Arrhythmogenic inherited heart muscle diseases in children

Journal of Electrocardiology Vol. 34 Supplement 2001

Arrhythmogenic Inherited Heart Muscle Diseases in Children

J. A. Towbin, MD,*†‡ and N. E. Bowles, PhD*

Abstract: The left ventricle (LV) plays a central role in the maintenance of health of children and adults due to its role as the major pump of the heart. In cases of LV dysfunction, a significant percentage of affected individuals develop signs and symptoms of congestive heart failure, leading to the need for therapeutic intervention. Therapy for these patients include anticongestive medications and, in some, placement of devices such as aortic balloon pump or left ventricular assist device, or cardiac transplantation. In the majority of patients the origin is unknown, leading to the term idiopathic dilated cardiomyopathy. During the past decade, the basis of LV dysfunction has begun to unravel. In approximately 30% to 40% of cases, the disorder is inherited; autosomal dominant inheritance is most common (although X-linked, autosomal recessive and mitochondrial inheritance occurs). In the remaining patients, the disorder is presumed to be acquired, with inflammatory heart disease playing an important role. In the case of familial dilated cardiomyopathy, the genetic basis is beginning to unfold. To date, 2 genes for X-linked familial dilated cardiomyopathy (dystrophin, G4.5) have been identified and 4 genes for the autosomal dominant form (actin, desmin, lamin A/C, ␦-sarcoglycan) have been described. In 1 form of inflammatory heart disease, coxsackievirus myocarditis, inflammatory mediators, and dystrophin cleavage play a role in the development of LV dysfunction. This review describes the molecular genetics of LV dysfunction and provide evidence for a “final common pathway” responsible for the phenotype. Key words: Dilated cardiomyopathy, congestive heart failure, arrhythmias.

Congestive heart failure (CHF) caused by myocardial dysfunction is a serious malady, which is a major cause of morbidity and mortality in children

and adults. In these conditions, ventricular arrhythmias commonly occur and in many instances result in sudden death. These disorders are the most common diseases leading to cardiac transplantation, with an associated cost of approximately $200 million/year (1). Cardiomyopathies are classified into 4 forms: a) dilated cardiomyopathy (DCM), b) hypertrophic cardiomyopathy (HCM), c) restrictive cardiomyopathy (RCM), and d) arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). DCM is most common, occurring in 60% of cases (2). The mortality rate in the United States because

From the *Departments of Pediatrics (Cardiology), †Molecular and Human Genetics, and ‡Cardiovascular Sciences, Baylor College of Medicine, Houston, TX. Reprint requests: Jeffrey A. Towbin, MD, Pediatric Cardiology, Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, TX 77030; e-mail: [email protected]. Copyright © 2001 by Churchill Livingstone® 0022-0736/01/340S-0023$35.00/0 doi:10.1054/jelc.2001.28859

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Fig. 1. Echocardiographic evidence of dilated cardiomyopathy Parasternal short axis echocardiogram demonstrating a dilated left ventricle (LV). In real-time, the contractile function is poor.

of cardiomyopathy is greater than 10,000 deaths per annum (3), with DCM being the major contributor to this death rate. DCM is the most common cause of CHF and although the overall incidence varies (4), it is believed that DCM occurs in at least 40 of 100,000 of the population (5–7). The prevalence and incidence of DCM appear to be increasing (7). Depending on the diagnostic criteria used, the annual incidence varies between 5 to 8 cases per 100,000 of the population (3,6 –10); the true incidence is probably underestimated by these figures, because many asymptomatic cases go unrecognized. Idiopathic DCM (IDCM) is characterized by increased ventricular size (ie, left ventricular or biventricular dilation) and reduced ventricular contractility (Fig. 1) in the absence of coronary artery disease, valvular abnormalities or pericardial disease (2). Mitral regurgitation is common, as is ventricular arrhythmias (Fig. 2), particularly ventricular tachycardia (VT), torsade de pointes (TdP),

and ventricular fibrillation (VF). Clinical features include the signs and symptoms of CHF, radiographic evidence of cardiomegaly (with or without pulmonary edema), and reduced cardiac contractility with (or without) ventricular dilation on echocardiography (11,12). Most commonly, IDCM presents between 18 to 50 years of age, but children of all ages and the elderly certainly may be affected. It is more frequently seen in men (2.5:1) and in African-Americans (2.5:1), but the causes of these differences are not well understood (3,13). The clinical course of DCM, almost regardless of origin, may be progressive, with nearly 75% of individuals reported to die within 5 years of diagnosis without transplantation. The cause of death is evenly divided between sudden death and pump failure. Longer survival has been accomplished more recently with improved medical therapies (ie, ACE inhibitors, ␤-blockers) and interventions (ie, implantable defibrillators, ventricular assist devices). DCM was believed to be inherited in a small percentage of cases (14 –16) until Michels et al. (5) showed that approximately 20% of probands had family members with echocardiographic evidence of DCM when family screening was performed. More recently, inherited, familial DCM (FDCM) has been shown to occur in over 30% of cases (17–19). In the remaining cases, a large percentage are acquired, with viral myocarditis playing a significant role (20 –23).

Clinical Genetics of Dilated Cardiomyopathy As noted, over 30% of patients with DCM have a familial form of disease (17–19). Autosomal dominant inheritance is the predominant pattern of transmission, with X-linked, autosomal recessive, and mitochondrial inheritance less common (17– 19,24). Mitochondrial inheritance is seen most commonly in childhood forms of FDCM, whereas X-linked and autosomal recessive forms are probably evenly mixed between childhood and adult forms of disease.

Fig. 2. Ventricular tachycardia in a patient with dilated cardiomyopathy.

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Molecular Genetics of Left Ventricular Dysfunction With Ventricular Arrhythmias Over the past decade, progress has been made in the understanding of the genetic origin of FDCM. Initial progress was made studying families with X-linked forms of DCM, with the autosomal dominant forms of DCM beginning to unravel over the past few years. In the case of X-linked forms of DCM, 2 disorders have been well characterized, X-linked cardiomyopathy (XLCM), which presents in adolescence and young adults, and Barth syndrome, which is most frequently identified in infancy.

X-Linked Cardiomyopathies X-Linked Dilated Cardiomyopathy First described in 1987 by Berko and Swift (25) as DCM occurring in men in the teen years and early 20s with rapid progression to death because of CHF or VT/VF or transplantation, these patients are distinguished by elevated serum creatine kinase muscle isoforms (CK-MM). Female carriers tend to develop mild to moderate DCM in the fifth decade and the disease is slowly progressive. Towbin et al. (26) were the first to identify the disease-causing gene on chromosome Xp21 (Fig. 3) and characterize the functional defect. In this report, the dystrophin gene was shown to be responsible for the clinical abnormalities and protein analysis by immunoblotting showed severe reduction or absence

Fig. 3. X-linked cardiomyopathies. The chromosomal positions of the genes for XLCM (Xp21; dystrophin) and Barth syndrome (Xq28; G4.5) are shown.

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of dystrophin protein in the heart of these patients. These findings were later confirmed by Muntoni et al. (27) when a mutation in the muscle promoter and exon 1 of dystrophin was identified in another family with XLCM. Subsequently, multiple mutations have been identified in dystrophin in patients with XLCM (28 –32). Dystrophin is a cytoskeletal protein which provides structural support to the myocyte by creating a lattice-like network to the sarcolemma (33). In addition, dystrophin plays a major role in linking the sarcomeric contractile apparatus to the sarcolemma and extracellar matrix (34 –37). Furthermore, dystrophin is involved in cell signaling, particularly through its interactions with nitric oxide synthase (38). The dystrophin gene is responsible for Duchenne and Becker muscular dystrophy (DMD/BMD) when mutated as well (39). These skeletal myopathies present early in life (DMD is diagnosed before age 12 years while BMD is seen in teenage men older than 16 years of age) and the vast majority of patients develop DCM before the 25th birthday. In most patients, CK-MM is elevated similar to that seen in XLCM; in addition, manifesting female carriers develop disease late in life, similar to XLCM. Furthermore, Immunohistochemical analysis demonstrates reduced levels (or absence) of dystrophin, similar to that seen in the hearts of patients with XLCM. Murine models of dystrophin deficiency show abnormalities of muscle physiology based on membrane structural support abnormalities. In addition to the dysfunction of dystrophin, mutations in dystrophin secondarily affect proteins that interact with dystrophin. At the amino-terminus (N-terminus), dystrophin binds to the sarcomeric protein actin, a member of the thin filament of the contractile apparatus. At the carboxy-terminus (C-terminus), dystrophin interacts with ␣-dystroglycan, a dystrophin-associated membrane-bound protein which is involved in the function of the dystrophinassociated protein complex (DAPC), which includes ␤-dystroglycan, the sarcoglycan subcomplex (␣, ␤, ␥, ␦, and ␧ sarcoglycan), syntrophins, and dystrobrevins. In turn, this complex interacts with ␣2laminin and the extracellular matrix. Like dystrophin, mutations in these genes lead to muscular dystrophies with or without cardiomyopathy, supporting the contention that this group of proteins are important to the normal function of the myocytes of the heart and skeletal muscles (36,40 – 41). In both cases, mechanical stress (42) appears to play a significant role in the age-onset dependent dysfunction of these muscles. The information gained from the studies on XLCM, DMD, and BMD, led us

154 Journal of Electrocardiology Vol. 34 Supplement 2001 to hypothesize that DCM is a disease of the cytoskeleton/sarcolemma which affects the sarcomere (43).

Barth Syndrome Initially described as X-linked cardioskeletal myopathy with abnormal mitochondria and neutropenia by Neustein et al. (44) and Barth et al. (45), this disorder typically presents in male infants as CHF associated with neutropenia (cyclic) and 3-methylglutaconic aciduria (46). Mitochondrial dysfunction is noted on EM and electron transport chain biochemical analysis. Echocardiographically these infants typically have left ventricular dysfunction with left ventricular dilation, endocardial fibroelastosis, or a dilated hypertrophic left ventricle. In some cases, these infants succumb because of CHF, sudden death, VT/VF, or sepsis caused by leukocyte dysfunction. The majority of these children survive infancy and do well clinically, although DCM usually persists. In some cases, cardiac transplantation has been performed. Histopathologic evaluation typically demonstrates the features of DCM, although endocardial fibroelastosis may be prominent and the mitochondria are abnormal in shape and abundance. The genetic basis of Barth syndrome was first described by Bione et al. (47) who cloned the disease-causing gene, G4.5, found on chromosome Xq28 (Fig. 3). This gene encodes a novel protein called tafazzin, whose function is not currently known. However, mutations in G4.5 result in a wide clinical spectrum (48 – 49), which includes apparent classic DCM, hypertrophic DCM, endocardial fibroelastosis (EFE), or left ventricular noncompaction (LVNC) (50,51). In the latter case, the left ventricular noncompaction is characterized by deep trabeculations giving the appearance of a “spongiform” myocardium (Fig. 4) (51). The mechanisms responsible for this clinical heterogeneity are not currently known. Another gene has recently been identified by our group for autosomal dominant LVNC associated with congenital heart disease. This gene, ␣-dystrobrevin, is another of the DAPC components (52).

Autosomal Dominant Dilated Cardiomyopathy The most common form of inherited DCM, these patients present as classic “pure” DCM or DCM

Fig. 4. Left ventricular noncompaction. Echocardiogram with Doppler tissue imaging demonstrating the highly trabeculated left ventricle with deep recesses in the free wall creating the appearance of holes in the myocardium (arrows) in left ventricular noncompaction or “spongy myocardium”. LV⫽left ventricle.

associated with conduction system disease (CDDC). In the latter case, patients usually present in their twenties with mild conduction system disease, which can progress to complete heart block over decades. DCM usually presents late in the course but is out-of-proportion to the degree of conduction system disease. The echocardiographic and histologic findings in both subgroups are classic for DCM, although the conduction system may be fibrotic in patients with CDDC. In both groups of DCM patients, VT, VF, and TdP occur and may result in sudden death. Genetic heterogeneity exists for autosomal dominant DCM with 7 genetic loci mapped for pure DCM and five loci for CDDC. In the case of pure DCM, the genetic loci identified thus far include 3 mapped by our group (1q32, 5q33, 10q21-23) (53–55), as well as those mapped to chromosome 2q31, 2q35, 9q13-22, 15q14, 1q3, and 14q11 (56 – 60) by others. Five of these genes are now known: actin (chromosome 15q14-linked) (59), desmin (chromosome 2q35) (57), ␦-sarcoglycan (chromosome 5q33) (54), troponin T (chromosome 1q3), and ␤-myosin heavy chain (chromosome 14q11) (60). Cardiac actin is a sarcomeric protein that is a

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member of the sarcomeric thin filament interacting with tropomyosin and the troponin complex. As previously noted, actin plays a significant role in linking the sarcomere to the sarcolemma via its binding to the N-terminus of dystrophin. Interestingly, the mutations in actin which resulted in DCM as described by Olson et al. (59) appear to be directly involved in the binding of dystrophin while mutations in the sarcomeric end of actin result in hypertrophic cardiomyopathy (61). The DCM-causing mutations are believed to result in DCM by causing force transmission abnormalities. Further, actin interacts in the sarcomere with TnT and ␤-MHc, two other genes resulting in either DCM or HCM depending on the position of the mutation (62,63). Desmin is a cytoskeletal protein that forms intermediate filaments specific for muscle. This musclespecific 53 kDa subunit of class III intermediate filaments forms connections between the nuclear and plasma membranes of cardiac, skeletal and smooth muscle. Desmin is found at the Z lines and intercalated disk of muscle and its role in muscle function appears to involve attachment or stabilization of the sarcomere. Mutations in this gene appear to cause abnormalities of force and signal transmission similar to that believed to occur with actin mutations (57). Another recently identified DCM-causing gene was reported by our group (54) to be ␦-sarcoglycan, a member of the sarcoglycan subcomplex of the DAPC. This gene encodes for a protein involved in stabilization of the myocyte sarcolemma as well as signal transduction. Mutations identified in familial and sporadic cases resulted in reduction of the protein within the myocardium. As seen in pure autosomal dominant DCM, genetic heterogeneity also exists for CDDC. To date, CDDC genes have been mapped to chromosomes 1p1-1q1 (64), 2q14-21 (65), 3p25-22 (66), and 6q23 (67). Thus far the only gene identified was recently reported by Fatkin et al. (68) and Brodsky et al. (69) to be lamin A/C on chromosome 1q21 which encodes a nuclear envelope intermediate filament protein. The mechanisms responsible for the development of DCM and conduction system abnormalities is currently unknown. Interestingly, all of the genes identified for inherited DCM are also known to cause skeletal myopathy. In the case of dystrophin, mutations cause DMD and BMD (36), and sarcoglycan mutations cause limb girdle muscular dystrophy (LGMD2F) (33,65,70,71). Lamin A/C has been shown to cause autosomal dominant Emery-Dreifuss muscular dys-

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trophy (EDMD) (72–74), and actin mutations are associated with nemaline myopathy (75). Desmin, G4.5, and ␣-dystrobrevin mutations also have associated skeletal myopathy (76 –79) suggesting that cardiac and skeletal muscle function is interrelated and that possibly the skeletal muscle fatigue seen in patients with DCM with and without CHF may be caused by primary skeletal muscle disease and not related to the cardiac dysfunction. It also suggests that the function of these muscles has a “final common pathway” and that cardiologists and neurologists should consider evaluation of both sets of muscles. Further support for this concept comes from studies of both humans and animal models. Maeda et al. (80) identified absence of the metavinculin transcript in the heart of a patient with idiopathic DCM and confirmed the metavinculin abnormality by immunoblot, which demonstrated the absence of metavinculin protein in the myocardium. Metavinculin plays a role in attaching the sarcomere to the cardiomyocyte membrane by complexing with nonsarcomeric actin microfilaments complexed with other cytoskeletal proteins such as talin, vinculin and ␣-actinin, which are linked to cadherin or to the integrin receptor. Mutations in ␦-sarcoglycan in hamsters result in cardiomyopathy (81– 83), and mutations in all sarcoglycan subcomplex genes in mice cause skeletal and cardiac muscle disease (84 – 86). Mutations in other DAPC genes as well as dystrophin in murine models also consistently demonstrate abnormalities of skeletal and cardiac muscle function. Arber et al. (87) also produced a mouse deficient in muscle LIM protein (MLP), a structural protein that links the actin cytoskeleton to the contractile apparatus. The resultant mice develop severe DCM, CHF and disruption of cardiac myocyte cytoskeletal architecture. Recently, human abnormalities in MLP have been seen in DCM patients as well (88 – 89). Finally, Badorff et al. (90) has shown that the DCM that develops after viral myocarditis has a mechanism similar to the inherited forms. By using coxsackievirus B3 (CVB3) infection of mice, the authors showed that the CVB3 genome encodes for a protease (enteroviral protease 2A), which cleaves dystrophin at the third hinge region of dystrophin, resulting in force transmission abnormalities and DCM. Interestingly, a similar dystrophin mutation which affects the first hinge region of dystrophin in patients with XLCM was previously reported by our laboratory (26), showing a consistent mechanism of DCM development, abnormalities of the cytoskeleton/sarcolemma and sarcomere.

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Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/ARVC) is a primary disorder of the right ventricle in which fibrofatty infiltration of the right ventricular wall occurs, with dilation and systolic dysfunction of the right ventricle resulting (Fig. 5) (91–93). Ventricular arrhythmias commonly occur (usually left bundle branch VT and VF) and sudden death, particularly in athletes, is an important and tragic outcome in many patients (93). Genetics A significant percentage of patients with ARVD/ ARVC have autosomal dominant transmission of disease (94). In one subgroup of patients with ARVD/ARVC associated with wooly hair, palmoplantar keratosis and autosomal recessive inheritance, known as Naxos disease because of its identification in individuals from Naxos, Greece, the disorder is complex (95). ARVD/ARVC is another genetically heterogeneous disorder with genetic loci identified for pure ARVD/ARVC [chromosomes 14q24 (96), 1q42 (97), 14q12-22 (98), 2q35 (99), 10p12-14 (100), 3p23 (101) and one locus for Naxos disease (chromosome 17q21) (95). A gene for Naxos disease has recently been identified, and mutations in plakoglobin, an intermediate filament, desmosomal protein, has been reported (102). Another desmosomal protein, desmoplakin, has also been identified in patients with Naxos-like disease with affected LV

Fig. 5. Arrhythmogenic right ventricular dysplasia (ARVD). Diagnostic angiograms demonstrating right ventricular thinning, outflow tract dilation and aneurysmal pouching.

(103). Although this protein type could be involved in cases of pure ARVD/ARVC, it is not clear whether the pure form of this disease and the complex phenotype of Naxos disease are interchangeable. Another possibility that is to be considered is that ARVD/ARVC is a primary rhythm disorder with secondary cardiomyopathy. To date, all inherited ventricular arrhythmias have been found to occur due to mutations in ion channelencoding genes (104). In long QT syndrome (prolonged QT interval corrected for heart rate, abnormal T waves, VT/TdP), 5 genes have been identified, including two potassium channel ␣-subunits (KVLQT1, HERG), 2 potassium channel ␤-subunits (minK, MiRP1), and the cardiac sodium channel ␣-subunit (SCN5A). Brugada syndrome, a disorder in which ST-segment elevation in V1–V3, right bundle branch block, and VT/VF is classically seen, is also due to ion channel-encoding gene mutations (SCN5A), suggesting that ventricular arrhythmias are ion channelopathies (104 –105). Therefore, the possibility that ARVD/ARVC could be caused by ion channel abnormalities, particularly calcium channel genes, was considered. Recently, Tiso et al. (106) reported mutations in the ryanodine receptor (RyR2), which encodes a protein integral to calcium channel function, as the cause of chromosome 1q42-linked ARVD2, suggesting ARVD to be a primary rhythm disorder. Interestingly, RyR2 was subsequently shown to cause catecholamine-sensitive VT (bi-directional VT), further supporting this concept (107) Corrado et al. (100) has shown that ST-segment elevation in V1–V3 commonly occurs in ARVD, similar to the ECG abnormalities in Brugada syndrome, another primary electrical disorder (108).

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Hypertrophic Cardiomyopathy Hypertrophic cardiomyopathy is a complex cardiac disease with unique pathophysiological characteristics and a great diversity of morphologic, functional, and clinical features (110,111). Although hypertrophic cardiomyopathy has been regarded largely as a relatively uncommon cardiac disease, the prevalence of echcocardiographically defined hypertrophic cardiomyopathy in a large cohort of apparently healthy young adults selected from a community-based general population was reported to be as high as 0.2% (112). Familial disease with autosomal dominant inheritance predominates.

Clinical Aspects of Familial Hypertrophic Cardiomyopathy Observations of myocardial diseases that can reasonably be interpreted as hypertrophic cardiomyopathy were made in the middle of the last century at the Hospital La Salpeˆtrie`re in Parish by A. Vulpian, who called what he saw at the macroscopic level a “re´tre´cissement de l’orifice ventriculoaortique” or “sub-aortic stricture” (113). However, it was only in the 1950s that the unique clinical features of hypertrophic cardiomyopathy were systemically described. It is characterized by left and/or right ventricular hypertrophy, which is usually asymmetric and which can affect different regions of the ventricle. The interventricular septum is most commonly affected, with or without involvement of anterior wall or the posterior wall in continuity (Fig. 6). A particular form of regional involvement affects the apex but spares the upper portion of the septum (apical hypertrophy) (110). Typically, the left ventricular volume is normal or reduced. Systolic gradients are common. Typical morphological changes include myocyte hypertrophy and disarray surrounding the areas of increased loose connective tissue. Patients with HCM frequently report a reduced exercise capacity and functional limitation. Although the pathophysiological features of the disease that contribute to this limitation are complex to increased filling pressures and a failure to augment cardiac output during exercise, leading to exertional symptoms. Arrhythmias and premature sudden deaths are common (111–112).

Fig. 6. Hypertrophic cardiomyopathy. Echocardiographic features of hypertrophic cardiomyopathy with thickened interventricular septum and left ventricular posterior wall. In real-time, hypercontractile function is notable. The left ventricular chamber (LV) is small and the right ventricle (RV) size is also reduced.

Mapping of Familial Hypertorphic Cardiomypathy Genes The first gene for familial hypertrophic cardiomyopathy (FHC) was mapped to chromosome 14q11.2q12 by using genome-wide linkage analysis in a large Canadian family (114). Soon afterwards, FHC locus heterogeneity was reported (115,116) and subsequently confirmed by the mapping of the second FHC locus to chromosome 1q3 and of the third locus to chromosome 15q2 (117,119). Carrier et al. (120) mapped the fourth FHC locus to chromosome 11p11.2. Four other loci were subsequently reported, located on chromosomes 7q3 (122), 3p21.2-3p21.3 (123), 12q23-q24.3, (124) 19p13.2-q13.2, (125) and 15q14 (126). Several other families are not linked to any known FHC loci, indicating the existence of additional FHCcausing genes.

Gene Identification in Familial Hypertrophic Cardiomyopathy All the disease-causing genes code proteins that are part of the sarcomere, which is a complex structure with an exact stoichiometry and multiple

158 Journal of Electrocardiology Vol. 34 Supplement 2001 sites of protein-protein interactions: 3 myofilament proteins, the ␤-myosin heavy chain (␤-MyHC), the ventricular myosin essential light chain 1 (MLC1s/v) and the ventricular myosin reguloatory light chain 2 (MLC-2s/v); 4 thin filament proteins, cardiac actin, cardiac troponin T (cTnT), cardiac troponin I (cTnI), and ␣-tropomyosin (␣-TM); and finally 1 myosin-binding protein, the cardiac myosin binding protein C (cMyBP-C). Each of these proteins is encoded by multigene families, which exhibit tissue specific, developmental, and physiologically regulated patterns of expression (127).

Thick Filament Proteins Myosin subunits. Myosin is the molecular motor that transduces energy from the hydrolysis of ATP into directed movement and that, by doing so, drives sarcomere shortening and muscle contraction. Cardiac myosin consists of two heavy chains (MyHC) and 2 pairs of light chains (MLC), referred to as essential (or alkali) light chains (MLC-1) and regulatory (or phosphorylatable) light chains (MLC-2), respectively (128). The myosin molecule is highly asymmetric, consisting of two globular heads joined to a long rod-like tail. The light chains are arranged in tandem in the head-tail junction. Their function is not fully understood. Neither myosin light chain type is required for the adenosine triphosphatase (ATPase) activity of the myosin head, but they probably modulate it in the presence of actin and contribute power stroke. Mutations have been found in the heavy chains and in the 2 types of ventricular light chains (127). Concerning the heavy chains, the ␤ isoform (␤MyHC) is the major isoform of the human ventricle and of slow-twitch skeletal fibers. It is encoded by MYH7. At least 50 mutations have been found in unrelated families with FHC, and three hot spots for mutations were identified, codons 403, 719, and 741. All but three of these mutations are missense mutations located either in the head or in the head-rod junction of the molecule. The 3 exceptions are two 3 bp deletions that do not disrupt the reading frame, 1 of codon 10 and the other of codon 930, and a 2.4 kb deletion in the 3 region. In the kindred with the latter mutation, only the proband had developed clinically diagnosed hypertrophic cardiomyopathy at a very late age of onset (59 years). As for the light chains, the isoforms expressed in the ventricular myocardium and in the slow-twitch muscles are the so-called ventricular myosin regulatory light chains (MLC-2 s/v) encoded by MYL2, and the ventricular myosin essential light chain

(MLC-1 s/v) encoded by MYL3. They both belong to the superfamily of EF-hand proteins. Two missense mutations have been reported in MYL3, and five in MYL2 (124). Myosin-binding protein C. (MyBP-C) is part of the thick filaments of the sarcomere, being located at the level of the transverse stripes, 43 nm apart, seen by electron microscopy in the sarcomere A band. Its function is uncertain, but, for a decade, evidence has existed to indicate both structural and regulatory roles. Partial extraction of cMyBP-C from rat skinned cardiac myocytes and rabbit skeletal muscle fibers alters Ca2⫹-sensitive tension (129), and it has shown that phosphorylation of cMyBP-C alters myosin cross-bridges in native thick filaments, suggesting that cMyBP-C can modify force production in activated cardiac muscles. The cardiac isoform is encoded by the MYBPC3 gene. We have recently determined its organization and sequence (130). Gautel et al. (131) showed that 3 distinct regions are specific to the cardiac isoform: the NH2-terminal domain C0 Ig-I containing 101 residues, the MyBP-C motif (a 105-residue stretch linking the C1 and C2 Ig-I domains), and a 28residue loop inserted in the C5 Ig-I domain (131). It was recently shown that cMyBP-C is specifically expressed in the heart during human and murine development (132,133). Twenty-seven MYBPC3 mutations have been identified in unrelated families with FHC. Seventeen of them result in aberrant transcripts that are predicted to encode COOH-terminal truncated cardiac MyBP-C polypeptides lacking at least the myosin-binding domain. Seven others result in mutated or deleted proteins without disruption of the reading frame: 5 are missense mutations in exons 6, 17, 21, and 23, 1 is a splice donor site mutation in intron 27, and 1 is a 18-residue duplication in exon 33. Finally, three mutations are predicted to produce either a mutated protein or a truncated one: 2 are missense mutations in exon 15 and 17 and 1 is a branch point mutation in intron 23.

Thin Filament Proteins The thin filament contains actin, the troponin complex and tropomyosin. The troponin complex and tropomyosin constitute the Ca2⫹-sensitive switch that regulates the contraction of cardiac muscle fibers. Mutations were found in ␣-TM and in 2 of the subunits of the troponin complex: cTnI, the inhibitory subunit, and cTnT, the tropomyosinbinding subunit.

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␣-TM is encoded by TPM1 . The cardiac isoform is expressed both in the ventricular myocardium and in fast twitch skeletal muscles (134). It shares the overall structure of other tropomyosins that are rod-like proteins that possess a simple dimeric ␣-coiled-coil structure of other tropomyosins that are rod-like proteins that possess a simple dimeric ␣-coiled-coil structure in parallel orientation along their entire length (134). Four missense mutations were found in unrelated FHC families. Two of them, A63V and K70T, are located in exon 2b within the consensus pattern of sequence repeats of ␣-TM and could alter tropomyosin binding to actin. Mutations D175N and E180G are both located within constitutive exon 5, in a region near the C190 and near the calcium-dependent TnT binding domain. cTnT is encoded by TNNT2. In human cardiac muscle, multiple isoforms of cTnT have been described which are expressed in the fetal, adult and diseased heart, and which result from alternative splicing of the single gene TNNT2 (135,136). The precise physiological relevance of these isoforms is currently poorly understood, but the organization of the human gene has been partially established allowing precise identification the position of the mutations within exons, including those alternatively spliced during development; it also enables an amino acid numbering that reflects the full coding potential of human TNNT2 (137,138). Eleven mutations have been found in unrelated FHC families, 3 of which are located in a hot spot (codon 102). Ten mutations are missense ones located between exons 9 and 17, 1 mutation is a 3 bb deletion located in exon 12 that does not disrupt the coding frame, and the last is located in the intron 16 splice donor site and is predicted to produce a truncated protein in which the C-terminal binding sites are disrupted. cTnI is encoded by TNNI3. The cTnI isoform is expressed only in cardiac muscles (139). Cooperative binding of cTnI to actin-tropomyosin is a unique properly of the cardiac variant (review in ref 140). Six mutations were recently identified. Five are missense mutations located in exons 7 and 8, and 1 is a K183D mutation that does not disrupt the coding frame. ␣-Cardiac actin (ACTC) was recently identified as a cause of FHC. Mogensen et al. (124) studied a family with heterogeneous phenotypes, ranging from asymptomatic with mild hypertrophy to pronounced septal hypertrophy and left ventricular outflow tract obstruction. By using linkage analysis and mutation screening, the gene was mapped to chromosome 15q14 with a lod score of 3.6 and a

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missense mutation (G3 T in position 253 exon 5) resulted in an Ala295Ser amino acid substitution. The mutation is localized at the surface of actin in proximity to a putative myosin-binding site. This mutation, which causes FHC, differs from the two mutations reported to cause dilated cardiomyopathy (DCM) (141). These missense mutations were localized in the immobilized end of actin that crossbinds to the anchor polypeptides in the Z bands.

Genotype-Phenotype Relations in Familial Hypertrophic Cardiomyopathy The pattern and extent of left ventricular hypertrophy in patients with HCM vary greatly even in first-degree relatives and a high incidence of sudden death is reported in selected families. An important issue therefore is to determine whether the genotype heterogeneity observed in FHC accounts for the phenotypic diversity of the disease. However, the results must be seen as preliminary, because the available data relate to only a few hundred individuals, and it is obvious that although a given phenotype may be apparent in a small family, examining large or multiple families with the same mutation is required before drawing unambiguous conclusions. Several concepts nevertheless begin to emerge, at least for mutations in the MYH7, TNNT2, and MYBPC3 genes. For MYH7, it is clear that prognosis for patients with different mutations varies considerably (review in ref 142). For example, the R403Q mutation appears to be associated with markedly reduced survival (143), whereas some others, such as V606M, appear more benign (144). The disease caused by TNNT2 mutations is usually associated with a 20% incidence of non penetrance, a relatively mild and sometimes subclnical hypertrophy, but a high incidence of sudden death which can occur even in the absence of significant clinical left ventricular hypertrophy (120,145,146). In 1 family with a TNNT2 mutation, however, penetrance is complete, echocardiographic data show a wide range of hypertrophy and there was no sudden cardiac death (138). Mutations in MYBPC3 seem to be characterized by specific clinical features with a mild phenotype in young subjects, a delayed age at the onset of symptoms and a favorable prognosis before the age of 40 (147). Genetic studies have also revealed the presence of clinically healthy individuals carrying the mutant allele, which is associated in first-degree relatives with a typical phenotype of the disease. Several

160 Journal of Electrocardiology Vol. 34 Supplement 2001 mechanisms could account for the large variability of the phenotypic expression of the mutations: the role of environmental differences and acquired traits (eg, differences in lifestyle, risk factors, and exercise) and finally the existence of modifier genes and/or polymorphisms that could modulate the phenotypic expression of the disease. The only significant results obtained so far concern the influence of the angiotensin I converting enzyme insertion/deletion (ACE I/D) polymorphism. Association studies showed that, compared to a control population, the D allele is more common in patients with hypertrophic cardiomyopathy and in patients with a high incidence of sudden cardiac death (148,149). It was recently shown that the association between the D allele and hypertrophy is observed in the case of MYH7 R403 codon mutations, but not with MYBPC3 mutation carriers (150) raising the concept of multiple genetic modifiers in FHC.

Final Common Pathway Hypothesis Clearly, FHC of adults is a disease of the sarcomere. Similarly, patients with other cardiac disorders, such as familial dilated cardiomyopathy (FDCM) and familial ventricular arrhythmias (ie, long QT syndromes and Brugada syndrome) have been shown to have mutations in genes encoding a consistent family of proteins. In familial ventricular arrhythmias, ion channel gene mutations (ie, ion channelopathy) have been found in all cases thus far reported. In FDCM, cytoskeletal protein-encoding genes and sarcomeric proteins have been speculated to be causative (ie, cytoskeletal/sarcomyopathy) (37,109). Hence, the final common pathways of these disorders include ion channels and cytoskeletal proteins, similar to the sarcomyopathy in HCM (151). Intermediate disorders, such as ARVD/ ARVC appear to connect the primary electrical and primary muscle disorders mechanistically. Although it is not yet certain what the underlying pathways and targets are for restrictive cardiomyopathy, hints have been forthcoming. Desmin and other intermediate filament proteins appear to be at play in restrictive cardiomyopathy as has been shown in animal models. In addition, it appears that cascade pathways are involved directly in some cases (ie, mitochondrial abnormalities in HCM, DCM) while secondary influences are likely to result in the wide clinical spectrum seen in patients with similar mutations. In HCM, mitochondrial and metabolic influences are probably important. Addi-

tionally, molecular interactions with such molecules as calcineurin, sex hormones, growth factors, amongst others, are probably involved in development of clinical signs, symptoms, and age of presentation. In the future, these factors are expected to be uncovered, allowing for development of new therapeutic strategies. Relevance The relevance of the hypothesis is its ability to classify disease entities on a molecular and mechanistic basis (151). This re-classification of disorders on the basis of molecular abnormalities such as “dystrophinopathies,” “ion channelopathies,” “sarcomyopathies,” or “cytoskeletopathies” could lead to more focused approaches to gene discovery and future therapeutic interventions. For instance, on the basis of the understanding of the molecular aspects of long QT syndrome, we considered the possibility that all ventricular arrhythmias are the result of ion channel abnormalities. On the basis of the hypothesis, we studied the possibility that the cardiac sodium channel gene (SCN5A) was mutated in patients with the idiopathic ventricular fibrillation disorder called Brugada syndrome, identifying mutations in three separate, unrelated families. Use of this hypothesis for disorders such as inherited DCM is likely to more narrowly focus efforts at gene identification. In the near future when the human genome project is completed, this will allow for investigations to more rapidly identify disease responsible genes. Once the genes and the mechanisms causing the clinical phenotype and natural history are known, improved pharmacologic therapies based on the actual disease mechanism can be produced and used. At that time, the impact of molecular genetics on clinical practice and patient care will become fully evident.

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