Molecular Genetics of Left Ventricular Dysfunction

J.A. Towbin*^1,2,3 and N.E. Bowles¹

Departments of Pediatrics (Cardiology),¹ Molecular and Human Genetics,² and Cardiovascular Sciences³, Baylor College of Medicine, Houston, TX USA  

*Address correspondence to this authors at the Pediatric Cardiology, Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, TX 77030 USA; Phone: (713) 798-7342; Fax: (713) 798-8085; Email: jtowbin@bcm.tmc.edu  

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 (CHF), 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 (LVAD), or cardiac transplantation. In the majority of patients the etiology is unknown, leading to the term idiopathic dilated cardiomyopathy (IDC).

During the past decade, the basis of LV dysfunction has begun to unravel. In approximately 30-40% 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 (FDCM), the genetic basis is beginning to unfold. To date, two genes for X-linked FDCM (dystrophin, G4.5) have been identified and four genes for the autosomal dominant form (actin, desmin, lamin A/C, d-sarcoglycan) have been described. In one form of inflammatory heart disease, coxsackievirus myocarditis, inflammatory mediators and dystrophin cleavage play a role in the development of LV dysfunction.

In this review, we will describe the molecular genetics of LV dysfunction and provide evidence for a “final common pathway” responsible for the phenotype.

INTRODUCTION

Congestive heart failure (CHF) due to myocardial dysfunction is a serious malady which is a major cause of morbidity and mortality in children and adults. 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 four forms: (1) dilated (DCM), (2) hypertrophic (HCM), (3) restrictive (RCM), and (4) arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). DCM is most common, occurring in 60% of cases [2]. The mortality rate in the United States due to 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 40/100,000 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 5-8 cases/100,000 population [3,6-10]; the true incidence is probably underestimated by these figures, since many asymptomatic cases go unrecognized.

 Idiopathic DCM (IDCM) is characterized by increased ventricular size (i.e., left ventricular or biventricular dilation) and reduced ventricular contractility in the absence of coronary artery disease, valvular abnormalities or pericardial disease [2]. Mitral regurgitation is common “Figure (1)”, as is ventricular arrhythmias. 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 18-50 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 etiology, may be progressive, with nearly 75% of individuals reported to die within five 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 (i.e., ACE inhibitors, b-blockers) and interventions (i.e., implantable defibrillators, ventricular assist devices).

Dilated cardiomyopathy 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, while X-linked and autosomal recessive forms are probably evenly mixed between childhood and adult forms of disease.

MOLECULAR GENETICS OF LEFT VENTRICULAR DYSFUNCTION

Over the past decade, progress has been made in the understanding of the genetic etiology 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, two 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 (XLCM)

First described in 1987 by Berko and Swift [25] as DCM occurring in males in the teen years and early twenties with rapid progression from CHF to death 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 and colleagues (1993) were the first to identify the disease-causing gene and characterize the functional defect [26]. In this report, the dystrophin gene was shown to be responsible for the clinical abnormalities and protein analysis by immunoblotting demonstrated severe reduction or absence of dystrophin protein in the heart of these patients “Figure (2)”. 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 males older than 16 years of age) and the vast majority of patients develop DCM before the 25^th 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 demonstrate abnormalities of muscle physiology based on membrane structural support abnormalities. In addition to the dysfunction of dystrophin, mutations in dystrophin secondarily affects proteins which 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 b-dystroglycan, a dystrophin-associated membrane-bound protein which is involved in the function of the dystrophin-associated protein complex (DAPC), which includes a-dystroglycan, the sarcoglycan subcomplex (a, b, g, d, and e sarcoglycan), syntrophins, and dystrobrevins “Figure (3)”. In turn, this complex interacts with a2-laminin 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 of 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 to hypothesize that DCM is a disease of the cytoskeleton/sarcolemma [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 due to CHF/sudden death or sepsis due to 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. 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]. In the latter case, the left ventricular noncompaction is characterized by deep trabeculations giving the appearance of a “spongiform” myocardium “Figure (4)” [51]. The mechanisms responsible for this clinical heterogeneity are not currently known.

AUTOSOMAL DOMINANT DILATED CARDIOMYOPATHY

The most common form of inherited DCM, these patients present as classic “pure” DCM or DCM associated with conduction system disease (CDDC). In the latter case, patients usually present in the 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.

Genetic heterogeneity exists for autosomal dominant DCM with seven genetic loci mapped for pure DCM and five loci for CDDC. In the case of pure DCM, the genetic loci identified thus far include three mapped by our group (1q32, 5q33, 10q21-23) [52-54], as well as those mapped to chromosome 2q31, 2q35, 9q13-22, and 15q14 by others [55-58]. Three of these genes are now known: actin (chromosome 15q14-linked) [58], desmin (chromosome 2q35) [56], and d-sarcoglycan (chromosome 5q33) [53].

Cardiac actin is a sarcomeric protein that is a 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 [58] appear to be directly involved in the binding of dystrophin while mutations in the sarcomeric end of actin result in hypertrophic cardiomyopathy [59]. The DCM-causing mutations are believed to result in DCM by causing force transmission abnormalities.

Desmin is a cytoskeletal protein that forms intermediate filaments specific for muscle. This muscle-specific 53kDa 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 [56].

The most recently identified DCM-causing gene was reported by our group [53] to be d-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 [60], 2q14-21[61], 3p25-22 [62], and 6q23 [63]. The only gene thus far identified was recently reported by Fatkin et al [64] and Brodsky et al [65] 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] while sarcoglycan mutations cause limb girdle muscular dystrophy (LGMD2F) [33,61,66-67]. Lamin A/C has been shown to cause autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) [68-70] while actin mutations are associated with nemaline myopathy [71]. Desmin and G4.5 mutations also have associated skeletal myopathy [72-74] 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 due to 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 [75] 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 a–actinin, which are linked to cadherin or to the integrin receptor. Mutations in d-sarcoglycan in hamsters result in cardiomyopathy [76-78] while mutations in all sarcoglycan subcomplex genes in mice cause skeletal and cardiac muscle disease [79-81]. 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 [82] 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 [83-84]. Finally, Badorff et al [85] has shown that the DCM that develops after viral myocarditis has a mechanism similar to the inherited forms. 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], demonstrating a consistent mechanism of DCM development, abnormalities of the cytoskeleton/sarcolemma.

ARRHYTHMOGEHIC RIGHT VENTRICULAR DYSPLASIA/CARDIOMYOPATHY (ARVD/ARVC)

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 [86-88]. Ventricular arrhythmias commonly occur and sudden death, particularly in athletes, is an important and tragic outcome in many patients [88].

Genetics

A significant percentage of patients with ARVD/ARVC have autosomal dominant transmission of disease [89]. 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 [90].

ARVD/ARVC is another genetically heterogeneous disorder with genetic loci identified for pure ARVD/ARVC [chromosomes 14q24 [91], 1q42 [92], 14q12-22 [93], 2q35 [94], 10p12-14 [95], 3p23 [93] and one locus for Naxos disease (chromosome 17q21) [90]. The only gene identified thus far is for Naxos disease, where mutations in plakoglobin, an intermediate filament, desmosomal protein, has been reported [97]. 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 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 channel-encoding genes [98]. In long QT syndrome, five genes have been identified, including two potassium channel a-subunits (KVLQT1, HERG), two potassium channel b-subunits (minK, MiRP1), and the cardiac sodium channel a-subunit (SCN5A), Brugada syndrome, a disorder in which ventricular fibrillation occurs, is also due to ion channel-encoding gene mutations (SCN5A), suggesting that ventricular arrhythmias are ion channelopathies [98-99]. Therefore, it is possible that ARVD/ARVC could be caused by ion channel abnormalities, particularly calcium channel genes.

FINAL COMMON PATHWAY HYPOTHESIS

Clearly, hypertrophic cardiomyopathy (HCM) of adults is a disease of the sarcomere "Figure (5)". Similarly, patients with other cardiac disorders, such as familial dilated cardiomyopathy (FDCM) and familial ventricular arrhythmias (i.e., 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 (i.e., ion channelopathy) have been found in all cases thus far reported. In FDCM, cytoskeletal protein-encoding genes have been speculated to be causative (i.e., cytoskeletal myopathy) [37, 100]. Hence, the final common pathways of these disorders include ion channels and cytoskeletal proteins, similar to the sarcomyopathy in HCM. Although it is not yet certain what the underlying pathways and targets are for restrictive cardiomyopathy (RCM) and ARVD, hints have been forthcoming. Desmin and other intermediate filament proteins appear to be at play in RCM while cell-cell adhesion proteins (i.e., desmosomes, adherens junction proteins) or possibly ion channels are involved in ARVD. In addition, it appears that cascade pathways are involved directly in some cases (i.e., 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 “Figure (5)”. Additionally, 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. 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 are known, 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 utilized. At that time, the impact of molecular genetics on clinical practice and patient care will become fully evident.

REFERENCES

[1]        O’Connell, J.B. and Bristow, M.R. (1994) J. Heart. Lung Transplant., 13, S107-S112.

[2]        Report of the (1995) World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of Cardiomyopathies. Circulation, 93, 841-842, 1996.

[3]        Gillum, R.F. 1986 Am. Heart J., 111, 752-755.

[4]        Abelman, W.H. and Lorrell, B.H. (1989) J. Am. Coll. Cardiol., 13, 1219.

[5]        Michels ,V.V., Moll, P.P., Miller, F.A., Tajik, A.J., Chu, J.S., Driscoll, D.J., Burnett, J.C., Rodeheffer, R.J., Chesebro, J.H. and Tazelaar, H.D. (1992) N. Engl. J. Med., 326, 77-82.

[6]        Codd, M.B., Sugrue, D.D., Gersh, B.J. and Melton, L.J. (1989) Circulation, 80, 564-572.

[7]        Williams, D.G. and Olsen, E.G.J. (1985) Br. Heart J., 54, 153-155.

[8]        Bagger, J.P., Baandrup, U., Rasmussen, J., Moller, M. and Vesterlund, T. (1984) Br. Heart J., 52, 327-331.

[9]        Arola, A., Jokinen, E., Ruuskanen, O., Saraste, M., Pesonen, E., Kuusela, A.-L., Tikanoja, T., Paavilanen, T. and Simell, O. (1997) Am. J. Epidemiol., 146, 385-393.

[10]      Dec, GW. and Fuster, V. (1994) N. Engl. J. Med., 331, 1564-1575.

[11]      Towbin, J.A. (2000 In,  The Molecular and Clinical Genetics of Cardiac Electrophysiological Disease. Berul CI, Towbin JA, Eds. Kluwer Academic Publishers. Chap 13,  pp 195-218.

[12]      Wynn, J. and Braunwald, E. (1987) In,  Heart Disease,  A Textbook of Cardiovascular Medicine. Braunwald E, Ed. W.B. Saunders.

[13]      Coughlin, S.S., Szklo, M., Baughman, K. and Pearson, T.A. (1990) Am. J. Epidemiol., 131, 48-56.

[14]      Michels, V.V., Driscoll, D.J. and Miller, F.A., Jr. (1985) Am. J. Cardiol., 55, 1232-1233.

[15]      Valentine, H.A., Hunt, S.A., Fowler, M.B., Billingham, M.E. and Schroeder, J.S. (1989) Am. J. Cardiol., 63, 959-963.

[16]      Mestroni, L., Miani, D., DiLenarda, A., Silvestri, F., Bussani, R., Filippi, G. and Camerini, F. (1990) Am. J. Cardiol., 65, 1449-143.

[17]      Keeling, P.J. and McKenna, W.J. (1994) Herz, 19, 91-96.

[18]      Baig, M.K., Goldman, J.H., Caforio, A.L.P., Coonar, A.S., Keeling, P.J. and McKenna, W.J. (1998) J. Am. Coll. Cardiol., 31, 195-201.

[19]      Grunig, E., Tasman, J.A., Kucherer, H., Franz, W., Kubler, W. and Katus, H.A. (1998) J. Am. Coll. Cardiol., 31, 186-194.

[20]      Bowles, N.E. and Towbin, J.A. (2000) Curr. Infect. Dis. Rep., 2, 134-140.

[21]      Kasper, E.K., Agema, W.R., Hutchins, G.M., Deckers, J.W., Hare, J.M. and Baughman, K.L. (1994) J. Am. Coll. Cardiol., 23, 586-590.

[22]      Towbin, J.A. (1999) Pediatr. Clin. North Am., 46, 289-312.

[23]      Towbin, J.A., Bowles, K.R. and Bowles, N.E. (1999) Nature Med., 5, 266-267.

[24]      Towbin, J.A. (1993) Biochem. Med. Metab. Biol., 49, 285-320.

[25]      Berko, B.A. and Swift, M. (1987) N. Engl. J. Med., 316, 1186-1191.

[26]      Towbin, J.A., Hejtmancik, J.F., Brink, P., Gelb, B.D., Zhu, X.M., Chamberlain, J.S., McCabe, E.R.B. and Swift, M. (1993) Circulation, 87, 1854-1865.

[27]      Muntoni, F., Cau, M., Ganau, A., Congiu, R., Arvedi, G., Mateddu, A., Marrosu, M.G., Cianchetti, C., Realdi, G. and Cao, A. (1993) N. Engl. J. Med., 329, 921-925.

[28]      Milasin, J., Muntoni, F., Severini, C.M., et al. (1996) Hum. Mol. Genet., 5, 73-79.

[29]      Ortiz-Lopez, R., Li, H., Su, J., Goytia, V. and Towbin, J.A. (1997) Circulation, 95, 2434-2440.

[30]      Ferlini, A., Galie, N., Merlini, L., Sewry, C., Branzi, A. and Muntoni, E. (1998) Am. J. Hum. Genet., 63, 436-460.

[31]      Yoshida, K., Nakamura, A., Yazak, M., et al. (1998) Hum. Molec. Med., 7, 1129-1132.

[32]      Franz, W.-M., Muller, M., Muller, A.J., Herrmann, R., Rothmann, T., Cremer, M., Cohn, R.D., Voit, T. and Katus, H.A. (2000) Lancet, 355, 1781-1785.

[33]      Hoffman, E.P., Brown, R.H. and Kunkel, L.M. (1987) Cell, 51, 919-928.

[34]      Meng, H., Leddy, J.J., Frank, J., Holland, P. and Tuana, B.S. (1996) J. Biol. Chem., 271, 12364-12371.

[35]      Kaprielian, R.R., Stevenson, S., Rothery, S.M., Cullen, M.J. and Severs, N.J. (2000) Circulation, 101, 2586-2594.

[36]      Campbell, K.P. (1995) Cell, 80, 675-679.

[37]      Cox, G.F. and Kunkel, L.M. (1997) Curr. Opin. Cardiol., 12, 329-343.

[38]      Chang, W.J., Iannaccone, S.T., Lau, K.S., Masters, B.S., McCabe, T.J., McMillan, K., Padre, R.C., Spencer, M.J., Tidball, J.G. and Stull, J.T. (1996) Proc. Natl. Acad. Sci. USA, 93, 9142-9147.

[39]      Koenig, M., Hoffman, E.P. and Bertelson, C.J., et al. (1987) Cell, 50, 509-517.

[40]      Klietsch, R., Ervasti, J.M., Arnold, W., Campbell, K.P. and Jorgensen, A.O. (1993) Circ. Res., 72, 349-360.

[41]      Ozawa, E., Yoshida, M., Suzuki, A., Mizuno, Y., Hagiwara, Y. and Noguchi, S. (1995) Hum. Mol. Genet., 4, 1711-1716.

[42]      Petrof, B.J., Shrager, J.B., Stedman, H.H., Kelly, A.M. and Sweeny, H.L. (1993) Proc. Natl. Acad. Sci. USA, 90, 3710-3714.

[43]      Towbin, J.A. (1998) Curr. Opin. Cell Biol., 10, 131-139.

[44]      Neustein, H.D., Lurie, P.R., Dahms, B. and Takahashi, M. (1979) Pediatrics, 64, 24-29.

[45]      Barth, P.G., Scholte, H.R., Berden, J.A., Van der Klei-Van Moorsel, J.M., Luyt-Houwen, I.E., Van 't Veer-Korthof, E.T., Van der Harten, J.J. and Sobotka-Plojhar, M.A. (1983) J. Neurol. Sci., 62, 327-355.

[46]      Kelley, R.I., Cheatham, J.P., Clark, B.J., et al.(1991)  J. Pediatr., 119, 738-747.

[47]      Bione, S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G. and Toniolo, D. (1994) Nature Genet., 8, 323-327.

[48]      D’Adamo, P., Fassone, L., Gedeon, A., Janssen, E.A., Bione, S., Bolhuis, P.A., Barth, P.G., Wilson, M., Haan, E., Orstavik, K.H., Patton, M.A., Green, A.J., Zammarchi, E., Donati, M.A. and Toniolo, D. (1997) Am. J. Hum. Genet., 61, 862-867.

[49]      Johnston, J., Kelley, R.I., Feigenbaum, A., Cox, G.F., Iyer, G.S., Funanage, V.L. and Proujansky, R. (1997) Am. J. Hum. Genet., 61, 1053-1058.

[50]      Bleyl, S.B., Mumford, B.R., Thompson, V., Carey, J.C., Pysher, T.J. and Chin, T.K, Ward, K. (1997) Am. J. Hum. Genet., 61, 868-872.

[51]      Chin, T.K., Perloff, J.K., Williams, R.G., Jue, K. and Mohrmann, R. (1990) Circulation, 82, 507-513.

[52]      Durand, J.B., Bachinski, L.L., Bieling, L., Czernuszewicz, G.Z., Abchee, A.B., Yu, Q.T., Tapscott, T., Hill, R., Ifegwu, J. and Marian, A.J. (1995) Circulation, 92, 3387-3389.

[53]      Tsubata, S., Bowles, K.R., Vatta, M., Zintz, C., Titus, J., Muhonen, L., Bowles, N.E. and Towbin, J.A. (2000) J. Clin. Invest., 106, 655-662.

[54]      Bowles, K.R., Gajarski, R., Porter, P., Goytia, V., Bachinski, L., Roberts, R., Pignatelli, R. and Towbin, J.A. (1996)  J. Clin. Invest., 98, 1355-1360.

[55]      Siu, B.L., Nimura, H., Osborne, J.A., Fatkin, D., MacRae, C., Solomon, S. and Benson, D.W. (1999) Circulation, 99, 1022-1026.

[56]      Li, D., Tapscott, T., Gonzalez, O., et al. (1999) Circulation, 100, 461-464.

[57]      Krajinovic, M., Pinamonti, B., Sinagra, G., Vatta, M., Severini, G.M., Milasin, J., Falaschi, A., Camerini, F., Giacca, M. and Mestroni, L. (1995) Am. J. Hum. Genet., 57, 846-852.

[58]      Olson, T.M., Michels, V.V., Thibodeau, S.N., Tai, Y.S. and Keating, M.T. (1998) Science, 280, 750-752.

[59]      Mogensen, J., Klausen, I.C., Pedersen, A.K., Egeblad, H., Bross, P., Kruse, T.A., Gregersen, N., Hansen, P.S., Baandrup, U. and Borglum, A.D. (1999) J. Clin. Invest., 103, R39-R43.

[60]      Kass, S., MacRae, C., Graber, H.L., Sparks, E.A., McNamara, D., Boudoulas, H., Basson, C.T., Baker, P.B. III; Cody, R.J., Fishman, M.C., et al. (1994) Nat. Genet., 7, 546-551.

[61]      Jung, M., Poepping, I., Perrot, A., Ellmer, A.E., Wienker, T.F., Dietz, R., Reis, A. and Osterziel, K.J. (1999) Am. J. Hum. Genet., 65, 1068-1077.

[62]      Olson, T.M. and Keating, M.T. (1996) J. Clin. Invest., 97, 528-532.

[63]      Messina, D.N., Speer, M.C., Pericak-Vance, M.A. and McNally, E.M. (1997) Am. J. Hum. Genet., 61, 909-917.

[64]      Fatkin, D., Christe, M.E., Aristizabal, O., McConnell, B.K., Srinivasan, S., Schoen, F.J., Seidman, C.E., Turnball, D.H. and Seidman, J.G. (1999) J. Clin. Invest., 103, 147-153.

[65]      Brodsky, G.L., Muntoni, F., Miocic, S., Sinagra, G., Sewry, C. and Mestroni, L. (2000) Circulation, 101, 473-476.

[66]      Jung, D., Duclos, F., Apostal, B., et al. (1996) J. Biol. Chem., 271, 32321-32329.

[67]      Nigro, V., de Sa Moreira, E., Piluso, G., Vainzof, M., Belsito, A., Politano, L., Puca, A.A., Passos-Bueno, M.R. and Zatz, M. (1996) Nat. Genet., 14, 195-198.

[68]      Bonne, G., DiBarletta, M.R., Varnous, S., et al. (1999) Nat. Genet., 21, 285-288.

[69]      Di Barletta, R., Ricci, E., Galluzzi, G., Tonali, P., Mora, M., Morandi, L., Romorini, A., Voit, T., Orstavik, K.H., Merlini, L., Trevisan, C., et al. (2000) Am. J. Hum. Genet., 66, 1407-1412.

[70]      Muchir, A., et al. (2000) Hum. Mol. Genet., 9, 1453-1459.

[71]      Nowak, K.J., Wattanasirichaigoon, D., Goebel, H.H., Wilce, M., Pelin, K., Donner, K., Jacob, R.L., Hubner, C., Oexle, K., Anderson, J.R., et al. (1999) Nat. Genet., 23, 208-212.

[72]      Bione, S., D'Adamo, P., Maestrini, E., Gedeon, A.K., Bolhuis, P.A. and Toniolo, D. (1996) Nat. Genet., 12, 385-389.

[73]      Goldfarb, L.G., Park, K.-Y., Cervenakova, L., Gorokhova, S., Lee, H.-S., Vasconcelos, O., Nagle, J.W., Semino-Mora, C., Sivakumar, K. and Dalakas, MC. (1998) Nat. Genet., 19, 402-403.

[74]      Dalakas, M.C., Park, K.-Y.., Semino-Mora, C., Lee H.S., Sivakumar, K. and Goldfarb L.G. (2000) N. Engl. J. Med., 342, 770-780.

[75]      Maeda, M., Holder, E., Lowes, B., Valent, S. and Bies, R.D. (1997) Circulation, 95, 17-20.

[76]      Nigro, V., Okazaki, Y., Belsito, A., Belsito, A., Piluso, G., Matsuda, Y., Politano, L., Nigro, G., Ventura, C., Abbondanza, C., Molinari, A.M., et al. (1997) Hum. Mol. Genet., 6, 601-607.

[77]      Sakamoto, A., Ono, K., Abe, M., Jasmin, G., Eki, T., Murakami, Y., Masaki, T., Toyo-oka, T. and Hanaoka, F. (1997) Proc. Natl. Acad. Sci. USA, 94, 13873-13878.

[78]      Sakamoto, A, Abe, M. and Masaki, T. (1999) FEBS Lett., 447, 124-128.

[79]      Araishi, K., Sasaoka, T., Imamura, M., Noguchi, S., Hama, H., Wakabayashi, E., Yoshida, M., Hori, T. and Ozawa, E. (1999) Hum. Mol. Genet., 8, 1589-1598.

[80]      Coral-Vazquez, R., Cohn, R.D., Moore, S.A., Hill, J.A.., Weiss, R.M., Davisson, R.L., Straub, V., Barresi, R., Bansal, D., Hrstka, R.F., Williamson, R. and Campbell, K.P. (1999) Cell, 98,  465-474.

[81]      Hack, A.A., Cordier, L., Shoturma, D.I., Lam, M.Y., Sweeney, H.L. and McNally, E.M. (1999) J. Cell, Biol., 142, 1279-1287.

[82]      Arber, S., Hunter, J.J., Ross, J. Jr., Hongo, M., Sansig, G., Borg, J., Perriard, J.C., Chien, K.R. and Caroni, P. (1997) Cell, 88, 393-403.

[83]      Zolk, O., Caroni, P. and Bohm, M. (2000) Circulation, 101, 2674-2677.

[84]      Katz, A.M. (2000) Circulation, 101, 2672-2673.

[85]      Badorff, C., Lee, G.H., Lamphear, B.J., et al. (1999) Nat. Med., 5, 320-326.

[86]      Marcus, F.I., Fontaine, G., Guiraudon, G., Frank, R., Laurenceau, J.L., Malergue, C. and Grosgogeat, Y. (1982) Circulation, 65, 384-398.

[87]      Corrado, D., Basso, C., Thiene, G., McKenna, W.J., Davies, M.J., Fontaliran, F., Nava, A., Silvestri, F., Blomstrom-Lundquist, C., Wlodarska, E.K., Fontaine, G. and Camerini, F. (1997) J. Am. Coll. Cardiol., 30, 1512-1520.

[88]      Thiene, G., Nava, A., Corrado, D., Rossi, L. and Pennelli, N. (1988) N. Engl. J. Med., 318, 129-133.

[89]      Nava, A., Thiene, G., Canciani, B., Scognamiglio, R., Daliento, L., Buja, G.F., Martini, B., Stritoni, P. and Fasoli, G. (1988) J. Am. Coll. Cardiol., 12, 1222-1228.

[90]      Coonar, A.S., Protonotarios, N., Tsatsopoulou, A., Needham, E.W.A., Houlston, R.S., Cliff, S., Otter, M.I., Murday, V.A., Mattu, R.K. and McKenna, W.J. (1998) Circulation, 97, 2049-2058.

[91]      Rampazzo, A., Nava, A., Danieli, G.A., Buja, G.F., Daliento, L., Fasoli, G., Scognamiglio, R., Corrado, D. and Thiene, G. (1994) Hum. Mol. Genet., 3, 959-962.

[92]      Rampazzo, A., Nava, A., Erne, P., Eberhard, M., Vian, E., Slomp, P., Tiso, N., Thiene, G. and Danieli, G.A. (1995) Hum. Mol. Genet., 4, 2151-2154.

[93]      Severini, G.A., Krajinovic, M., Pinamonti, B., Sinagra, G., Fioretti, P., Brunazzi, M.C., Falaschi, A., Camerini, F., Giacca, M. and Mestroni, L. (1996) Genomics, 31, 193-200.

[94]      Rampazzo, A., Nava, A., Miorin, M., Fonderico, P., Pope, B., Tiso, N., Livolsi, B., Lerman, B., Thiene, G. and Danieli, G.A. (1997) Genomics, 45, 259-263.

[95]      Li, D., Ahmad, F., Gardner, M.J., Weilbaecher, D., Hill, R., Karibe, A., Gonzalez, O., Tapscott, T., Sharratt, G.P., Bachinski, L.L. and Roberts, R. (2000) Am. J. Hum. Genet., 66, 148-156.

[96]      Ahmad, F., Li, D., Karibe, A., Gonzalez, O., Tapscott, T., Hill, R., Weilbaecher, D., Blackie, P., Furey, M., Gardner, M., Bachinski, L.L. and Roberts, R. (1998) Circulation, 98, 2791-2795.

[97]      McKoy, G., Protonotarios, N., Cosby, A., Tsatsopoulou, A., Anastasakis, A., Coonar, A., Norman, M., Baboonian, C., Jeffery, S. and McKenna, W.J. (2000) Lancet, 355, 2119-2124.

[98]      Vatta, M., Li, H. and Towbin, J.A. (2000) Curr. Opin. Cardiol., 15, 12-22.

[99]      Towbin, J.A. (2000) J. Cardiovasc. Electrophysiol., 11, 601-602.

[100]    Bowles, N.E., Bowles, K.R. and Towbin, J.A. (2000) Herz, 25, 168-175.