Hypertrophic cardiomyopathy: from molecular and genetic mechanisms to clinical management

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1 European Heart Journal Supplements (2001) 3 (Supplement L), L43 L50 Hypertrophic cardiomyopathy: from molecular and genetic mechanisms to clinical management Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, U.K. Molecular genetic research in hypertrophic cardiomyopathy (HCM) has shown that this heart muscle disorder, which was previously considered idiopathic, is caused by a wide diversity of mutations that affect the cardiac contractile proteins. With this information, it is now possible to explore molecular genetic diagnosis, recalibration of clinical diagnostic tools and criteria, and genotype phenotype correlations. However, the biggest potential benefit is that a detailed understanding of the disease pathway may lead to disease-modifying treatments. Demonstration of the mutations in cardiac contractile protein genes has focused attention on alterations in contractility. However, no unifying abnormality of contractility is apparent; rather, the defects point to an inefficiency of ATP usage in the sarcomere. The very recent finding of HCM-causing mutations in a regulatory subunit of AMP-activated protein kinase strongly supports the hypothesis that the unifying abnormality in this condition is an inability to maintain normal ATP availability in the myocardium during times of stress. This conclusion should ultimately lead to new approaches to therapy and to further consideration of the role of altered myocardial energetics in other forms of heart muscle disease. (Eur Heart J Supplements 2001; 3 (Suppl L): L43 L50) 2001 The European Society of Cardiology Key Words: AMP kinase, cardiomyopathy, contractile proteins, energetics, genes Introduction Familial hypertrophic cardiomyopathy (HCM) has been the subject of intense molecular genetic research over the past decade. In the first instance, this condition was chosen for study because it appeared likely to be genetically tractable, because it was known to be a singlegene disorder. Thereafter, research efforts have, if anything, increased because new molecular insights have raised many new questions and created opportunities to evaluate the application of molecular genetics in cardiology. HCM was the first primary idiopathic disorder affecting the heart to be understood at a molecular level, and so has been something of a test case, illustrating the strengths and weaknesses of genotype phenotype predictions in cardiac disease. In brief, nine disease genes have been identified that encode sarcomeric proteins [1 8]. This has led to the suggestion that, in the broadest terms, HCM is a disease of the sarcomere [2]. HCM-causing mutations have been found in genes that encode components of the thick filament (e.g. myosin heavy chain and light chains, myosin binding protein C) and the thin filament (e.g. cardiac troponin T [TnT] and troponin I, tropomyosin, actin). Studies of the Correspondence: Hugh Watkins, Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DV, U.K X/01/0L $35.00/0 expression of the mutant gene products, which are principally caused by single amino acid substitutions encoded by missense mutations, have implicated the incorporation of mutant protein within the sarcomere as the cause of disease [9 11]. There are, in general, a number of anticipated benefits that might arise from a molecular understanding of a primarily inherited disorder (Fig. 1). Most straightforward, the molecular information can be expected to improve management of the condition itself; this is therefore important for the more common inherited disorders such as HCM [12]. Second, even relatively rare genetic experiments of nature can provide new biological insights of much wider relevance by pointing out new disease pathways. Third, geneticists working with single-gene disorders have been learning how to handle genetic information clinically. This often proves more complex than might be expected, and is an important reminder that the predictive nature of susceptibility genes in complex traits may be limited. In reference to the disease itself, there are also a number of different avenues through which benefit may be anticipated following the identification of the underlying disease genes. These include molecular genetic diagnosis and molecular genetic classification, whereby genotype phenotype correlations may be used to predict natural history or response to treatment. Potentially most important is the hope that new disease genes and pathways will be implicated that will yield ultimately a new rational approach to disease-modifying therapy The European Society of Cardiology

2 L44 About disease in question Molecular diagnosis Genetic classification, predict: natural history response to treatment Learn New biological insight Fundamental mechanisms Disease modifying treatment Relevance to acquired diseases Use of genetic information Practical issues Ethical issues Figure 1 A summary of the rationale behind molecular genetic analysis of inherited disorders as models of a wider disease spectrum. In order to address the benefits of molecular progress in HCM, each of these areas is reviewed to assess their current impact and future potential. Molecular genetic diagnosis Re-evaluation of existing clinical tools and criteria It is important to emphasize that the information that arises from the relatively modest number of genotyped families studied for the purposes of clinical and molecular genetic research has already dramatically altered clinical perceptions and practice. Key observations that were not apparent until mutation-carrying individuals within families could be studied include the following. First, the disease is nearly always familial, and de novo mutations that cause sporadic disease account for a minority of cases. Even if an individual does have sporadic disease due to a de novo mutation, then the mutation is carried in that individual s germ line and their offspring have the usual one in two risk for inheriting the genetic defect. Second, penetrance is incomplete, even in adults, and this varies by mutation type [3,13]. Thus, the reason why clinical studies suggested that only 50 60% of individuals had familial disease was that the affected parent often had only minor features that were insufficient to make the diagnosis on the basis of conventional criteria. Finally, the age of onset of detectable clinical abnormalities is broader than had been suspected. It remains the case that hypertrophy is apparent in childhood in only a minority, and in most patients it typically becomes apparent during adolescence; in other families, however, detectable ECG and echocardiographic abnormalities can arise in mid or late adult life. Most typically this is a feature of HCM that is attributable to mutations in the gene that encodes myosin binding protein C [14], but this has also been described with other disease genes. Notably, phenotypic conversions can occur over a relatively small period of time (a few years) in mid adult life. An important development resulting from the above observations has been the rederivation of diagnostic criteria for HCM. This has been quite effective in defining disease status in individuals with an affected first-degree relative [15], because their prior probability of HCM is one in two, and so the problems of false-positive diagnosis when applying non-stringent criteria are manageable. Difficulties still exist in defining minimal criteria for HCM in unrelated individuals without a family history. It should be recognized that standard criteria (e.g. diastolic wall thickness >15 mm, or septum to posterior wall asymmetry >1 3) will miss a substantial proportion of affected individuals (that proportion varying considerably with specific genotype). New clinical diagnostic modalities Just as the presence or absence of mutations has been an important gold standard for evaluating ECG and echocardiographic criteria, it can be anticipated that new clinical tools will be derived in the future because the opportunity now exists for defining sensitivity and specificity in a genetically affected population. These may potentially include more refined interpretation of the surface ECG, although attempts to do this to date have been disappointing (e.g. in analysis of signal-averaged ECGs). Invasive ECG procedures currently hold more promise, for example in the quantification of ECG fractionation [16]. Imaging modalities include tissue-phase characterization by echocardiogram [17] and, potentially, magnetic resonance imaging with spectroscopy. The latter is likely to be of growing importance with increased use of magnetic resonance for defining anatomy and function, and with increased emphasis on the energetic abnormalities of HCM (see below). Attention is also likely

3 HCM: from molecular and genetic mechanisms to clinical management L45 to be paid to skeletal muscle characteristics, because only a subset of HCM disease gene proteins are expressed in skeletal muscle, and non-invasive tools to detect skeletal involvement hold at least theoretical value in narrowing the search for the underlying mutation. Direct application of molecular genetic diagnosis This is an area of clear growth for the future, because technological advances will continue to reduce the current obstacles that arise from the diversity of disease genes and mutations. Because many families will ultimately be found to have a private mutation (i.e. not previously reported), direct screening of the full coding sequence of each disease gene will probably remain necessary. Even though technologies for typing known variants have advanced, it is unlikely that a DNA chip that tests for a defined list of mutations would be very useful because the total number of such mutations would be huge. Furthermore, there are concerns that compound, or complex, mutations may exist, such that identification of a known variant may not accurately characterize the abnormalities in a given family. In particular, double mutations in the same copy of a disease gene (in cis) would systematically confound genotype phenotype correlations [18]. Fortunately, detection of mutation by systematic screening is now semiautomated and relatively efficient in those research laboratories that conduct it routinely. Transfer from a research-based phase to a research and development phase of genetic testing is imminent. Transfer to routine service laboratories may then follow. Indications for molecular diagnosis Some important limitations are inherent in the predictive value of the presence or absence of a mutation that may continue to restrict the indications for testing, even when there are no other technological obstacles. First, a mutation needs to be found in the DNA of an affected member of a family before any conclusions can be drawn; failure to find such a mutation does not mean that one is not present. As a result, DNA detection in apparently unaffected surviving relatives after an individual has died suddenly is of limited value. The clinical value of autopsy tissue samples is clear, although current trends may limit collection of such samples. Second, providing certainty that an identified mutation is actually responsible for causing the disease is not as trivial as might have been expected a few years ago, and some examples exist of mutations that were believed to cause HCM and later shown to be very rare nonsegregating polymorphisms. Strict attention to the criteria of proven cosegregation with disease and presumed or proven biological impact remain key. The FHC Mutation Database [19] seeks to provide an authoritative source of validated mutations. Once the mutation in any individual family is identified, then genotyping of all relatives is technically very straightforward, but not always clinically straightforward. If a mutation is known to be disease causing, then demonstrating its absence in a family member provides conclusive evidence that they do not carry the disease, they will not develop it and neither will their descendants. This provides a greater degree of certainty than clinical testing ever can, and allows young adults who are at risk to cease long-term follow up. Unfortunately, the converse situation is not so simple; the presence of the mutation confirms that an individual is at risk for manifesting the condition and that their offspring will have a one in two chance of inheriting the mutation. However, the proportions of mutation carriers that will manifest detectable abnormalities, or clinically important abnormalities, vary from family to family and from mutation to mutation. Accordingly, the positive predictive accuracy is not absolute, and the use of this information should vary with the specific context. Current practice Mutation detection, and indeed the use of simple clinical tools for screening family members [20], is best conducted after careful counselling. Mutation detection in adults who have an abnormal heart, which may be due either to HCM or to a confounding diagnosis (e.g. in hypertensive persons or athletes), is generally straightforward and useful. Similarly, mutation detection would commonly be recommended in an at risk adolescent or young adult (i.e. a member of a known HCM family) who had developed equivocal clinical abnormalities. In contrast, caution is warranted in at risk individuals who have completely normal studies. In this instance genetic counselling is particularly necessary because clinical intervention would likely not be warranted, with the result that the potential downside of pre-symptomatic diagnosis might outweigh the benefits. Thus, molecular diagnosis in young individuals with completely normal clinical studies is probably only warranted in families with a high incidence of sudden death and with instances of serious consequences of disease in individuals with little or no hypertrophy. Future use The one development that would dramatically change the situation would be the advent of disease-modifying treatments. At present, the available drug, surgical and device treatments for HCM aim to limit complications of the disease rather than to modify the underlying disease process, and as such are reserved until both clinically apparent abnormalities and demonstrable risk factors are present. The great hope of biological and functional efforts in HCM is that new bases for intervening in the underlying

4 L46 disease process itself will be developed. If this occurs, then it may be anticipated that initiation of treatment in advance of demonstrable electrophysiological abnormalities or cardiac hypertrophy will probably be most effective. In such an instance, molecular genetic diagnosis of presymptomatic mutation carriers will be of paramount importance. Molecular genetic classification Max LVWT (a) 24 ± 8 16 ± 5 Great hopes are pinned on the ways in which a molecular taxonomy of disease may refine, or even individualize, treatment for a broad range of cardiovascular diseases. For example, this is the basis of enormous efforts in the field of pharmacogenetics, in which it is hoped that common genetic polymorphisms will predict individual responses to drugs and the risk for adverse effects, and ultimately lead to tailored therapies according to an individual s disease type and pharmacogenetic variants. Accordingly, the relatively simple test case of a single-gene disorder such as HCM offers an important opportunity to review the nature of genotype phenotype correlations. In general, experience in single-gene disorders has been that such correlations do exist and are often useful for broad classification and for gaining a biological understanding, but tend not to have great utility for prediction in individual patients. This should perhaps send a note of caution to those who have high expectations for the application of molecular taxonomy in complex trait disorders. Genotype phenotype studies in HCM have revealed predominantly quantitative differences between the phenotypes that are associated with different disease genes and different mutations within them (Fig. 2) [3,13,14,21,22]. Examples of qualitative phenotypic differences have recently been reported (e.g. HCM with Wolff Parkinson White syndrome [23] may have a particular genetic basis; see below), but these examples are in the minority. Nevertheless, the quantitative differences are important in that they explain some of the clinical heterogeneity that is so characteristic of HCM and because some of these differences are of material significance to patient management. Circumstances in which genotype phenotype correlations are useful Knowledge of an underlying mutation will generally be useful for disease classification where the phenotypic result is consistent, clinically important and not otherwise detectable by clinical tools. Probably the best example is afforded by the subset of HCM families in which disease results from mutations in the gene that encodes cardiac TnT. Initial reports suggested that these mutations were associated with a strikingly poor prognosis and a high incidence of sudden death, despite below average hypertrophy. Up to 20% of adults with pathogenic TnT mutations did not have demonstrable hypertrophy at all [3], WT 10 ± 2 MHC TnT Figure 2 Maximum left ventricular wall thickness (LVWT; by echocardiography) of mutation carriers with a variety of myosin heavy chain (MHC) mutations and a variety of troponin T (TnT) mutations, with their unaffected relatives as wild-type controls (WT). The two disease genes are associated with quantitative differences in phenotype, with clinically important consequences (as many individuals with TnT mutations have normal, or near normal, wall thickness despite the high risk for sudden death). However, within each group there is a wide range of phenotype, illustrating the limitations of genotype phenotype correlations for individual prediction. Primary data are from Watkins and coworkers [3,21]. and yet at least some without hypertrophy are still at risk for sudden death [24]. Subsequent studies have largely shown these associations to be consistent [25], although, as expected in any heterogeneous disorder, there are exceptions [26]. Thus, although the TnT subgroup is generally in the minority, it does appear to identify individuals who are at significant risk for adverse events. It also identifies families in which direct molecular typing may be of major importance, given the ambiguous clinical studies that characterize these families. The proportion of TnT mutations in a panel of probands with HCM is strongly influenced by the way in which the families are ascertained. In referral practices that see families that have been referred because of instances of sudden death, TnT mutations appear to be prevalent (e.g %). These therefore represent an important exception to data-sets derived from other mutation classes that suggest that hypertrophy and risk are broadly correlated [27]. Even where family studies show a consistent genotype phenotype correlation, the effect of the mutation on disease manifestation in a given individual still shows variability. A variant in a disease gene is interpreted on the genetic background of a given patient, and is further modified by environmental and perhaps random (stochastic) influences. Thus, a particular genotype may be quite helpful in predicting cohort risk as it applies to a particular family, but within this cohort there will always be individual outliers. Existing data suggest that the genotype phenotype correlations in HCM are more

5 HCM: from molecular and genetic mechanisms to clinical management L47 consistent for prognosis and risk for sudden death than they are in the distribution or extent of hypertrophy. It is perhaps not surprising that the pathways that lead to hypertrophy give scope for multiple modifier genes and modifying environmental influences. Will a molecular genetic classification ever replace a clinical one? For the all promise of the additional insights that might arise from a molecular taxonomy, most would not anticipate that a clinical classification could become redundant, based on the assumption that there would always be important aspects of the clinical disease that would not be predicted by genotype. More recently, new genetic findings in HCM have pointed to the fact that molecular genetic classifications can be in conflict with classifications that are based on clinical features. This is because not only can many genes cause one clinical entity, but mutations in a given gene can also give rise to quite divergent entities. Thus, HCM has been labelled a disease of the sarcomere, and classical disease genes are now typified by those such as beta-myosin heavy chain and cardiac TnT. In apparently clear contrast, the quite different clinical entity of dilated cardiomyopathy has been attributed to diseases of the cytoskeleton, intermediate filament and nuclear envelope (mutations in genes that encode components of the dystrophin complex, actin, desmin and lamin A/C, as reviewed by Seidman and Seidman [28] ). However, missense mutations in beta-myosin heavy chain and cardiac TnT genes have now convincingly been shown also to cause primary dilated cardiomyopathy inherited as an autosomal defect [29]. Clearly, a simple genetic classification based on the disease gene would lump together significantly different disease states, without (as yet) providing any useful biological insight to justify it. It may be that this is just an issue regarding the level of detail at which the genetic classification is based; a more sophisticated analysis may subsequently show that certain mutation types group together to produce the different diseases. Equally, however, this may not be so because the diverging disease pathways may result from epistatic influences. Of note, inbred strains of hamster that are characterized by either HCM or dilated cardiomyopathy, and that have been longstanding animal models for these conditions, share an identical mutation in the δ- sarcoglycan gene [30], emphasizing the deficiencies of simply classifying on the basis of the primary underlying mutation. New biological insights Although much of the focus of current human genetic investigation appears to be placed on the predictive potential of genetic variants, this is, as illustrated above, not always as useful as perhaps supposed. In contrast, the primary rationale for investing in a reverse genetics approach to gain an understanding of the basis of genetically determined or genetically influenced diseases is to understand better the underlying biology. Genetic traits powerfully select for defects in key biological components: rate-limiting enzymes, nodal points in pathways and key domains of proteins. The vast majority of DNA variants are not sufficient to produce a distinctive phenotype; where they are and where such a phenotype is stringently selected (e.g. in the case of specified disease), the reductionist approach of genetics can be expected to identify key abnormalities in critical genes. Where this approach is used in a disorder that was previously poorly understood at a fundamental level, there is every chance that the newly implicated disease gene will identify a previously unknown protein or pathway, with the promise of providing novel therapeutic targets. Even where the genetic variant is responsible for a rare disease, or a rare form of a common disease, identification of novel therapeutic targets can be of far wider application. Perhaps the most convincing precedent in cardiovascular disease is the ultimate discovery of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors ( statins ); this arose directly from a molecular understanding of the low-density lipoprotein receptor pathway, which was implicated by rare mutations that cause familial hypercholesterolaemia. Thus, one might predict that the most significant impact arising from an understanding of the molecular basis of HCM will come from molecular and cellular dissection of the disease pathway. This can be illustrated in terms of both fundamental (and potentially unrelated) insights into the function of the proteins that are mutated in this disease and in an understanding of the final common pathway that unifies the diversity of disease genes. Fundamental blue skies insights Some of the mutations that have been shown to cause HCM are easily explicable in terms of the known function of the region of the contractile protein affected. However, diseasecausing mutations that affect proteins, or parts of proteins, of previously unknown function direct attention to these regions and provide a route to dissect new molecular mechanisms. For example, mutations in the S2 region of beta-myosin heavy chain led to the demonstration that this part of the protein directly interacts with another component of the sarcomere myosin binding protein C which reveals previously unrecognized mechanisms for the regulation of cardiac contractility [31]. Similarly, functional studies of cardiac TnT mutants reveal abnormalities in actin myosin interactions, and again document a previously unrecognized regulatory role for cardiac TnT [32]. Additionally, studies of the downstream consequences of specific alterations in contractility illustrate the potential results of attempts to manipulate contractility pharmacologically. A notable example would be the impact of mutations in alpha-tropomyosin, in which the net result is a calcium-sensitizing effect [10]. These mutations probably

6 L48 Mitochondrial mutations Friedreich s ataxia Fatty acid oxidation defects Sarcomeric protein gene mutations AMP-activated protein kinase (AMPK) mutations Disordered ATP production Inefficient ATP usage Failure to protect from ATP depletion Inability to maintain normal ATP availability in the cardiomyocyte Hypertrophic cardiomyopathy phenocopies Hypertrophic cardiomyopathy Hypertrophic cardiomyopathy + WPW Figure 3 Pathogenesis of several genetically unrelated conditions that all produce features of hypertrophic cardiomyopathy (HCM). The recent demonstration [36] that mutations other than those in sarcomeric protein genes can cause HCM has important implications for disease mechanisms, because mutations in AMP-activated protein kinase (AMPK) corroborate the central role of abnormal cardiac energetics. Thus, the findings in this subset of patients with HCM (clinically associated with Wolff Parkinson White [WPW] syndrome in some individuals) will probably redirect thinking regarding potential therapies in the more common sarcomeric forms of HCM. enhance systolic contractility but interfere with diastolic relaxation, and thus show that any pharmacological calcium-sensitizing agent that simply shifts the force calcium activation curve to the left is likely to have a deleterious effect in the long term. molecular causes could really be used to devise interventions in the disease pathway. Equally, it was unclear whether HCM would indeed be a widely useful genetic model of cardiac hypertrophy, with relevance to acquired forms of heart muscle disease. Do contractile protein mutations act by a final common pathway of altered contractility? In order to realize the potential for new therapies for HCM (and perhaps mechanistically related conditions), much effort has been invested in gaining an understanding of the final common pathway by which the contractile proteins cause the disease. Surprisingly, conflicting findings have emerged that show that the diverse disease genes have quite different impacts on contractility (for review [33] ). Thus, early studies focused on the role of mutant myosin proteins, and these yielded (in the main) consistent evidence that mutations in the myosin heavy chain impair contractility, slowing the velocity of contraction and diminishing force output [9]. It thus appeared that, at least in this instance, HCM was a compensatory response to an initially hypo-contractile insult. Mutations in other classes of HCM disease gene, however, produce diametrically opposed results. For example, mutations in alpha-tropomyosin and cardiac troponin-i impair relaxation and, if anything, have a hyper-contractile phenotype [10,34,35]. These findings suggested that there was not a unifying abnormality of contractility that was central to HCM, and left much uncertainty as to how an understanding of these different A novel class of hypertrophic cardiomyopathy disease gene providing new insights into pathogenesis One abnormality that the diverse contractile protein mutations do appear to share is that they all result in inefficiency of ATP usage in the sarcomere (Fig. 3). For example, myosin mutations produce an internal drag (as normal and mutant myosin motors are arrayed in series), whereas TnT mutations appear to mechanically uncouple the cross-bridge cycle, resulting in lower force generation per ATP molecule used [32]. These observations allowed a hypothesis to be developed that proposes that the critical abnormality in HCM is a relative deficiency of ATP (probably only in certain cellular compartments under conditions of stress). This would be sufficient to impair calcium-handling, because the calcium reuptake pumps are the other main energy users in the cardiomyocytes [37]. The resultant elevation in intracellular calcium could be expected to induce calcium-dependent hypertrophic signalling, as well as predisposing to arrhythmia. Until recently the only supportive data for this model have been indirect. These include molecular insights into some of the phenocopies of HCM, showing that defects in ATP production in the myocyte results in an HCM-like

7 HCM: from molecular and genetic mechanisms to clinical management L49 phenotype (e.g. in mitochondrial mutations, Friedreich s ataxia and defects in fatty acid oxidation [38 40] ). Very recently, direct genetic evidence has confirmed that a primary defect in ATP homeostasis can indeed cause HCM. Mutations have been described that affect a component of AMP-activated protein kinase (AMPK), which is a master regulator of cell response to metabolic stress. AMPK has been likened to a fuel gauge of the cell [41]. When ATP levels fall, the kinase is activated and thereby inhibits ATP-using synthetic pathways (e.g. cholesterol synthesis) and activates glucose uptake, glycolysis and fatty acid oxidation [42]. We have recently shown that mutations in a regulatory subunit of AMPK are responsible for autosomal-dominant HCM in families with a severe form of the condition associated, in some individuals, with Wolff Parkinson White syndrome [36]. Although these families may represent a relatively small minority of all HCM, the biological significance of this new class of mutation should radically alter our thinking regarding the pathogenesis of the condition. Considerable support has now been provided for the central role of myocardial energetics in HCM. This indicates that the HCM phenotype can result from failure to synthesize enough ATP (e.g. mitochondrial mutations and other metabolic syndromes); failure to use ATP for force generation efficiently ( sarcomeric HCM); or failure to protect the cell from ATP depletion during times of stress (AMPK mutations). This unifying model will suggest new avenues for intervention. Strategies to avoid critical over-expenditure of ATP in the heart, to enhance energy reserve or to enhance efficiency of ATP usage must be explored. Small molecule agents already exist that manipulate AMPK activity. Such approaches, combined with appropriate experiments in genetically manipulated mouse models, may provide a route to the first disease-modifying treatment for HCM. Finally, a new model of HCM, in which the disease phenotype results from a primary impairment of myocardial energetics, may have widespread implications for other myocardial disorders. These findings validate the importance of altered myocardial energetics as being potentially causative of human disease. Thus, the well characterized energetic abnormalities that have been described in unselected series of patients with dilated cardiomyopathy and heart failure, and which are known to correlate with prognosis [43], may indicate that HCM will ultimately be an important model for acquired conditions. Any therapeutic strategies that are shown to be effective in HCM may be widely applicable. The experimental work of the author s laboratory that is discussed in this review was supported by the British Heart Foundation and the Wellcome Trust. References [1] Geisterfer-Lowrance AAT, Kass S, Tanigawa G et al. A molecular basis for familial Hypertrophic cardiomyopathy: a β cardiac myosin heavy chain gene missense mutation. Cell 1990; 62: [2] Thierfelder L, Watkins H, MacRae C et al. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell 1994; 77: [3] Watkins H, McKenna W, Thierfelder L et al. Mutations in the genes for cardiac troponin T and α-tropomyosin in hypertrophic cardiomyopathy. N Engl J Med 1995; 332: [4] Watkins H, Conner D, Thierfelder L et al. Mutations in the cardiac myosin binding protein-c on chromosome 11 cause familial Hypertrophic cardiomyopathy. Nat Genet 1995; 11: [5] Poetter K, Jiang H, Hassanzadeh S et al. Mutations in either the essential or regulatory light chains are associated with a rare myopathy in human heart and skeletal muscle. Nat Genet 1996; 13: [6] Kimura A, Harada H, Park JE et al. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet 1997; 16: [7] Mogensen J, Klausen IC, Pedersen AK et al. Alpha-cardiac actin is a novel disease gene in familial hypertrophic cardiomyopathy. J Clin Invest 1999; 103: [8] Satoh M, Takahashi M, Sakamoto M et al. Structural analysis of the titin gene in Hypertrophic cardiomyopathy: identification of a novel disease gene. Biochem Biophys Res Commun 1999; 262: [9] Lankford EB, Epstein ND, Fananapazir L, Sweeney HL. Abnormal contractile properties of muscle fibers expressing beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy. J Clin Invest 1995; 95: [10] Bottinelli R, Coviello DA, Redwood CS et al. A mutant tropomyosin that causes hypertrophic cardiomyopathy is incorporated in vivo and associated with an increased calcium sensitivity. Circ Res 1998; 82: [11] Watkins H, Seidman CE, Seidman JG et al. Expression and functional assessment of a truncated cardiac troponin T that causes hypertrophic cardiomyopathy. Evidence for a dominant negative action. J Clin Invest 1996; 98: [12] Maron BJ, Gardin JM, Flack JM et al. Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults. Circulation 1995; 92: [13] Bonne G, Carrier L, Richard P et al. Familial hypertrophic cardiomyopathy: from mutations to functional defects. Circ Res 1998; 83: [14] Niimura H, Bachinski LL, Sangwatanaroj S et al. Human cardiac myosin binding protein C mutations cause late-onset familial cardiac hypertrophy. N Engl J Med 1998; 338: [15] McKenna WJ, Spirito P, Desnos M et al. Experience from clinical genetics in hypertrophic cardiomyopathy: proposal for new diagnostic criteria in adult members of affected families. Heart 1997; 77: [16] Saumarez RC, Slade AK, Grace AA et al. The significance of paced electrogram fractionation in hypertrophic cardiomyopathy. A prospective study. Circulation 1995; 91: [17] Nagueh SF, Kopelen HA, Lim DS et al. Tissue Doppler imaging consistently detects myocardial contraction and relaxation abnormalities, irrespective of cardiac hypertrophy, in a transgenic rabbit model of human hypertrophic cardiomyopathy. Circulation 2000; 102: [18] Blair E, Price SJ, Baty CJ et al. Mutations in cis can confound genotype-phenotype correlations in hypertrophic cardiomyopathy. J Med Genet 2001; 38: [19] FHC Mutation Database: Heart/fhc [20] Yu B, French JA, Jeremy RW et al. Counselling issues in familial hypertrophic cardiomyopathy. J Med Genet 1998; 35: [21] Watkins H, Rosenzweig A, Hwang DS et al. Characteristics and prognostic implications of myosin missense mutations in familial Hypertrophic cardiomyopathy. N Engl J Med 1992; 326:

8 L50 [22] Epstein ND, Cohn GM, Cyran F et al. Differences in clinical expression of Hypertrophic cardiomyopathy associated with two distinct mutations in the beta-myosin heavy chain gene. A Leu908Val mutation and a Arg403Gln mutation. Circulation 1992; 86: [23] MacRae CA, Ghaisas N, Kass S et al. Familial hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome maps to a locus on chromosome 7q3. J Clin Invest 1995; 96: [24] Varnava A, Baboonian C, Davison F et al. A new mutation of the cardiac troponin T gene causing familial hypertrophic cardiomyopathy without left ventricular hypertrophy. Heart 1999; 82: [25] Corfield VA, Moolman JC, Posen B et al. Sudden death due to troponin T mutations. J Am Coll Cardiol 1997; 29: [26] Anan R, Shono H, Kisanuki A et al. Patients with familial hypertrophic cardiomyopathy caused by a Phe110Ile missense mutation in the cardiac troponin T gene have variable cardiac morphologies and a favorable prognosis. Circulation 1998; 98: [27] Spirito P, Bellone P, Harris KM et al. Magnitude of left ventricular hypertrophy and risk of sudden death in hypertrophic cardiomyopathy. N Engl J Med 2000; 342: [28] Seidman JG, Seidman C. The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell 2001; 104: [29] Kamisago M, Sharma SD, DePalma SR et al. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N Engl J Med 2000; 343: [30] Sakamoto A, Ono K, Abe M et al. Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, deltasarcoglycan, in hamster: an animal model of disrupted dystrophin-associated glycoprotein complex. Proc Natl Acad Sci USA 1997; 94: [31] Gruen M, Gautel M. Mutations in the β-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin binding protein-c. J Mol Biol 1999; 286: [32] Sweeney HL, Feng HS, Yang Z, Watkins H. Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: insights into disease pathogenesis and troponin function. Proc Natl Acad Sci U S A 1998; 95: [33] Redwood CR, Moolman-Smook JC, Watkins H. Properties of mutant contractile proteins that cause hypertrophic cardiomyopathy. Cardiovasc Res 1999; 44: [34] Bing W, Knott A, Redwood C et al. Effect of hypertrophic cardiomyopathy mutations in human cardiac muscle alphatropomyosin (Asp175Asn and Glu180Gly) on the regulatory properties of human cardiac troponin determined by in vitro motility assay. J Mol Cell Cardiol 2000; 32: [35] Elliott K, Watkins H, Redwood CS. Altered regulatory properties of human cardiac troponin I mutants that cause hypertrophic cardiomyopathy. J Biol Chem 2000; 275: [36] Blair E, Redwood C, Ashrafian H et al. Mutations in the γ 2 subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet 2001; 10: [37] Spindler M, Saupe KW, Christe ME et al. Diastolic dysfunction and altered energetics in the alphamhc403/+ mouse model of familial hypertrophic cardiomyopathy. J Clin Invest 1998; 101: [38] Merante F, Tein I, Benson L, Robinson BH. Maternally inherited hypertrophic cardiomyopathy due to a novel T-to-C transition at nucleotide 9997 in the mitochondrial trna (glycine) gene. Am J Hum Genet 1994; 55: [39] Puccio H, Koenig M. Recent advances in the molecular pathogenesis of Friedreich ataxia. Hum Mol Genet 2000; 9: [40] Bonnet D, Martin D, De Lonlay P et al. Arrhythmias and conduction defects as presenting symptoms of fatty acid oxidation disorders in children. Circulation 1999; 100: [41] Hardie DG, Carling D. The AMP-activated protein kinase: fuel gauge of the mammalian cell? Eur J Biochem 1997; 246: [42] Kemp BE, Mitchelhill KI, Stapleton D et al. Dealing with energy demand: the AMP-activated protein kinase. Trends Biochem Sci 1999; 24: [43] Neubauer S, Horn M, Cramer M et al. Myocardial phosphocreatine-to-atp ratio is a predictor of mortality in patients with dilated cardiomyopathy. Circulation 1997; 96: