| | 156th ENMC International Workshop: Desmin and protein aggregate myopathies, 9–11 November 2007, Naarden, The NetherlandsReceived 4 April 2008 1. Introduction  The fifth workshop [1], [2], [3], [4] devoted to desmin, the intermediate filament of striated muscle cells and one of the first components identified among accruing proteins in protein aggregate myopathies (PAM), assembled 19 clinicians and scientists from seven European countries and the USA. The aims of the workshop were to assess recent advances in this group of increasingly recognised neuromuscular conditions, to correlate genetic, clinical, morphological and imaging findings and to consider therapeutic aspects. 2. Background Opening the workshop, Hans H. Goebel (Mainz/Germany), co-chairman and co-convener of this workshop concentrated on the increasing recognition of desmin-related myopathies, now also called myofibrillar myopathies, as a major subgroup among the PAM. This group nosographically grew out of morphological observations of abnormal accumulations in muscle fibres, first identified by electron microscopy and later better characterised by immunohistochemistry as containing desmin and numerous other proteins. These lesions had earlier been labelled cytoplasmic bodies, spheroid bodies, or desmin plaques. When mutations in the desmin gene were identified in some of these cases a molecular basis for some of these neuromuscular conditions was defined and this group was designated as desminopathies indicating the importance of these mutations in the desmin gene. From this point, further work on moving from a morphologically identified protein to the mutation in its gene was affirmed and subsequently expanded when other proteins were found accumulating in muscle fibres and aggregates of proteins. Since the fourth desmin-related ENMC-sponsored workshop in 2003, myotilinopathies, filaminopathies, ZASPopathies, αB crystallinopathies, and recently, valosin-containing proteinopathies, defined by mutations in the respective genes, were added to the desminopathies and, thus, now encompass a major subgroup of PAM. Several neuromuscular centres have assembled cohorts of patients and families with PAM, the majority of which are of autosomal-dominant inheritance with variable frequencies of the individual disease entities. Mouse models and in vitro studies have not only added to the understanding of these PAM but also contributed new information on the basic function of the individual proteins and their metabolic and binding partners in the muscle fibre. Protein aggregation in these PAM is most likely evidence of faulty extralysosomal degradation of proteins. Several of these are mutant ones, giving rise to their respectively named proteinopathies. They involve the catabolism of proteins and, therefore, constitute catabolic PAM. Catabolic PAM are largely adult or even late-adult onset neuromuscular conditions. A recent development not further discussed in this workshop, is the recognition of other PAM marked by accumulation of actin filament aggregates and granular myosin aggregates, the former members of the nemaline myopathies, the latter termed hyaline body myopathy and now renamed myosinopathy. These forms of PAM may have anabolic, i.e. synthetic defects of actin and myosin filament formation and their integration in sarcomeres. These conditions which are also marked by aggregation of few if any additional proteins, mainly develop in early childhood. It therefore follows that protein aggregation in muscle fibres is now recognised as an important pathogenetic factor in PAM. These diseases share some characteristics with protein aggregate encephalopathies, such as Alzheimer, Parkinson, and Huntington diseases as well as protein aggregate neuropathies, such as giant axonal neuropathy. Apart from the afore-mentioned desminopathies and others, aggregation of proteins appears to occur whenever sarcomere integrity is impaired. Cores of central cores, multi-minicores, and target/targetoid types show accretion of multiple proteins. Caps in cap disease and ragged red fibres owing to subsarcolemmal accumulation of abnormal mitochondria are further examples of protein aggregation. Moreover, in inclusion body myositis (IBM), aggregation of diverse proteins is a hallmark of the disease, perhaps implying it is a myodegenerative disease rather than an inflammatory condition. IBM is the best known example of a non-genetic, i.e. sporadic PAM. Other cases of PAM for which an underlying genetic defect cannot be identified probably fall into two categories: Those which after exclusion of mutations in known genes may still have originated from mutations in as yet unidentified genes and those which are truly non-genetic, non-familial sporadic degenerative neuromuscular diseases like non-genetic IBM. Working on this premise, presentations concerning protein aggregate degradation in relation to oxidative stress and autophagy supplemented presentations on individual PAM and their proteins. 3. Desmin and protein aggregation myopathies (PAM) 3.1. Review In his retrospective talk, Michel Fardeau presented the data collected from the princeps family in which, by electron microscopic study of the muscle biopsies the accumulation of a dense granulofilamentous (g.f.) material was discovered within the muscle fibres, in 1978 [5], i.e 2 years before the description of the intracellular desmin cytoskeletal network by Lazarides [6]. Presence of desmin within this dense material was demonstrated first by quantitative biochemical assay [7], then by immunofluorescent staining with desmin antibodies. Analysis of the desmin gene localised in 2q35 failed to find any mutation; further studies revealed linkage not to chromosome 2, but to chromosome 11, and a mutation was found in the gene coding for the chaperone protein αB-crystallin [8]. Several points were stressed in this presentation. Clinically, there was the presence of spotty white deposits at slit-lamp examination of the lens in several patients of this family. Morphologically, the dense g.f. material was present in every muscle fibre of the biopsies. In the less affected muscle fibres, without evidence of any myofibrillar disorganization, the g.f. material was visible as small dense dots facing the Z-lines, but without any continuity between these dots and the Z-lines. These data were compared, during the discussion, with the data reported on the few other cases of αB-crystallinopathy since the original publication [9], [10]. Duygu Selcen from the Mayo Clinic indicated that the term myofibrillar myopathies (MFM) is the morphologic denominator of a group of dominant disorders associated with myofibrillar degradation that begins at the Z-disc, accumulation of the arising degradation products, congophilia of some of these products, and appearance of multiple proteins in abnormal fibre regions. The most frequently and strongly expressed proteins are myotilin, desmin, αB-crystallin, and dystrophin. The abnormal muscle fibres are best identified in trichrome-stained frozen sections that harbour pleomorphic amorphous, granular, or hyaline structures as well as vacuoles containing membranous material. In a cohort of 82 MFM patients investigated at the Mayo Clinic, the disease typically presented with slowly progressive distal and/or proximal weakness. Cardiomyopathy was present in 19% and evidence for neuropathy in 50%. The EMG was typically myopathic and associated with abnormal electrical irritability, including myotonic discharges. Mutation analysis in different laboratories to date revealed mutations in different Z-disc associated proteins. In 82 patients studied at the Mayo clinic [11], 14%, 10%, 7%, 3%, and 4% harboured mutations in ZASP (a Z-band alternatively spliced PDZ motif-containing protein) [12], myotilin, desmin, αB-crystallin, and filamin C, respectively. Because Z-disc changes are present in many muscle diseases, Dr Selcen suggests the following minimal criteria for diagnosis of MFM: (1) Pleomorphic abnormal fibre regions with characteristic tinctorial properties in trichrome stained sections. (2) Vacuolar change in some fibres in most cases. (3) Immunolocalization of multiple proteins in the pleomorphic fibre regions. (4) Congophilia of some hyaline deposits in most cases. Denise Paulin and colleagues summarised information on “Synemin and other desmin-associated proteins”. Interaction between intermediate filaments (IF) and other cytoskeletal elements and organelles are coordinated by intermediate filament associated proteins (IFAPs). Desmin named also skeletin (52 kDa) is the main IF protein in mature striated muscle and it is found at the sarcolemma, Z-lines, neuromuscular junction (NMJ) and myotendinous junction (MTJ) [13]. Several other IF or IFA proteins are characterised in muscle: vimentin (54kDa), nestin (178kDa), syncoilin (64kDa), and synemin (180, 150, 41kDa), all containing the coiled-coil domains typical of IF proteins. The classification of proteins as IF has been debated over the years. Thus synemin and paranemin (nestin, or tanabin) which were originally identified as IFAPs have now been dubbed IF [14]. The vestigial 10 amino-acid head domain of synemin (as nestin) suggests that it is incapable of self-assembly and requires a partner such as vimentin or desmin for incorporation into filaments. Syncoilin has an N-terminal head domain with no homology with any known protein and cannot assemble to form a filament alone, but can form heterofilaments. Synemin and syncoilin are linked to desmin and their association is strongly related to the presence of desmin. Syncoilin is a member of the dystrophin associated complex via its interaction with dystrobrevin and is selectively lost at the Z lines of desmin deficient muscle and accumulates at the sarcolemma [15]. The synemin gene is located on human chromosome 15. It encodes three isoforms produced by alternative splicing [16], according to the type of tissues: muscles, lens, astrocytes, and neurons [17]. Synemin interacts directly with vinculin in vivo and is promoted by 4–5 biphosphate [18]. Synemin, too, interacts with α-actinin, α-dystrobrevin, utrophin, and dystrophin. The tail domain of synemin provides a binding site for both desmin and α-actinin, thus integrating IF and the actin system at costameres and Z-lines in striated muscle. Several studies have screened many groups of DRM patients and have found synemin and syncoilin associated with muscle protein aggregates in human desminopathies [11], [19], [20]. Plectin (500kDa) is a cytolinker protein the absence of which leads to skin blistering and muscular dystrophy. Using plectin isoform-specific knock-out mouse models it was found that desmin aggregates accumulate in distinct cytoplasmic compartments dependent on which plectin isoform is missing. Plectin isoforms 1d, 1b, 1f and l specifically anchor desmin IF to Z discs mitochondria costameres and nuclei respectively. Mutations in the plectin gene cause as in the desmin null mice mitochondrial dysfunction, fibre degeneration and progressive muscular dystrophy. The association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a serine residue in the C-terminal extremity of desmoplakin, which can potentially be phosphorylated, affects its association with desmin; mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakin-desmin interaction [21]. Together the data suggest that IF networks employ multiple mechanisms to regulate their organization sometimes incorporating spacer IFAPs into the core of polymer or recruiting peripheral proteins from which the polymer may dissociate during remodelling events. Caroline Sewry summarised the clinical and pathological data on molecularly confirmed cases of PAM in the UK, kindly provided by several colleagues in various UK centres. All cases had been submitted for molecular analysis on the basis of the clinical and pathological data. Twelve cases from 5 families (additional family members not yet analysed) had mutations in desmin, 8 in myotilin, 2 in ZASP, and 2 in VCP genes. Two additional cases had ‘double trouble’, one with a mutation in the gene for desmin and a de novo mutation in the lamin A/C gene; and the other a mutation in both the genes for desmin and myotilin. Several other possible cases have been identified but are not yet molecularly confirmed. Various mutations in the desmin gene were identified. Two cases from different, unrelated families had a Lys449Thr change, and both showed a reduction of calpain-3 on immunoblot. A previously reported change in one allele (Arg 490 Trp) of the calpain-3 gene was identified in one of these cases. The second case has not yet been screened for changes in the calpain-3 gene, and calpain -3 has not yet been studied in other affected members of these families. The significance of this finding is therefore unclear. All the myotilin mutations had the Ser60Cys change identified in exon 2, and the VCP mutation was the common mutation in exon 5. There was a positive family history, compatible with dominant inheritance, in all the desminopathies and in 7 out of 8 myotilinopathies. In common with other studies, the cases with mutations in the desmin gene tended to have an earlier onset (13–50years) than those with mutations in the myotilin gene (53–72years). Onset in the VCP cases was about 30years. Cardiac involvement was common in the desmin cases but rare in the myotilin cases. Weakness was often distal and proximal, and in the myotilinopathies distal weakness was sometimes greater than proximal weakness. Serum creatine kinase levels were variable but often showed a mild to moderate elevation, and EMG was myopathic. One case showed a neurogenic pattern at a later stage, with myotonic discharges. The pathological features included those previously published which were variable in degree, ranging from mild to ‘dystrophic’ with a wide range in fibre size, fibrosis and an increase in adipose tissue. Vacuoles were not a consistent feature. The accumulation of desmin was variable and in some desminopathy cases had a punctuate appearance. Granulofilamentous material was seen in the desminopathy cases that were studied with electron microscopy. In the myotilinopathies accumulation of protein such as desmin and myotilin was often pronounced. Other features of note were the presence of ring fibres in one case with a myotilin mutation, an abundance of multiple internal nuclei in several cases, and the presence of isolated multiple split fibres resembling myotendinous junctions. The significance of this is not clear. 3.2. Desmin Anna Fidzianska and Patrick Vicart identified a desmin R355P mutation in a 44-year-old patient with familial cardiac and skeletal myopathy. Two types of desmin storage were observed in skeletal muscle. By Gomori trichrome staining, small punctate dark inclusions were mainly located in fast fibres while a spiderweb-like network was observed in slow muscle fibres. At the ultrastructural level abnormal desmin aggregation was characterised in fast fibres by the presence of round or ellipsoid shaped bodies located in the interior or on the periphery of muscle fibres with well preserved architecture. Round bodies contained granular dense material incorporated in thin curled filaments. Abundant accumulation of granulofilamentous material in subsarcolemmal and intermyofibrillar spaces as well as at the level of the Z-line was observed in slow muscle fibres. Ample accumulation of truncated desmin aggregates led to destructive changes in the architecture of slow muscle fibres. In peripheral regions of such muscle fibres, small collections of tubulofilaments (TFs) appeared. They were surrounded by myelin-like structures and cellular debris. Diffuse and extensive accumulation of granulofilamentous material identical to that observed in slow skeletal muscle fibres was present in cardiomyocytes. Immunohistochemical analysis revealed that punctuate inclusions in fast fibres were strongly positive for desmin while in slow muscle fibres as well as in cardiomyocytes diffuse intracellular distribution of desmin immunoreactivity was seen. These results suggest that the R355P desmin mutation exerts different effects on the arrangement of truncated desmin in slow and fast muscle fibres. These structural differences may be a consequence of differences in their function, architecture, innervation as well as various amounts of desmin protein [22] demonstrated that slow muscle had a 2-fold higher amount of desmin compared to fast muscle. The most surprising findings seen in our study is the appearance of TFs in slow fibres with desmin storage. Anna Kaminska and colleagues discovered diversity of cardiomyopathy phenotypes caused by mutations in desmin. They had conducted an analysis of entries in MedLine on the topics “desmin mutation” and “desmin-related cardiomyopathy” as of June 30, 2007. Sufficient information on 92 patients had been extracted. The results (Table 1) showed that 73% of patients with molecularly verified desminopathy showed cardiac involvement. In 38% of the 92 cases, cardiomyopathy was the early and leading or even the sole disease manifestation. Of 47 patients who underwent complete cardiovascular assessment with echocardiography, 27 (57%) were diagnosed with dilated cardiomyopathy (DCM), 13 (28%) with restrictive cardiomyopathy (RCM), and 7 (15%) with hypertrophic cardiomyopathy (HCM). Atrioventricular conduction abnormality is a frequent feature in desminopathy patients attributed to the fact that the heart conduction system is rich in desmin. A combination of DCM with conduction defects was described in 16 patients, and a combination of RCM with conduction abnormalities in 11. Based on these data, RCM by itself and RCM plus atrioventricular block are frequent but not the most common desminopathy phenotypes. One of the possible explanations for different desmin mutations causing either progressive skeletal myopathy with no cardiac manifestations, or cardiac myopathy characterised as DCM or RCM, could involve type and location of the mutation within the functional desmin protein. It has been conclusively shown that desmin myopathies are caused by mutations in several functional parts of desmin, each resulting in a distinct effect on filament assembly [23]. Data presented showed that patients with mutations in the 2B segment of desmin have primarily skeletal muscle involvement, while those carrying mutations in 1B and tail domains predominantly develop a more ominous cardiac disease. Cochran–Armitage test confirmed this trend. The difference in disease phenotype distribution in patients with 1B mutations vs. those with mutations in the 2B domain are highly significant (p=0.0039). The difference between the patients with tail vs. 2B mutations is less striking, but meaningful (p=0.049). They concluded that the location of the causative mutation exerts a significant influence on phenotypic characteristics. There is also a significant tendency for a higher frequency of RCM in patients with mutations in the 1B vs. the tail domains (p=0.0076). Patients with skeletal and cardiac myopathy have been identified among myofibrillar myopathies and caused by mutations in αB-crystallin [8], [10], myotilin [11], [24], or LDB3-ZASP [12]. Maggie Walter spoke about “Desminopathies without typical morphological features”. In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named ‘scapuloperoneal syndrome type Kaeser’. By genetic analysis of the original kindred, a heterozygous missense mutation of the desmin gene R350P was discovered cosegregating with the disorder, recently described [25]. Examination of 15 patients from five unrelated families harbouring R350P, including the original kindred described by Kaeser, showed large clinical variability, even within the same family, covering scapuloperoneal, limb girdle, and distal phenotypes with variable cardiac or respiratory involvement. While males do not have a higher overall incidence of cardiac or pulmonary involvement compared to females, risk for sudden death or fatal respiratory failure seems to be higher in males. Therefore, gender-related factors or modifier genes may be involved in determining disease onset and severity. Usually, desminopathies are diagnosed through muscle histology. Interestingly, histopathological examination of the biceps brachii muscle of the index patient from the original Kaeser family showed very limited myofibrillar changes or protein aggregation which was hardly demonstrated by desmin and filamin-c immunohistochemistry [26]. Therefore, there is a wide morphologic spectrum of findings, ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. Mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy with LGMD, distal, or scapuloperoneal phenotype, even if specific myofibrillar pathology is absent. As a next step, they wanted to know if there are other MFM without their specific pathology. A total of 205 unrelated, adult patients with clinical evidence for a myopathy in scapuloperoneal, distal or limb girdle distribution were screened for mutations in the myotilin gene. They found MYOT S60F in six independent patients, all presenting with a late onset distal myopathy. Interestingly, all 6 patients had been histologically classified as sporadic IBM. However, myofibrillar changes were not found in any of the 6 patients, desmin immunohistochemistry was performed in two of these patients, but did not reveal any conspicuous protein accumulation (unpublished). In contrast, 4/57 patients with myofibrillar myopathy described by Selcen and Engel [27] showed a mutation at S60. Therefore, there is differing pathology with or without typical features of MFM and protein aggregation along with an identical pheno- and genotype. As a contradicting finding, MFM was suspected in a 53-year-old female patient. Muscle biopsy showed a degenerative myopathy with rimmed vacuoles without inflammation. Ultrastructurally, filamentous inclusions indicative of IBM were reported, desmin staining was suggestive of MFM. Onset of symptoms started at age 33 years with foot extensor weakness, CK was elevated to 500 U/l; she showed slow progression of weakness for the last 20 years. During the course of the disease, proximal leg muscles and finger extensors became affected. So far, there is mild pulmonary involvement, but no cardiac involvement. Mutations in desmin, myotilin, ZASP, and VCP genes were excluded. Surprisingly, FSHD genetics showed a shortened fragment of 38kB confirming diagnosis of FSHD (unpublished). Therefore, testing for FSHD in adult-onset dominant myopathies with distal involvement, even if facial weakness is absent, is recommended. So far, different diseases which may show a scapuloperoneal phenotype have been genetically identified: FSHD, IBMPFD due to VCP mutations, MFM due to mutations in DES, MYOT, FLNC, X-linked recessive myopathy due to FHL1 mutations, myosin storage myopathy (MSM) due to MYH7 mutations, EDMD2/LGMD1B due to mutations in Lamin A/C, and the neurogenic Davidenkow’s syndrome, which was recently found to be allelic with HNPP (hereditary neuropathy with pressure palsy) due to a PMP22 deletion. However, the scapuloperoneal phenotype is not specific for these diseases, all forms can also present with predominant distal or limb-girdle involvement. Some questions remain unsolved – does morphological examination for MFM actually lead to the diagnosis? Desmin staining may not be sensitive enough, because there are primary and secondary desminopathies without desmin accumulation and it may not be specific enough, since there are other, genetically distinct myopathies showing desmin accumulation occasionally. Therefore, better tools may be needed for diagnosis and a different definition of MFM, including genetic and/or clinical data. Kristl Claeys, Michel Fardeau and Gisela Stoltenburg reported on “Ultrastructural findings in myofibrillar myopathies (MFM)”. They studied the ultrastructural characteristics in patients with MFM and differentiated between the MFM subtypes using electron microscopic findings. The ultrastructure of muscle tissues in 17 patients with different genetically proven MFMs were analyzed. This group included 8 patients with a mutation in desmin (S2I, L338R, L392P, T442I, R454W), 5 patients belonging to the same family with an αB-crystallin mutation (R120G), 3 unrelated patients with a ZASP mutation (A165V) and 1 patient with a mutation in the myotilin gene (S60F). The ultrastructural findings in desminopathies and αB-crystallinopathies were very similar and consisted of electron-dense granulofilamentous material and sandwich formation. They differed in the presence of early apoptotic nuclear changes in αB-crystallinopathies. ZASPopathies were characterised by filamentous bundles and floccular accumulations of thin filamentous material. The presence of tubulofilamentous inclusions with diameters between 15 and 18nm in sarcoplasm and myonuclei in combination with filamentous bundles was characteristic for myotilinopathy. Common features in the four MFM subgroups were the focal character of the alterations among and within the muscle fibres, Z-disc streaming and more extensive flag-like semidense extensions oriented perpendicularly to the Z-disc, and the presence of (autophagic) vacuoles. The authors concluded that the distinct ultrastructural features in the MFM subgroups can be used to guide the genetic analysis in patients with MFM, and that ultrastructural data should be included in the diagnostic workup in patients with MFM. 3.3. Desmin-associated myopathies/myofibrillar myopathies Montse Olivé and Isidro Ferrer spoke on “New clinical, morphological, radiological, and genetic aspects of myofibrillar myopathies.” Thirty-two Spanish patients suffering from MFM were described. Seven patients (from 6 families) had mutations in the desmin gene, 19 patients (from 14 families) had myotilin mutations and three other patients (from 1 family) had ZASP mutations. In the remaining 3 patients, no mutations were found in any of the known MFM genes. All desmin mutations were autosomal dominant except for a single sporadic mutation. Four missense mutations and a single amino acid deletion in the desmin gene were identified. Clinically, patients with desminopathy presented at a mean age of 24years. Cardiac involvement manifested with conduction blocks; hypertrophic, or, less often, restrictive cardiomyopathy was observed in all of the cases, and was the initial symptom in two. Distal weakness was greater than proximal one at presentation, but proximal weakness was present later. During the course of the illness 4 patients developed respiratory insufficiency. Ankle contractures were common. Nasal voice and dysphagia were common. No case had peripheral neuropathy. EMG was myopathic with spontaneous activity at rest. Muscle CT scan was useful and showed a characteristic pattern with an early involvement of the semitendinosus, sartorius and gracilis muscles at mid-thigh level, and peroneal group and anterior tibialis muscle at the mid-leg. Muscle biopsies showed patches of cytoplasmic or subsarcolemmal amorphous inclusions strongly reacting for desmin, αB-crystallin, filamin C, dystrophin and phosphorylated neurofilaments, revealed with the SMI 31 antibody among others. Importantly, myotilin was weakly expressed. Small rimmed vacuoles were frequently observed. Ultrastructurally, accumulation of granulofilamentous material was universally present. Similar abnormalities were seen in the explanted heart from one desminopathy patient. Some patients with myotilin mutations had a dominant pattern of inheritance, but mostly the disease was sporadic. Molecular studies revealed four different myotilin mutations, all of them in exon 2. The disease started at a mean age of 69 years. Foot drop was the initial symptom in most of the cases, but some presented with a combination of proximal and distal weakness and still others presented with proximal weakness. Ankle contractures were common. Nasal voice, cardiac involvement and respiratory insufficiency were extremely rare. A single patient had peripheral neuropathy. Except for this case, EMG showed myopathic changes with prominent spontaneous activity at rest. A characteristic pattern of muscle involvement which clearly differed from that observed in desminopathy was found on muscle CT scans. This was characterised by the early involvement of the semimembranosus and biceps femoris muscles at thigh level and soleus muscle at mid-leg level. Pathological studies revealed the presence of polymorphic eosinophilic inclusions, displaying strong thioflavine T positivity, and strongly reacting for myotilin, desmin, αB-crystallin, filamin C, dystrophin, and phosphorylated neurofilaments, among others. Cytoplasmic bodies, spheroid bodies and large numbers of rimmed and non-rimmed vacuoles were present in the majority of cases. Focal inflammatory infiltrates were observed in six cases. By EM, streaming of Z-lines, large numbers of autophagic vacuoles were seen in most of the cases, as well as accumulation of compacted and fragmented filaments and dense material. Finally, three members of a family with a ZASP mutation presented at a mean age of 59 years with distal muscle weakness in lower extremities, later involving proximal muscles of lower limbs and distal muscles of upper extremities. None of them had cardiomyopathy, peripheral neuropathy or respiratory insufficiency. The morphological abnormalities observed in the muscle biopsies closely resembled those observed in patients with myotilin mutations. A comparative analysis of this series of patients indicates that major differences exist between myotilinopathies and desminopathies regarding the age of onset, the pattern of muscle involvement on muscle CT scans, the presence of cardiac and respiratory involvement, and the type of myofibrillar inclusions. By contrast, myotilinopathies and ZASPopathies show marked clinical, radiological and morphological similarities. 3.3.1. B-crystallin Patrick Vicart and colleagues reported on three mutations (R120G, Q151X, 464ΔCT) in the small heat shock protein (sHsp) αB-crystallin (αBC) which have been found to cause inherited myofibrillar myopathy [28]. αBC forms homo-dimers, hetero-dimers with other sHsps, and larger oligomers. In an effort to elucidate the molecular basis for the associated myopathy, they have determined for these mutant αBC proteins (i) the formation of aggregates in transfected cells, (ii) the partition into different subcellular fractions, (iii) the phosphorylation status, and (iv) the ability to interact with themselves, with wild-type αBC, and with other sHsps that are abundant in muscle. They found that all three αBC mutants have an increased propensity to form cytoplasmic aggregates in transfected cells and an increased proportion of phosphorylation of their three phosphorylation sites when compared to the wild-type protein. While wild-type αBC partitioned essentially into the fractions of cytosol and membranes/organelles, mutant αBC proteins partitioned additionally into the nuclear and cytoskeletal fractions. Using various protein interaction assays, including quantitative fluorescence resonance energy transfer measurements in live cells, they found abnormally changed interactions of the various αBC mutants with wild-type αBC, themselves and the other sHsps Hsp20, Hsp22, and possibly with Hsp27. The collected data suggest that each αBC mutant has a characteristic pattern of abnormal interaction properties. These abnormal properties of the αBC mutants identified are likely to contribute to a better understanding of the slow-going manifestation and of the clinical heterogeneity of the associated myopathy in patients. 3.3.2. Myotilin Olli Carpén reviewed the current knowledge on the biological functions of myotilin, one of the Z-disc proteins mutated in PAM. Myotilin, together with palladin and myopalladin, forms a small subfamily of Ig-domain containing proteins [29]. Expression of myotilin is restricted to striated muscle. Myotilin interacts with several structural Z-disc components, including α-actinin, FATZ (calsarcin), filamin C, and actin. It has a strong actin filament bundling and stabilizing function [30]. At present it is unclear, how single missense mutations in the N-terminal part of myotilin lead to dominantly inherited PAM. Further insight into the functional interplay between proteins causing PAM is provided by findings showing that myotilin directly interacts with ZASP. The interaction is regulated by phosphorylation of the myotilin C-terminus thus suggesting a mechanism by which the Z-disc architecture can be modulated during physiological or pathological stress responses. Interestingly, targeted deletion of myotilin in mice does not cause an obvious phenotype. The mice have a normal life span, and their muscle structure and function are preserved throughout life [31]. On the other hand, transgenic mice expressing one of the human mutations (myotilin T57A) reproduce the morphological and functional features of human myotilinopathy [32]. These results indicate that there may be functional redundancy within the myotilin protein family and that the presence of mutant myotilin is more harmful than loss of protein. The former conclusion is also supported by preliminary findings indicating that combined deletion of myotilin and palladin in mice results in muscle defects. 3.3.3. Filamin Dieter O. Fürst and colleagues discussed “Filamin C and associated proteins in neuromuscular disease”. Filamin C (FLNC) is a large actin binding protein in cross-striated muscle cells located mainly at the myofibrillar Z-disc. Previous work has indicated that FLNC is a sensitive indicator of alterations in the context of various neuromuscular diseases [33], [34]. More recently, they have identified an FLNC mutation in myofibrillar myopathy (MFM) families (8130 GrA; W2710X). The mutation affects the C-terminal dimerization domain of FLNC, and recombinant protein comprising this portion of FLNC revealed reduced beta-sheet content of the mutant rendering the protein more prone to proteolysis and thermal denaturation in vitro. The truncation also leads to aberrant dimerization resulting in the formation of large aggregates. As a consequence, muscle fibres of affected patients display massive cytoplasmic protein aggregates containing filamin, Z-disc-associated- and even sarcolemmal proteins. Transfection of mutant FLNC constructs into cultured cells also resulted in the formation of protein aggregates. In summary, these data provide a first mechanistic explanation for abnormal protein aggregation in FLNC-associated MFM. In extension of the above-mentioned work, they have begun to screen a panel of biopsies from patients with various neuromuscular diseases with a panel of myofibrillar antibodies. Interestingly, only the two filamin-associated proteins in and XIRP2 exhibit a similarly striking disease-associated subcellular redistribution. These findings suggest that antibodies specific for filamin C, Xin, and XIRP2 may be excellently suited as diagnostic markers. Rudolf A. Kley and Matthias Vorgerd discussing “The phenotype of myofibrillar myopathy associated with p.W2710X mutation in filamin C in 31 German patients” had examined 31 patients from four German families to evaluate the phenotype of filaminopathy [35], a novel form of myofibrillar myopathy (MFM) [36]. All patients harboured the same p.W2710X mutation in the dimerization domain of the filamin C gene on chromosome 7q32.1. Haplotype analyses strongly indicate a founder mutation. First clinical symptoms mostly occurred in the 5th decade of life (mean age, 44±6 years; range, 24–57 years). Nine affected persons died at a mean age of 66 years. The distribution of slowly progressive muscle weakness was evaluated in 28 patients: in 25 patients, weakness was more pronounced proximally than distally as seen in limb-girdle muscular dystrophies (LGMD), in one patient proximal and distal weakness was equally present, one patient presented with a clearly distal myopathy and one patient with weakness of the neck flexors. Five patients had slight involvement of facial muscles and 10 patients reported back pain as an initial symptom. About 14/31 patients suffered from weakness of respiratory muscles. One patient reported hypaesthesia in distal lower extremities but neurographic measurements were inconspicuous in 14/14 patients. EMG studies in 16 patients revealed spontaneous activity (12/16) and myogenic low amplitudes (4/16). Cardiac evaluation of 25 patients showed abnormalities in 9 patients comprising right bundle branch block (3/25), left ventricular hypertrophy (3/25), diastolic dysfunction (2/25) and atrial flutter (1/25) indicating cardiac involvement in filaminopathy. Serum CK levels varied from normal up to 10-fold of the upper limit. Magnetic resonance imaging studies showed a similar distribution of lipomatous changes in 10 patients: the semimembranosus, biceps femoris, soleus, and adductor magnus muscles were most affected whereas the rectus femoris, gracilis, and sartorius muscles were relatively spared. The histological features were typical of MFM. The intracellular aggregates in abnormal fibres were composed of a variety of proteins including filamin C, desmin, myotilin, αB-crystallin, Xin, dystrophin and sarcoglycans. A decrease of oxidative enzyme activities and fibre hypertrophy occurred at an early stage, whereas dystrophic changes were present in advanced stages of filaminopathy. In conclusion, the phenotype of the German filaminopathy cohort is characterised by clinical features similar to LGMD and by myopathological changes typical of MFM. 3.3.4. IBMPFD Rolf Schröder reported on the clinical, genetic, biochemical, and myopathological findings in IBMPFD (inclusion body myopathy associated with Paget disease of the bone (P) and frontotemporal dementia), which is caused by mutations of the human valosin-containing protein (VCP) gene on chromosome 9p13-p12. This rare autosomal dominant multisystem disorder is morphologically characterised by VCP- and ubiquitin-positive protein aggregates in neuronal and striated muscle cells [37], [38]. There are several links between IBMPFD and myofibrillar myopathies. IBMPFD skeletal muscle was demonstrated to contain muscle fibres with increased subsarcolemmal and cytoplasmic desmin and αB-crystallin immunoreactivity and further electron microscopic analysis revealed granulofilamentous material, which represents the characteristic ultrastructural hallmark of desmin-positive PAM [37]. On the other hand, cytoplasmic protein aggregates in genetically proven desminopathies and myotilinopathies also stained positive for VCP. In contrast to desmin and αB-crystallin mutants, transfection experiments using mutant R93C-, R155H-, R155C-VCP, and wild-type VCP did not lead to abnormal protein aggregate formation. These findings implicate that cell transfection models are of limited value in studying mutant VCP-associated protein aggregate formation [37]. The protein aggregate pathology in IBMPFD and various forms of myofibrillar myopathies has been attributed to an impairment of the proteolytic function of the ubiquitin-proteasome protein degradation system, which exerts a central role in the quality control of proteins and removal of improperly folded proteins. Though the exact molecular pathogenesis of IBMPFD awaits further elucidation, preliminary data indicate that mutant VCP interferes with the processing of tetra-ubiquitinated proteins by the 26s proteasome. 3.4. Protein degradation Isidro Ferrer and Montse Olivé outlined “Oxidative stress, the proteasome and abnormal aggregation in MFM”. The processes leading to protein aggregation in MFM are not well understood, although abnormalities of the ubiquitin–proteasome system (UPS), including expression of immunoproteasome subunits in abnormal muscle fibres, co-localization of clusterin and the aggresome marker γ-tubulin, as well as p62, ubiquitin, and, importantly, the misreading-resultant mutant ubiquitin UBB1+ which may block the UPS, are associated with aggregate formation in MFM. Misfolding and aggregation of proteins are facilitated by oxidation and nitration. Increased levels of glycation-end products (AGE), N-carboxymethyl-lysine and N-carboxyethyl-lysine, malondialdehyde-lysine, 4-hydroxynonenal, and nitrotyrosine are also found in MFM. Furthermore, aberrant expression of glycoxidation and lipoxidation markers, as well as of neuronal, inducible and endothelial nitric oxide synthases and superoxide dismutase 2 are present in muscle fibres containing protein aggregates in myotilinopathies and desminopathies. Furthermore AGE, ubiquitin and p62 co-localize in several muscle fibres in MFM [39]. By using single immunohistochemistry, double-labeling immunofluorescence and confocal microscopy, monodimensional gel electrophoresis and Western blotting, and bidimensional gel electrophoresis, in-gel digestion and mass spectrometry, desmin was demonstrated to be a major target of oxidation and nitration in both desminopathies and myotilinopathies. Modified desmin can be considered to be an additional element in the pathogenesis of MFM. In addition to desmin, pyruvate kinase muscle splice form M1 is oxidised in muscule tissues, thus supporting complemental mitochondrital damage, at least in some cases of myotilinopathy [40]. Together, these observations support a link between oxidative stress, protein aggregation and abnormal protein deposition in MFMs. Finally, several neuron-related proteins such as ubiquitin carboxy-terminal hydrolase L1 (UCHL1), synaptosomal-associated protein 25 (SNAP-25), synaptophysin and α-internexin accumulate in aberrant protein aggregates in myotilinopathy. We have determined that the neuron-restrictive silencer factor (NRSF/REST), a transcription factor expressed in non-neuronal tissues repressing the expression of several neuronal genes, is reduced in myotilinopathies. Moreover, NRSF cell transfection reduces UCHL1, SNAP25, synaptophysin and α-internexin mRNA levels, whereas siNRSF transfection increases UCHL1 and synaptophysin mRNA levels. Chromatin immunoprecipitation assays have shown that NRSF interacts with the UCHL1 promoter, and in silico analysis of the UCHL1 gene promoter sequence has predicted three potential NRSE sites. These findings show abnormal regulation of NRSF/REST as a mechanism associated with the aberrant expression of selected neuron-related proteins in myotilinopathy, which in turn accumulate in abnormal protein aggregates [41]. Benedikt Schoser discussed “Autophagy in skeletal muscle” [42]. Restrained vacuolar changes are common morphological features in acquired and inherited myopathies. At least, there are two common types of vacuoles in skeletal muscle, (i) rimmed and (ii) round vacuoles. Both have an autophagic character. Rimmed vacuoles seem to arise from abnormal nuclear pyknosis or apoptosis and are a consequence of nuclear membrane breakdown. In distinction, originating from the endoplasmic reticulum, modified through the trans-Golgi network, lysosomes are organelles of 400–500nm, and the hallmark of the round lysosomal-autophagic vacuoles. Both rimmed and round vacuoles principally belong to the autophagic pathway of long-lived protein degradation. In general, the ubiquitine/proteasome-mediated degradation pathway seems to have a high degree of specificity, whereas the lysosomal-autophagic degradation pathway is more non-selective in nature. Nevertheless, misfolded proteins like aggregates may also undergo autophagic degradation. In all types of MFM/PAM, beside well-known granulofilamentous inclusions, autophagic vacuoles are noticed. This autophagic build-up may be of principal importance in these late-onset diseases. Decline of protein clearance may unwrap gene mutation by long-term cellular autophagic build-up, combined with an overflow activation of the proteasome-mediated pathway and may lead to a toxic threshold, unmasking the mutational lesion load in cell anabolism and catabolism by finally enhancing apoptosis and necrosis in the dying muscle fibre. For monitoring, electron microscopy, LC3 immunohistology, and Western blotting seem to be valid markers of autophagy in skeletal muscle. Identification of novel inductors of autophagy in myopathies may encapsulate future treatment options. 4. Conclusion Among the PAM discussed at this workshop, different genetic forms, i.e. desminopathies, myotilinopathies, etc. based on several cohorts of patients from different neuromuscular centres could be identified and tentatively separated on clinical, neuroimaging, electron microscopic, and immunohistochemical grounds to facilitate subsequent mutational analysis in individual patients, establishing genotype–phenotype and genotype–morphotype correlations. While course-associated or gene-related therapies are not available, recommendations for management, especially of disease-complicating clinical features such as cardiac and respiratory problems as well as physiotherapy were discussed. Given the consistent presence of these complications in all series of patients, recommendations on management in these diseases should include surveillance for complications in these systems and intervention as appropriate. 5. Future perspectives The data provided from such a large number of patients mean that it is now timely to address the morphological criteria and guidelines by which a myofibrillary myopathy might be diagnosed by biopsy. Likewise, guidelines for management in diagnosis, especially addressing life-threatening cardiac and pulmonary complications as well as physiotherapy can be developed. Patient registries for MFM were discussed at this workshop and at a subsequent ENMC workshop on registries for rare disorders as a first step towards trial readiness in this population group. Finally, genetic and electron microscopic laboratories which may aid in genetic and ultrastructural studies will be identified in countries of workshop participants and beyond in Europe. 6. List of participants 1.Bushby, Kate (ENMC, Baarn, The Netherlands) 2.Boersen, Annette (ENMC, Baarn, The Netherlands) 3.Carpén, Olli (Turku, Finland) 4.Claeys, Kristl (Paris, France) 5.Fardeau, Michel (Paris, France) 6.Ferrer, Isidro (Barcelona, Spain) 7.Fidzianska, Anna (Warsaw, Poland) 8.Fürst, Dieter (Bonn, Germany) 9.Goebel, Hans H. (Mainz, Germany) 10.Kaminska, Anna (Warsaw, Poland) 11.Kley, Rudolf (Bochum, Germany) 12.Olivé, Montse (Barcelona, Spain) 13.Paulin, Denise (Paris, France) 14.Schoser, Benedikt (Munich, Germany) 15.Schröder, Rolf (Erlangen, Germany) 16.Selcen, Duygu (Rochester (MN), USA) 17.Sewry, Caroline (London, UK) 18.Stoltenburg, Gisela (Paris, France) 19.Vicart, Patrick (Paris, France) 20.Walter, Maggie (Munich, Germany) (Complete addresses of the individual workshop participants are available from the ENMC workshop organisers.) Acknowledgements This workshop was made possible by the financial support of the European Neuromuscular Centre (ENMC) and its main sponsors and associated members: –Association Française contre les Myopathies (France) –Deutsche Gesellschaft für Muskelkranke (Germany) –Telethon Foundation (Italy) –Muscular Dystrophy Campaign (UK) –Muskelsvindfonden (Denmark) –Prinses Beatrix Fonds (The Netherlands) –Schweizerische Stiftung für die Erforschung der Muskelkrankheiten (Switzerland) –Österreichische Muskelforschung (Austria) –Vereiniging Spierziekten Nederland (The Netherlands) –Asociación Española contra las Enfermedades Neuromusculares (Spain) –In addition we are grateful to Ms. Anelies Zittersteijn and Mr. Maarten Rector for their organisational support. We also thank Mrs. Astrid Wöber for both organisational pre-workshop help and editorial assistance, and Prof. Kate Bushby for her thorough review of the manuscript. References [1]. [1]Goebel HH, Fardeau M. 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[42]. [42]Schoser BG. Autophagic changes in skeletal muscle – towards the enigma of rimmed and round vacuoles. Clin Neuropathol; in press. Johannes Gutenberg University, Medical Center, Langenbeckstrasse 1, 55131 Mainz, Germany  Corresponding author. Tel.: +49 6131 177033; fax: +49 6131 176606.
PII: S0960-8966(08)00108-9 doi:10.1016/j.nmd.2008.04.008 © 2008 Published by Elsevier Inc. | |
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