Molecular genetic basis of sudden cardiac death

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1 Cardiovascular Pathology 10 (2001) Molecular genetic basis of sudden cardiac death Jeffrey A. Towbin* Departments of Pediatrics (Cardiology), Cardiovascular Sciences and Molecular and Human Genetics, Texas Children s Hospital and Baylor College of Medicine, One Baylor Plaza, Room 333E, Houston, TX 77030, USA Received 31 July 2001; accepted 2 August 2001 Abstract In this review, the up-to-date understanding of the molecular basis of disorders causing sudden death will be described. Two arrhythmic disorders causing sudden death have recently been well described at the molecular level, the long QT syndromes (LQTS) and Brugada syndrome, and in this article we will review the current scientific knowledge of each disease. A third disorder, hypertrophic cardiomyopathy (HCM), a myocardial disorder causing sudden death, has also been well studied. Finally, a disorder in which both myocardial abnormalities and rhythm abnormalities coexist, arrhythmogenic right ventricular dysplasia (ARVD) will also be described. The role of the pathologist in these studies will be highlighted. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Long QT syndrome; Brugada syndrome; Idiopathic ventricular fibrillation; Ion channels; Sodium channel; Potassium channel; ARVD, arrhythmogenic right ventricular dysplasia; HCM, hypertrophic cardiomyopathy; Sarcomere 1. Introduction Sudden cardiac death in the United States occurs with a reported incidence of greater than 300,000 persons per year [1]. Although coronary heart disease is a major cause of death, other etiologies contribute to this problem. In many of these nonischemia related cases, autopsies are unrevealing. Interest in identifying the underlying cause of the death in these instances has been focused on cases of unexpected arrhythmogenic death, which is estimated to represent 5% of all sudden deaths. In cases in which no structural heart disease can be identified, the long QT syndrome (LQTS), ventricular preexcitation and idiopathic ventricular fibrillation (IVF) or Brugada syndrome [2] (characterized by STsegment elevation in the right precordial leads with or without right bundle branch block [RBBB]) are most commonly considered as likely causes. The most common myocardial disorder causing sudden death, hypertrophic cardiomyopathy (HCM), is characterized by thick heart muscle and abnormal diastolic function. Arrhythmogenic right ventricular dysplasia (ARVD) is also a significant cause of sudden death but is associated with fibrosis and * Tel.: ; fax: address: jtowbin@bcm.tmc.edu (J.A. Towbin). fatty infiltration of the right ventricle, as well as electrical instability. The purpose of this paper is to describe the current understanding of the clinical and molecular genetic aspects of inherited diseases in which arrhythmias and sudden death are prominent features, and highlight the role of the pathologist in identifying these disorders. 2. The LQTS 2.1. Clinical description The LQTS are inherited or acquired disorders of repolarization identified by the electrocardiographic (ECG) abnormalities of prolongation of the QT interval corrected for heart rate (QTc) above ms, relative bradycardia, T wave abnormalities and episodic ventricular tachyarrhythmias [3], particularly torsade de pointes. The inherited form of LQTS is transmitted as an autosomal dominant or autosomal recessive trait. Acquired LQTS may be seen as a complication of various drug therapies or electrolyte abnormalities. Whether the abnormality is genetic-based or acquired, the clinical presentation is similar. In many cases, the initial presentation is syncope while in other cases an evaluation includes an ECG, leading to the diagnosis. Tragically, some individuals have sudden death as their initial presenting feature and this /01/$ see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S (01)

2 284 J.A. Towbin / Cardiovascular Pathology 10 (2001) has been reported to occur in families in some cases. Typically, autopsy evaluation identifies a normal structural heart with no apparent cardiac cause of death Clinical genetics Two differently inherited forms of familial LQTS have been reported. The Romano Ward syndrome is the most common of the inherited forms of LQTS and appears to be transmitted as an autosomal dominant trait [4,5]. In this disorder, the disease gene is transmitted to 50% of the offspring of an affected individual. However, low penetrance has been described and therefore gene carriers may in fact have no clinical features of disease [6]. Individuals with Romano Ward syndrome have the pure syndrome of prolonged QT interval on ECG with the associated symptom complex of syncope, sudden death and, in some patients, seizures [7,8]. Occasionally, other noncardiac abnormalities such as diabetes mellitus [9,10], asthma [11] or syndactyly [12] may also be associated with QT prolongation. LQTS may also be involved in some cases of Sudden Infant Death Syndrome (SIDS) [13 15]. The Jervell and Lange-Nielsen syndrome (JLNS) is a relatively uncommon inherited form of LQTS. Classically, this disease has been described as having apparent autosomal recessive transmission [16,17]. These patients have the identical clinical presentation as those with Romano Ward syndrome, but also have associated sensorineural deafness [16 18]. Clinically, patients with JLNS usually have longer QT intervals as compared to individuals with Romano Ward syndrome, and also have a more malignant course. Recently, Priori and colleagues reported autosomal recessive cases of Romano Ward syndrome [19], thus, changing one of the sina-qua-nons of JLNS. 3. Gene identification in Romano Ward syndrome Analysis of the predicted amino acid sequence of KVLQT1 suggests that it encodes a potassium channel a subunit with a conserved potassium-selective pore-signature sequence flanked by six membrane-spanning segments similar to shaker-type channels [21,24,25]. A putative voltage sensor is found in the fourth membranespanning domain (S4) and the selective pore loop is between the fifth and sixth membrane-spanning domains (S5 and S6) (Fig. 1). Biophysical characterization of the KVLQT1 protein confirmed that KVLQT1 is a voltage-gated potassium channel protein subunit, which requires coassembly with a b subunit called mink to function properly [24,25]. Expression in either KVLQT1 or mink alone results in either inefficient or no current development. When mink and KVLQT1 are coexpressed in either mammalian cell lines or Xenopus oocytes, however, the slowly activating potassium current [I Ks ] is developed in cardiac myocytes [24,25]. Combination of normal and mutant KVLQT1 subunits forms abnormal I Ks channels, and these mutations are believed to act through a dominant-negative mechanism (the mutant form of KVLQT1 interferes with the function of the normal wild-type form through a poison pill -type mechanism) or a loss-of-function mechanism (only the mutant form loses activity) [26]. Since KVLQT1 and mink form a unit, mutations in mink could also be expected to cause LQTS, and this fact was subsequently demonstrated (see section on LQT5) [27] HERG: the LQT2 gene After the initial localization of LQT2 to chromosome 7q35 36 by Jiang et al. [28] candidate genes (i.e., genes encoding proteins that could cause repolarization abnormalities if mutated, such as ion channels, modulators of ion channels, members of the sympathetic nervous system, etc.) in this region were analyzed. HERG (Human Ether-a-go-go- Related Gene), a cardiac potassium channel gene which was 3.1. KVLQT1 or KCNQ1: the LQT1 gene The first of the genes mapped for LQTS, termed LQT1, required 5 years from the time that mapping to chromosome 11p15.5 was first reported [20] to gene cloning. This gene, originally named KVLQT1, but more recently called KCNQ1, is a novel potassium channel gene that consists of 16 exons, spans approximately 400 kb, and is widely expressed in human tissues including heart, inner ear, kidney, lung, placenta and pancreas, but not in skeletal muscle, liver or brain [21]. In the original report, 11 different mutations (deletion and missense mutations) were identified in 16 LQTS families, establishing KVLQT1 as LQT1. To date, more than 100 families with KVLQT1 mutations have been described. Although most of the mutations are private (i.e., only seen in one family), there is at least one frequently mutated region (called a hotspot ) of KVLQT1 [21 23]. Fig. 1. (A) KVLQT1 and mink, which constitute the I Ks potassium channel. (B) HERG and MiRP1, which constitute the I Kr potassium channel. (C) The cardiac I Na sodium channel encoded by SCN5A. The mutations causing LQT and Brugada syndrome are shown.

3 J.A. Towbin / Cardiovascular Pathology 10 (2001) originally cloned from a brain cdna library [29] and found to be expressed in neural-crest derived neurons [30], microglia [31], a wide variety of tumor cell lines [32] and the heart [33], was one of the candidates evaluated and found to be mutated in patients with clinical evidence of LQTS [33]. Six LQTS-associated mutations were identified in HERG, including missense mutations, intragenic deletions and a splicing mutation. Later, Schulze-Bahr et al. [34] reported a single base pair deletion and a stop codon mutation in HERG, confirming this to be LQT2. Currently, this gene is thought to be the second most common gene mutated in LQTS (second to KVLQT1). As with KVLQT1, private mutations, which are scattered throughout the entire gene without clustering preferentially, are seen. HERG consists of 16 exons and spans 55 kb of genomic sequence [33]. The predicted topology of HERG (Fig. 1) is similar to KVLQT1. Unlike KVLQT1, HERG has extensive intracellular amino-and-carboxyl termini, with a region in the carboxyl-terminal domain having sequence similarity to nucleotide binding domains (NBD). Electrophysiological and biophysical characterization of expressed HERG in Xenopus oocytes established that HERG encodes the rapidly activating delayed rectifier potassium current I Kr [35,36]. Electrophysiological studies of LQTSassociated mutations showed that they act through either a loss-of-function or a dominant-negative mechanism [37]. In addition, protein trafficking abnormalities have been shown to occur [38,39]. This channel has been shown to coassemble with b-subunits for normal function, similar to that seen in I Ks. McDonald et al. [40] initially suggested that the complexing of HERG with mink is needed to regulate the I Kr potassium current. Bianchi et al. [41] provided confirmatory evidence that mink is involved in regulation of both I Ks and I Kr. Recently, Abbott et al. [42] identified MiRP1 as a b-subunit for HERG (see section on MiRP1) SCN5A: the LQT3 gene The positional candidate gene approach was also used to establish that the gene responsible for chromosome 3-linked LQTS (LQT3) is the cardiac sodium channel gene SCN5A [43,44]. SCN5A is highly expressed in human myocardium, but not in skeletal muscle, liver or uterus [45,46]. Recently, it has been found to be expressed in brain [47]. It consists of 28 exons that span 80 kb and encodes a protein of 2016 amino acids with a putative structure that consists of four homologous domains (DI DIV), each of which contains six membrane-spanning segments (S1 S6) similar to the structure of the potassium channel a subunits (Fig. 1) [34 36]. Linkage studies with LQT3 families and SCN5A initially demonstrated linkage to the LQT3 locus on chromosome 3p21 24 [45,46] and three mutations, including one 9-bp intragenic deletion (DK 1505 P 1506 Q 1507 ) and two missense mutations (R 1644 H and N 1325 S), were also identified in six LQTS families [45,46]. All three mutations were expressed in Xenopus oocytes and it was found that all mutations generated a late phase of inactivation-resistant, mexiletineand tetrodotoxin-sensitive whole-cell currents through multiple mechanisms [48,49]. Two of the three mutations showed dispersed reopening after the initial transient, but the other mutation showed both dispersed reopening and long-lasting bursts [49]. These results suggested that SCN5A mutations act through a gain-of-function mechanism (the mutant channel functions normally, but with altered properties such as delayed inactivation) and that the mechanism of chromosome 3-linked LQTS is persistent noninactivated sodium current in the plateau phase of the action potential. Later, An et al. [50] showed that not all mutations in SCN5A are associated with persistent current and demonstrated that SCN5A interacted with b-subunits mink: the LQT5 gene mink (IsK or KCNE1) was initially localized to chromosome 21 (21q22.1) and found to consist of three exons that span approximately 40 kb. It encodes a short protein consisting of 130 amino acids and has only one transmembrane-spanning segment with small extracellular and intercellular regions [26,27,51]. When expressed in Xenopus oocytes, it produces potassium current that closely resembles the slowly activating delayed-rectifier potassium current I Ks in cardiac cells [51,52]. The fact that the mink clone was only expressed in Xenopus oocytes and not in mammalian cell lines raised the question whether mink is a human channel protein. With the cloning of KVLQT1 and coexpression of KVLQT1 and mink in both mammalian cell lines and Xenopus oocytes, it became clear that KVLQT1 interacts with mink to form the cardiac slowly activating delayed-rectifier I Ks current [24,25]; mink alone cannot form a functional channel, but induces the I Ks current by interacting with endogenous KVLQT1 protein in Xenopus oocytes and mammalian cells (Fig. 1). More recently, Bianchi et al. [41] showed that mutant mink results in abnormalities of I Ks, I Kr and protein trafficking abnormalities. McDonald et al. [40] showed that mink also complexes with HERG to regulate the I Kr potassium current. Splawski et al. [27] demonstrated that mink mutations cause LQT5 when they identified mutations in two families with LQTS. In both cases, missense mutations (S74L and D76N) were identified which reduced I Ks by shifting the voltage dependence of activation and accelerating channel deactivation. This was supported by the fact that a mouse model of mink-defective LQTS was also created [53]. The functional consequences of these mutations includes delayed cardiac repolarization and, hence, an increased risk of arrhythmias MiRP1: the LQT6 gene MiRP1, the mink-related peptide 1, or KCNE2 is a novel potassium channel gene recently cloned and characterized by Abbott et al. [42]. This small integral membrane subunit protein assembles with HERG (LQT2) to alter its

4 286 J.A. Towbin / Cardiovascular Pathology 10 (2001) function and enabling full development of the I Kr current (Fig. 1). MiRP1 is a 123 amino acid channel protein with a single predicted transmembrane segment similar to that described for mink [51]. Chromosomal localization studies mapped this KCNE2 gene to chromosome 21q22.1, within 79 kb of KCNE1 (mink) and arrayed in opposite orientation [42]. The open reading frames of these two genes share 34% identity and both are contained in a single exon, suggesting that they are related through gene duplication and divergent evolution. Three missense mutations associated with LQTS and ventricular fibrillation were identified in KCNE2 by Abbott et al. [42] (Fig. 1) and biophysical analysis demonstrated that these mutants form channels that open slowly and close rapidly, thus, diminishing potassium currents. In one case, the missense mutation, a C to G transversion at nucleotide 25 which produced a glutamine (Q) to glutamic acid (E) substitution at codon 9 (Q9E) in the putative extracellular domain of MiRP1, led to the development of torsade de pointes and ventricular fibrillation after intravenous clarythromycin infusion (i.e., drug-induced). Therefore, like mink, this channel protein acts as a b-subunit but, by itself, leads to ventricular arrhythmia risk when mutated. These similar channel proteins (i.e., mink and MiRP1) suggest that a family of channels exist which regulate ion channel a-subunits. The specific role of this subunit and its stoichiometry remains unclear and is currently being hotly debated. 4. Genetics and physiology of autosomal recessive LQTS (JLNS) Neyroud et al. [54] reported the first molecular abnormality in patients with JLNS when they reported on two families in which three children were affected by JLNS and in whom a novel homozygous deletion-insertion mutation of KVLQT1 in three patients was found. A deletion of 7 bp and an insertion of 8 bp at the same location led to premature termination at the C-terminal end of the KVLQT1 channel. At the same time, Splawski et al. [55] identified a homozygous insertion of a single nucleotide, which caused a frameshift in the coding sequence after the second putative transmembrane domain (S2) of KVLQT1. Together, these data strongly suggested that at least one form of JLNS is caused by homozygous mutations in KVLQT1. This has been confirmed by others [26,56,57]. As a general rule, heterozygous mutations in KVLQT1 cause Romano Ward syndrome (LQTS only), while homozygous (or compound heterozygous) mutations in KVLQT1 cause JLNS (LQTS and deafness). The hypothetical explanation suggests that although heterozygous KVLQT1 mutations act by a dominant-negative mechanism, some functional KVLQT1 potassium channels still exist in the stria vascularis of the inner ear. Therefore, congenital deafness is averted in patients with heterozygous KVLQT1 mutations. For patients with homozygous KVLQT1 mutations, no functional KVLQT1 potassium channels can be formed. It has been shown by in situ hybridization that KVLQT1 is expressed in the inner ear [55], suggesting that homozygous KVLQT1 mutations can cause the dysfunction of potassium secretion in the inner ear and lead to deafness. However, it should be noted that incomplete penetrance exists, and not all heterozygous or homozygous mutations follow this rule [7,19]. As with Romano Ward syndrome, if KVLQT1 mutations can cause the phenotype, it could be expected that mink mutations could also be causative of the phenotype (JLNS). Schulze-Bahr et al. [58], in fact, showed that mutations in mink result in JLNS syndrome as well, and this was confirmed subsequently [55]. Hence, abnormal I Ks current, whether it occurs due to homozygous or compound heterozygous mutations in KVLQT1 or mink result in LQTS and deafness. 5. Brugada syndrome 5.1. Clinical aspects of Brugada syndrome The first identification of the ECG pattern of RBBB with ST-elevation in leads V1 V3 was reported by Osher and Wolff [59]. Shortly thereafter, Edeiken [60] identified persistent ST-elevation without RBBB in 10 asymptomatic males and Levine et al. [61] described ST-elevation in the right chest leads and conduction block in the right ventricle in patients with severe hyperkalemia. The first association of this ECG pattern with sudden death was described by Martini et al. [62] and later by Aihara et al. [63]. This association was further confirmed in 1991 by Pedro and Josep Brugada [64] who described four patients with sudden and aborted sudden death with ECGs demonstrating RBBB and persistent ST-elevation in leads V1 V3. In 1992, these authors characterized what they believed to be a distinct clinical and ECG syndrome [2]. The finding of ST-elevation in the right chest leads has been observed in a variety of clinical and experimental settings and is not unique or diagnostic of Brugada syndrome by itself. Situations in which these ECG findings occur include electrolyte or metabolic disorders, pulmonary or inflammatory diseases, abnormalities of the central or peripheral nervous system. In the absence of these abnormalities, the term idiopathic ST-elevation is often used and may identify Brugada syndrome patients. The ECG findings and associated sudden and unexpected death has been reported as a common problem in Japan and Southeast Asia where it most commonly affects men during sleep [65]. This disorder, known as Sudden and Unexpected Death Syndrome (SUDS) or Sudden Unexpected Nocturnal Death Syndrome (SUNDS), has many names in Southeast Asia including bangungut (to rise and moan in sleep) in the Philippines, nonlaitai (sleep death) in Laos, lai-tai (died

5 J.A. Towbin / Cardiovascular Pathology 10 (2001) during sleep) in Thailand and pokkuri (sudden and unexpectedly ceased phenomena) in Japan. General characteristics of SUDS include young, healthy males in whom death occurs suddenly with a groan, usually during sleep late at night. No precipitating factors are identified and autopsy findings are generally negative [66]. Life-threatening ventricular tachyarrhythmias as a primary cause of SUDS has been demonstrated, with VF occurring in most cases [67] Clinical genetics of Brugada syndrome Most of the families thus far identified with Brugada syndrome have apparent autosomal dominant inheritance [68 71]. In these families, approximately 50% of offspring of affected patients develop the disease. Although the number of families reported has been small, it is likely that this is due to under-recognition as well as premature and unexpected death [71,72] Molecular genetics of Brugada syndrome In 1998, our laboratory reported the findings on six families and several sporadic cases of Brugada syndrome [73]. The families were initially studied by linkage analysis using markers to the known ARVD loci and linkage was excluded. Candidate gene screening using the mutation analysis approach of single strand conformation polymorphism (SSCP) analysis and DNA sequencing was performed and SCN5A was chosen for study, based on the suggestions of Antzelevitch [71,72,74 79]. In three families, mutations in SCN5A were identified [73] including (1) a missense mutation (C-to-T base substitution) causing a substitution of a highly conserved threonine by methionine at codon 1620 (T1620M) in the extracellular loop between transmembrane segments S3 and S4 of domain IV (DIVS3 DIVS4), an area important for coupling of channel activation to fast inactivation; (2) a two nucleotide insertion (AA), which disrupts the splice-donor sequence of intron 7 of SCN5A; and (3) a single nucleotide deletion (A) at codon 1397, which results in an in-frame stop codon that eliminates DIIIS6, DIVS1 DIVS6 and the carboxy-terminus of SCN5A (Fig. 1). Mutations have also been found in SCN5A in children with sudden cardiac death [80]. Biophysical analysis of the mutants in Xenopus oocytes demonstrated a reduction in the number of functional sodium channels in both the splicing mutation and onenucleotide deletion mutation, which should promote development of reentrant arrhythmias. In the missense mutation, sodium channels recover from inactivation more rapidly than normal. Subsequent experiments conducted in modified HEK cells revealed that at physiological temperatures (37 C) reactivation of the T1620M mutant channel was actually slower, while inactivation of the channel was importantly accelerated. These alterations leave the transient outward current unopposed and thus able to effect an all-ornone repolarization of the action potential at the end of Phase 1 [81]. Failure of the sodium channel to express, as with the insertion and deletion mutations, results in similar electrophysiological changes. Reduction of the sodium channel I Na current causes heterogeneous loss of the action potential dome in the right ventricular epicardium, leading to a marked dispersion of depolarization and refractoriness, an ideal substrate for development of reentrant arrhythmias. Phase 2 reentry produced by the same substrate is believed to provide the premature beat necessary for initiation of the VT and VF responsible for symptoms in these patients. Interestingly, however, Kambouris et al.[82] identified a mutation in essentially the same region of SCN5A as the T1620M mutation (R1623H), but the clinical and biophysical features of this mutation was found to be consistent with LQT3 and not Brugada syndrome. Hence, there clearly remains a gap in our understanding of these entities. 6. ARVD ARVD is characterized by fatty infiltration of the right ventricle, fibrosis and ultimately thinning of the wall with chamber dilatation [83]. It is the most common cause of sudden cardiac death in the young in Italy [84] and is said to account for about 17% of sudden death in the young in the Unites States [85]. Rampazzo et al. [86] mapped this disease in two families, one to 1q42 q43, and the other on chromosome 2q32 and a third locus was mapped to 14q12 [87]. A large Greek family with ARVD and Naxos disease was recently mapped to 17q21 [88]. Two loci responsible for ARVD in North America were recently mapped at 3p23 [89] and the other at 10p12 [90]. This is a very devastating disease since the first symptom is often sudden death. ECG abnormalities include inverted T waves in the right precordial leads, late potentials and right ventricular arrhythmias with LBBB. This is compounded by the great difficulty in making the diagnosis even when occurring in a family with the disease history. Since the disease affects only the right ventricle, it is difficult to detect. There is no diagnostic definitive standard. The right ventricular biopsy is definitive when positive, but often gives a false negative since the disease initiates in the epicardium and spreads to the endocardium of the RV free wall making it inaccessible to biopsy. A consensus diagnostic criterion was developed which includes right ventricular biopsy, MRI, echocardiography and electrocardiography [91]. Recently, a gene was identified for ARVD2, the ryanodine receptor [92]. This is consistent with ARVD being an ion channel disorder. 7. Brugada syndrome and ARVD Controversy exists concerning the possible association of Brugada syndrome and ARVD, with some investigators arguing that these are the same disorder or at least one is a forme-fruste of the other [68,93 97]. However, the classic

6 288 J.A. Towbin / Cardiovascular Pathology 10 (2001) echocardiographic, angiographic and magnetic resonance imaging findings of ARVD are not seen in Brugada syndrome patients. In addition, Brugada syndrome patients typically are without the histopathologic findings of ARVD. Further, the morphology of VT/VF differs [69]. 8. HCM HCM is a complex cardiac disease with unique pathophysio-logical characteristics and a great diversity of morphologic, functional and clinical features [98,99]. Although HCM has been regarded largely as a relatively uncommon cardiac disease, the prevalence of electrocardiographically defined HCM in a large cohort of apparently healthy young adults selected from a community-based general population was reported 3 years ago to be as high as 0.2% [100]. Familial disease with autosomal dominant inheritance predominates. 9. Clinical aspects of familial hypertrophic cardiomyopathy (FHC) Observations of myocardial diseases that can reasonably be interpreted as HCM were made in the middle of the last century at the Hospital La Salpêtrière in Paris by A. Vulpian[101], who called what he saw at the macroscopic level a rétrécissement de l orifice ventriculoaortique or subaortic stricture. It was however only in the 1950s that the unique clinical features of HCM 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. A particular form of regional involvement affects the apex, but spares the upper portion of the septum (apical hypertrophy) [98]. 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. Though 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 [99,100]. 10. Mapping of FHC genes The first gene for FHC was mapped to chromosome 14q11.2 q12 using genome-wide linkage analysis in a large Canadian family [102]. Soon afterwards, FHC locus heterogeneity was reported [103,104] and subsequently confirmed Fig. 2. Representation of the thick and thin muscle filaments, showing the chromosomal map for each of the structural components. by the mapping of the second FHC locus to chromosome 1q3 and of the third locus to chromosome 15q2 [105,106]. Carrier et al. [107] mapped the fourth FHC locus to chromosome 11p11.2. Four other loci were subsequently reported, located on chromosomes 7q3. [108] 3p21.2 3p21.3 [109], 12q23 q24.3 [109], 19p13.2 q13.2 [110] and 15q14 [111] (Fig. 2). Several other families are not linked to any known FHC loci, indicating the existence of additional FHC-causing genes. 11. Gene identification in FHC All the disease-causing genes code proteins that are part of the sarcomere, which is a complex structure with an exact stoichiometry and multiple sites of protein protein interactions: three myofilament proteins, the b-myosin heavy chain (b-myhc), the ventricular myosin essential light chain 1 (MLC-1s/v) and the ventricular myosin regulatory light chain 2 (MLC-2s/v); four thin filament proteins, cardiac actin, cardiac troponin T (ctnt), cardiac troponin I (ctnl) and a-tropomyosin (a-tm); and finally one 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 [112] 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 two pairs of light chains (MLC), referred to as essential (or alkali) light chains (MLC-1) and regulatory (or phosphorylatable) light chains (MLC-2), respectively [113]. The myosin molecule is highly asymmetric, consisting of

7 J.A. Towbin / Cardiovascular Pathology 10 (2001) 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 two types of ventricular light chains [112]. Concerning the heavy chains, the b isoform (b-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 three exceptions are two 3-bp deletions that do not disrupt the reading frame, one of codon 10 and the other of codon 930, and a 2.4-kb deletion in the three regions. In the kindred with the latter mutation, only the proband had developed clinically diagnosed HCM 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 MYL Myosin-binding protein C (MyBP-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 Ca 2+ - sensitive tension [114], 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 [115]. Gautel et al.[116] showed that three distinct regions are specific to the cardiac isoform: the NH 2 -terminal domain C0 lg-l containing 101 residues, the MyBP-C motif (a 105- residue stretch linking the C1 and C2 lg-l domains), and a 28-residue loop inserted in the C5 lg-l domain. It was recently shown that cmybp-c is specifically expressed in the heart during human and murine development [117,118]. Twenty-seven MYBPC3 mutations have been identified in unrelated families with FHC (Fig. 2). 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: five are missense mutations in exons 6, 17, 21 and 23, one is a splice donor site mutation in intron 27 and one is an 18-residue duplication in exon 33. Finally, three mutations are predicted to produce either a mutated protein or a truncated one: two are missense mutations in exons 15 and 17 and one is a branch point mutation in intron Thin filament proteins The thin filament contains actin, the troponin complex and tropomyosin. The troponin complex and tropomyosin constitute the Ca 2+ -sensitive switch that regulates the contraction of cardiac muscle fibers. Mutations were found in a-tm and in two of the subunits of the troponin complex: ctnl, the inhibitory subunit, and ctnt, the tropomyosinbinding subunit. a-tm is encoded by TPM1. The cardiac isoform is expressed both in the ventricular myocardium and in fast twitch skeletal muscles [119]. It shares the overall structure of other tropomyosins that are rod-like proteins that possess a simple dimeric a-coiled-coil structure of other tropomyosins that are rod-like proteins that possess a simple dimeric a-coiled-coil structure in parallel orientation along their entire length [119]. 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 a-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 [120,121]. 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 [122,123]. Eleven mutations have been found in unrelated FHC families, three of which are located in a hot spot (codon 102). Ten mutations are missense ones located between exons 9 and 17, one mutation is a 3-bp 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. ctnl is encoded by TNNl3. The ctnl isoform is expressed only in cardiac muscles [124]. Cooperative binding of ctnl to actin-tropomyosin is a unique property of the cardiac variant. Six mutations were recently identified. Five

8 290 J.A. Towbin / Cardiovascular Pathology 10 (2001) are missense mutations located in exons 7 and 8, and one is a K183D mutation that does not disrupt the coding frame. a-cardiac actin (ACTC) was recently identified as a cause of FHC. Mogensen et al. [111] studied a family with heterogeneous phenotypes, ranging from asymptomatic with mild hypertrophy to pronounced septal hypertrophy and left ventricular outflow tract obstruction. Using linkage analysis and mutation screening, the gene was mapped to chromosome 15q14 with a LOD score of 3.6 and a missense mutation (G! 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) [125]. These missense mutations were localized in the immobilized end of actin that crossbinds to the anchor polypeptides in the Z bands. 12. Genotype phenotype relations in FHC 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 vary considerably. For example, the R403Q mutation appears to be associated with markedly reduced survival [126], whereas some others, such as V606M, appear more benign [127]. The disease caused by TNNT2 mutations is usually associated with a 20% incidence of nonpenetrance, a relatively mild and sometimes subclinical hypertrophy, but a high incidence of sudden death which can occur even in the absence of significant clinical left ventricular hypertrophy [128,129]. In one family with a TNNT2 mutation, however, penetrance is complete, echocardiographic data show a wide range of hypertrophy and there was no sudden cardiac death [122]. 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 [ ]. 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 mechanisms could account for the large variability of the phenotypic expression of the mutations: the role of environmental differences and acquired traits (e.g., 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 HCM and in patients with a high incidence of sudden cardiac death [134,135]. 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 [136], raising the concept of multiple genetic modifiers in FHC. 13. 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 (i.e., LQTS 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 and sarcomeric proteins have been speculated to be causative (i.e., cytoskeletal/sarcomyopathy) [137]. Hence, the final common pathways of these disorders include ion channels and cytoskeletal proteins, similar to the sarcomyopathy in HCM. 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 RCM, hints have been forthcoming. Desmin and other intermediate filament proteins appear to be at play in RCM as has been shown in animal models. 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. Additionally, molecular interactions with such molecules as calcineurin, sex hormones, growth factors, among 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

9 J.A. Towbin / Cardiovascular Pathology 10 (2001) reclassification 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 LQTS, 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 IVF 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 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. 14. The role of the pathologist in identifying the cause of death In patients with LQTS and Brugada syndrome, autopsy evaluation demonstrates normal structural hearts and no obvious cause of death. Autopsies of patients with HCM and ARVD, on the other hand, provide diagnostic clues in many cases but in other questions remain. In the current molecular era, it behooves the pathologist to think ahead by freezing myocardial samples and, when possible, obtaining blood for DNA extraction. Although not as reliable as frozen specimens, appropriately fixed (formalin or paraffin) tissue may also be used for molecular analysis and, therefore, should be saved and properly catalogued. Despite previous statements to the contrary, fixed tissue can be used for genetic screening and, in the case where the autopsy is diagnostic, serves as the best entre into studies of families with possible inherited disorders. The more tissue available, the more likely the study can be pursued and genetic mutations discovered. In addition, appropriately preserved specimens may be used for functional studies such as Western blots and immunohistochemistry while frozen specimens may be used for RNA work including Northern blots, and is better suited for functional studies as well. 15. Summary Ventricular arrhythmias appear to result from ion channel abnormalities. 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