Harnessing Molecular Genetics for the Diagnosis and Management of Hypertrophic Cardiomyopathy

Unexplained cardiac hypertrophy, the diagnostic criterion for hypertrophic cardiomyopathy (HCM), occurs in 1 in 500 adults. Insights into the genetic cause and molecular pathophysiology of HCM are reshaping clinical paradigms for diagnosis and treatment of this common myocardial disorder. Human genetic studies have established that dominant mutations in the proteins that comprise the contractile apparatus (the sarcomere) cause HCM. With the current availability of clinical gene-based diagnostics, pathogenic mutations in affected patients can be defined, which can suggest a clinical course and allow definitive preclinical identification of family members at risk for HCM. Genetic discoveries have also fostered mechanistic investigations in model organisms that are engineered to carry human HCM mutations. Novel therapeutic targets have emerged from these fundamental studies and are currently under clinical assessment in humans. The combination of contemporary gene-based diagnosis with new strategies to attenuate disease development and progression is changing the natural history of life-long cardiac symptoms, arrhythmias, and heart failure from HCM.

Hypertrophic cardiomyopathy (HCM) is a heritable disorder characterized by increased heart mass and abnormal diastolic function. The remarkable anatomic features of HCM (Figure 1) were first recognized more than 200 years ago, but the contemporary understanding of this disorder is based on integrated investigations into its histopathology, myocardial hemodynamics, and genetics (1–4).

Figure 1.

Remodeling in hypertrophic cardiomyopathy (HCM). The mass of the normal myocardium (A) is increased in HCM because of ventricular hypertrophy, which can be asymmetric (B), concentric (C), or focal (not shown). Diastolic dysfunction in HCM leads to atrial enlargement and increases risk for atrial clots (C), an important cause of thromboembolic events in HCM. The normal myocyte histology (D) is perturbed in HCM, with marked enlargement and disarray of myocytes (E; hematoxylin and eosin staining). Masson trichrome staining (F) reveals increased amounts of interstitial fibrosis (blue).

Recognition of familial clustering for HCM allowed the application of genetic strategies that ultimately defined its molecular causes. Investigators have identified 13 genes and more than 900 unique mutations (5–7), which makes clinical genetic testing for HCM possible. Molecular diagnosis discriminates between HCM and other hypertrophic heart disorders with distinct clinical courses and thus provides information about expected disease course. Genetic testing also definitively identifies family members who have inherited a mutation and are at high risk for HCM. Finally, knowledge of genetic causes of HCM has fueled mechanistic insights and therapeutic targets. Translation of these into clinical trials is underway. Here, we consider how molecular genetic discoveries provide new opportunities for predictive and personalized medicine in HCM.

HCM Pathology

Left ventricular hypertrophy (8), the anatomic hallmark of HCM (Figure 1, A to C), varies considerably in extent and distribution among affected persons. The disorder usually causes asymmetric hypertrophy that particularly involves the interventricular septum, but hypertrophy can also be limited to the apex or be widespread throughout the ventricle (3, 8). Histopathologic manifestations of HCM include enlarged myocytes with prominent nuclei, disorganized cell-to-cell contacts (myocyte disarray), and increased fibrosis (Figure 1, D to F) that can be interstitial and diffuse as well as focal (4). These anatomic findings contribute to diastolic dysfunction in HCM, which propels atrial remodeling and predisposes affected persons to arrhythmias (9).

Genetics causes of HCM

Most HCM is caused by a dominant gene mutation. One half of all offspring of affected persons will inherit the HCM mutation (Figure 2) and are at very high risk for the disease. Young carriers of the mutation often have no clinical manifestations, and symptoms evolve insidiously as hypertrophic remodeling occurs with aging (3). Because of the age-related penetrance of HCM, absence of disease at 1 evaluation cannot exclude subsequent development. Serial clinical evaluations or genetic testing of family members at risk for HCM are therefore important.

Figure 2.

Pedigree analysis of a family with HCM reveals autosomal dominant inheritance and age-related penetrance. Symbols denote sex (square, male; circle, female) and clinical status (solid, HCM; open, unaffected; slashed, deceased; gray, status unknown). Analyses of the DNA of sarcomere genes (see Table) identified a causal mutation in the β-myosin heavy-chain gene in the proband (arrow), which allowed gene-based diagnosis of all family members. The same mutation was found in adults with clinical evidence of HCM (+) but was absent in unaffected adults (−). Note that 5 children have pre-clinical HCM: They carry the β-myosin heavy-chain mutation but have no evidence of disease.

Mutations in 13 unique genes cause HCM (Table) (5) and account for approximately 75% of familial HCM (10). Mutations predominantly occur in genes that encode proteins within the sarcomere (7), the contractile unit of myocytes. These mutations usually affect thick and thin sarcomere filament proteins (11) and less commonly affect sarcomere-associated or Z-disc proteins that modulate or transmit sarcomere forces.

Table 1.

Genetic Causes of Cardiac Hypertrophy

			Gene	Clinical
Disease	Location	Protein	Symbol	Features
Hypertrophic	Sarcomere:	β-myosin heavy chain	MYH7	Variable hypertrophy
Cardiomyopathy	Thick filament	Myosin-binding protein C	MYBPC3	Prognosis good
		Regulatory myosin light chain	MYL2	Atrial arrhythmias
		Essential myosin light chain	MYL3	↑ with disease duration
		Titin	TTN	Ventricular arrhythmias
	Thin filament	Troponin T	TNNT2	uncommon
		Troponin I	TNNT3	Heart failure uncommon
		α–Tropomyosin	TPM1
		Actin	ACTC1
	Z-disc	Muscle LIM protein	CSRP3	Variable Hypertrophy
				+ Skeletal myopathy
		Telethonin	TCAP	Variable hypertrophy
		Myozenin 2	MYOZ2	Variable hypertrophy
	Intercalated disc	Vinculin	VCL	Variable hypertrophy
Metabolic	Cytosol	Adenosine mono-phosphate-	PRKAG2	Variable hypertrophy
Cardiomyopathy		activated protein kinase, γ2		Atrial arrhythmias; Progressive
		subunit		Conduction System Disease
	Lysosome	Lysosome-associated	LAMP2	Massive hypertrophy
		membrane protein-2		Ventricular arrhythmias
				common
				Heart failure common
	Lysosome	α-Galactosidase A	GLA	Variable hypertrophy
				Renal disease common

Most HCM mutations are “private,” or unique to a patient and his or her family members. Unrelated HCM families usually have different mutations (12), although a few are shared among specific populations. For example, 4% of persons with South Asian ancestry carry a common HCM mutation (13). However, defining the causal mutation in any given patient usually requires detailed sequencing of all HCM genes (14). This daunting technical challenge is met by newly developed DNA sequencing strategies, and genetic diagnosis of HCM is now clinically available at several certified molecular diagnostic laboratories, which are listed at the National Center for Biotechnology Information GeneTests website, www.genetests.org. Defining the pathogenic mutation in an affected family member allows subsequent definition and low-cost assessment of the mutation status in all relatives. Carriers of the mutation are at high risk for HCM (Figure 2), whereas mutation-negative persons are at no risk for the disease and do not need serial clinical evaluations.

Linking Genotype with Clinical Manifestations in HCM

The age at which signs and symptoms of HCM present is influenced by the causal gene mutation (5, 7). Clinical manifestations of HCM caused by β-myosin heavy chain or troponin T mutations typically begin near adolescence (12, 15). In contrast, myosin-binding protein C mutations, particularly those that foreshorten the protein, trigger HCM after a protracted period of clinical quiescence that may extend into middle age (16, 17).

Different genetic causes of HCM do not correlate with the extent or distribution of hypertrophy, with a few notable exceptions. Troponin T mutations generally cause substantially less hypertrophy than other HCM genes, and gene-based diagnosis is particularly helpful in determining the status of persons at risk for inheriting these mutations (15). The different morphologic patterns of hypertrophy in HCM (asymmetric, concentric, or apical) do not relate to underlying genotype, with the exception of a unique actin mutation that uniformly produces apical hypertrophy (18). The factors that account for morphologic patterning remain unknown.

The natural history of HCM includes progressive dyspnea and angina. These symptoms reflect a poorly compliant myocardium, increased ventricular diastolic pressure, and impaired diastolic filling (3, 8, 19, 20). Myocardial ischemia occurs in HCM despite normal coronary artery anatomy, due in part to intramural vascular remodeling and impaired myocardial relaxation that impedes diastolic blood flow (21). Approximately 5% of patients with HCM develop profound diastolic dysfunction that is ultimately accompanied by diminished systolic performance (22).

Patients with a defined sarcomere protein gene mutation have substantially worse cardiac outcomes than those in whom the cause of HCM remains unknown (23). In addition, particular HCM mutations (24), compound mutations (25), and a mutation that is prevalent among patients with Indian ancestry (13) have been shown to substantially increase the propensity for heart failure.

Arrhythmias in HCM

Cardiac arrhythmias are a common and potentially life-threatening complication of HCM. Supraventricular arrhythmias markedly increase with disease duration, and 25% of patients ultimately develop atrial fibrillation, which contributes to both morbidity and premature death due to thromboembolic events (3, 9). Ventricular arrhythmias can cause sudden death in HCM (26, 27), and the efficacy of implanted cardioverter defibrillators in terminating these life-threatening arrhythmias (28) makes proactive identification of patients with HCM who are at high risk for sudden death an important but difficult clinical issue. Traditional clinical parameters that define patients at increased risk for sudden death include syncope, substantial left ventricular outflow tract gradient (≥50 mm Hg), massive left ventricular hypertrophy (wall thickness ≥30 mm), exercise-induced hypotension, and sudden deaths in first-degree relatives (20). This last parameter is noteworthy, because all affected members of a family share an identical genetic mutation that causes HCM. The predictive value of sudden death in relatives implies that some HCM mutations convey greater risk for these events. This hypothesis is being studied by the compilation of sudden death events that occur in patients with different HCM mutations (24, 29–31) and by analysis of models of HCM.

Mechanistic Insights from Models of HCM

Knowledge of the genetic causes of HCM has allowed the development of models that carry human mutations (32–36). Studies of these models demonstrate that HCM mutations produce stable proteins that are incorporated into the sarcomere along with the normal protein derived from the nonmutated allele. The biophysical properties of sarcomeres that contain mutant β-myosin heavy chains indicate that HCM mutations enhance contractile properties. Myosin mutations increase force generation, adenosine triphosphate (ATP) hydrolysis, and actin–myosin sliding velocity (37, 38). Sarcomeres that contain mutant troponin T proteins, such as HCM myosin mutations, also increase these properties (39, 40). Together, these data indicate that the hypertrophy that characterizes HCM is not a compensatory response to diminished contractile function. However, models of HCM also show delayed myocardial relaxation that occurs before the onset of hypertrophy (41). Diastolic dysfunction therefore seems to be a direct consequence of HCM mutations.

Hearts from models of HCM progressively accumulate myocardial fibrosis in the same manner as human patients (32–36). These models have proven useful for determining whether reducing myocardial fibrosis improves the intrinsic relaxation deficits produced by HCM mutations. In one study (42), an angiotensin II type 1 receptor inhibitor (candesartan) significantly reduced myocardial fibrosis, collagen deposition, and hypertrophy in mice with HCM compared with placebo (P < 0.001). A second protocol examined N-acetylcysteine (mycomyst), which protects against oxidative stress, and demonstrated reduced HCM pathology (43). Assessment of these therapeutic strategies in human HCM is underway.

Altered Calcium Homeostasis in HCM

A critical mechanistic question in HCM is why mutations that increase biophysical properties of sarcomere proteins cause hypertrophy. Although we do not completely understand the process, biochemical studies hint that calcium signaling is a critical trigger of hypertrophy. Studies of mice that carry a human mutation in myosin heavy chains (44), troponin T (45), or regulatory myosin light chains (46) revealed abnormal calcium cycling in myocytes before overt histopathologic changes occurred in the myocardium. Abnormal calcium cycling is also a prominent feature of other cardiomyopathies (47, 48).

Calcium cycling (Figure 3) is essential for cardiac function. During depolarization, calcium enters myocytes through the voltage-gated L-type Ca²⁺ channels on the cell membrane and triggers cardiac contraction. In response, the sarcoplasmic reticulum releases calcium through the cardiac ryanodine receptor (RyR2), a process termed calcium-induced calcium release. Increased cytosolic levels produce a shift in calcium from troponin I to troponin C, which releases the inhibition of actin–myosin ATPase activity. Hydrolysis of adenosine triphosphate (ATP) supplies energy that enables myosin to slide along actin and transmit force to the sarcolemma and extracellular matrix. With completion of contraction, calcium recycles back to the sarcoplasmic reticulum by means of an ATPase-dependent pump and is also extruded outside of myocytes by means of the Na⁺/Ca²⁺ exchanger.

Figure 3.

HCM remodeling pathways. Ca²⁺ enters myocyte through the voltage-gated L-type Ca²⁺ channel and triggers Ca²⁺-induced Ca²⁺ release (CICR). Ca²⁺ is released from the sarcoplasmic reticulum (SR) through the RyR2 complexes that are composed of cardiac ryanodine receptor, calsequestrin, junctin, triadin, and sorcin. When one contractile protein in the sarcomere carries an HCM mutation (red star), sarcomere contractility increases and relaxation diminishes. This results in abnormal Ca²⁺ cycling with slower Ca²⁺ reuptake of the SR by the Sarco/Endoplasmic Reticulum Ca²⁺-ATPase (SERCA) pump and reduced Ca²⁺ content in the SR (blue arrow). Abnormal Ca²⁺ cytosolic concentration and mechanical dynamics of the mutant sarcomere stimulate signaling pathways in the nucleus that promote myocyte growth, premature myocyte death, and increased myocardial fibrosis. In model organisms that carry a human HCM mutation, early normalization of Ca²⁺ handling during the preclinical phase of disease attenuated the subsequent development of hypertrophy and fibrosis.

Myocytes from the hearts of patients with HCM have lower levels of calcium in the sarcoplasmic reticulum (Figure 3) and altered levels or activities of key calcium-binding proteins, including ryanodine receptors, calsequestrin, triadin, junctin, cardiac sarcoplasmic reticular Ca²⁺–ATPase, and phospholamban (44–46). These biochemical changes may promote diastolic dysfunction in HCM. In addition, abnormal calcium cycling increases the probability for after-depolarization in myocytes, which is a cellular substrate for cardiac arrhythmias (49).

Because calcium abnormalities predate hypertrophic remodeling in HCM models, research has addressed whether early intervention with pharmacologic agents that normalized calcium levels might reduce the emergence of hypertrophic remodeling. To test this hypothesis, mice engineered to carry a human HCM mutation in myosin were treated with diltiazem, an L-type calcium channel blocker. With early administration of the drug, levels of calcium-binding proteins and sarcoplasmic reticulum calcium content were normalized. Continued treatment significantly attenuated the emergence of hypertrophy and development of hemodynamic abnormalities (P < 0.001) (50). These preclinical studies raise the potential for identifying persons at risk for HCM through gene-based diagnosis, and early pharmacologic interventions may delay or attenuate development of disease.

Arrhythmias have also been studied in mice with HCM. Electrophysiologic tests of mice with mutations associated with high risk in humans show more ventricular arrhythmias than those in mice with low-risk HCM mutations (30, 34), which implies that specific HCM mutations may increase sudden death risk. Other components of HCM pathophysiology that contribute to life-threatening arrhythmias remain incompletely understood. Because postmortem examination of victims of sudden death sometimes reveals striking amounts of myocardial fibrosis (51, 52), fibrosis has been proposed as a contributor to arrhythmias (53, 54). Although rigorous analysis of this hypothesis is difficult in human patients, the relevance of fibrosis, disarray, and cardiac hypertrophy to arrhythmia susceptibility has been assessed in mice that carry human HCM mutations. Only the amount of hypertrophy, not the fibrotic load or myocyte disarray, was found to correlate with inducible ventricular arrhythmia (55). This both confirms the observation that marked hypertrophy increases risk for sudden death in HCM and implies that strategies that limit hypertrophy may also reduce the risk for sudden death.

Established and Emerging Medical Strategies for HCM

Clinical management of HCM is focused on relief of symptoms. β-adrenergic inhibitors and L-type calcium channel blockers lower heart rate, prolong diastolic filling, diminish left ventricular outflow tract gradients, and improve HCM symptoms (3, 8, 56). In patients with substantial outflow tract obstruction, surgical septal myectomy (57–59) or percutaneous alcohol ablation (60–62) relieve symptoms and may also reduce sudden death risk. Although effective, these traditional management strategies apply to clinically overt HCM. Newer paradigms have emerged on the basis of pathophysiologic studies of models of HCM and the opportunity for disease prevention in persons with inherited HCM mutations.

Because angiotensin blockade decreased interstitial fibrosis and collagen deposition in mice with HCM, clinical studies have assessed angiotensin-1 receptor antagonists in nonobstructive HCM in humans (63, 64). Reductions in hypertrophy that range from 6% to 15% have been reported, with the greatest benefit occurring in patients with myosin mutations (64). If replicated, this result may indicate that knowledge of the genetic cause of HCM may help direct treatment strategies.

Other novel strategies aim to prevent development of HCM and are predicated upon gene-based diagnosis. Within families, genetic testing typically defines mutation carriers with established HCM and other, usually younger, asymptomatic mutation carriers without hypertrophy (Figure 2). Detailed clinical investigation of preclinical carriers with different mutations (65) indicate electrocardiographic abnormalities (QRS widening and T-wave inversion), as well as substantially lower diastolic relaxation properties (assessed by Doppler-tissue-imaging) (P < 0.001) and substantially higher ejection fractions (P < 0.001) than are seen in age-matched relatives without mutations. These observations indicate that hypertrophy is not the earliest manifestation of HCM.

The identification of prehypertrophic manifestations of HCM mutations raises the question of whether early disruption of the signals triggered by these mutations might attenuate ventricular remodeling and reduce symptoms. Clinical studies aim to answer this by interventions that target sarcomere mutation carriers without hypertrophy. The first predictive human genetics and prevention trial in HCM, “Treatment of Preclinical Hypertrophic Cardiomyopathy With Diltiazem” (http://clinicaltrials.gov/ct2/show/record/NCT00319982), illustrates this strategy. This single-center trial, which is based on studies of models of HCM in which early restoration of calcium homeostasis limited disease development, will use diltiazem to normalize calcium cycling. A pilot cohort (50 prehypertrophic patients with any sarcomere mutation identified by gene-based diagnostics) treated with diltiazem or placebo will be analyzed for signs of the emergence of HCM (electrocardiographic abnormalities, diastolic dysfunction, and hypertrophy). Results are expected in 2013.

With the ongoing efforts to discover signaling molecules that are activated by sarcomere gene mutations in model organisms, other targets for new HCM therapies should be anticipated. Combined with gene-based diagnosis of HCM for patients at risk, these fundamental mechanistic insights may someday allow strategies to prevent the development of this disease.

Additional Genetic Causes of Cardiac Hypertrophy

Although they are less common than sarcomere protein gene mutations, other human gene mutations can cause hypertrophy that mimics HCM (Table). Recognition of these is important because of their distinct manifestations and outcomes that affect clinical care. For example, mutations in PRKAG2, which encodes the γ2 subunit of adenosine monophosphate–dependent protein kinase (66–69), produce hypertrophy and progressive electrophysiologic abnormalities (ventricular pre-excitation followed by conduction system disease) that necessitate pacemaker implantation. Mutations in the X-linked gene LAMP2 (66), which encodes lysosome-associated membrane protein-2, cause massive hypertrophy with malignant ventricular arrhythmias and rapidly progressive heart failure, primarily in young males (70). Genetic testing distinguishes between HCM and these other conditions, allows interventions to limit adverse events, and enables family counseling.

Final Perspectives

Mutations in sarcomere genes are common causes of HCM. Discovering these molecular causes provides the basis for clinical genetic diagnostics, information that definitely establishes the risk for HCM in family members. For the subset of HCM mutations with defined clinical manifestations, genetic information can assist in management strategies. Through analyses of fundamental pathogenic mechanisms in HCM models engineered to carry human mutations, new approaches to prevent development of HCM have emerged. Translation of research into clinical trials in humans is underway. The integration of genetics to predict HCM and preemptive strategies to prevent HCM represents a new paradigm for personalized genetic medicine.