA only from the ovum. A confounding problem is establishing the causal relation of mitochondrial DNA mutations to HCM, due to the presence of enormous polymorphism in the mitochondrial DNA and a gradual increase in mitochondrial DNA mutations over time. Furthermore, the mitochondrial DNA content of different cells varies (heteroplasmy), with some cells showing mutations and others lacking that particular mutation.

No chromosomal locus has been identified for familial DCM⁹⁰ ; however, DCM occurring in association with conduction defects⁹¹ has been mapped to chromosome 1p-1q, but the gene has not yet been identified. Several diseases of muscle, such as Duchenne's muscular dystrophy^{92 93 94} or myotonic dystrophy,^{95 96} that affect the heart secondarily induce DCM, as does the X-linked cardiomyopathy syndrome.⁹⁷

The hypertrophy observed with acquired disorders (eg, from hypertension or from other inherited defects) does not show the myocyte or sarcomere disarray that is the hallmark of HCM previously referred to as IHSS. HCM as defined by the phenotype of myocardial hypertrophy in the absence of an increased load is also seen in overexpression studies in transgenic animals. Overexpression of calmodulin in the transgenic mouse induced a hypertrophic response in the myocardium.⁹⁸ Similarly, overexpression of insulin-like growth factor–1, Ras, and Myf5 genes in transgenic mice also produced a phenotype of hypertrophy.^{99 100} However, no disruption of sarcomeric structure, a characteristic finding in HCM, was reported in these studies. Soon, through overexpression of transgenes or elimination of defective genes through homologous recombination, one can expect to identify the molecular basis for why cardiac hypertrophy occurs in one disease and dilatation occurs in another.

	Perspective on the Pathogenesis of HCM

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
Results from the in vitro studies strongly indicate that the primary defect is inherent in the mutant ß-MHC and is manifested as impaired contractility. Because there are so many different mutations in the ß-MHC gene, it is likely that mechanism(s) by which the mutations lead to impaired contractility will also be multiple. Until such hypotheses are confirmed in vivo, however, it is reasonable to assume that impaired contractility is the primary defect, regardless of the mechanism. How does such a defect lead to the hallmark of this disease as seen in the pathology of the myocardium, ie, hypertrophy, myocyte and sarcomere disarray, increased systolic function, and diastolic dysfunction?

The studies in the COS cells suggest filament assembly is impaired; however, given that sarcomeres do not form in COS cells, even with normal myosin, no implications for sarcomere formation can be deduced. Results of studies in adult feline cardiac myocytes may reflect induced breakdown of the formed sarcomeres, or the breakdown in sarcomere may reflect the normal turnover of myosin, with the defect being the inability to regenerate new sarcomeres. The defective protein may destabilize the sarcomere through either increased turnover or impaired binding of myosin to actin or through impaired interaction with other proteins necessary for structural integrity. What is the stimulus for increased protein synthesis (hypertrophy) by the myocyte? We have shown previously that the ß-MHC protein synthesized by the heart from an individual with HCM appears normal, as is the ratio of ß-MHC to actin or to -MHC.¹⁰¹ In the studies in adult cardiac myocytes, it was also evident that the filament formation was normal. It is reasonable to assume that the increased breakdown of ß-MHC protein provides the stimulus for compensatory hypertrophy. Presumably, the growth stimulus is highly localized and may be modulated by autocrine factor(s) given that hypertrophy is localized, in most patients, primarily to the interventricular septum. Despite the hallmark of extensive sarcomere and myocyte disarray, there is no clue as to why this occurs or why it is primarily localized to the ventricular septum. Similarly, there is no obvious reason why systolic ventricular function is hyperdynamic. Elucidation of the molecular basis for the pathogenesis of this disease must provide a rationale for three puzzling consistent features of the pathology: (1) predominance of hypertrophy in the septum, (2) sarcomere and myocyte disarray, and (3) the supernormal systolic function. The diastolic stiffness or decreased compliance is expected with hypertrophy whether primary or compensatory, but these other features are not typically seen in compensatory hypertrophy such as that observed after pressure or volume overload.

	Significance of Identification and Characterization of HCM Mutations and Their Impact on the Practice of Cardiology

Top Introduction Clinical Epidemiology Genetic Basis of HCM... ß-MHC Protein and Its... ß-MHC Gene... Cardiac Troponin T and... Evidence That ß-MHC... Genotype-Phenotype Correlation... Influence of Environmental... Structure-Function Analysis of... Evidence That Hypertrophy Is... Response of the Heart... Perspective on the Pathogenesis... Significance of Identification... Glimpses Into the Future... References

 
Identification of the mutations responsible for HCM will provide an important diagnostic armamentarium. The present diagnosis of HCM occurs through echocardiographic detection of cardiac hypertrophy. This usually is not evident until puberty or later, whereas a genetic diagnosis is possible before or at any time after birth and requires only a blood sample. A characteristic of HCM is that the development of hypertrophy and its phenotypic expression are age dependent. This also is true for the diagnosis of HCM late in life for mutations that are associated with a low penetrance. Also, diseases such as hypertensive HCM in the elderly are indistinguishable from HCM by clinical criteria.¹⁹ Because HCM is an autosomal dominant disease, only half of the offspring of an affected individual will inherit the disease, and the other half will be normal. Thus, genetic screening through identification of the mutation will identify the individuals at risk of developing the disease before and independent of the presence of symptoms or the development of hypertrophy.

Genetic diagnosis will also provide prognostic information, as shown, particularly for the risk of SCD. This is particularly important as SCD often is the first manifestation of HCM and occurs frequently in apparently healthy young individuals.^{16 17} HCM is the most common cause of death in young competitive athletes.¹⁷ Although the clinical parameters of lack of syncope, lack of inducibility by electrophysiological testing, and lack of ischemia (in children) carry a favorable prognosis, none of the parameters when present alone or in combination have the desirable positive predictability. The need to determine whether an individual is at risk of subsequently developing HCM and possible SCD is greatest in children before the development of cardiac hypertrophy. The ability to identify and separate individuals with the mutation who are at risk of developing the disease from those without the mutation and therefore not at risk of developing the disease provides additional information that will supplement clinical ability to manage these patients. The indication for interventions such as implantable defibrillators should be decided after risk stratification based on many clinical as well as genetic variables that influence the outcome in patients with HCM. Therefore, genetic screening of members of a family affected with HCM will determine who is normal and who is likely to develop the disease as well as provide important prognostic data of particular importance for the children and asymptomatic individuals.

Screening for known mutations in individuals from a family affected with HCM is feasible but tedious, time consuming, and expensive. However, this is only possible for known mutations, whereas in a family in whom the disease is not due to a known mutation, chromosomal mapping and subsequent identification of the gene are required. Our preliminary studies indicate that the ß-MHC mutation is responsible for the occurrence of HCM in fewer than 20% of families with HCM.⁴⁵ It is expected, however, that the techniques for mass genetic screening to identify known mutations will soon be automated. Thus, it is very likely that within the next few years, physicians will be able to routinely screen and identify genotype-positive individuals. Ultimately, if therapy such as gene transfer becomes available, genotyping will, of course, be essential.