Neuromuscular Disorders 13 (2003) Workshop report

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1 Neuromuscular Disorders 13 (2003) Workshop report 108th ENMC International Workshop, 3rd Workshop of the MYO-CLUSTER project: EUROMEN, 7th International Emery-Dreifuss Muscular Dystrophy (EDMD) Workshop, September 2002, Naarden, The Netherlands Gisèle Bonne a, *, Rabah Ben Yaou a, Christophe Béroud b, Giuseppe Boriani c, Sue Brown d, Marianne de Visser e, Denis Duboc a,f, Juliet Ellis g, Irena Hausmanowa-Petrusewicz h, Giovanna Lattanzi i, Luciano Merlini j, Glenn Morris k, Francesco Muntoni d, Grzegorz Opolski l, Yigal M. Pinto m, Federica Sangiuolo n, Daniela Toniolo o, Richard Trembath p, Jop H. van Berlo m, Anneke J. van der Kooi e, Manfred Wehnert q a Inserm UR582 (ex 523), Institut de Myologie, Bâtiment Babinski, G.H. Pitié-Salpétrière, 47, boulevard de l Hôpital, Paris Cedex 13, France b Laboratoire de Génétique Moléculaire et Chromosomique, IURC, Montpellier, France c Institute of Cardiology, University of Bologna, Bologna, Italy d Department of Paediatrics and Neonatal Medicine, Imperial College School of Medicine, Hammersmith Campus, London, UK e Department of Neurology, Academic Medical Centre, Amsterdam, The Netherlands f Service de Cardiologie, GH Cochin, Paris, France g Randall Center, Kings College, London, UK h Neuromuscular Unit, M.R.C. Polish Academy of Sciences, Warsaw, Poland i ITOI, Unit of Bologna, c/o IOR, Bologna, Italy j Istituto Ortopedico Rizzoli, Neuromuscular Unit, Bologna, Italy k MRIC, North East Wales Institute, Wrexham, UK l Department of Internal Medicine and Cardiology Medical University of Warsaw, Warsaw, Poland m Department of Cardiology, University Hospital Maastricht, Maastricht, The Netherlands n Dipartimento di Biopatologia e Diagnostica per Immagini, Rome, Italy o Istituto di Genetica Biochimica ed Evoluzionistica, CNR (IGBE-CNR), Pavia, Italy p Division of Medical Genetics, University of Leicester, Leicester, UK q Institute of Human Genetics, Greifswald, Germany 1. Introduction The ENMC consortium on Emery-Dreifuss Muscular Dystrophy (EDMD) held its 7th meeting in Naarden during the weekend of September 13 15, It was attended by 23 participants from six countries, which included France, Germany, Italy, the Netherlands, Poland and the United Kingdom. This workshop was sponsored by the European Communities and represented the third meeting of the Myo- Cluster project EUROMEN (EUROpean Muscle Envelope Nucleopathies). This meeting focused on three main areas: (1) new * Corresponding author. Tel.: þ ; fax: þ address: g.bonne@myologie.chups.jussieu.fr (G. Bonne). clinical insights in laminopathies and emerinopathies: whereas mutations in the emerin gene (EMD) give rise to one phenotype, the X-linked form of EDMD, up to six phenotypes are associated with mutations in the lamin A/C gene (LMNA). The clinical spectrum of these disorders was discussed with a special emphasis on the tissue-specificity of the various laminopathies and their possible overlaps; a special emphasize was made on the cardiac diseases with the aim to give recommendations for cardiac management of these disorders; (2) the genetic spectrum of these disorders including the current status of the mutation databases of EMD and LMNA genes; and (3) the fundamental aspects of these diseases with the analysis of the cellular consequences of mutations in EMD and LMNA genes /03/$ - see front matter q 2003 Published by Elsevier Science B.V. doi: /s (03)

2 G. Bonne et al. / Neuromuscular Disorders 13 (2003) Clinical aspects 2.1. Wide clinical spectrum of Laminopathies: an update of new phenotypes Within the last year, two new phenotypes were reported to be due to LMNA mutation, an autosomal recessive form of Charcot-Marie-Tooth type 2 disease (AR-CMT2) and the mandibuloacral-dysplasia (MAD). These two new phenotypes of laminopathy, both autosomal recessive, are still tissue-specific disorders affecting either the peripheral nerve for AR-CMT2 or the bone/skin/adipose tissue for MAD Charcot-Marie-Tooth type 2 disorders (AR-CMT2) Rabah Ben Yaou (Paris, France) and Gisèle Bonne (Paris, France), in the name of Nicolas Levy (Marseille, France) who was not able to attend the meeting, presented respectively the clinical and the genetics features of the fifth phenotype linked to LMNA mutations [1]. In order to identify the genes involved in autosomal recessive CMT2 (AR-CMT2), homozygosity mapping and candidate genes screening were performed on consanguineous Algerian families affected with AR-CMT2. Linkage to the 1q21.2 q21.3 region was evidenced in three families with all affected patients sharing an ancestral homozygous haplotype inside the linkage interval and marker D1S2721 being in linkage disequilibrium with the disease. A homozygous LMNA mutation (C892T), leading to R298C substitution was identified in all affected patients. A further analysis of CMT2 families from Algeria identified the same homozygous mutation in patients from seven more unrelated families; this further confirmed that R298C is a founder mutation in Algeria. Clinically, the age of onset was in the second decade, with distal wasting and weakness more prominent in the lower than in the upper limbs, and areflexia. Proximal wasting and weakness was noticed in six and kyphoscoliosis in three patients. Pes cavus was common (7/9). Motor nerve conduction velocity was normal or slightly reduced and sensory nerve action potential was decrease or absent clearly reflecting an axonal degenerative process. A sural biopsy from patient and sciatic nerves from lmna 2/2 mice were examined by electron microscopy. In human, a marked loss of myelinated and unmyelinated fibers was observed, with absence of large myelinated fibers. Lesions of axons and myelin, highly similar to those observed in human CMT2 were also observed in sciatic nerves of lmna 2/2 mice. Deeper morphological and functional explorations are in process on patients and lmna KO and KI mice, and will give new hints towards a better understanding of nervemuscle interactions, and to the question as to whether mutations in the ubiquitously expressed lamin A/C proteins have tissue-specific or, vice versa, pleiotropic effects Mandibuloacral dysplasia (MAD) Luciano Merlini (Bologna, Italy) reported on the MAD story, the sixth laminopathy phenotype. MAD is a rare autosomal recessive condition characterized by short stature, under-developed lower jaw and clavicles and resorption of terminal phalanges, small face with beaked nose, high-pitched voice, stiff joints, hair loss, and mottled pigmentation of the skin. Among a total of 30 reported patients, three recently described, had loss of fat from the upper and lower extremities, and one of them had insulin resistance and diabetes mellitus. Reviewing the literature Merlini noticed that the first report of this condition described a patient native of Italy who in addition to mandibular hypoplasia and acroosteolysis showed after the age of 10 increase in subcutaneous tissue in a buffalo hump distribution. The picture of this patient showed the classic lipodystrophy picture with fat loss of limbs and trunk and accumulation in the face and neck [2]. In the paper this peculiar appearance was described but not recognized as expression of partial lipodystrophy. For this reason it was thought that lamin A/C was a good candidate for MAD. Merlini examined two patients from the five consanguineous MAD families with nine patients all from a sparsely populated area from central Italy identified by Giuseppe Novelli. The patients showed no detectable muscle wasting or weakness and creatine kinase levels were normal. Patients reported no cardiac symptoms and electrocardiograms were normal. Muscle magnetic resonance imaging showed a normal muscle appearance but complete loss of subcutaneous fat in trunk and limbs, marked accumulation around the neck, and normal intra-abdominal fat distribution. Insulin resistance and diabetes mellitus were shown in these patients. Federica Sangiuolo (Rome, Italy) reported then, the genetic analyzes in these five consanguineous MAD families originated from Italy [3]. They mapped the MAD gene on human chromosome 1 in the region where LMNA was localized and defined a conserved haplotype present in all affected patients never observed in controls. Sequence analysis of LMNA revealed a G1580A transition that cause R527H change. All patients were homozygotes for this mutation. The mutation is located in exon 9, which encodes for the C-terminal domain of the protein. The R527H change probably alters the surface structure of the protein, and its fundamental binding sites, since arginine residues in position 527 forms critical hydrogen bonds with DNA. The mutation was dated using the Q method, calculating the allele frequencies of a marker located outside the diseasebearing haplotype (D1S305) among normal and mutated chromosomes. This analysis has revealed that R527H is originated during the early middle age (XI century), about 40 generations ago (935 years ago). All Italian MAD families come from a small area of Central Italy, strongly suggesting again the origin of the mutation from a common founder. To confirm the pathogenetic effect of this mutation, cultured skin fibroblasts from a patient (homozygous for R527H) and his unaffected mother (heterozygous) were analyzed by immunofluorescence analysis, using a lamin

3 510 G. Bonne et al. / Neuromuscular Disorders 13 (2003) A/C specific antibody. Patient nuclei clearly show a lobulation of nuclear envelope with a honeycomb labelling of lamins A/C, while in the control nuclei the lamin localized at nuclear envelope. This pattern was similar to the one already described in autosomal dominant EDMD patients. The complexity of the MAD phenotype suggest that other genes are involved in pathogenesis of the disorder. To identify additional anomalies in expression of other genes, a microarray analysis was established, containing about 5000 cdnas mainly expressed in the skin since MAD is considered as dermal dysostosis. Preliminary results revealed at least 31 ESTs (Expressed Sequence Tags) up-regulated and 16 down-regulated. Studies are in progress to characterize these ESTs. MAD is therefore now classified as new type of laminopathy characterized by a variety of phenotypic changes that include skeletal and skin defects Patients with no EMD or LMNA mutation but affected with a cardiomyopathy and myopathy compatible with a laminopathy or emerinopathy Francesco Muntoni reported on two families with a combination of cardiac and muscle involvement in whom a laminopathy was excluded. The first family was a three-generation family: the propositus is a 36-years-old male who presented at the age of 19 with syncopes. An atrio-ventricular conduction defect was documented on him and he was implanted with a pacemaker. In the last 10 years he started to complain about breathless following minimal exertion and fatigability. His serum CK was elevated at 900 IU/l. His mother and maternal grandmother were similarly affected and had died of sudden death (his mother died despite pacing). On examination he had mild wasting of the deltoid and calf muscles, mild tightness of the Achilles tendon and elbows and weakness in elbow extension, hip flexion and abduction, knee flexion and foot dorsiflexion. A muscle biopsy was myopathic, with variability in fibre size because of scattered very small fibers. Immunocytochemistry showed occasional fibres with desmin accumulation, but normal expression of dystrophin, lamin A and emerin. The EMD and LMNA genes were studied in Paris and found to be normal; his desmin gene is being studied in USA and the preliminary report suggests this is normal. The second is a dominant family (mother and son). The propositus is a 20-year-old male whose complains are of muscle cramps and fatigue on exercise. He has mild muscle hypertrophy (quads, triceps, calves) and minimal proximal muscle weakness. His cardiac examination is normal so far, but his serum CK are elevated ( ) and a muscle biopsy is myopathic. His mother had similar features but in addition developed a dilated cardiomyopathy leading to cardiac transplant when 35. Her CK are elevated ( ). The LMNA and EMD genes were excluded in this family as well Cardiac disease in emerinopathy and laminopathy LMNA mutation and unusual cardiac phenotype Muntoni presented an intriguing family in which the two affected members (mother and son) demonstrated severe cardiac involvement followed by proximal myopathy. The mother presented in the 3rd decade of life with dilated cardiomyopathy followed by skeletal muscle weakness. Her condition progressed rapidly shortly after giving birth to a male infant and she developed heart failure leading to death at the age of 35. Skeletal and cardiac muscle biopsies showed myopathic features and mild accumulation of desmin. The boy remained asymptomatic until his late teens when he presented with severe hypertrophic cardiomyopathy. The cardiomyopathy rapidly progressed and the patient, who developed mild muscle weakness in the meantime, required a cardiac transplant at the age of 21. Muscle and heart biopsies showed fibrosis, myopathic changes and accumulation of desmin. The explanted heart showed extensive replacement fibrosis, extensive myocyte and myofibrillar disarray consistent with hypertophic cardiomyopathy and randomly scattered myocytes containing accumulation of desmin. Since direct sequencing excluded the primary involvement of the desmin gene, mutational analysis of LMNA was performed in this patient in Paris. Gisèle Bonne and Pascale Richard identified a mutation in exon 11 (1930C. T) leading to R644C. The same mutation has been identified in Paris in another case from London with cardiomyopathy, as well as reported in a patient with dilated cardiomyopathy [4]. The confusing picture is however given by the screening of the maternal DNA: this failed to identify the LMNA mutation, suggesting that she was affected by a different disorder. This case therefore highlights the possibility that a hypertrophic cardiomyopathy can be associated with a mutation in the LMNA gene. However, it is likely that the mixed genetic background of this patient played a significant role in determining the hypertrophic response Cardiac sudden death in laminopathies Denis Duboc and Rabah Ben Yaou (Paris) reported on the results of a retrospective study on sudden death in laminopathies performed in collaboration with the Euromen consortium. The circumstances of death have been reviewed in 27 families carrying LMNA mutation and including at least one unequivocally affected and deceased member. Among the 154 affected members, 69 died in different circumstances: 32 died suddenly, 14 at the end stage of heart failure, three from heart transplant rejection and 18 died in unknown circumstances. Among the 32 patients who died suddenly at an age ranging from 20 to 70 (M ¼ 45), 12 had pacemaker implanted for conduction abnormalities and five had a left ventricular ejection fraction $45%. In fact, pacemaker does not prevent sudden death in these patients in whom ventricular arrhythmia was suspected to be the cause of death. Complete electrophysiological (EP, invasive

4 G. Bonne et al. / Neuromuscular Disorders 13 (2003) and non-invasive) testing is indicated to detect atrioventricular conduction defects but also ventricular vulnerability. Implantable cardioverter defibrillator (ICD) is highly recommended for these patients especially if pacemaker is needed. Larger indication of ICD implantation has to be definitely discussed for every patient carrying Lamin A/C gene mutation. To illustrate the situation, the cardiological history of three patients carrying LMNA mutation and who required ICD implantation was reported. The first patient (carrying DK260 LMNA mutation) had a moderate EDMD phenotype. She had slight left ventricular (LV) dysfunction, several premature atrial and ventricular contractions and arrhythmia during EP testing. An ICD was implanted. Multiple chocks secondary to ventricular tachycardia have been detected after ICD implantation. The second patient (R453W LMNA mutation) had a severe EDMD phenotype (contractures, loss of ambulation and respiratory failure). Her cardiac assessment revealed conduction abnormalities with 1st and 2nd degree atrio-ventricular block (AVB I and II), slight LV and RV dysfunction, and several premature atrial and ventricular contractions. An ICD was implanted and no electrical event was detected until now. The third patient (R50P LMNA mutation) had also a severe EDMD phenotype (severe contractures, loss of ambulation). He had atrial fibrillation associated with AVB I and II and bradycardia, with slightly reduced left ventricular ejection fraction. After an episode of stroke of thromboembolic origin and despite normal EP testing, pacemaker was indicated and an ICD was implanted. In the same line, Jop H. van Berlo and Yigal M. Pinto (Maastricht, The Netherlands) reported a retrospective study of the literature on sudden death not prevented by pacemaker therapy in patients with lamin A/C mutations. They pooled the clinical data of 20 publications reporting carriers of mutations in lamin A/C gene. In total, 299 carriers of a LMNA mutation have been published, 163 males, with a mean age at presentation of 31 years. The overall penetrance of heart failure was reported in 26%. This increased with age to 64% after the age of 50. Conduction system disease was reported overall in 61%, which increased with age to 91% after the age of 30. The overall pacemaker implantation rate was 28% with an increase with age to a pacemaker implantation rate of 44% after the age of 30. Seventy-six patients (25.4%) had died at a mean age of 46.4 years. 38% of pacemaker carriers had died versus 21% of patients without a pacemaker. More than 50% of pacemaker carriers had died suddenly. Similarly, over 43% of those without a pacemaker suffered sudden death. In conclusion, carriers of LMNA mutations show an unexpected high risk of premature sudden death. This risk is not related to heart failure, which is reported far less frequent. Importantly, sudden death seems to be caused by lethal tachy-arrhythmias given the failure of pacemaker therapy. Therefore, similarly as Duboc and Ben Yaou conclusion, the recommendation proposed is that when carriers of a LMNA mutation need a pacemaker, an automated cardioverter defibrillator should be considered. Two manuscripts from Ben Yaou et al. and Yigal et al. are currently submitted to promote the latter message Longitudinal study of the cardiac disease in XL- and AD-EDMD Giuseppe Boriani (Bologna, Italy), in collaboration with Luciano Merlini, reported on a series of 18 patients with both X-linked and autosomal dominant EDMD who were followed in Bologna. This longitudinal study showed that while atrial standstill is known to be a typical manifestation of EDMD from a young age, atrial fibrillation and flutter often combined with atrio-ventricular block should also be recognized as frequent arrhythmic expressions of the disease, which can occur at various stages of its clinical manifestation. Moreover, this series of patients suggests that in the presence of either atrial standstill or of atrial fibrillation/flutter, there is a risk of cerebral thromboembolism, and particularly of ischemic stroke due to emboli of cardiac origin. Stroke can be the first clinical manifestation of EDMD in young adults, and can frequently be disabling. Thus, even though no specific guidelines for administration of aspirin or anticoagulants are currently available for EDMD, antithromboembolic prophylaxis is probably required even in young EDMD patients affected by atrial fibrillation/flutter or standstill. Early implant of a pacemaker is advisable when important bradyarryhthmias appear, irrespective of the symptoms. The indication to couple defibrillation capabilities (i.e. to implant a cardioverter defibrillator) is left to physician judgment and will probably increase for AD-EDMD. No case of sudden death was observed among patients with X-linked EDMD at long-term follow-up after pacemaker implant (median follow-up 9 years, range 1 24 years). Implantation of a device at a relatively young age requires special attention regarding potential long-term complications (lead fracture, failure of the system, etc.). This series of patients highlights the broad spectrum of clinical expression of EDMD, which may also include severe heart failure, requiring heart transplant. No apparent relationship between the degree and the severity of muscular and cardiac involvement was evident in this series of patients Cardiac pacing in EDMD patients Grzegorz Opolski and Irena Hausmanowa-Petrusewicz (Warsaw, Poland) reported on cardiac pacing in EDMD patient during period between 1976 and 2002, 13 patients from 44 families with EDMD. The study included 11 males and two females. At the time of implantation the mean age of males was 27 (21 34 years). The age of both females was 41 and 42 years, respectively. Eleven patients were presented with emerinopathy and two patients with laminopathy. While history of presyncope was observed in only four patients, no syncope was reported. Sinus node dysfunction was observed in all patients. Tachycardia-bradycardia

5 512 G. Bonne et al. / Neuromuscular Disorders 13 (2003) syndrome (mostly paroxysmal and permanent atrial fibrillation AF) occurred in 11 and four patients, respectively. Atrio-ventricular block (mainly I or II degree) was observed in three patients. Single-chamber ventricular (VVI) and dual-chamber atrio-ventricular (DDD) pacemakers have been implanted in ten and three patients respectively. Initially, six patients were qualified to DDD pacing. However, during implantation two patients presented with very high atrial pacing thresholds exceeding five volts (indicating diminished atrial tissue excitability). In a one of these cases a pathological lengthening of the latency time following the testing impulse (more than 100 ms) occurred. In two other patients a paroxysmal AF during the implantation procedure initiated by a mechanical pacing or provoked by an electrical pacing during the examination was observed. Finally, only three patients have had a DDD system implanted. During the mean 12-year follow-up period no patients died. In one patient complete loss of capture was observed, due to the appearance of the Twiddler s Syndrome 3 months after implantation with withdrawal of the electrode from the right ventricle. This complication was caused by lack of supporting-muscle tissue surrounding the pacemaker pocket. In conclusion, diminished excitability of the atrial tissue towards electrical pacing together with electrical instability of the atrium leading towards paroxysmal AF makes the introduction of DDD pacing in EDMD patients practically impossible. In EDMD patients with emerinopathy, pacemaker implantation for significant bradyarrhythmia seems to be an effective method in prevention of sudden cardiac death. 3. Genetics aspects 3.1. LMNA and EMD UMD databases: tools for analysis of phenotype/genotype and genotype/phenotype relations Christophe Beroud (Montpellier, France) presented the concept of Universal Mutation Database (UMD) mutation databases. To handle and exploit the numerous data collected by the French Clinical and Research network for EDMD and other nucleopathies and the EUROMEN consortium on emerinopathy and laminopathy, Locus Specific DataBase (LSDB) was developed. It was adapted from the UMD package [5] previously used for many LSDBs such as p53, APC, FBN1, LDLR, VHL, MEN1, ATP7B. The specific features of the present software are: (i) access to the various routines via internet; and (ii) inclusion of multi-parametric analytic tools allowing optimized searching of correlations. This requires a full implementation of relevant clinical, para-clinical and biological features. A specific module have been developed to display phenotype-genotype and genotype-phenotype correlations. The database of EMD and LMNA genes mutations was developed using the Universal Mutation Database tool. They contain all mutations localized either in the coding sequence or in the intronic splice site junctions of both genes. In order to standardize the numbering system used to describe mutations, the UMD-EMD and UMD- LMNA software use the nomenclature recommendations from HUGO [6]. The current versions of the databases contain 537 entries for UMD-LMNA and 84 entries for UMD-EMD. When the same mutation from the same sample was reported in more than one article, only the first report was taken into account. For each mutation, information is provided at several levels: at the gene and the protein levels, at the patient and clinical levels and at the molecular level. The software package contains routines for the analysis of the databases developed with the 4th dimension from 4D. The use of the 4D language gives access to optimized multicriteria research and sorting tools to select records from any field. Moreover, all routines already developed for other UMD databases [5] were added to the EMD and LMNA package with the addition of new routines like: (1) Potential Stop codons, a new function which displays all codons from a specific exon that can be mutated in a stop codon by a single substitution; (2) Phenotype-Genotype analysis give an easy access to phenotype-genotype correlations. In a first step, the number of records with a specific symptom is sorted according to the description of this symptom. In a second step, the user can choose some of these symptoms to visualize the distribution of genotypes associated with each phenotype; and (3) Genotype-Phenotype analysis is similar to the previous function. The user can choose a particular genotype from a list and display the associated phenotypes. All these routines can be applied to the overall or to a subset of each database. Gisèle Bonne (Paris, France) reported then the current status of both databases. UMD-LMNA database to date contains up to 537 entries collected from the EUROMEN consortium and from the literature, all entries being made by Anne Helbing-Leclerc (Paris). This corresponds to 537 individuals carrying a LMNA mutation, to a total of 124 different mutations and to six different pathologies (AR- and AD-EDMD, LGMD1B, DCM-CD, FPLD, AR-CMT2, and MAD [1,3,7]). Among the 537 individuals, 283 (53%) were identified with EUROMEN consortium, with for most of them (86%) a laminopathy affecting striated muscles (EDMD/LGMD1B/DCM-CD), 121 individuals (45%) reported in the literature present a partial lipodystrophy. Fifty-six out of the 124 LMNA mutations were identified within the consortium in Paris and are still unpublished. These 124 mutations were identified in 136 families and 120 isolated cases. Most of them (80%) are missense mutation, the remaining being in frame deletion/insertion, nonsense mutation, deletion/insertion with frameshift and splice site mutations. Two hotspot mutations were found: (1) R453W represented 11% of all laminopathy cases, always identified in patients presenting EDMD phenotype and represents 16% of all EDMD with a LMNA mutation; and (2) R482W/Q/L

6 G. Bonne et al. / Neuromuscular Disorders 13 (2003) represented 13% of all laminopathy cases, always identified in patients presenting partial lipodystrophy (FPLD) and represents 84% of all FPLD. Finally, whereas mutations leading to striated muscle laminopathy (EDMD/LGMD1B/DCM-CD) are spread all along the LMNA gene, mutation leading to FPLD are restricted to the end of the molecule with the R482 hotspot and other mutation with exons 8 and 11. If the UMD-LMNA database is almost updated, the UMD-EMD database entries are still in progress, this work being performed by Dominique Recan (Paris, France). To date it contains 84 entries collected at Cochin Hospital (Paris). Dominique Recan is currently completing the UMD-EMD entries with the EMD mutations reported in the literature and/or in the former STA database set up by Jones Yates [8]. The present 84 entries correspond to 31 different mutations identified in 29 families and three isolated cases. Seventy-seven of these mutations (87%) are null mutations, the four remaining mutation being two missense and two in frame deletions. No hot spot mutation was identified for EMD. At the present time, the two databases contains mainly genetic data. In order to explore completely the new routine of the UMD software developed for LMNA and EMD, i.e. phenotype/genotype and genotype/phenotype relations, clinical information of high quality for each individual are necessary. Rabah Ben Yaou (Paris, France) presented and proposed a new version the clinical form called Myo- Cluster form, used so far within the consortium, to collect all clinical information from patients carrying either EMD or LMNA mutations. This new version is adapted for its use with the UMD software to provide pertinent clinical symptoms of high quality for phenotype/genotype and genotype/phenotype relations. All symptoms present in the Myo-Cluster form were discussed one by one and for each of them decision was made within the consortium to leave or to remove them. New items were also implemented to the new clinical form. A consensus was reach to used starting from this ENMC workshop, this new mini-myocluster form to collect clinical information from patients carrying EMD or LMNA mutations Sequence analysis of LMNA in different groups of patients Daniela Toniolo reported the work of Michal Vytopil (Pavia, Italy). One hundred sixty six patients were collected that could be divided into four groups: typical EDMD, isolated heart disease, isolated muscular dystrophy, and idiopathic hyperckemia. All patients were positive for emerin immunohistochemistry. They were screened for LMNA and those negative were also screened for EMD mutations. Despite the phenotypic heterogenity, the presence of heart involvement and age of onset are distinguishing feature separating EDMD cases with LMNA mutation from LMNA/EMD negative. Frequency of LMNA mutations in isolated heart disease is very low (3 5%), underscoring the high heterogeneity of the cardiac phenotype and a role of LMNA in this group of disorders. Among the patients analyzed four families harboured missense mutations in the LMNA gene that were inherited with a pattern of incomplete penetrance. This emphasize the significance of mutational analysis in relatives of sporadic cases of laminopathies, as asymptomatic carriers may face high risk of sudden cardiac death Other genes for EDMD? Manfred Wehnert (Greiswald, Germany) reported the screening of potential candidate gene that could be implicated in classical phenotype of EDMD. Routine mutational analysis in patients with EDMD and related phenotypes as conducted in four laboratories (G. Bonne, D. Recan, D. Toniolo, M. Wehnert) revealed that approx. 60% of the cases are not linked to EMD or LMNA indicating that further genes are involved in EDMD. Unfortunately, almost no large families or sufficient numbers of sib pairs are available for linkage or association studies to identify such genes. Thus a functional candidate gene approach has been suggested. M. Wehnert presented preliminary data of a mutational screening in 12 candidate genes. Genes encoding peptides exclusively expressed in skeletal muscle and heart or those located in the nuclear membrane or lamina that interact with emerin or lamin A/C or have structural similarities to emerin or lamin A/C were considered as functional candidates. Particularly, the following genes have been investigated in 56 EDMD patients: SMPX (small muscular protein has been mapped to the X-chromosome), FLNC (filamin C), LMNB1 (lamin B1), LMNB2 (lamin B2), LBR (lamin B receptor), LAP1 (laminaassociated peptide 1), LAP2 (lamina-associated peptide2), MAN1 (man1), NRM (nurim), BAF Barrier-to-autointegration factor), PSME3 (proteasome activator subunit 3) DDX16 (DEAD/H-box polypeptide). SMPX, LMNB2, LAP1 and BAF turned out to be monomorphic and thus further to be excluded as candidates for EDMD. In the residual genes, 21 novel DNA variations have been identified in patients and defined by sequencing. The frequency of these DNA variations has been estimated in 400 alleles of a German control population. There were intronic variations, frequent SNPs (Single Neucleotide Polymorphisms) and a dinucleotide short tandem repeat among those variations not likely to cause the disease. Therefore additional genes (MAN1, LMNB1, PSME3, LBR) could be excluded as candidates for EDMD Partially unique exonic DNA-variations causing amino acid exchanges have been found in FLNC, LAP2, NRM and DDX16. But the segregation of these variations could not be confirmed because the variation did not segregate with the EDMD phenotype or there were no DNA samples available from the patient s relatives. However the latter four genes should still be considered as candidates and tested in further

7 514 G. Bonne et al. / Neuromuscular Disorders 13 (2003) patients to approve or disapprove them to be involved in EDMD. Additionally, further candidate genes should be tested. 4. Fundamental aspects of nuclear function and structure and consequences of LMNA and EMD mutations 4.1. Mapping epitope of lamin A/C monoclonal antibodies Glenn Morris (Wrexham, UK) provided detailed characterization of five new monoclonal antibodies against lamin A/C prepared for the EUROMEN project. Epitope mapping using phage-displayed peptide libraries showed that all five antibodies recognize a short sequence within a surface region of the lamin A/C common tail. This epitope sequence includes R482 commonly affected by lipodystrophy mutations and confirms the surface accessibility of this site (Nguyen thi Man, Ryan, Pham and Morris, unpublished data). He also presented biochemical and yeast two-hybrid evidence for an interaction of emerin with a splicingassociated factor, YT521-B (Wilkinson, Sharma, Manilal, Holt, Stamm, Wilson and Morris, ms. submitted). He suggested that the emerin-lamin A/C complex may have a role in tissue-specific gene expression by organizing factors required for transcription and processing of mrna in relation to chromatin Creation of mammalian tissue cell culture lines as models for XL- and AD-EDMD Juliet Ellis (London, UK) reported the construction of stably transfected mouse myoblasts and Chinese hamster ovary fibroblasts expressing mutations found in either X- or AD-EDMD. Cell lines expressing either lamin A or lamin C missense mutations have been successfully constructed, which mimic molecular defects observed in patent biopsy samples. Molecular defects seen include mis-localization of exogenous lamin A and of endogenous emerin and lamin C. Cell lines expressing both lamin A and C mutations are currently being constructed. Attempts have been made to construct cell lines expressing the emerin null phenotype, but this resulted in a non-viable phenotype. Cell lines containing endogenous wild-type emerin and exogenous modified forms of emerin have been successfully made. This cell line should provide a cell model for the X-linked EDMD carrier phenotype, as well as providing functional data on emerin. The cell lines are being used to understand the molecular defects behind EDMD, and whether there is any correlation between the molecular defects and the large variation in clinical phenotype seen in this condition. We have recently shown that emerin is phosphorylated at five sites, three of which are tyrosine kinase sites. Phosphorylation of these residues controls the efficiency of both emerin and lamin A/C localization at the nuclear envelope. Sue Brown (London, UK), using a newly published protocol that utilizes hyaluronidase treatment in conjunction with electroporation to attain a high transfer efficiency [9], introduced mutant lamin A constructs conjugated with EGFP (Green Fluorescence Protein) into the tibialis anterior muscle of immuno-competent mice. Using this methodology up to 40% of the fibers are transfected and EGFP labeled lamin A, both mutant and wild type, are successfully incorporated into the lamina of muscle fiber nuclei. These preliminary experiments were carried out in immuno-competent mice and so were relatively short term since after 1 week the immune system begins to eliminate the foreign EGFP protein. Ongoing work will now seek to examine the longterm effects of the presence of the mutant protein in nude mice Emerin interactions in differentiated myocytes Emerin is a nuclear envelope protein whose biological function remains to be elucidated. In this study, Giovanna Lattanzi (Bologna, Italy) analyzed emerin interactions in differentiating C2C12 myoblasts. She show that an emerinactin complex, which includes nuclear alpha and beta actins, is formed, but it is not stable, in cycling myoblasts, while it is strongly stabilized at the late stages of myotube differentiation. The stabilization of the emerin-actin complex is mediated by a protein phosphatase activity, as demonstrated by pull-down assay with a GFP-tagged actin. In the attempt to identify the enzyme involved in this event, they were able to demonstrate that the A-kinase anchoring protein AKAP149, protein phosphatase 1 and c-amp-dependent protein kinase associate with emerin-lamin-actin complex in myotubes. They further demonstrated that the interaction between emerin and actin also occurs in mature mouse skeletal and cardiac muscle. These results provide evidence that emerin inter-molecular interactions are dynamically regulated in myogenic cells. A study aimed to evaluate the protein-protein interactions here shown in normal human muscle and Emery-Dreifuss tissue is now in progress Laminopathy of adipose tissue: partial lipodystrophy which mechanisms are involved? R. C. Trembath (London, UK) reported on the recent study on the possible mechanism of LMNA linked to FPLD. Novel molecular mechanisms responsible for the maintenance of normal adipocyte function has recently been gained to the characterization of a number of conditions characterized by the reduction or absence of subcutaneous adipose tissue [10]. Of the inherited causes of lipodystrophy, mutations have been reported in a diverse range of genes whose functions in the maintenance of adipocyte cell activity are now being explored. LMNA is mutated in FPLD characterized by variable but significant insulin resistance, absence of fat deposits in the trunk and limbs, but retention of adipocity in the neck and face [11 13].

8 G. Bonne et al. / Neuromuscular Disorders 13 (2003) Investigation of the expression and subcellular localization of FPLD lamin A mutations demonstrated no abnormality compared to wild type [14]. The FPLD mutations in contrast to those causing muscle disease are tightly clustered within the C-terminal domain of lamin A/C. In a yeast two hybrid interaction screen they identified protein specifically interacting with the C-terminal domain of lamin A. Amongst a group of novel lamin A interacting proteins they have identified the sterol response element binding proteins 1 and 2 (SREBPs). Specificity of interaction with the SREBP adipocyte differentiation factors was confined through in vitro glutaltione S transferase pull down and in vivo co-immuno precipitation studies, and characterized a region on the N-terminal transcription factor domain of SREBP1 as the likely binding site to lamin A. Importantly, at least one of the reported AD-EDMD mutations was also shown to interfere with this interaction. The physiological relevance of this interaction requires further study, but supports a potential role for altered binding of the adipocyte differentiation factor, SREBP1 to lamin A in the pathogenesis of lipodystrophy [14]. 5. List of participants Rabah Ben Yaou (Paris, France) Christophe Béroud (Montpellier, France) Gisèle Bonne (Paris, France) Giuseppe Boriani (Bologna, Italy) Susan Brown (London, UK) Marianne De Visser (Amsterdam, The Netherlands) Denis Duboc (Paris, France) Juliet Ellis (London, UK) Irena Hausmanowa-Petrusewicz (Warsaw, Poland) Giovanna Lattanzi (Bologna, Italy) Eugenio Mercuri (London, UK) Luciano Merlini (Bologna, Italy) Glenn Morris (Wrexham, UK) Francesco Muntoni (London, UK) Grzegorz Opolski (Warsaw, Poland) Yigal Pinto (Maastricht, The Netherlands) Federica Sangiuolo (Rome, Italy) Caroline Sewry (London, UK) Beril Talim (Ankara, Turkey) Daniela Toniolo (Pavia, Italy) Richard Trembath (Leicester, UK) Jop Van Berlo (Maastricht, The Netherlands) Anneke Van Der Kooi (Amsterdam, The Netherlands) Manfred Wehnert (Greifswald, Germany) Acknowledgements This workshop was made possible thanks to European Community Grant (QLG1-CT ) Myolcuster- Euromen and to logistic 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), Osterreichische Muskelforschung (Austria), Vereniging Spierziekten Nederland (The Netherlands), and ENMC associate member: Muscular Dystrophy Association of Finland. References [1] De Sandre-Giovannoli A, Chaouch M, Kozlov S, et al. Homozygous Defects in LMNA. Encoding Lamin A/C Nuclear-Envelope Proteins, Cause Autosomal Recessive Axonal Neuropathy in Human (Charcot- Marie-Tooth Disorder Type 2) and Mouse. Am J Hum Genet 2002;70: [2] Young LW, Radebaugh JF, Rubin P, Sensenbrenner JA, Fiorelli G, McKusick VA. New syndrome manifested by mandibular hypoplasia, acroosteolysis, stiff joints and cutaneous atrophy (mandibuloacral dysplasia) in two unrelated boys. Birth Defects Orig Artic Ser 1971;7: [3] Novelli G, Muchir A, Sangiuolo F, et al. Mandibuloacral dysplasia is caused by a mutation in LMNA encoding lamins A/C. Am J Hum Genet 2002;71: [4] Genschel J, Bochow B, Kuepferling S, Ewert R, Hetzer RHL, Schmidt H. A R644C mutation within lamin A extends the mutations causing dilated cardiomyopathy. Hum Mutat 2001;17:154. [5] Beroud C, Collod-Beroud G, Boileau C, Soussi T, Junien C. UMD (Universal mutation database): a generic software to build and analyze locus-specific databases. Hum Mutat 2000;15: [6] den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 2000;15:7 12. [7] Bonne G, Capeau J, Recan D, et al. 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. Neuromuscul Disord 2002;12: [8] Yates JR, Wehnert M. The Emery-Dreifuss Muscular Dystrophy Mutation Database. Neuromuscul Disord 1999;9:199. [9] McMahon JM, Signori E, Wells KE, Fazio VM, Wells DJ. Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase increased expression with reduced muscle damage. Gene Ther 2001;8: [10] Garg A, Peshock RM, Fleckenstein JL. Adipose tissue distribution pattern in patients with familial partial lipodystrophy (Dunnigan variety). J Clin Endocrinol Metab 1999;84: [11] Jackson SN, Howlett TA, McNally PG, O Rahilly S, Trembath RC. Dunnigan-Kobberling syndrome: an autosomal dominant form of partial lipodystrophy. QJM 1997;90: [12] Shackleton S, Lloyd DJ, Jackson SN, et al. LMNA, encoding lamin A/C, is mutated in partial lipodystrophy. Nat Genet 2000;24: [13] Cao H, Hegele RA. Nuclear lamin A/C R482Q mutation in Canadian kindreds with Dunnigan-type familial partial lipodystrophy. Hum Mol Genet 2000;9: [14] Lloyd DJ, Trembath RC, Shackleton S. A novel interaction between lamin A and SREBP1: implications for partial lipodystrophy and other laminopathies. Hum Mol Genet 2002;11: