Workshop report. 1. Introduction

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1 Neuromuscular Disorders 12 (2002) Workshop report 82nd ENMC international workshop, 5th international Emery Dreifuss muscular dystrophy (EDMD) workshop, 1st Workshop of the MYO-CLUSTER project EUROMEN (European muscle envelope nucleopathies), September 2000, Naarden, The Netherlands q Gisèle Bonne a, *, Jaqueline Capeau b, Marianne De Visser c, Denis Duboc a,d, Luciano Merlini e, Glenn E. Morris f, Francesco Muntoni g, Dominique Recan h, Caroline Sewry i, Stefano Squarzoni j, Colin Stewart k, Beril Talim e, Anneke van der Kooi c, Howard Worman l, Ketty Schwartz a a INSERM UR523-Institut de Myologie, Bâtiment Babinski, G.H. Pitié-Salpétrière, 47, Boulevard de l Hôpital, Paris Cedex 13, France b INSERM UR402-Hôpital Saint-Antoine, Paris, France c Amsterdam Medical Center, Amsterdam, The Netherlands d Service de Cardiologie, Hôpital Cochin, Paris, France e Istituto Ortopedico Rizzoli, Neuromuscular Unit, Bologna, Italy f MRIC, North East Wales Institute, Wrexham, UK g Hammersmith Hospital, ICSM, Dubowitz Neuromuscular Unit, London, UK h Laboratoire de Génétique Médicale, Hôpital Cochin, Paris, France i Robert Jones and Agnes Hunt Orthopedic NHS Trust, Oswestry, UK j Istituto di Citomorfologia NP CNR c/o IOR, Bologna, Italy k Laboratory of Cancer and Developmental Biology, NCI-FCRDC, Frederick, MD, USA l College of Physicians and Surgeons of Columbia University, New York, NY, USA 1. Introduction The first workshop of the MYO-CLUSTER, project EUROMEN (European muscle envelope nucleopathies), was held in Naarden on 15 and 16 September It was attended by 22 clinical and basic scientists from France, Italy, Germany, The Netherlands, UK and USA. The EUROMEN project points towards the application of molecular genetic advances for the understanding and management of Emery Dreifuss muscular dystrophies (EDMD) and associated skeletal and cardiac phenotypes. The aims of this q Other workshop participants: Professor Alan E.H. Emery, Devon, UK; Dr Anne Helbling-Leclerc, INSERM U523-Institut de Myologie, Paris, France; Dr France Leturcq, Laboratoire de Génétique Médicale, Hôpital Cochin, Paris, France; Dr Nadir Maraldi, Istituto di Citomorfologia NP CNR c/o IOR, Bologna, Italy; Dr Daniela Toniolo, IBGE-CNR, Pavia, Italy; Dr J. Andoni Urtizberea, Institut de Myologie, Paris, France; Dr Corinne Vigouroux, INSERM UR402-Hôpital Saint-Antoine, Paris, France; Professor Manfred Wehnert, Institute of Human Genetics, Greifswald, Germany. * Corresponding author. Tel.: ; fax: address: g.bonne@myologie.chups.jussieu.fr (G. Bonne). project are characterization of the phenotype of individuals affected by EDMD and associated phenotypes, development of diagnostic tools that will significantly help the study of muscle biopsies, identification of new loci/genes involved, and creation of in vitro and in vivo models of various clinical entities defined by genetic studies. The scientific coordinator of this project is Ketty Schwartz from INSERM U523, Paris, France [1]. This workshop was in fact the 82nd European Neuromuscular Centre (ENMC) international workshop and the fifth workshop devoted to EDMD; the main focus was the autosomal dominant form of the disease. 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. Two main modes of inheritance, X-linked (X-EDMD) and autosomal dominant (AD- EDMD) occur in EDMD; however, a rare autosomal recessive mode of inheritance had also been reported [2]. At the third ENMC workshop in 1998, it was reported that the locus for AD-EDMD was on 1q11 q23. Subsequently, in /02/$ - see front matter. PII: S (01)

2 188 G. Bonne et al. / Neuromuscular Disorders 12 (2002) , the nuclear lamin A/C (LMNA) gene at this locus was found to be responsible for AD-EDMD [3]. Since this first implication of LMNA, four disorders have now been reported to be caused by mutations in lamin A/C gene: AD- EDMD, dilated cardiomyopathy with conduction-system disease (DCM-CD) [4,5], autosomal dominant limb-girdle muscular dystrophy with AV conduction disturbances (LGMD1B) [6] and Dunnigan-type familial partial lipodystrophy (FPLD) [7]. The meeting opened with a synopsis of the clinical and molecular genetic spectrum of these four disorders. Then, different diagnostic tools were discussed. Finally, the role of lamins A/C mutations was discussed with the help of in vitro analysis of lamin emerin interactions and the study of mice lacking A-type lamins. 2. Clinical and molecular genetic spectrum of disorders due to mutations of lamin A/C gene 2.1. Genetic data Gisèle Bonne presented the update spectrum of mutations identified and reported in the lamin A/C gene. In the context of the EUROMEN program, a large number of DNA samples from patients (most of them having been diagnosed as EDMD) were collected and screened for mutation in LMNA gene by PCR/SSCP/DHPLC/sequencing methods in the two laboratories of INSERM U523 (Paris) and IGBE-CNR (Pavia). LMNA mutations were identified in 18 AD-EDMD families, three LGMD1B families, 39 isolated EDMD cases and one isolated case of DMD-CD ([3,5,6,8,9] and unpublished data). In parallel with this work, mutations were reported in DCM-CD and FPLD. A total of 48 mutations were reported in LMNA, out of which 31 mutations were identified in EDMD ([8 11] and unpublished data), four in LGMD1B [6,10], eight in DCM-CD ([4,5,10] and unpublished data) and seven in FPLD [7,12 14]. Most of the mutations are missense mutations (41/48), four are inframe deletions of a codon and three are nonsense mutations, i.e. one introduction of a stop codon, one base pair deletion and one splice donor site mutation, the two latter introducing a frameshift and a premature stop codon. One mutation was reported to be transmitted as an autosomal recessive trait, with healthy parents carrying the mutation as heterozygote and the affected child carrying the mutation at homozygous state [9]. Two mutations (Q6X and DTaa320) were reported to lead to different phenotypes within the same family [5,10]. In a family carrying Q6X mutation, AD-EDMD and isolated DCM-CD were reported [5], as for DT in aa320 mutation, EDMD, LGMD and isolated DCM-CD were reported among members of the same family [10]. Moreover, a marked inter- and intrafamilial variability in the clinical expression of LMNA mutations exists, which points towards a significant role of possible modifier genes in the course of this disease [8,9]. With the exception of FPLD mutations that are restricted to two specific locations in exons 8 and 11 [7,12 14], there was no clear correlation between the phenotype and the type or localization of the mutations within the gene Clinical data Cardiomyopathy isolated or associated with muscular dystrophy Denis Duboc reported the cardiac investigations of the large French family in which was identified the first LMNA mutation (Q6X). The cardiac evaluation was performed for 54 living relatives, among whom 17 members presented with a cardiomyopathy and only five of these individuals had clinical manifestations of EDMD. They retrospectively determined the cause of death of 15 deceased family members, eight of whom had died suddenly, two as a first and single manifestation of the disease. The six other cases had histories of arrhythmias and left ventricular dysfunction before dying suddenly, and three of them died despite the prior implantation of a permanent pacemaker. The mean age of onset of cardiac symptoms among affected living family members was 33 years (range years), and the first symptoms were due to atrioventricular conduction defects (sometimes prolonged PR interval) or sinus dysfunction, requiring the implantation of permanent pacemakers in seven cases for severe conduction disturbances. Myocardial dysfunction accompanied by ventricular arrhythmias developed rapidly in the course of the disease, and resulted in severe dilated cardiomyopathy requiring cardiac transplantation in three cases. Denis Duboc proposed that in patients presenting a life-threatening familial or sporadic cardiac restricted phenotype similar to that described in this family, mutations in LMNA should be looked for. And in the genotypically affected individuals, cardiological and electrophysiological follow-up should be performed in order to prevent sudden death, which could occur rapidly in the time-course evolution of such disease. These data were published recently in Bécane et al. [5]. Francesco Muntoni presented a family with a frameshift mutation in exon 6 (DTaa320) that he has been able to study together with Professor Luisa Mestroni. The phenotype in this family was interesting: while two members had a classical EDMD phenotype with cardiac involvement, their father had an isolated cardiomyopathy with conductionsystem disease (DCM-CD); a paternal uncle had features of LGMD. This family was reported in Brodsky et al. [10]. Luciano Merlini reported on the early onset of conduction defects in AD-ADMD. A 14-year-old boy with the classic EDMD phenotype showed ventricular ectopic beats as the first manifestation of cardiac involvement. As a comparison to AD-EDMD, he also reported on a 25-year follow-up of an X-linked EDMD patient. From a functional point of view, he had mild weakness, which began in his teens and contin-

3 G. Bonne et al. / Neuromuscular Disorders 12 (2002) ued into his forties, which then later progressed to moderate. He performed manual work until his fifties. In his late sixties, he was still ambulant at home. From the cardiac point of view, at 43 years he had sinoatrial block and marked bradycardia, and a pacemaker was implanted. An episode of atrial fibrillation was shown at age 51. Frequent atrial fibrillation was recorded at 55. At 56, Holter showed persistent atrial fibrillation. At 57, echocardiography showed mild reduction in left ventricular contractility. At 67 the ejection fraction (EF) was 35%, with left ventricular and right atrial dilatation. This patient showed conduction disorder successfully treated with a pacemaker; he subsequently developed persistent atrial fibrillation and dilated cardiomyopathy EDMD phenotype Francesco Muntoni presented the clinical spectrum of patients with AD-EDMD studied at the Hammersmith Hospital; he drew the attention of the participants to the fact that he worked in a paediatric neuromuscular centre, and that therefore it is likely that his experience reflected patients with more severe disease course than those seen in other centres. He indicated that the condition can have its onset in infancy; he reported the case of several children who were already symptomatic in the second year of life with difficulties in walking because of a combination of proximal and distal weakness, with frequent foot drop. These severely affected children also have significant muscle wasting in humero-peroneal distribution at presentation and tightness of the Achilles tendons. Serum CK is usually markedly elevated in these cases and they may follow a severe disease course, losing the ability to walk unsupported in the first decade of life. The most severely affected case in his series was a 4-year-old boy who presented at the age of 13 months with inversion of both feet, scapular winging and scapuloperoneal muscle wasting. He now walks with difficulty with leg support and has already shown signs of cardiac involvement (one episode of ventricular tachicardia during a general anaesthetic at the age of 28 months and one episode of cardiac failure during pneumonia at the age of 4 years). Although the severe weakness of this case was exceptional, in a large series recently reported [8] a significant number of patients (17%) followed a relatively severe disease course with loss of ambulation. This appears different from that observed in XL-EDMD, in which such a severe disease course has only been reported in exceptional cases. Most patients in his paediatric series developed signs of cardiac involvement characterized either by a conduction-system disease, or left ventricular dysfunction, or both, before the age of 20 years. The majority of patients followed a milder disease course, with onset in the first decade of life with contractures and weakness with mild to moderate tendency to progress over the years. Serum CK levels were variable (from normal to moderately elevated) in these patients, with higher levels in individuals between the age of 4.5 and 35 years. Francesco Muntoni also presented the clinical features of an adult who presented with mild proximal weakness, atrial fibrillation, cataracts and hypothyroidism in whom a mutation in exon 11 of the LMNA gene was identified by Bonne et al. This lady lacked the typical contractures and muscle wasting observed in patients with EDMD; she did not have any evidence of lipodystrophy either LGMD1B phenotype: AD-LGMD with AV conduction disturbances A.J. van der Kooi presented three families with an autosomal dominantly inherited neuromuscular disease, characterized by slowly progressive limb-girdle weakness and life-threatening cardiac involvement. Sixty-five living members of the three families underwent neurological, cardiological and biological investigations. A diagnosis of LGMD was established in at least the propositus based on clinical, EMG, and histological data, and careful exclusion of other causes of limb-girdle syndrome. Thirtyfive members were diagnosed as having LGMD: 34 subjects (aged years) were symptomatic, one was identified on the basis of elevated CK (25 normal value). Age of onset varied from 4 to 38 years. The main clinical feature was symmetrical weakness and wasting of the hip and thigh muscles causing moderate impairment in the sixth or seventh decade. Weakness of the upper limbs was observed around the age of 30. Only eight had weakness of the lower legs, and in three mild facial weakness was present. Calf enlargement was seen in six patients. Contractures, if present, were either minimal or a late symptom. Neck or spinal rigidity was absent. CK was normal in ten and elevated (up to 25 ) in 25 patients. EMG and muscle biopsy were consistent with a mild muscular dystrophy. Cardiological analysis disclosed AV conduction disturbances, presenting as AV-block, bradycardia, syncopal attacks or sudden death from the third decade onwards in 21 LGMD patients. There was a significant relation between the severity of AV conduction disturbances and age. Dilated cardiomyopathy was present in two individuals. One 27-year-old individual showed alternating first- and second-degree AV-block on his ECG, but no muscle weakness, in the presence of a markedly elevated serum CK activity (25 normal value). He died of dilated cardiomyopathy 3 years after his first visit. Skeletal muscle CT showed fatty degeneration of predominantly paravertebral, gluteal, thigh, and calf muscles FPLD phenotype: familial partial lipodystrophy Missense mutations of the lamin A/C gene, LMNA, have been recently identified in Dunnigan-type familial partial lipodystrophy (FPLD), which belongs to a group of genetic or acquired diseases associating abnormalities in adipose tissue body repartition and insulin resistance. FPLD is the only lipodystrophic syndrome with a known molecular

4 190 G. Bonne et al. / Neuromuscular Disorders 12 (2002) basis. Clinical signs appear after puberty and are associated with a lack of adipose tissue in the limbs, buttocks and trunk with sometimes fat accumulation in the neck and face, signs linked to major insulin resistance (acanthosis nigricans, hyperandrogenism in females, muscular hypertrophy and altered glucose tolerance or diabetes), liver steatosis and hypertriglyceridemia with an increased risk of acute pancreatitis. Jacqueline Capeau presented the work of her group in St-Antoine Hospital (Paris). They sequenced the LMNA coding region from patients presenting with FPLD or other forms of lipodystrophy. They identified two heterozygous mutations in exon 8, R482W and R482Q, in FPLD patients (six families and one individual). Heterozygous mutations at the 482nd lamin A/C residue, either R482W, Q, or L represent 87% of the genetic alterations described in FPLD; the other mutations reported concern exon 8 of the gene: G465D (one family) and R486N (two families); one family harboured a R582H mutation (exon 11). In addition, their group found a novel heterozygous mutation (R584H) in exon 11, encoding specifically the lamin A isoform, in a patient with typical FPLD. All the affected residues belong to highly conserved lamin C and/ or A protein sequences. All mutations are heterozygous and transmitted among generations. No de novo mutations were reported. Clinical and biochemical investigations in FPLD patients revealed that the expression and the severity of the phenotype were markedly dependent upon sex, female patients being more markedly affected. In subjects with generalized lipoatrophy, either congenital (13 cases) or acquired (14 cases), or Barraquer Simon syndrome (another partial lipodystrophy) (two cases), the entire LMNA coding sequence was normal [12]. In their patients with LMNA mutations, no skeletal or cardiac muscular sign of Emery Dreifuss disease was present. One individual with FPLD (R482W) is currently investigated for a proximal muscular weakness Conclusions There appears to be significant clinical diversity between patients with LGMD1B and AD-EDMD phenotype to justify the different naming of these clinical entities at the moment, although their apparently identical genetic defect is somehow confusing. Similarly, there does not appear to be a clear-cut correlation between the location of the mutation and the isolated cardiac involvement vs. the phenotype also involving skeletal muscle. Finally, there does not appear to be a clear-cut correlation between the location of the mutation in the LMNA gene and the clinical severity of the disease; on the contrary, identical mutation could lead to substantial difference in the severity of the phenotype. The only established correlation between the genotype and the phenotype regards FPLD in which 90% mutations were localized in exon 8 of the LMNA gene. 3. How to optimize diagnostic tools 3.1. Cardiac investigations Excluding the lipodystrophies, cardiomyopathy appears to be the striking feature of nucleopathies: it occurs in every phenotype whereas the skeletal involvement is inconsistent and variable. The cardiac involvement represents the Sword of Damocles over the head of every affected patient, even in an infra-clinical stage of the disease. A particular program of clinical, paraclinical and biological follow-up of the cardiomyopathy will be established through the EUROMEN project and was presented by Denis Duboc. The aims of the cardiac evaluation are: (i) to validate, by re-assessment of genetically proven EDMD patients, the clinical and electrical diagnosis criteria of the disease that were described in the large French family [5]; (ii) to apply specific tools for the early detection of cardiomyopathy, i.e. Doppler tissue imaging will apply in asymptomatic patients with infra-echographic cardiomyopathy; (iii) to establish an appropriate clinical and electrophysiological follow-up of the patients presenting first cardiac symptoms (syncope, acute dyspnea, arrhythmia), i.e. to apply systematically in these cases Holter monitoring (48 h), and electrophysiological testing (EP testing). These results will be correlated to the evaluation of the myocardial function performed by ECG and radionuclide ventricular EF. Along the same lines, in order to detect myocardial involvement before detection of functional abnormalities early in the course of the disease, coronary reserve measurement performed by positron emission tomography (PET) will be proposed (in the centre in which this experiment could be performed); (iv) to develop investigations by cardiac magnetic resonance imaging (MRI), in order to highlight new markers of arrhythmia: lipid atrial or ventricular infiltration, dyskinesis or thinning down of the right ventricular wall, abnormal features of the left ventricular wall; (v) to evaluate the benefit of therapies currently used in the prevention and the treatment of several states of the disease. The aims of this cardiac analysis are to establish correlations between the cardiac involvement and the genetic findings, prognosis criteria and recommendations for relevant follow-up and therapy. The particular endpoint will be to clearly determine the indication of implantable cardioverter (ICD) to prevent cardiac sudden death in this population Skeletal muscle imaging Francesco Muntoni presented the results of muscle imaging using MRI obtained in collaboration with Dr Mercuri at the Hammersmith Hospital. Eight patients with mutationally proven diagnosis of AD-EDMD had MRI scanning of their leg muscles. Three patients with XL- EDMD were also studied. In all patients with AD-EDMD studied, there was a characteristic pattern of muscle invol-

5 G. Bonne et al. / Neuromuscular Disorders 12 (2002) vement of the calf muscles with predominant involvement of the medial head of the gastrocnemius while the lateral head was relatively spared. This pattern of muscle involvement occurred in isolation in mildly affected patients while was present as part of an overall more diffuse involvement in the patients with more advanced disease. This pattern appears to be specific to AD-EDMD, as patients with the X-linked variant showed a different pattern of involvement. Two other patients of interest were briefly presented; one was a lady with the exon 11 mutation and the proximal weakness. Her muscle only showed some mild proximal involvement, a very different picture from that seen in EDMD patients. The other was an 11-year-old boy who had been assessed very recently; the pattern of muscle involvement with MRI was more similar to that observed in the X-linked form of the disease, but emerin immunolabelling was normal in the muscle biopsy. Mutation analysis of both the emerin and LMNA gene is in progress for him. Beril Talim presented the results of muscle computerized tomography (CT) examination in ten EDMD patients (six AD and four X-linked). Age of examination was between 14 and 64 years for AD and between 16 and 63 years for X- linked cases. There was early and progressive involvement of paravertebral and posterior calf muscles, with subsequent involvement of vasti, hamstrings and adductor magnus. Rectus femoris, sartorius and gracilis were spared. Anterior lateral lower leg muscles were usually affected to a lesser degree compared to posterior calf muscles, but there was selective involvement of the peroneals in one case. An X- linked EDMD patient showed sparing of the lateral head of the gastrocnemius while its medial head and soleus were moderately affected. Asymmetrical involvement was present in some patients. One AD-EDMD patient had moderate to severe substitution of the posterior calf muscles on one side while the other side was completely normal; in one X-linked EDMD patient there was unilateral sparing of the vastus lateralis. Muscle CT examination is a useful technique for showing the pattern of selective muscle involvement in EDMD. However, there is no distinct pattern of involvement in AD or X-linked forms of the disease Muscle biopsy evaluation: histological, histochemical, immunocytochemical and electron microscopy techniques Caroline Sewry presented pathological data on the skeletal muscle biopsies of nine cases from seven families with identified mutations in the lamin A/C gene, aged 2 35 years at the time of biopsy. The severity of pathological change was moderate in most cases and the features were mainly myopathic rather than dystrophic, as necrosis and increased connective tissue were rare. The most common features were variation in fibre size, increase in internal nuclei, and selective involvement or predominance of fibres with high oxidative enzyme activity. A few fibres had particularly high oxidative enzyme content. One severely affected child showed a little necrosis in the first biopsy (quadriceps), and this was more evident in a second sample (tibialis anterior) obtained a few years later. Immunocytochemistry detected an age-related secondary reduction of laminin b1 on the muscle fibres in adolescent and adult cases. Although this is neither a specific nor a consistent feature, it may be a useful diagnostic marker when present. Nuclear labelling with antibodies to lamin A, A/C and emerin did not reveal any detectable differences from controls. Electron microscopy in a few cases revealed some abnormal distribution of heterochromatin in many fibre nuclei, but the significance of this is not yet certain. These data have been submitted for publication [15]. Stefano Squarzoni presented electron microscopy of X- linked and AD-EDMD cases. This study revealed structural nuclear alterations both in the X-linked EDMD (one case) and in AD-EDMD (three cases). The most relevant changes were represented by loss of contact between the peripheral heterochromatin and nuclear lamina, focal absence of heterochromatin along the nuclear envelope and hypercondensation of the residual heterochromatin. In skeletal muscle nuclei from an X-linked EDMD patient, nuclear lamina thickening was also present, while in the AD- EDMD form, a looser inter-chromatin texture and a nonuniform nuclear pore distribution were observed. These alterations were detected in about 10% of the nuclei of cells bearing a preserved cytoplasm. As the available EDMD samples for electron microscopy studies are very few, a method has been developed to observe samples, which had previously been frozen for routine cryostat sectioning, at the electron microscope level. The preservation of the cellular structures obtained by this method appears very good, provided that the initial freezing of the tissue had been performed carefully. Stefano Squarzoni developed further approaches in the study of the X-linked form, such as oral exfoliative cytology and the blood smear immunohistochemistry. These demonstrated that circulating platelets contain emerin, localized (by electron microscopy immunohistochemistry and Western blot) both to cytoskeletal and to lipid-bound structures. The possible role of emerin in the cell metabolism was also evaluated in two models: the differentiation process of muscle cultured cells and the regeneration of animal skeletal muscle. In both cases, an up-regulation of emerin has been detected; moreover, the presence of this protein in the cytoplasm of differentiation-committed cells and in myotubes has been observed. These results suggest an involvement of emerin in the muscle differentiation, most probably in connection with cytoplasmic functions Western blot analysis as diagnostic tool in nucleopathies France Leturcq and Dominique Recan reported their experience with Western blot analysis. During the last 4 years, about 80 EDMD patients were referred to LBGM

6 192 G. Bonne et al. / Neuromuscular Disorders 12 (2002) (Laboratoire de Biochimie et Génétique Moléculaire) at Cochin Hospital in order to screen emerin expression and, more recently, lamin A/C expression. As peripheral blood samples were more accessible than muscle biopsies, and as lymphoblastoid cell lines (LCL) establishment was available in Cochin Hospital cell bank, France Leturcq optimized emerin Western blot on LCL rather than on biopsies. Moreover, pellets of 50 million lymphoblastoid cells (i.e. 50 million nuclei) give more homogeneous and more total proteins, for reliable quantitative studies, than a muscle biopsy. In 19 cases (25%) emerin was absent. In all of them, Dominique Recan identified a mutation in the emerin gene. Among the remaining 58 emerin positive cases, LMNA gene was fully investigated in 23 patients and 17 LMNA mutations were characterized in the INSERM U523 laboratory. These results suggest that the screening of emerin by Western blot, as a first step of the molecular diagnostic process, will direct further molecular investigations and the LMNA gene is the first candidate to be analyzed if emerin is positive. In none of these cases were evident quantitative and qualitative differences in emerin observed on LCL Western blots. Polyclonal and monoclonal antibodies against lamin A/C from J.C. Courvalin (Institut J. Monod, Paris) and North East Wales Institute (NEWI), respectively, were used for Western blots. As muscle immunohistochemistry experiments showed normal lamin labelling in LMNA patients, comparative Western blots were performed from several controls and LMNA mutant LCL samples, by double labelling using emerin and lamin A/C Ab. More than 30 samples were analyzed. Preliminary results show that: (i) lamin A/C expression/labelling are different in muscle and in LCL; and (ii) lamin A/C expression/labelling seems reduced in some LMNA patients. To confirm these first results, further investigations will now be intended in order to (i) standardize the method by the use of other mab as internal controls in a multiplex Western blot in association with a reliable automatized quantification method, and (ii) analyze further control samples and other available LMNA mutants. 4. Basic aspects of nuclear envelope proteins structure and function 4.1. The nuclear envelope and the nuclear envelopathies Howard Worman cloned the human lamin A/C gene and worked on the identification and characterization of inner nuclear membrane proteins and their genes. He presented the basic aspects of the nuclear envelope as well as hypotheses regarding possible pathogenic mechanisms of the nuclear envelopathies. The vertebrate nuclear lamina is a protein meshwork associated with the nuclear face of the inner nuclear membrane (INM). It provides anchoring sites for chromatin domains and is an important determinant of inter-phase nuclear architecture. The major components of the lamina are the intermediate filament-like proteins, the nuclear lamins. The lamins are grouped into two classes, A-type and B-type. Three genes encode nuclear lamins in humans; lamins A, C, A(delta)10 (A-type lamins) and C2 arise by alternative RNA splicing and are encoded by the LMNA gene on chromosome 1q Lamin B1 is encoded by the LMNB1 gene on chromosome 5q and lamins B2 and B3 are encoded by LMNB2 on chromosome 19p13.3. Lamin A is synthesized as a precursor, prelamin A, which is farnesylated and subsequently processed by endoproteolysis to a shorter form. A nuclear protein called Narf has also been identified that specifically associates with prenylated prelamin A; however, its function is not clear [16]. In inter-phase cells, six integral membrane proteins are specifically localized to the inner nuclear membrane: LBR, LAP1 (A, B, C isoforms), LAP2 (seven isoforms, some nucleoplasmic), emerin, nurim and MAN1. LBR, LAP1, LAP2 and emerin interact with lamins and/or chromatin. Protein sequence analysis has identified a conserved globular module of approximately 40 amino acids which has been termed the LEM domain because it is found in LAP2, emerin and MAN1. It is not clear how mutations in emerin cause XL-EDMD or how mutations in lamins A/C cause the AD-EDMD as well as DCM-CD, LGMD1B 1B and FPLD. Several hypotheses can be proposed, including: (i) increased susceptibility to apoptosis as nuclear lamins and inner nuclear membrane proteins are substrates for capases; (ii) regulation of cell type-specific gene expression by chromatin inner nuclear membrane interactions, a possible example being the interaction of LBR with HP1 chromatin proteins; (iii) increased susceptibility to mechanical stress as the nuclear lamina and inner nuclear membrane may be connected to the cytoskeleton via nuclear pore complexes; (iv) functions of inner nuclear membrane proteins in locations other than the nuclear envelope in some cells as suggested by the controversial localization of emerin to cardiac muscle intercalated discs. In preliminary studies, H. Worman s group has examined the targeting of 15 lamin A with point mutations in patients with muscular dystrophy and partial lipodystrophy in transfected C2C12 cells. Most target appropriately to the inner nuclear membrane, but three induced the formation of markedly abnormal nuclear envelopes containing aggregates of mutant and endogenous lamins EDMD pathophysiology and nuclear signalling Nadir Maraldi, after a presentation of nuclear signalling, proposed a hypothesis for the pathophysiology of EDMD in relation to nuclear signalling. The involvement of two nuclear proteins, emerin and lamin A/C, in EDMD raises new questions about the mechanism of muscle cell degen-

7 G. Bonne et al. / Neuromuscular Disorders 12 (2002) eration, which might be different from that of muscular dystrophy due to membrane-associated proteins. Knowledge of the molecular structure and activities of the cell nucleus has been significantly improved recently and can account for some aspects of EDMD pathophysiology. In fact, the control of gene expression through the release of template restriction depends on the chromatin organization that, in turn, is modulated by interactions with the nuclear matrix. Moreover, the nucleus is not only the final target of extracellular signals, but, in response to them, activates an autonomous signalling pathway based on inositol lipids, which are phosphorylated and hydrolyzed at specific intranuclear sites. The intra-nuclear signalling system is involved in key functions, such as initiation of DNA replication and control of transcript splicing, as well as in the heterochromatin decondensation, which represents a prerequisite of gene transcription. The chromatin remodelling complexes (CRC), in fact, have been demonstrated recently to be constituted by a nuclear actin isoform, whose polymerization is modulated by the signalling system constituted by inositol lipids linked to profilin at the nuclear matrix level. As a consequence, the reduced or altered expression of structural nuclear matrix proteins, such as lamin A/C and emerin, may affect chromatin arrangement and gene expression. This can result in modified patterns of development that mainly affect some cell lineages of common origin and prevent correct mechanisms of differentiation of satellite cells in the adult Lamin emerin interaction Glenn Morris showed how the BIAcore biosensor could be used for quantitative studies of interactions between emerin and lamin A. The NEWI group prepared lamin A with a known FPLD mutation (R482Q) by in vitro mutagenesis. Nuclear targeting of lamin A in transfected COS-1 cells, human skeletal muscle cells or mouse adipocyte cell cultures (pre- and post-differentiation) was not detectably affected by the mutation. Quantitative in vitro measurements of lamin A interaction with emerin using the biosensor also showed no effect of mutation. The results show that the loss of function of R482 in lamin A/C in FPLD does not involve loss of ability to form a nuclear lamina or to interact with the nuclear membrane protein, emerin. They also showed that a Q133H mutation in the nuclear targeting region of emerin did not prevent emerin localization to the nuclear membrane. These data are currently in press [17] Loss of A-type lamin function results in muscular dystrophy and the subcellular relocalization of emerin To determine the function of the A-type lamins in embryogenesis, the group of Colin Steward and Brian Burke derived mice null for A-type lamin expression. Colin Stewart presented recent data obtained with lmna 2/2 mice [18]. Development of the homozygotes to weaning appears normal. Thereafter, they cease to grow and develop a stiff walking posture with splaying of the hind legs and a hunched appearance. By 5 6 weeks, the homozygotes start to die with all having perished by 8 weeks. Histologically, the lmna 2/2 mice are characterized by a marked absence of white fat (WAT) and the skeletal musculature, particularly the paravertebral, distal limb, pharyngeal and tongue, are dystrophic. The hearts are abnormal with many atrophic and abnormal myocytes with vacuolated cytoplasm. Mice heterozygous for the lmna mutation are overtly normal at 6 10 months with minimal evidence of dystrophy. Embryonic fibroblast and liver cell nuclei from the homozygotes exhibit an abnormal highly irregular and often polarized morphology, with the loss of lamin B, and other INM-associated proteins such as Lap2 and Nup153 from one pole. Emerin is normally anchored to the INM, where it interacts with the A-type lamins and is entirely localized to the nucleus. In lmna 2/2 null fibroblasts emerin is redistributed between the endoplasmic reticulum in the cytoplasm and the INM. In tissues from lmna 2/2 mice, emerin exhibited differential relocalization according to the tissue, from a marked reduction in tongue epithelium, diminished nuclear localization in skeletal myocytes to a polarized location in cardiac myocytes. Emerin localization to the INM therefore depends on the lamins, although its exact cellular distribution may be modified by other as yet unidentified tissue-specific factors. Although the lmna null mice have no WAT they are not insulin resistant and are tolerant to glucose indicating that they may not be lipodystrophic at this age. There are also indications that their hearts are enlarged, particularly in those that survive to 8 weeks. Whether the homozygous lmna 2/2 mice develop all three pathologies is at present unclear. A full understanding of these phenotypes will only become available with the derivation of novel mouse lines carrying individual point mutations in the lmna gene or that are null for emerin. Acknowledgements This workshop was made possible thanks to European Community grant (QLG ) MYO-CLUSTER- EUROMEN (Application of molecular genetic advances to the understanding and management of Emery Dreifuss muscular dystrophies and associated skeletal and cardiac phenotypes) and to the logistic support of the European Neuromuscular Centre (ENMC). References [1] Merlini L. Myo-Cluster. Neuromusc Disord 2001;11: [2] Emery AEH. Emery Dreifuss muscular dystrophy a 40 year retrospective. Neuromusc Disord 2000;10: [3] Bonne G, Di Barletta MR, Varnous S, Becane H, Hammouda EH, Merlini L, Muntoni F, Greenberg CR, Gary F, Urtizberea JA, Duboc D, Fardeau M, Toniolo D, Schwartz K. Mutations in the gene encod-

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