138th ENMC Workshop: Nemaline Myopathy, 20–22 May 2005, Naarden, The Netherlands
Article Outline
- 1. Introduction
- 2. Clinical
- 3. Pathology
- 4. Genetics
- 5. Animal and tissue culture models and pathobiology
- 6. Nemaline myopathy convention 2004
- 7. Future directions
- 8. Participants
- Acknowledgements
- References
- Copyright
1. Introduction
Twenty-one doctors and scientists from nine countries gathered in Naarden, The Netherlands, for the 6th ENMC workshop on nemaline myopathy. The focus of the Consortium, now that five genes have been identified for nemaline myopathy, is changing to analysing the pathobiology of the mutant proteins and working towards developing effective therapies.
2. Clinical
Carina Wallgren-Pettersson, Helsinki, gave an initial description of a newly discovered distal myopathy caused by homozygous missense mutations in the nebulin gene. The muscle weakness in these patients differed from that of other patients with nebulin mutations in lacking the proximal component, and there appeared to be histological differences also.
Claudio Graziano, Bologna, reported the case of a 2-year-old patient with nemaline myopathy and hypertrophic cardiomyopathy (HCM), where an ACTA1 mutation has been identified. There are only a few patients with HCM in the context of nemaline myopathy described in the medical literature and to our knowledge this is the first one where an ACTA1 mutation is present. The proband is the second son of healthy unrelated parents, born at term of an uneventful pregnancy and with no complications at birth. Both he and his brother are carriers of an apparently balanced chromosome translocation t(6;9). Family history is negative for myopathy, cardiomyopathy and sudden death; echocardiography has been performed on close relatives and results are in the normal range.
The proband was referred at 2 months of age for a cardiac murmur and echocardiography showed a non-obstructive HCM of the septum and posterior left ventricle. A muscle biopsy was performed at 6 months of age due to a delay of motor milestones and hypotonia, and nemaline myopathy was diagnosed. Currently, at 2 years of age, the child is not able to stand, he has reduced facial expressions and frequent episodes of bronchopneumonia but speech development is normal.
HCM is diagnosed when the interventricular septum/posterior wall thickness ratio exceeds 1.3. At age 6 months, our patient's interventricular septum measured 6.7
mm and the posterior wall thickness was 4.6, giving a ratio of 1.46. The HCM in our patient seems non-progressive but follow up is being performed every 6 months. Biochemical analysis of the ACTA1 mutated product is ongoing.
Kathryn North, Sydney, reviewed the clinical and pathological phenotype of patients with congenital fibre type disproportion (CFTD) and mutations in ACTA1 [1]. The three CFTD ACTA1 mutations described to date caused a severe congenital lethal disorder in the absence of myofibrillar disorganisation on histopathology. Further studies are being performed to identify the pathogenetic basis of the severe muscle weakness. Mutant actin is expressed at similar levels to wild type actin in skeletal muscle from these patients and is incorporated into the thin filament, suggesting a dominant negative effect. Expression of the GFP-labelled mutant actin in myoblasts suggest that these specific mutants are less likely to form aggregates of actin compared with mutants resulting in nemaline rod pathology. The CFTD actin mutants do not differ from wild type actin in their polymerisation into thin filaments. Molecular modelling suggests that the CFTD actin mutations may affect the interaction of the actin thin filament with tropomyosin and affect the mechanics of muscle contraction rather than affecting muscle structure.
3. Pathology
3.1. Actin filament aggregate myopathy
Hans H. Goebel, Mainz, described actin filament aggregate myopathy (AFAM), a combination of actin filament aggregation within muscle fibres and a mutation in the ACTA1 gene causing a congenital myopathy which, so far, has only been recorded in children, often right after birth. Nine patients have been described and two additional patients were presented at this workshop. Only three out of these nine published patients have survived their first year of life, one of them having the same missense mutation as the longest surviving patient. The condition appears autosomal dominant with almost all patients having de novo missense mutations and one showing a two amino-acid duplication.
Morphologically, actin filament aggregates form masses of varying size, often beneath the plasmalemma, and are sharply demarcated from surrounding, often intact sarcomeres. Apart from actin, they may contain nebulin and tropomyosin as well as α-B-crystallin, suggesting impaired integration into intact sarcomeres and, perhaps, thereby, aggregation in separate, displaced foci of actin filaments.
As ACTA1 mutations are encountered in some 20–25% of patients with nemaline myopathy, AFAM thus forming a subgroup of nemaline myopathy, it is not surprising to find, in patients with AFAM, sarcoplasmic rods, intranuclear rods, both, or none. The heart appears to be clinically and pathologically intact.
The myopathy with actin accumulation described by Edström and co-workers [2] is clinically of adult onset and genetically caused by a titin kinase mutation [3]. Cardiac actin filament aggregation in cardiac myocytes, based on mutations in the ACTC gene, is seemingly not known. Actin aggregation in neuronal and glial cells has recently been found in a peculiar form of fatal juvenile dystonia [4].
3.2. Cap disease
Andoni Urtizberea, ENMC, described a consanguineous family with two sibs affected by cap disease and their cousin with nemaline myopathy.
3.3. Complex I and nemaline myopathy
Nigel Laing, Perth, summarised the findings published by Lamont et al last year [5] of three patients with nemaline bodies and co-incident complex I deficiency. He updated the information that homozygosity for the nebulin exon 55 Ashkenazi mutation has now been identified in one of these three patients.
Martin Lammens, Nijmegen, described a child with the classical features of nemaline rod myopathy with a severe complex I deficiency in a muscle biopsy. The deficiency was not expressed in fibroblasts. A missense mutation was detected in the skeletal muscle alpha actin gene (ACTA1) in this patient.
Other members of the consortium stated they had further patients with nemaline myopathy combined with complex I deficiency, however, secondary complex I deficiency has been observed in several muscle disorders. There was general discussion, in the light of Edna Hardeman's results, on signalling relationships between the sarcomere, mitochondria and nucleus and possible relationships between nemaline myopathy and complex I deficiency in rare cases and the need for further investigation of this, including re-analysis of patients with identified mutations.
3.4. Skeletal actin null patients
Caroline Sewry, Oswestry and London, summarized the pathological data on the Pakistani case homozygous for an ACTA1 null mutation in exon 4 (p.Asp179fs189X). This is the only patient null for ACTA1 known to be still living, the other three identified patients having died in early infancy. The biopsy taken at 2 months of age showed abnormal variation in fibre size with type 1 hypotrophy, some peripheral vacuolation and core-like areas devoid of oxidative enzyme stain in some fibres. Rods were not apparent with the Gomori trichrome technique, although some fibres were a little red. However, rods were clearly seen with electron microscopy. Electron microscopy also showed disrupted Z lines but a normal hexagonal arrangement of thin filaments and the presence of sarcomeres. A zebra body was also observed, which may be significant, as similar structures have been observed in the flight muscles of actin mutant Drosophila. Immunohistochemical studies of the ACTA1 null patient using isoform-specific antibodies showed the absence of skeletal muscle actin, but the presence of the cardiac actin isoform in every fibre. This was confirmed by immunoblots (performed by Kristen Nowak) that showed the skeletal muscle from this child had more cardiac actin than a deceased Spanish patient also homozygous for an ACTA1 null mutation. The cardiac isoform of troponin I was also abundant. The cardiac isoform of actin is present in fetal muscle and the switch to the skeletal isoform occurs in utero. The possible beneficial effect of the cardiac actin was discussed.
3.5. Myofibrillar myopathy and nemaline myopathy
Olli Carpén, Turku, introduced recent developments in myofibrillar myopathy (MFM, also termed desmin-related myopathy or desmin storage disease), a heterogeneous disorder characterized by a pathological pattern of myofibrillar dissolution associated with accumulation of myofibrillar degradation products and ectopic expression of multiple Z disc-associated proteins. In addition to the previously known desmin and α-B-crystallin genes, two novel MFM genes, myotilin [6] (also mutated in LGMD1A) and ZASP/cypher [7] were described recently. Both novel genes encode Z-disc proteins and interact with multiple Z-disc components. In addition to the reported cases, novel myotilin and ZASP patients have been identified. All known disease-causing myotilin and ZASP alterations are missense mutations that are inherited in an autosomal dominant manner. Although the clinical phenotype in MFM differs from that of the typical form of nemaline myopathy (MFM being a late-onset disorder) there is overlap in the morphological changes and many of the nemaline body components are also accumulated in MFM aggregates. This raises the possibility of common pathogenetic pathways between nemaline myopathy and MFM.
Pradeep Luther, London, described his work done together with Todd Herron and Sian Harding on ventricular myocytes from the explant heart from a patient with dilated cardiomyopathy. Examination of these myocytes by electron microscopy showed frequent occurrence of enlarged, spindle-shaped Z-bands varying in width from 100 to 400 nm. Dispersed within the myocytes, nemaline rod structures were also found. The contractile activity of the myocytes was found to be greatly reduced compared to non-failing hearts. The study suggests that a nemaline phenotype attenuates ventricular myocyte contractile activity and likely contributes to poor function at the level of the whole heart.
Pradeep Luther, London, also described his comparisons of the ultrastructure of nemaline bodies in genetically defined patients done together with Yohan Chalabi, Edward Morris, Cathy Timson, John Squire and Caroline Sewry. It is now well known that nemaline myopathy is caused by mutations in actin and actin-related proteins of the sarcomere. However we do not know how similar or different is the ultrastructure of the nemaline bodies that characterise these myopathies. In this study, electron microscopy was used to investigate the ultrastructure of nemaline bodies from several patients with actin or nebulin mutations. For each sample, the primary lattice view was obtained by carefully tilting the sections in the electron microscope. By Fourier filtering these views, the group showed that in these samples the mean views of the nemaline rods were very similar.
Lars-Eric Thornell, Umeå, summarised his work on the composition and ultrastructure of nemaline rods. In Sweden the cases of nemaline myopathies have not been systematically studied, however a common effort among the neuropathologists to do so has been initiated. During the last 25 years, cases of nemaline myopathy have been studied using cryoultramicrotomy [8]. This method includes fixation with mild chemical crosslinking of proteins followed by antifreeze treatment and sectioning at low temperature. Semithin sections can be used for immunohistochemistry whereas ultrathin sections are negatively stained and viewed in the electron microscope. This method allows the group to show that the nemaline rods vary in composition and show different crystalline patterns. Their hypothesis is that the different patterns are related to different genetic backgrounds. They now collect DNA from these patients to be able to determine which gene defects are the cause of the different types of nemaline rods. Screening of the actin gene will first be done in A. Oldfors' laboratory and, if negative, followed by screening of the nebulin gene in C. Wallgren-Pettersson's laboratory.
4. Genetics
4.1. Update on the nebulin gene
Alan Beggs, Boston, Carina Wallgren-Pettersson, Helsinki and Nigel Laing, Perth, summarised their findings in other populations analysed for the presence of the exon 55 nebulin deletion first described in the Ashkenazim [9]. The Perth series identified one homozygous patient and two heterozygous patients out of 128 patients, the Helsinki series 3 heterozygotes out of 144 patients, and the Boston series 5 homozygous cases out of 112 from around the world, not overlapping with the other two cohorts. All the homozygous cases at least appear to have Ashkenazi inheritance.
Vilma-Lotta Lehtokari, Helsinki, presented a current overview of nebulin mutations identified to date. Mutation detection in nemaline myopathy remains a problem. This is mainly due to nebulin, one of the principal causative genes, being one of the largest genes known in man. The gene encoding nebulin (NEB) measures 249kb in length and consists of 183 exons. Using SSCP analysis, the Helsinki group has previously identified 18 nebulin mutations in 18 families. These mutations were all found in the last 42 of the 183 exons. Included in the present study were 15 Finnish families with nemaline myopathy and 21 families from other countries. Actin (ACTA1) mutations had been excluded. In six of these families, one of the causative mutations had already been identified. Using dHPLC permitted the analysis of 159 of the 183 exons. The group detected six novel mutations in Finnish patients and 24 novel mutations in other families. Each mutation was identified in one of the unaffected parents also. Thus, to date, in this series of 36 families, a total of 30 hitherto unpublished mutations were identified. The majority of the nebulin mutations identified to date have been frameshift mutations due to small deletions or insertions. Nonsense and splice site mutations are quite common as well, while missense mutations are rare. dHPLC offers efficient identification of heterozygous mutations and appears promising for routine mutation screening of NEB. However, it will require a complementary method for the detection of large deletions or duplications.
Katarina Pelin, Helsinki, described a mini-gene approach for the testing of the effects of putative splice-site mutations in the nebulin gene. About 30% of the mutations identified in NEB are considered to cause abnormal pre-mRNA splicing, i.e. exon skipping or inclusion of intronic sequence. However, muscle RNA is seldom available from the patients carrying the mutations, complicating the verification of the mutation at the RNA level. The group has developed a method based on nebulin minigene constructs, which are transfected into cultured myoblasts for assessment of splicing. A PCR product spanning three exons and two introns of NEB is cloned into pcDNA3.1 (Invitrogen) expression vectors, which are transfected into C2C12 myoblasts. The myoblasts are cultured for 20–24h and RNA is extracted. The splicing of the minigene is assessed by RT-PCR, agarose gel electrophoresis and sequencing. Results from three different minigenes, all carrying a point mutation at intron position +5 of a 5′splice site, showed skipping of the preceding exon. The control minigenes were correctly spliced. Nebulin minigene constructs are useful for testing putative splice mutations when patient RNA is unavailable.
Kati Donner, Helsinki, reported on her studies of alternative splicing of the nebulin gene [10]. There are four regions with alternatively spliced exons, exons 63–66, 82–105, 143–144 and 166–177, giving rise to a number of different transcripts. These transcripts were studied with the aid of reverse transcription (RT)-PCR, cloning, and sequencing. To study the expression of mouse exons 127 and 128, corresponding to exons 143 and 144 in human, the group used quantitative real-time PCR. Exons 143–144 in human and 127–128 in mouse give rise to two different transcripts varying between muscle types and between muscles of different developmental stages. The lengths of the transcripts encoded by exons 63–66 differ between fetal and adult human muscles. Exons 166–177 express at least twenty different transcripts in adult human tibialis anterior muscle alone. Preliminary results indicate that alternative splicing of exons 82–105 also give rise to a number of different transcripts. Extensive alternative splicing of NEB may explain why NM patients with homozygous truncating mutations, contrary to expectations, show expression of the carboxy terminus of the nebulin protein [11].
4.2. Update on the ACTA1 gene
Claudio Graziano, Bologna, and his group had collected samples from 20 independent Italian families with patients affected by nemaline myopathy. Mutation screening of the ACTA1 gene by direct sequencing of the coding sequence showed mutations in seven out of 17 families and all were of de novo origin.
Worth noting is that two patients with an ACTA1 mutation have/had an affected monozygotic twin and pregnancy was complicated by the loss of a twin in utero in a patient where ACTA1 screening is not yet complete. Thus, twin pregnancies appear common at least in the Italian ACTA1 population. There are no data on ACTA1 expression in the early days of human embryonic development, but it may now be speculated that the presence of an ACTA1 mutation could result in altered cytoskeletal dynamics.
Nigel Laing gave an update on ACTA1 mutations known to the Perth Laboratory. At the start of the meeting the Perth laboratory knew of 112 mutations in 82 different amino-acid residues of the mature actin protein. With the addition of Claudio Graziano's newly described mutations at the workshop, this total increased to 116 mutations in 84 residues. The Perth laboratory had overall identified 99 mutations in 498 probands, giving an average percentage of 19.9%. In some ethnic groups, notably the Japanese cohort examined, there is perhaps a lesser rate of mutation suggesting possible founder effects, as with the Amish slow troponin T (TNNT1) nonsense mutation [12] and the Ashkenazi nebulin exon 55 deletion [9]. There was also discussion of the relationship between mutations in actin and congenital fibre type disproportion, with a fourth ACTA1 variation identified in a fourth patient with congenital fibre type disproportion.
4.3. Chromosome 15 nemaline myopathy with core-like areas
Nigel Laing gave an update on the efforts to identify the gene for autosomal dominant nemaline myopathy with core-like areas linked to chromosome 15 [13]. Work has still not shown any indication of a shared haplotype between the two families that provided the original linkage. No other families have been recognized with this phenotype and added to the cohort to try to reduce the size of the linkage region. The linkage region contains 52 genes. The most likely candidate genes are fast alpha-tropomyosin (TPM1), annexin II (ANXA2) and talin 2 (TLN2). Genomic sequencing demonstrated no variations in TPM1. Analysis of ANXA2 and TLN2 is virtually complete.
5. Animal and tissue culture models and pathobiology
At the previous workshop, Kristen Nowak, Perth, spoke about her plans to investigate a possible therapy for nemaline myopathy and other congenital myopathies caused by mutations in the skeletal muscle alpha-actin gene. There is preliminary evidence that suggests that diluting out the mutant actin protein, perhaps by over-expressing an alternative gene, could ameliorate skeletal actin disease. Cardiac actin, the main actin in adult heart, is also the predominant striated actin protein expressed in skeletal muscle during development, and is 99% identical to skeletal actin. Dr Nowak spent two years in Professor Kay Davies' laboratory making a series of transgenic mouse models to explore the possibility of over-expressing cardiac actin as a potential therapeutic approach. She has now returned to Perth to conduct further investigations of the mouse lines: two strains that over-express cardiac actin in mature muscle, two strains that express a skeletal actin mutant transgene, and another that expresses this mutant skeletal actin fused to enhanced green fluorescent protein (EGFP). Preliminary pathological analyses performed in collaboration with Professor Caroline Sewry suggest that the over-expressing cardiac actin mice do not appear to have any adverse effects. Contrastingly, those mice that express the mutant actin-EGFP protein show large osmiophilic bodies at the position of Z-lines that resemble nemaline bodies by electron microscopy. Kristen Nowak is to cross the various mouse lines to establish whether the disease phenotypes in the mutant skeletal actin mice (and additionally those that are null for skeletal actin created by Prof Lessard's laboratory, Cincinnati [14]), are overcome by the presence of over-expressed cardiac actin.
Steve Marston, London, together with his colleagues, has developed methods for studying the functional consequences of nemaline myopathy mutations in actin. These include assays for protein folding; monomer stability; polymerisation; motor properties (interaction with myosin); Ca2+-regulatory properties in the presence of tropomyosin and troponin, and changes in binding to Z-disc proteins. Isolation methods have been developed for obtaining pure actin from small biopsy samples, from transgenic mouse models and from baculovirus/Sf9 expression. The group has examined two mutations in biopsies, two in transgenic mouse lines and four expressed in baculovirus/Sf9. Substantial differences in polymerisation, motor activity and alpha-actinin binding, characteristic for each mutation and independent of source, were observed.
Actin filaments containing the D286G mutation, extracted from transgenic mice developed by Kristen Nowak (above) support crossbridge cycling 18% faster than wild-type; they are less stable than WT and have a lower affinity for alpha-actinin. The K336E mutation described by Claudio Graziano (above) that causes myopathy and HCM was studied using biopsy material. The mutant actin constituted 28% of muscle actin and the extracted actin has a 15% slower sliding speed than WT, reduced co-operativity and appears to have considerably reduced alpha-actinin binding affinity.
In future work, the group hopes to be able to relate the molecular phenotype to the specific pathology of each mutation, an approach that they have successfully applied to mutations causing cardiomyopathies.
Edna Hardeman, Sydney, described candidate proteins involved in linking structural and metabolic abnormalities in mouse models for nemaline myopathy. The TPM3(Met9Arg) and ACTA1(His40Tyr) mouse models for nemaline myopathy, in addition to having structural pathologies, potentially have metabolic abnormalities as evidenced by glycogen accumulations and increased mitochondrial number and size. Candidate molecules that could play a role in linking these two aspects of nemaline myopathy are members of the Four and a Half LIM Domain protein family, FHL1 and FHL3. It was found that LIM proteins are located both at sites of actin-based filaments (Z line, M-band) and subsarcolemal accumulations of mitochondria in adult muscles. In the muscles of nemaline mice, FHL3 protein level is increased and FHL3 is present within nemaline rods. This may reflect its inhibitory role in actin bundling and account for the presence of sarcomeric disruptions in nemaline muscles. FHL1 protein level is also increased in nemaline muscles and its location also changes. It translocates from cytoplasmic structures into the nuclei of myofibres. This is reminiscent of immature muscles and may indicate areas that have undergone repair. The association of both FHL1 and FHL3 with subsarcolemal mitochondria is diminished in nemaline muscles, suggesting a role for these proteins in altered metabolic properties.
Biljana Ilkovski, Sydney, presented data on a three- generation autosomal dominant family with intranuclear rod myopathy and a V163M mutation in ACTA1. The three family members are mildly affected with variability in age of onset. Analysis of the patient muscle biopsies showed positive labelling of rods with α-actinin 2 and phalloidin (specific for all forms of filamentous actin). Rods did not label with antibodies raised against total actin and sarcomeric actin antibodies, suggesting antibody inaccessibility due to epitope masking. Expression of EGFP-tagged V163M mutant actin in myoblasts results in aggregates in nuclei that are highly reminiscent of rods observed in the patient muscle. Electron microscopy confirmed the presence of these aggregates in the nuclei of muscle cells and showed that they have a filamentous structure. To determine how intranuclear rods form in muscle cells the group performed live cell imaging of EGFP-tagged V163L mutant protein in fibroblasts and myoblasts. The aggregates form inside the nucleus where they fuse and grow in size. They are highly dynamic structures that move rapidly within the nucleus and alter their shape by bending. The intranuclear aggregates were shown not to result from reduced export of the mutant actin through the CRM1 exporter, by using the nuclear export blocker leptomycin B. These results provide insights into the mechanisms of intranuclear rod formation and disease pathogenesis.
Muscle weakness in many nemaline myopathy patients can be exacerbated by prolonged periods of physical inactivity. Anthony Kee, Sydney, reported on a study examining running exercise as a means of improving recovery following muscle inactivity in the TPM3(Met9Arg)-mouse model of nemaline myopathy [15]. Physical inactivity was mimicked using a hind-limb immobilization protocol that results in fiber atrophy and severe muscle weakness. Following immobilization, the nemaline mice (NM) were weaker than wild-type (WT) mice but regained whole body strength with exercise training. This provides support for exercise as a means of alleviating disuse-induced weakness in nemaline patients. The disuse-induced weakness and the regain of strength with exercise in NM were associated with the respective formation and resolution of nemaline rods, suggesting a role for rods in muscle weakness. Muscles in NM did not show the typical features of muscle repair during chronic stretch immobilization of the soleus muscle (regeneration occurred with relative lack of centralized nuclei). This indicates that the process of regeneration in nemaline myopathy is different from the repair of normal and dystrophic muscle. This form of repair may be specific to diseases of the muscle thin filament.
6. Nemaline myopathy convention 2004
Drs Wallgren-Pettersson and Nowak reported on the Nemaline Myopathy Convention they attended in Toronto on 23rd and 24th October, 2004. The director of the convention was David McDougall who created and diligently runs the nemaline myopathy website at http:www.davidmcd.btinternet.co.uk. Twenty-eight nemaline myopathy patients and their families travelled from around Canada, the United States and even the United Kingdom to attend the convention, with many meeting a fellow “nemaliner” for the first time. The weekend was filled with talks and discussion on various aspects of nemaline myopathy such as medical care, current scientific research, genetics and counselling, parenting with a disability, non-invasive mechanical ventilation, growing up with nemaline myopathy, having a child with nemaline myopathy and even adaptive yoga! The Nemaline Myopathy Consortium wishes to congratulate David McDougall and the organising committee on a fantastic and truly memorable inaugural convention and hopes to continue and extend our relationship with this patient group.
7. Future directions
There was general discussion of the difficulties the Consortium had had in enrolling a sufficiently large cohort of patients in a proper clinical trial of l-tyrosine and the difficulties encountered in obtaining regulatory approval in multiple countries.
The need for a mutation database for the nemaline myopathy genes was discussed. It was decided to collaborate with the Universal Mutation Database to develop the necessary database.
The need to develop more efficient methods for finding mutations within the giant nebulin gene was recognised. Dr Andoni Urtizberea stated that this was not a problem faced solely in nemaline myopathy, but that large genes had to be screened for mutations in other diseases and that perhaps there needed to be a focus, possibly even an ENMC workshop, on methods for finding mutations in large genes.
8. Participants
Acknowledgements
This workshop was made possible thanks to the financial support of the European Neuromuscular Centre (ENMC) and its main sponsors: Association Francaise contre les Myopathies (France); Deutsche Gesellschaft für Muskelkranke (Germany); Telethon Foundation (Italy); Muscular Dystrophy Campaign (UK); Muskelsvindfonden (Denmark); Prinses Beatrix Fonds (Netherlands); Schweizerische Stiftung für die Erforschung von Muskelkrankheiten (Switzerland); Österreichische Muskelforschung (Austria); Vereniging Spierziekten Nederlands (Netherlands) and ENMC associate member: Asociacion Espanola contra las Enfermedades Neuromusculares (Spain).
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PII: S0960-8966(05)00290-7
doi:10.1016/j.nmd.2005.10.006
© 2005 Elsevier B.V. All rights reserved.
