Neuromuscular Disorders
Volume 11, Issue 4 , Pages 417-420, May 2001

81st ENMC International Workshop: 4th Meeting on Emery–Dreifuss Muscular Dystrophy 7th and 8th July 2000, Naarden, The Netherlands

Randall Centre for the Molecular Mechanism of Cell function, Kings College, New Hunts House, Guys Campus, London, SE1 1UL, UK

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1. Introduction 

Emery–Dreifuss muscular dystrophy (EDMD) is characterized by early contractures of the elbows, Achilles tendons and spine, slowly progressive muscle wasting and weakness with a predominantly humeroperoneal distribution and cardiomyopathy, usually presented as a heart block [1]. EDMD occurs as three modes of inheritance, X-linked (X-EDMD), autosomal dominant (AD-EDMD) and autosomal recessive (AR-EDMD) [2], which are clinically very similar. At the second ENMC workshop in 1996, it was reported that the X-linked EDMD locus had been precisely mapped to Xq28, the emerin gene (STA) isolated and now more than 90 emerin mutations have been identified which are freely accessible on the web (http://www.path.cam.ac.uk/emd/mutation/html). Subsequently, emerin was shown to be a single-spanning protein of the inner nuclear membrane [3], [4] in all tissues studied. Emerin's function is unknown, but it shares homology with a family of inner nuclear membrane proteins, known as lamina-associated polypeptides (LAPs), which are well studied. At the third ENMC workshop in 1998, a genome-wide linkage study reported that the locus for AD-EDMD was 1q11-q23. Subsequently, Bonne et al. [5] identified the nuclear lamin A/C (LMNA) gene at this locus to be responsible for AD-EDMD.

Despite extensive clinical studies spanning over 40 years, there are substantial unanswered questions, which Alan Emery (UK) highlighted in the introduction to the meeting. This directed the aims of the fourth ENMC workshop on EDMD:

•To highlight the clinical differences between X- and AD-EDMD.

•To discuss on whether the intra- and inter-familial phenotype variability seen in EDMD can be attributed to individual variations in nuclear architecture in muscle tissue.

•To examine the developmental expression of emerin and lamin A/C in both skeletal and cardiac muscle.

•To discuss the possible binding partners of emerin and lamin A/C in both skeletal and cardiac muscle.

•To discuss the role of emerin and lamin A/C and their protein interactions in nuclear envelope disassembly/reassembly in mitosis.

•To examine the functions of well characterized homologues of emerin to provide insight into the role of nuclear proteins in EDMD.

•To provide a progress report on the knock-out mice models for both emerin and lamin A/C.

•To examine muscle biopsies from EDMD patients, examining what effect the absence/modification of emerin and lamin A/C has on nuclear architecture.

•To discuss possible mechanisms which link the nuclear functions of emerin and lamin A/C to the development of the characteristic cytoskeletal abnormalities associated with muscular dystrophy.

Howard Worman (USA) provided an overview on nuclear membrane-associated components. Nuclear lamins belong to a class of intermediate filament proteins which form a cage-like structure underlying the inner nuclear membrane and have been shown to interact with both chromatin and inner nuclear membrane proteins. Nuclear lamin functions are well documented and diverse and are divided into A-type (A and C) and B-type (B1 and B2). Their functions include the regulation of nuclear architecture as well as being important in development and differentiation mediated events. Inner nuclear membrane proteins such as emerin, MAN1 and lamina-associated polypeptide (LAP) isoforms bind to the nuclear lamins. Together, they contribute to the structural framework of the nucleus. These three nuclear membrane proteins share a region of homology, referred to as the LEM domain, suggesting they possess at least some common functions.

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2. Clinical and genetic studies 

2.1. Intra- and inter-familial phenotype variability in EDMD 

Gisele Bonne (France) and Francesco Muntoni (UK) summarized the data of a large collaboration including themselves, Ketty Schwartz (France), Luciano Merlini (Italy), Kate Bushby (UK), Manfred Wehnert (Germany) and Daniela Toniolo (Italy) and reported the first large collection of clinical and genetic analysis on AD-EDMD families. The clinical features from 53 patients (36 members of six families and 17 sporadic cases) were reviewed [6]. Four main phenotypes were identified:-

1.An isolated cardiac phenotype: this was present in 12 members of a large French family, in which the other five members had typical AD-EDMD features.

2.An early onset and severe EDMD phenotype, with presentation in the first few years of life.

3.A ‘common’ phenotype, characterized by onset in the second part of the first decade of life.

4.A milder variant, with onset of weakness in the second or third decade was also rarely observed in 10% of cases.

Mutation analysis identified 18 mutations consisting of one nonsense, two codon deletions and 15 missense mutations within the region common to lamin A and C (exons 1–9). LMNA mutations arose de novo in 76% of the cases [6].

Irena Hausmanova-Petrusewicz (Poland) has twenty-four patients diagnosed with X-EDMD, fifteen with suspected AD-EDMD, and one patient is proven to be an autosomal recessive EDMD (lamin A/C) case. The intra-familial clinical variability in the AD-EDMD patients was more marked than in the X-EDMD patients, with some family members presenting with only the cardiomyopathy, others only with the myopathy, and some with only contractures, in agreement with the findings of Bonne and Muntoni. The patient with the autosomal recessive type presented with extremely strong contractures of cervical posterior joints, very slight ankle and elbow contractures and no muscle weakness nor atrophy.

The above studies all suggest that AD-EDMD patients show a wider range of disease severity compared with X-EDMD patients, both in the myopathy and cardiomyopathy. Isolated cardiac involvement is more prevalent, muscle weakness and disease course tend to be more severe in AD-EDMD than X-EDMD patients. There is a large number of de novo mutations in AD-EDMD, therefore genetic analysis should be considered even when clinical diagnosis is only suggestive of EDMD, because of the potential severity of the cardiomyopathy.

2.2. Histochemical studies on nuclear antigens in AD-EDMD 

Caroline Sewry (UK) presented a summary of morphological data on eight skeletal muscle biopsies from AD-EDMD patients. The degree of histological abnormalities was variable, but all cases showed variation in fibre size. Internal nuclei were common. Increased connective tissue deposition was rare and necrosis was a feature in only one case. A predominance of type 1 fibres, which had a smaller diameter, was seen. Both lamin A/C and emerin expression and localisation appeared similar to normal controls. The currently available antibodies against lamins are not suitable to make any accurate judgements on lamin expression levels. However, it would appear that lamin A/C and B1 are expressed at low levels in nuclei of muscle fibres and cardiomyocytes. In contrast, lamins A/C and B2 were readily detectable. Plasma membrane proteins, such as dystrophin, showed normal expression, but the extracellular matrix protein laminin β1, showed an age-related secondary reduction on the muscle fibres. This is not a consistent feature of all cases with AD-EDMD and it also occurs in other dominant myopathies. Immunocytochemistry of lamins is not, therefore, a useful tool for diagnosis for the AD-EDMD, but immunolabeling of laminin β1 can be a useful secondary indicator of disease progression.

2.3. Ultrastructural analysis on skeletal muscle from EDMD patients 

Anna Fidzianska (Poland) reported the focal loss of the nuclear membrane, chromatin blebbing and both chromatin and nucleoplasm extrusion into the extranuclear space, as the most characteristic finding in X-EDMD. In contrast, the inner nuclear membrane in a patient with AR-EDMD (lamin A/C) was well preserved. Loss of chromatin with rarefaction and paucity of nucleoplasm variations in shape and size was the most often feature in this form of disease. This kind of nuclear chromatin disorganization was correlated with lack of lamin A4 epitope (residues 572–646 of lamin A; C-terminal tail of lamin A only). Caroline Sewry (UK) also reported redistribution of the heterochromatin away from the inner nuclear membrane in several myonuclei in some AD-EDMD patients and suggested that anchoring may be affected or apoptosis may be involved. Overall, myofibrillar structure was well preserved.

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3. Molecular studies on nuclear proteins associated with EDMD 

3.1. Muscle differentiation 

Juliet Ellis (UK) reported the differential expression of emerin in the mouse myoblast cell line C2C12, which mimic satellite cell differentiation in vivo. Total emerin expression levels slightly increase upon serum withdrawal induced differentiation. More interestingly, under conditions whereby mitogenesis is prolonged a novel isoform of emerin is observed.

3.2. Muscle development 

Susan Brown (UK) presented data indicating that alterations in expression levels of nuclear envelope-associated components accompany myogenesis. Analysis of human fetal muscle at 12 weeks of gestation demonstrated that lamins A, C, B and emerin were expressed, as observed by both immunoblotting and immunohistochemistry. Lamin A, C, B1 and B2 and emerin exhibited the strongest labeling in newly formed myotube nuclei, with generally less being associated with the surrounding single myoblasts nuclei.

3.3. Cell cycle-mediated events 

Emerin and lamin A/C co-localize during interphase and at various stages of mitosis [7]. It has been suggested that emerin participates in the re-organisation of the nuclear envelope at the end of mitosis. Clemens Mueller-Reible (Germany) reported that micro-injection of emerin polyclonal antibodies into the interphase nuclei of Hep-G2 cells, had no effect on mitosis but hindered the final stages of cytokinesis. Marie-Christine Dabauvalle (Germany) reported that lamin A and lamin C are both unstable and both expressed at lower levels in lymphoblastoid cell lines derived from AD-EDMD patients than from normal controls. Protein instability was cell cycle dependent.

3.4. Binding partners of emerin and lamin A/C 

Co-immunoprecipitation of emerin with both A- and B-type lamins has been previously reported [8] but this could be initiated by a third protein or other macromolecular complex. A direct interaction between recombinant emerin and lamin A/C molecules using biomolecular interaction analysis was reported by Sushilal Manilal (UK) and Glenn Morris (UK). They reported that the lamin-binding site was reported to within the first 188 amino acids of emerin. Chris Hutchison (UK) identified that it is the lamin tail domain, which binds to emerin. In addition, emerin preferential binds to lamin C over A, and equally to lamin A and B1. In addition he also reported that emerin is localized to the inner nuclear membrane subsequent to lamin filament formation, where it is then anchored by lamin C. Kathy Wilson (USA) reported that emerin binds to Barrier-Autointegration-Factor (BAF) a DNA-binding protein of unknown function and that mutations in the LEM-domain region disrupt this binding. Further mutation analysis suggests that lamins bind to the middle region of emerin. She also reported the cloning of the emerin gene from C. elegans [9].

3.5. Mouse models for EDMD 

There have been no reports to date on the generation of mouse models which accurately represent X-EDMD. Colin Stewart (USA) reported the derivation of mice in which the A-type lamins have been eliminated by gene targeting, to produce either homozygous or heterozygous offspring [10]. Both types of mice developed to term with no overt abnormalities, however, post-natal growth was associated with the development of a severe muscular dystrophy phenotype in only the homozygous mice. At the molecular level the −/− mice possessed alterations in nuclear morphology. The nuclear envelope assumed an irregular shape and herniated appearance. There was also a thinning or loss of heterochromatin at discrete regions of the nuclear face of the inner nuclear membrane. Emerin was partially lost from the nuclear membrane, and appeared to be more cytoplasmic, with a distribution similar to that exhibited by ER resident proteins. The +/− mice exhibited these molecular alterations to a much less extent.

Transfection in of lamin A cDNA into mouse embryonic fibroblasts from −/− mice restored emerin to its nuclear membrane localisation. This provides further evidence for a role of lamin A in the correct localisation of emerin, but additional factors have to be involved, because nuclear envelope-associated emerin was not uniformly lost within the tissues of the −/− mice. In the heart, the ventricular muscle was the most severely compromised, although myocyte involvement was non-uniform.

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4. Nuclear proteins related to or associated with emerin/lamin A/C 

One way to further our understanding the function of emerin is to examine the functions of the LAP2 isoforms, which share regions of identity with emerin and which are already very well functionally characterized. In the LAP2 family, LAP2α is a component of nucleoskeletal structures throughout the nuclear interior, and not localized to the inner nuclear membrane. Roland Foisner (Austria) reported that LAP2α binds specifically to lamins A and C through its C-terminal 75 amino acids, throughout the nucleus and presented evidence suggesting that it may target lamins A and C to intranuclear sites during post-mitotic assembly. The C-terminal region common to both lamin A and lamin C, contains the binding site for LAP2α [11].

This data suggest that lamin A/C can occur in two different complexes in interphase cells; the lamin-emerin complex at the nuclear periphery and the lamin-LAP2α complex in the nuclear interior. It is currently unclear whether these complexes exist as independent entities, or whether there is an exchange of lamin A/C between peripheral and intranuclear sites. We can therefore speculate that any disturbance to the lamin A/C-LAP2α complexes in the nuclear interior or to the lamin A/C-emerin complexes at the periphery could cause different molecular and possibly clinical phenotype of this disease.

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5. Conclusions 

This meeting has illustrated for the first time that AD-EDMD exhibits a wider range of disease severity compared to X-EDMD patients, which is reflected at the molecular level by the different structural abnormalities in nuclear architecture between the two groups of patients. To date, lamin A/C protein expression levels in AD-EDMD patients appear to be normal and correctly localized, suggesting that either the normal allele is compensating for lack of expression from the mutant allele, or mutant lamin A/C is expressed and localized normally. It is also possible that heterodimers consisting of mutant lamin A/C and wild type lamin A/C may exist. Without antibodies which specifically recognise only the mutant forms of lamin A/C, it is not possible to address this question. The composition of the emerin-lamin complex at the nuclear membrane has now been shown to include lamins A, B, C and BAF the exact composition at any one time being cell cycle dependent. In addition, lamin A/C also forms a complex with LAP2α in the nuclear interior, suggesting an additional pathway may be affected by the presence of lamin A/C mutations. Components of either of these complexes may interact with muscle-specific transcription factors, which are involved in controlling muscle cell integrity. A possible molecular mechanism explaining how the pathology is specific to striated muscle is that this tissue naturally has low levels of lamin B1 and therefore if also subjected to reduced levels of emerin/lamin A/C, the nuclear envelope may be unable to cope with prolonged mechanical stress which results from muscle contraction.

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6. List of workshop participants 

Gisele Bonne (France)

Susan Brown (UK)

Marie-Christine Dabauvalle (Germany)

Juliet A. Ellis (UK) organiser

Anna Fidzianska (Poland)

Roland Foisner (Austria)

Irena-Hausmanowa-Petrusewicz (Poland)

Christopher Hutchison (UK)

Sushila Manilal (UK)

Glenn Morris (UK)

Clemens Mueller-Reible (Germany)

Francesco Muntoni (UK)

Caroline Sewry (UK)

Colin Stewart (USA)

Kathy Wilson (USA)

Howard Worman (USA)

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Acknowledgements 

The workshop was made possible thanks to the financial support of the European Neuromuscular Centre (ENMC) and ENMC main sponsors:-Association Francaise contre les Myopathies (France); Deutsche Gesellschaft fur Muskelkranke (Germany); Telethon Foundation (Italy); Muscular Dystrophy Campaign (UK); Muskelsvindfonden (Denmark); Prinses Beatrix Fonds (The Netherlands); Schweizerische Stiftung fur die Erforschung der Muskelkrankheiten (Switzerland); Verein zur Erforschung von Muskelkrankheiten bei Kindern (Austria); Vereniging Spierziekten Nederland (The Netherlands) and ENMC associate member, Muscular Dystrophy Association of Finland. I would like to thank Dr A. Urtizberea (Research Director) and Mr. M. Rutgers for their help in co-ordinating this meeting.

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References 

  1. Emery AEH. Emery–Dreifuss muscular dystrophy-a 40 year retrospective. Neuromusc Disord. 2000;10:228–232
  2. Raffaele Di Barletta M, Ricci E, Galluzzi G, Tonali P, Mora M, Morandi L, et al.  Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive EDMD. Am J Hum Gen. 2000;66:1407–1412
  3. Nagano A, Koga R, Ogawa M, et al.  Emerin deficiency at the nuclear membrane in patients with EDMD. Nat Genet. 1996;12:254–259
  4. Manilal S, Nguyen thi Man , Sewry CA, Morris GE. The Emery–Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein. Hum Mol Genet. 1996;5:801–808
  5. Bonne G, Raffaele Di Barletta M, et al.  Mutations in the gene encoding lamin A/C cause autosomal dominant EDMD. Nat Genet. 1999;21:285–288
  6. Bonne G, Mercuri E, Muchir A, et al.  Clinical and molecular genetic spectrum of AD-EDMD due to mutations of the lamin A/C gene. Ann Neurol. 2000;48:170–180
  7. Dabauvalle M-C, Muller E, Ewald A, Kress W, Krohne G, Muller CR. Distribution of emerin during the cell cycle. Eur J Cell Biol. 1999;78:749–756
  8. Fairley EAL, Kendrick-Jones J, Ellis JA. The EDMD phenotype arises due to aberrant targeting and binding of emerin at the inner nuclear membrane J Cell Sci. 1999;112:2571–2582
  9. Lee KK, Gruenbaum Y, Spann P, Liu J, Wilson KL. C. elegans nuclear envelope proteins emerin, MAN1, lamin and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis. Mol Biol Cell. 2000;11:3089–3099
  10. Sullivan T, Escalante-Alcalde D, Bhatt H, et al.  Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J Cell Biol. 1999;147:913–919
  11. Vlcek S, Just H, Dechat T, Foisner R. Functional diversity of LAP2α and LAP2β in post-mitotic chromosomes association is caused by the α-specific targeting domain. EMBO J. 1999;18:6370–6384

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Neuromuscular Disorders
Volume 11, Issue 4 , Pages 417-420, May 2001

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