Congenital myopathies: diseases of the actin cytoskeleton

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1 Journal of Pathology Published online in Wiley InterScience ( DOI: /path.1648 Review Article Congenital myopathies: diseases of the actin cytoskeleton Emilie Clarkson, Celine F Costa and Laura M Machesky* School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK *Correspondence to: Professor Laura M Machesky, School of Biosciences, Division of Mol Cell Biology, University of Birmingham, Birmingham, B15 2TT, UK. l.m.machesky@bham.ac.uk Abstract Congenital myopathies are clinical and genetic heterogeneous disorders characterized by skeletal muscle weakness ranging in severity. Three major forms have been identified: actin myopathy, intranuclear rod myopathy, and nemaline myopathy. Nemaline myopathy is the most common of these myopathies and is further subdivided into seven groups according to severity, progressiveness, and age of onset. At present, five genes have been linked to congenital myopathies. These include α-actin (ACTA1), α- and β-tropomyosin (TPM3 and TPM2), troponin T (TNNT1), and nebulin (NEB). Their protein products are all components of the thin filament of the sarcomere. The mutations identified within these genes have varying impacts on protein structure and give rise to different forms of congenital myopathies. Greater understanding of muscle formation and cause of disease can be established through the study of the effect of mutations on the functional proteins. However, a major limitation in the understanding of congenital myopathies is the lack of correlation between the degree of sarcomeric disruption and disease severity. Consequently, great difficulty may be encountered when diagnosing patients and predicting the progression of the disorders. There are no existing cures for congenital myopathies, although improvements can be made to both the standard of living and the life expectancy of the patient through various therapies. Copyright 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: myopathy; muscle; actin; sarcomere; thin filament Introduction The term congenital myopathy (CM) has been defined in a number of ways by the medical profession. Originally, it was defined as any muscle disorder present at birth. The currently accepted definition of congenital myopathy describes a subset of primary myopathies made up of nemaline myopathy (NM), actin myopathy (AM), and intranuclear rod myopathy (IRM) (see Table 1) [1]. These myopathies are clinical and genetic heterogeneous disorders characterized by skeletal muscle weakness that ranges in severity from neonatal life-threatening disorders to mild muscle weakness in adulthood. In most cases, the disease is only slowly progressive; for example, a few patients may eventually lose the ability to walk. Occasionally, muscle weakness may progress quite rapidly, leading patients to experience serious breathing problems. Previously, the typical specific structural changes of skeletal muscle observed in other diseases, such as dystrophic, inflammatory, metabolic or mitochondrial abnormalities, were classified as part of the congenital myopathies. However, the presence of rods and aggregates of protein in muscle biopsies is used to distinguish NM, AM, and IRM from other neuromuscular disorders, and the fairly recent development of molecular genetics has led to a more accurate definition of the aetiology of congenital myopathies, a type of neuromuscular disorder where no neurological cause can be distinguished, leading to suspicion of a lesion in contractility. Such lesions have now been identified at the level of the gene and resulting mutant proteins have been studied in some cases, so that we can now speculate why certain defects arise. It is emerging that while myopathies can still largely be distinguished from each other on the basis of histopathology, the mutations that underlie certain classes of pathology must also be considered. In many cases, for example, mutations in the same gene cause different types of pathological features in muscle biopsies. Additionally, the severity of the disease is not always predictable on the basis of how disrupted the muscle tissue appears down the microscope (see ref 2). Thus, our understanding of myopathies is changing as the different mutations are uncovered and the definitions of different myopathies are constantly being revised to fit the new genetic data. However, pathology of biopsy specimens viewed in the light microscope with Gomori trichome staining (Figure 1) still remains a primary basis for definition of the disease. This poses a problem when predicting or identifying the different congenital myopathies, particularly the numerous rare congenital myopathies, since the pathology can be difficult to correlate with a specific disease, unless electron microscopic examination is also used. In this review, we have chosen to concentrate on AM, IRM, Copyright 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

2 408 E Clarkson et al Table 1. Classification of congenital myopathies on the basis of histopathology Classification Pathology Gene(s) Nemaline myopathy (NM) Sarcoplasmic nemaline bodies Thin filament Actin myopathy (AM) Excess thin filament deposits Usually ACTA1 Intranuclear rod myopathy (IRM) Rods or deposits in the nucleus Thin filament Table 1 shows the classification of the three main congenital myopathies discussed in this review. In each case, the defining pathology seen in biopsy samples is listed under Pathology. Genes encoding thin filament proteins include ACTA1, TPM2, TPM3, NEB, and TNNT1. Figure 1. Gomori trichome staining of nemaline myopathy muscle sample from a patient with a T66I mutation [31], showing cytoplasmic nemaline bodies, which are deposits of Z-line proteins. Photo courtesy of Biljana Ilkovski and NM, and the recent developments in diagnosis and understanding of these disorders. The most common and better understood of the three myopathies is nemaline myopathy (NM), first described in 1963 by two groups [3,4]. Derived from the Greek word nema, meaning thread, NM is typified by sarcoplasmic thread-like nemaline bodies or rods (Table 1). NM has an estimated incidence of 0.02 per 1000 live births worldwide [2]. Many NM cases occur sporadically but some exhibit either autosomal recessive or dominant patterns of inheritance. Actin myopathy (AM) is the term used to describe homogeneous filamentous insertions containing actin, occupying certain areas of sarcomeres, which would normally be part of the myofibrillar filament lattice (Table 1). Intranuclear rod myopathy (IRM) is characterized by the presence of giant rod bodies in the muscle cell nuclei (Table 1) [1]. Both AM and IRM have a much lower incidence than NM. Other rare congenital myopathies exist, but have not been well studied due to a limited number of examples [5,6]. A broad spectrum of mutations causing congenital myopathies (CMs) has been linked to the proteins of the thin filament of the sarcomere. For example, over 60 different missense mutations have been identified in the α-actin gene (ACTA1) [1]. In addition, mutations have been identified in slow α-tropomyosin (TPM3), β-tropomyosin (TPM2), nebulin (NEB), and slow troponin T (TNNT1) genes. Mutations in the actin gene have been most easily identified due to its small size compared with the large nebulin gene. Despite more disease-causing mutations known in ACTA1, the nebulin gene is thought to be responsible for the majority of CM-causing mutations. Furthermore, many other CM-causing mutations remain to be identified. Another gene responsible for nemaline rod formation in the sarcomeres is the skeletal muscle ryanodine receptor gene (RYR1), also linked to other diseases, such as central core disease [7 9]. Congenital myopathies broadly include three main disorders: central core disease, nemaline myopathy, and centronuclear (myotubular) myopathy. Central core disease (CCD) is a dominantly inherited disease characterized by the appearance of mild leg weakness in infancy. This weakness does not progress with age but leads to delay in locomotion or malignant hyperthermia. In CCD, as well as the presence of nemaline bodies, abnormal cores are found in muscle cells and run the length of the muscle fibre. These cores appear empty, as if there is no metabolic activity, and also lack mitochondria. The genetic cause of CCD is thought to be linked to mutations in the skeletal muscle ryanodine receptor gene, which is related to a calcium channel in the sarcoplasmic reticulum [7 9]. Myotubular (centronuclear) myopathy (MTM) is another inherited genetic disorder characterized by muscle weakness and hypotonia at birth [10 12]. However, it can also develop in adolescence or early adulthood. Different autosomal and X-linked forms exist and are caused by mutations in MTM1 and possibly MTM2 and MTM3. The MTM gene encodes a tyrosine phosphatase, myotubularin, which dephosphorylates D3-phosphorylated inositol lipids. In MTM, the muscle fibres have nuclei in the centre of the fibre instead of, or as well as, at the periphery of the muscle fibre, where they normally are. This results in loss of muscle strength, which gradually increases over time, eventually resulting in severe muscle weakness. Infants may also have ophthalmoplegia or paralysis of the eye muscles. There is a huge emphasis on the need to define the exact causes of congenital myopathies. It is essential for the correct approach to be taken in the diagnosis and counselling of patients with CMs and eventually developing specific directed therapies. Muscle structure and function While an in-depth discussion of sarcomeric proteins and the molecular structure of muscle is beyond

3 Congenital myopathies 409 the scope of this review, it is useful to provide a brief overview with some appropriate references to recent reviews. In particular, since we focus on CMs that comprise the thin filament, we provide a brief description of its organization within the sarcomere below. A much more extensive discussion, including how this relates to contraction, can be found in one of the following reviews [13 17]. Sarcomeres are specialized contractile, repeating structures within muscle cells. They comprise ordered arrays of actin-containing thin filaments and myosincontaining thick filaments. Several other associated structural and regulatory proteins are present to maintain the ordered myofibrillar array and coordinate contraction. In the striated muscle sarcomeres, the core of the thin filament, F- (filamentous) actin, is the structure mostly affected in CMs [1]. The thin filament also contains nebulin, tropomyosin, and the troponin complex. Sarcomeres are delimited by Z-lines. These are electron-dense bands, perpendicular to the myofibrils, which bisect the actin-containing I band and anchor the thin filaments (see Figure 2). Z-lines are generally widest in type 1 (slow) fibres and narrowest in type 2b (fast) fibres. The exact composition of Z-lines has not been determined. However, it is clear that α-actinin is a major structural component along with actin, Cap Z, and several recently identified proteins including telethonin, myotilin, and myozenin [2]. Muscle contractions are created by cyclical interactions between the actin and myosin filaments through the consumption of ATP. Hence the major role of the thin filaments is to transfer the forces developed by myosin to the Z-disc, which in turn carries the contraction throughout the myofibril. It is evident that actin is a very important component and is highly conserved throughout evolution. It is therefore not surprising that many mutations within its gene have been linked to CMs. It is a major part of the cytoskeleton and is found in all cells as polymerized F-actin. F-actin is a helical filament in which all monomer actins are orientated similarly, and in the sarcomere, with the growing end anchored in the Z-disk, capped by Cap Z and the pointed (non-growing) end capped by tropomodulin. Since the I A I Z M Z Actin α/β-tropomyosin Troponin T Troponin I Troponin C Nebulin Figure 2. An electron micrograph is shown next to a schematic representation of a sarcomere and the arrangement of major thin filament proteins is shown below. Many of these thin filament proteins are abnormal in congenital myopathies. The sarcomere is delimited by the Z-lines that bisect the actin thin filament-containing I bands. A bands contain the myosin thick filaments centred about the M-line. This figure is reprinted from Trends in Molecular Medicine, Vol 7, D Sanoudou and AH Beggs. Clinical and genetic heterogeneity in nemaline myopathy a disease of skeletal muscle thin filaments. pp , Figure 1, Copyright 2001 with permission from Elsevier

4 410 E Clarkson et al actin monomer crystal structure has been determined at high resolution, predictions can be made as to how mutations in actin will affect actin function (see ref 1). The actin monomer unit is divided into two domains by a cleft containing the bound nucleotide (ATP or ADP) and cation (Ca 2+ ) [18]. Domains are connected by only two strands of single polypeptide chains, known as the hinge, allowing intramolecular movement. Actin is further divided into sub-domains 1, 2, 3, and 4. In the filament, the monomer large domain consists of sub-domains 3 and 4, which are close to the filament axis. Sub-domains 1 and 2 are on the outer edge and make up the small domain [18]. Therefore, each actin monomer makes specific interactions with four neighbouring monomers. Once the actin is polymerized, ATP hydrolysis occurs, causing a conformational change that is important for F-actin stability. The assembly of sarcomeres and the regulation of actin filament length within them are a major area of study (see refs 15 and 19). Nebulin is thought to be one of the determinants of sarcomere length, as it spans the length of the thin filament. It is also a determinant of Z-disk width, as its C-terminus anchors into the Z-disk and the length of the C-terminus has been correlated with Z-disk width [20,21]. The control of contraction, through the regulation of actin and myosin, is carried out by the tropomyosins and the troponin complex. At rest, tropomyosin dimers lie along the actin filament in a potential myosinbinding site, sterically inhibiting myosin actin interactions. Tropomyosin position and movement are controlled by the troponin complex consisting of three subunits: TN-I (inhibitory), TN-T (tropomyosinbinding), and TN-C (Ca 2+ binding). Striated muscle activation occurs through the binding of Ca 2+, released from the sarcoplasmic reticulum, to troponin C of the troponin complex. Ca 2+ binding causes troponin C and associated troponin I to undergo conformational changes releasing troponin I, which was bound to actin. This allows the troponin complex (troponins C, I, and T) and tropomyosin (TPM) to move across the F-actin surface, thereby exposing the myosin binding sites. The activated actomyosin ATPase allows these myosin-binding sites of the actin filament to bind to the myosin heads, permitting contraction [2]. The complexity of the multi-protein interactions involved in muscle contraction means that mutations in only one component can and do cause muscle dysfunction. Congenital myopathies and their clinical phenotype Actin myopathy is diagnosed when there is the accumulation of actin filaments observed in a biopsy specimen of the muscle fibres. This usually is preceded by severe congenital muscle weakness and often leads to high mortality. Intranuclear myopathy is diagnosed when nemaline rods are found in the muscle nuclei and is also associated with severe congenital muscle weakness and high mortality. Nemaline myopathy occurs when sarcoplasmic rods are present. NM is a highly variable disease, ranging from severe congenital muscle weakness and high early mortality to mild disease compatible with survival into adulthood [1]. These three myopathies can show phenotypic overlap. For example, patients can have both accumulations of actin and nemaline bodies, or have both intranuclear and sarcoplasmic, extranuclear rod bodies. Thus, it is possible that patients may have both NM and AM, or both NM and IRM [1]. Subtypes of nemaline myopathy Since NM is the most common of the myopathies, the European Neuromuscular Centre (ENMC) International Consortium on NM has divided it into seven different subtypes: typical, mild, severe, intermediate, adult-onset, Amish, and other forms based on the severity of the disease, age of onset, and additional features. Sanoudou and Beggs [2] give an excellent description of the different sub-types, so we will provide only a brief list below. Typical NM This is the most common form and is generally autosomal recessive. The onset is from birth to infancy and patients generally have respiratory independence but are in some danger of nocturnal hypoxia or hypercarbia. Patients do have respiratory insufficiency throughout their lives and this is often the cause of death. Many patients can lead an active life, but some will need a wheelchair. Severe NM The onset is from birth and infants are dependent on a ventilator from birth. There are often no spontaneous movements and this form is often lethal in the first year. Intermediate NM This form is also present from birth to infancy and patients have respiratory independence by 1 year, but this form is defined by progressive deterioration and loss of ambulation by 11 years. Mild NM The mildest form of NM is present during childhood and generally these patients have normal motor milestones. This form can be progressive, and may be indistinguishable from the typical form at later stages. Adult-onset NM Onset of this form is in the third to sixth decade and it is progressive. Some inflammatory changes are seen

5 Congenital myopathies 411 upon muscle biopsy and it is speculated that this form may be distinctly different from the other forms in terms of pathophysiology. Amish NM This form is autosomal recessive, with neonatal onset, and is lethal during early childhood, usually due to respiratory insufficiency. Other forms Cases that do not fit these categories are listed as other but it is not clear whether these are distinct variants of NM or other diseases with associated rods. Pathophysiology of congenital myopathies The presence of nemaline rods in the sarcoplasm is adefining feature in the diagnosis of NM. They are abnormal, electron-dense structures emanating from Z-lines and extending along the long axis of thin filaments. They vary from 1 to 7 µm in length and from 0.3 to 2 µm in width. They are thought to be largely composed of α-actinin as well as several other Z-line proteins. The actual process behind nemaline rod formation is unknown but it is thought to arise through the perturbation of the ratio of functional actin and its binding proteins, as well as the alteration of the interactions between these proteins [22]. A three-dimensional reconstruction of rods has revealed adjacent actin filaments of opposite polarity crosslinked by α-actinin dimers. This implies that rod formation might involve an inability to terminate or cap the thin filament ends at Z-lines [2]. Rods are found predominantly in fibre type 1 muscles [2]. These muscles involve sustained muscle contractions responsible for posture. Type 1 fibres include the intercostal muscles, which is likely to correlate with respiratory problems observed in CM patients. Nonetheless, in a minority of cases, rods are found to be equally distributed in type 1 and 2 (slow and fast) [2]. Although the presence of rods usually indicates NM and/or IRM, they have also been observed in normal muscle and a variety of other neuromuscular disorders. The proportion of fibres containing rods can vary from less than 1% to virtually all fibres [2]. The distribution of nemaline rods can also vary between different muscles in a single patient and between different parts of the same muscle. The presence of intranuclear rods fairly consistently correlates with a more severe phenotype and is in that way diagnostic [1]. When prominent thin filament accumulations are reported, AM is diagnosed. This tends to arise due to mutations in the actin gene, but actin mutations can cause any combination of AM, IRM or NM [1]. It would be desirable to evaluate all muscle biopsies by electron microscopy (EM), but this is not generally possible, due to the complexity and expense of the technique. EM facilitates the identification of intranuclear rods and glycogen accumulation within the muscle. It has been suggested that the extent of disruption of sarcomeric structures on EM appears to correlate better with clinical severity of CMs than abnormalities observed under the light microscope [23]. Inheritance pattern There are different patterns of inheritance within the CMs depending on the nature of the mutation. Mutations are either autosomal recessive or autosomal dominant. In addition, some cases occur sporadically, due to de novo mutations. Mutations in a particular gene cannot predict the form of inheritance nor the severity of the disease, as each gene can give rise to dominant or recessive mutations and severe, typical or mild forms. ACTA1 mutations are the most well characterized to date and these are mostly dominant [1]. Amish NM is autosomal recessive, with a truncated TNNT1 (troponin T1) of which the protein is absent in homozygous patients, resulting in a lethal phenotype due to complete loss of troponin T [24]. Nebulin mutations are typically autosomal recessive thus far, and many patients have the typical phenotype, but few nebulin mutations have been identified due to the large size of the gene [20,25,26]. More specific details of each gene underlying CMs are outlined below. The genetics of CM mutations To date, mutations in five different genes encoding sarcomeric proteins have been reported to cause CM. When studying the mutations in the genes responsible, up to 50% of cases of CM are potentially due to mutations in the nebulin gene and only approximately 20% of patients have mutations in the ACTA1 gene. Mutations in tropomyosin 2, 3, and troponin T are less common, probably accounting for 5 10% of cases [27]. The remainder of the mutations have not yet been identified. Skeletal muscle α-actin gene (ACTA1) Over 60 different myopathy-causing mutations in actin are distributed through all six coding exons of ACTA1. No overlap has been found between the mutant ACTA1 residues that produce skeletal myopathies and those that cause cardiomyopathies, despite the proteins being 99% identical [1]. This finding facilitates the identification of different muscle myopathies. ACTA1 mutations cause AM, IRM, and NM. In a survey of 38 individuals with ACTA1 mutations, Beggs and colleagues [28] found that ACTA1 causes proportionally more severe myopathy than would be expected to occur randomly between the five NM-linked genes, and that ACTA1 mutations

6 412 E Clarkson et al probably account for about 25% of all CMs. They also highlight the first examples of incomplete penetrance, where three unaffected heterozygous individuals had a mutation (Val134Ala) that only showed as a CM in a fourth individual in this family [28]. However, when investigating the effects of functional null alleles of the ACTA1 gene, the recessive nature of an ACTA1 mutant, R39X, provided two important insights into ACTA1 functional genetics. (The R39X mutant is due to a nonsense mutation of codon 39 producing a premature translation termination sequence that gives rise to a severe NM phenotype.) Firstly, this mutant suggested that one wild-type ACTA1 gene copy is sufficient for normal actin function. Secondly, it suggested that the dominance of the vast majority of ACTA1 mutations is not due to an absence of all actin functions, but to dominant negative effects [1]. This indicates that mutant alleles produce toxic polypeptides that interfere with the activity of wild-type actin generated by the nonmutant gene copy. A similar conclusion was drawn from ACTA1 knockout mice, where the heterozygotes are apparently normal, but the homozygous nulls die during the neonatal period [29]. Furthermore, several of the myopathy-causing mutations give a dominant negative effect in Drosophila and yeast [1]. While little or no apparent correlation between phenotype and position of mutation has yet been found, Sparrow et al [1] have used the actin structure to predict what effect different myopathy mutations might have on actin function. They predicted that all AM mutations would affect nucleotide binding and/or hinge flexibility. Similarly, most of the IRM mutations were predicted to affect these too, while the remainders may be involved in the communication between events at the nucleotide pocket, hinge, and sub-domain 2 movement [1]. All AM-causing mutations are in subdomains 1 and 3, located close to the central hinge region at the base of the cleft and nucleotide-binding pocket. All the missense mutations identified occur in very highly conserved residues, but overall, the actin sequence is extremely well conserved in evolution and nearly every residue is probably crucial to some aspect of its function. The majority of ACTA1 mutations result in NM. The mutant residues observed tend not to occur on the surface of actin where the ABP-binding sites are situated. Hence, identifying their functional impact is difficult. Changes in G-actin conformation and actin actin contacts will affect polymerization and the stability, functions, and conformations of F-actin. Likewise, nucleotide exchange will be affected by hinge flexibility and interactions that stabilize cleft closure. Most actin polymerization functions are closely related to the binding and hydrolysis of nucleotide. Some of the mutations are predicted to affect interactions with other actin-binding proteins in the sarcomere. In particular, some could affect binding to tropomyosin or nebulin. There are no mutations in the putative large myosin-binding site, and only a few ACTA1 NM mutations can have a primary effect on actin αactinin interaction. This highlights that the formation of nemaline bodies is much more complicated than the suggested theory of formation through impaired actin α-actinin interactions in the Z-discs [1]. Only a few in vitro studies of myopathy mutant human actin function have been done and there is some agreement with predictions based on the 3D structure. Mostly, these analyses point to the heterogeneous nature of the different defects in actin function that are likely to result from the various mutations. Costa et al [30] studied 19 different mutations and found that they divided into four categories of severity based on biochemical and cell biological assays. The most severely defective were L94P and E259V mutations, which resulted in non-folding actin and thus totally non-functional protein. The recessive nature of these two mutations [1] demonstrates again that dosage is not likely to be important and that dominant mutations probably result from poisonous effects of mutant proteins. Beggs and colleagues demonstrate that cardiac actin is likely to account for at least some compensation for patients with effectively null mutations in skeletal actin [28]. A second, less severe, category of mutants (G15R, N115S, V163L, G182D) folded, but behaved as if they were more unstable on native gels and, in particular, as if they had nucleotide binding instabilities [30]. Several mutations resulted in a polymerization defect (I64N, I136M, G268C, R183G, Q263L, N280K, V370F, G268R, I357L) and a few of the mutations did not show any defects in the assays done (H40Y, M132V, R183C, D286G). Overall, no correlation was found between the type of defect and the severity or pathology of the disease, suggesting that it will not be straightforward to predict disease outcomes based on the type of functional defect in actin. However, expression of mutant actins in tissue culture fibroblasts did induce the formation of rods and aggregates that may be useful for studying the kinds of structures observed in NM muscle in vitro. Some examples of these structures are shown in Figure 3. A similar study by Cooper and colleagues characterized ten mutations in actin with respect to the phenotype of C2C12 myoblasts overexpressing the mutant protein, the expression of other actin cytoskeletal proteins in patient samples, and the fraction of the mutant actin that was soluble versus insoluble [31]. This study highlighted the importance of the balance between the expression of various cytoskeletal proteins in myopathy muscle, as the authors found reproducible variations in many other thin filament proteins in patient samples. In some cases, they saw up-regulation of cardiac actin. In many cases, they saw general increases in γ -filamin, α-actinin, myotilin, and desmin. They speculated that this may reflect accumulation of Z- line materials in nemaline bodies. Interestingly, very low levels or undetectable nebulin expression occurred in several patients, indicating that protein levels are not a reliable test of which gene might be mutated

7 Congenital myopathies 413 [31]. This study and that of Costa et al [30] show that it is promising to use cells in culture as a model, but that not all NM mutants form nemaline-like bodies. Clearly more characterization of the rods and aggregates formed and more conditions for the culture are needed to determine the relevance of this approach to patient samples. Another recent study characterized the M132V mutation in detail and found that actin with this mutation purified from patient muscle samples did not polymerize as well as wild-type actin, and co-polymers with wild-type actin made in the presence of phalloidin showed a change in the relative force produced when reconstituted with tropomyosin and troponin in in vitro motility assays [34]. This powerful new technique for analysis of patient actin directly from biopsy samples shows promise of yielding physiologically relevant information on actin defects in patients. It is interesting that Marston et al [34] and Costa et al [30] came to somewhat different conclusions on the M132V mutation; this will need to be resolved by further studies. Laing and colleagues [35] have recently reported the successful expression of mutant actins in a baculovirus system, which may prove useful for further biochemical studies as well. One of the most promising avenues of study for actin mutations may turn out to be in using transgenic mice to express the mutant proteins in muscle, but this has not yet been reported for actin mutations. Figure 3. Immunofluorescence micrographs of NIH3T3 fibroblasts expressing the nemaline myopathy mutant actins I64N and V163L. Filamentous actin is shown in green after phalloidin staining; mutant actin is shown in red after probing with anti-myc tag antibodies; and the nucleus is shown in blue following Hoechst staining. Numerous rods and aggregates of mutant actin can be visualized in both panels of cells, with the I64N rods not co-staining with phalloidin, but the V163L rods showing some co-staining. Scale bar = 25 µm. Photo credit Celine Costa in NM as the whole balance may be altered. This also indicates that the phenotype of NM is likely to result not only from the expression of mutant actin, but also from alterations of the expression of several proteins, perhaps emphasizing the need for global analyses (gene chips and proteomics) such as those currently being developed by the Beggs laboratory [32,33]. The expression of mutants in C2C12 cells resulted in the formation of rods and aggregates similar to those seen in patient samples in several cases, although some of the mutants behaved similarly to the wild-type actin Nebulin The nebulin (NEB) gene is thought to be responsible for the majority of cases of congenital myopathy. Nebulin is a giant filamentous protein with a molecular mass of kd and 6669 amino acid residues [20]. The large size of nebulin makes it difficult to study, so fewer mutations have been identified in nebulin than in smaller genes such as ACTA1. Nebulin is exclusively expressed in the thin filaments of skeletal muscle and it comprises approximately 3% of the total myofibrillar protein. One nebulin molecule spans the entire length of the thin filament and is likely to be a major determinant of the length of the thin filament. The C-terminal end anchors into the Z-disc, and is thought to be important in the assembly and integration of Z-discs within the sarcomeres. Linkage data imply that mutations in NEB might be causing the typical autosomal recessive form of CM, but nebulin mutations can potentially cause all of the different subtypes of NM [20]. Human nebulin is encoded by a 20.8 kb gene and the gene comprises approximately exons [20,26,36]. Several differentially expressed exons are present in the central part of the gene, encoding repeats, and in the 3 end of the gene, encoding the part of the protein, which attaches to the Z-disc. The various splice forms are thought to be important during development and a reduction in the overall diversity of splice variants may account for some myopathy

8 414 E Clarkson et al phenotypes. At present, very little is known about the expression of the different nebulin isoforms and splice variants. As of 2002, 18 mutations within the nebulin gene had been reported. Most of the mutations described to date (if not all) result in expression of the mutant protein, often with truncations or internal deletions, and likely in reduction of the isoform and splice form diversity of nebulin. For example, antibodies to the SH3 domain region of nebulin showed an absence of immunolabelling in some patients who were clearly positive for nebulin as labelled with other antibodies [25]. Many mutations are in the differentially expressed exons, and would thus result in the lack of specific variants of nebulin [20]. Thus, it is likely that reduced complexity of nebulin isoforms/splice variants, resulting from nonsense mutations within individual exons, causes problems during myofibrillogenesis and leads to myopathy. However, there is still much to learn about nebulin gene expression in muscle development, so this hypothesis awaits further testing. Nebulin is important in determination of the Z-disc width. The C-terminal repeat of nebulin that extends into the Z-disc differs between fast and slow muscles, and between fetal and adult muscles. In addition, Z-discs vary in structure and width in different muscle and fibre types as well as during development. A positive association has been described between the Z-disc width and the number of nebulin C-terminal repeats [20]. Nevertheless, nebulin cannot be the only Z-disc width determinant, the reason being that nebulin does not span the whole width of the disc, nor do nebulin molecules from opposite sarcomeres overlap within the Z-disc [21]. Therefore alterations within nebulin cannot solely be responsible for nemaline rod formation. The process must also involve other thin filament proteins and their connecting partners such as α-actinin. However, the mechanisms of tight control for the co-assembly of titin, nebulin and α-actinin, and actin are not fully understood [22]. Recently, a newly described protein, myopalladin, has been shown to bind to the nebulin SH3 domain and therefore link nebulin to α-actinin in the Z-disc. Further investigations are required to identify the impact of this interaction in mutated nebulin or myopalladin genes [37]. Tropomyosin Tropomyosins are expressed in most eukaryotic cells, where they interact with filamentous actin and are generally thought to restrict or regulate the interactions between actin and other actin-binding proteins. Skeletal muscle tropomyosins are better understood than non-muscle forms, and their main function in muscle appears to be to confer co-operativity in calcium signalling upon the troponin complex [38]. This allows the switching of one troponin by calcium to control actin monomer units in the filament. Mutations that cause myopathies may alter this co-operativity, or they could alternatively affect actin binding or other interactions. In vertebrates, different isoforms are expressed in skeletal muscle, cardiac muscle, smooth muscle, and non-muscle cells. In humans, these are encoded by at least four different genes, TPM1 4. The regions that differ from one another between isoforms appear to encode functional domains involved in head-to-tail binding and coiled-coil interactions of tropomyosin molecules and their binding to actin, caldesmon, tropomodulin, and troponin T [39,40]. Alternative splicing of TPM1 and TPM2 produces tissue-specific isoforms that are expressed in striated, smooth, and non-muscle cells. TPM1 and TPM3 encode the alpha subunit of the muscle tropomyosin alpha beta dimer, while TPM2 encodes the beta subunit. TPM1 is predominantly expressed in cardiac muscle and fast, type 2 skeletal muscle fibres, whereas TPM3 is predominantly expressed in slow, type 1 skeletal muscle fibres [41]. α-tropomyosin gene (TPM3) The TPM3 locus is on chromosome 1 and encodes the slow type 1 fibre-specific isoform of skeletal muscle α-tropomyosin [42]. TPM3 mutations give rise to both dominant and recessive NM. The first identified tropomyosin mutation in NM was a missense mutation (M9R) with autosomal dominant transmission, which has been found responsible for a late childhood-onset form of NM [43]. This mutation is thought to weaken actin affinity and cause local destabilization of the tropomyosin coiled-coil dimer [43]. Expression of the mutant protein in baculovirus gives a biologically active protein, which is very ineffective at binding to actin (at least 100 times less effective than wildtype) [44]. A transgenic mouse model for this mutation gives a very similar phenotype to the human disease, with nemaline rods and cytoplasmic bodies, muscle weakness, and hypertrophy of fast, glycolytic fibres [45]. Other mutations include a homozygous, nonsense mutation, which gave rise to a severe infantile form of NM. This patient likely had no functional slow α- tropomyosin, as the peptide encoded by the mutant gene would only have been 30 amino acids and this would be unlikely to have any structure or be stable, as a homologous peptide from the TPM1 gene was disordered and non-functional [46]. Limited sample availability prevented the detection of this peptide in muscle samples or the determination of its presence (or absence) in the apparently normal heterozygous parents. The nemaline bodies in the patient were present only in the type 1 fibres, consistent with the presence of the TPM3 product being restricted to these [46]. β-tropomyosin gene (TPM2) The TPM2 gene is situated on chromosome 9 and is expressed at high levels in skeletal muscle [41].

9 Congenital myopathies 415 Recently, two heterozygous dominant missense mutations and four polymorphisms have been identified in the β-tropomyosin gene (TPM2) in patients suffering from CM. These mutations appear to have arisen de novo and affect conserved amino acids [41]. The nemaline bodies produced due to these mutations were mainly found in the type 1 slow muscle fibres. TPM2 is more highly expressed in slow muscle fibres (65%) than in fast muscle fibres (29%) [41]. It is thought that the two TPM2 mutations observed may alter the actinbinding properties of the β-tropomyosin molecule and cause local disruption of the α-helix coiled-coil dimer [41]. Michele et al [47] studied two mutant tropomyosins (TPM1 gene product): A63V, which causes hypertrophic cardiomyopathy (HCM) but not NM, and M8R, which causes NM but not cardiomyopathy. Expression in cardiac myocytes caused abnormal muscle relaxation for both mutants. However, the two mutants behaved very differently, with the HCM mutation showing a slowed relaxation phase and the NM mutant appearing normal at 37 C but causing faster relaxation at a lower temperature of 30 C. It was speculated that at least some NM mutations may be functional in cardiac cells, but in skeletal muscles, where the environment is different, including lower temperatures of the extremities, these may cause NM symptoms. These differences may be (part of) the reason why NM mutations do not cause HCM and vice versa. More importantly, heart muscle contains only TPM1 α-tropomyosin, while skeletal muscle contains TPM2 and TPM3, so tissue specificity of the isoforms likely accounts for the reason why NM and HCM have different genetic causes (see ref 48). Although TPM3 and TPM2 mutations are a relatively rare cause of CM (3% roughly), the two small genes and transcripts have allowed rapid mutation testing, which has now become an important part of molecular genetic development in CMs [42]. Slow skeletal muscle troponin T (TNNT1) The troponin (Tn) complex functions to prevent actin myosin interaction in resting muscle (for reviews see refs 49 and 50). Three types of TnT genes, which are expressed in a tissue-specific manner, have been identified: TNNT1, 2, and 3. TNNT1 is expressed in slow skeletal muscle, TNNT3 in fast skeletal muscle, and TNNT2 in cardiac muscle. These are subject to alternate splicing, producing additional complexity. The cardiac TnT gene is expressed also in embryonic skeletal muscle. To date, only one TnT CM mutation has been discovered. This nonsense mutation causes the lethal phenotype known as Amish nemaline myopathy, which is an autosomal recessive disorder [51]. The mutation causes a complete loss of slow TnT, as neither the intact nor the truncated TnT protein is found in muscles of affected individuals. Cardiac TnT is thought to compensate for the loss until around age 2 3, when affected children typically die of respiratory insufficiency. The TnT mutant lacks the COOH-terminal T2 domain that binds TnC, TnI, and tropomyosin to form the core of the Ca 2+ -regulatory system [24]. The discovery that a lethal myopathy is caused by the loss of only one TnT isoform emphasizes the importance of TnT in the regulation of muscle contraction and the functionally differentiated fibretype specific isoforms. Alternatively, the promoters of the alternative (cardiac) gene may fail to compensate for the loss of one isoform after a while, evoking the question of whether alteration of the expression levels of these genes may be useful targets for therapy. Ryanodine receptor gene Certain mutations discovered in the skeletal muscle ryanodine receptor gene (RYR1) are also thought to give rise to nemaline bodies. Ryanodine receptors are calcium channels located on the sarcoplasmic reticulum. Muscle contraction results when calcium is released into the cytoplasm through these. The possibility of RYR1 gene mutations causing rod formation indicates that nemaline rods are not specific to CM. Rods are encountered in a number of conditions, including the autosomal dominant form of central-core disease, the mixed muscle disorder core-rod myopathy, and some cases of malignant hyperthermia [8,9,52,53]. Treatments At present, there is no cure, nor any drug treatment for congenital myopathies. Helpful measures can be taken to increase the patients quality of life, such as physiotherapy, the use of antibiotics to treat chest infections, artificial ventilation or naso-gastric tube feeding, and supportive care in the neonatal stage [54]. Another general therapeutic target for muscle wasting disorders is myostatin, a member of the TGF-beta family of growth factors (for a recent review see ref 55). Myostatin functions to inhibit normal muscle growth by suppressing myoblast proliferation and inhibition of cell-cycle progression. It also inhibits myoblast differentiation through decreased expression of transcription factors that promote differentiation. Myostatin inhibition shows some promise for increasing muscle mass and strength of the mdx mouse model and for decreasing degeneration, but whether it would work for congenital myopathies, or even in humans, remains to be tested. At present, there is no gene therapy for congenital myopathies, and since many of the mutations appear to produce a dominant negative protein, it is unlikely that these will be developed in the near future. However, several approaches are being explored for muscular dystrophy (reviewed in ref 56) which, if successful, might be applied to certain congenital myopathies. Myopathies resulting from a deficiency in functional protein might also be helped by activation of expression of compensating isoforms,

10 416 E Clarkson et al as in the case of cardiac actin and skeletal actin, but this awaits further testing in model systems. Further research The first step towards understanding the disease process in the various subgroups of congenital myopathies is the identification of the genes involved. Various techniques are currently being employed to analyse the protein products of the disease genes. Model systems have been developed in order to improve the understanding of the mechanisms of the disease process and to reveal potential therapeutic possibilities, including pharmacology [54]. Microarray technology is being applied to CMs to study how global gene expression patterns determine the downstream or secondary consequences of particular genetic mutations and to identify novel muscle genes for further study. These genomic and proteomic approaches are yielding important new insights into basic muscle biology as well as the pathophysiology of inherited muscle diseases [32,33,56,57,58]. From linkage analysis, it is likely that a (as yet unidentified) sixth gene is responsible for congenital myopathies. Recent work has been carried out on a possible candidate, the α-actinins, which are essential Z-line proteins. Additionally, current studies include several new sarcomeric proteins identified through interactions with α-actinin, including myopalladin [20]. The molecular basis of the disorders continues to be examined in tissue culture models and mouse models, as well as other model organisms. For example, the indirect flight muscles of Drosophila are often used to express mutant actin isoforms. Their effects on myofibrillogenesis can then be determined, followed by purification and biochemical characterization of the mutations [59,60]. These studies are still at a relatively early stage in terms of generating a complete picture for how certain mutations affect the biochemical and cell biological functions of any of the NM disease proteins. 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