Early onset X-linked Emery-Dreifuss muscular dystrophy resembling limb-girdle muscular dystrophy

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Abstracts

form of the disease within 35 years. The 18 severely affected patients who had a fragment size over 20 kb had a mean age of 48. In the mild group, only 4 patients (10%) carried a fragment shorter than 20 kb, as compared to the 31 patients in the severe group (63%). The mean age was 31 years in the patients with a fragment shorter than 20 kb, 41 years in the others (P < 0.0005). Our data indicate that in FSHD the severity of the clinical phenotype correlates with the pl3E-11 fragment size; therefore the molecular study may have an impact on the clinical prognosis and genetic counseling.

Keywords: FSHD; 4q35 deletion; Clinical phenotypes GP2.5 Penetrance of the FSHD mutation differs according to the D4Z4 repeat number C. de Tomaa, S. Chiron~, P. Laforetb, J.A. Urtizberea~, B. Eymard ~, M. Fardeau b, J-C. Kaplan ~, M. Jeanpierre ~

~INSERM 129 and Htpital Cochin, Paris, France, bU153, lnstitut de Myologie, Paris, France, CGenethon, Evry, France Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder. The estimated penetrance is 95%, but there is a remarkable variation in age at onset and in clinical severity. In patients probe p l 3 E l l invariably detects a deletion of an integral number of 3.3 kb repeated units (D4ZA) within a highly polymorphic EcoRI fragment at chromosome 4q35. Until the description of the specific DNA rearrangement at chromosome 4q35, it was impossible to know whether or not an isolated case represents a de novo mutation or was born from an asymptomatic carrier parent. For genetic counselling, we need for each number of D4Z4 repeats a non-biased estimation of disease severity and penetrance by studying patients relatives, with a special emphasis on patients parents who are old enough to give information on mutation penetrance. The D4Z4 locus in asymptomafic FSHD patients relatives has been extensively studied in a serie of 400 FSHD families referred for diagnosis. Recent mutations are observed only in patients with less than 5 repeats and the proportion of de novo mutations (up to 75% for 2-repeats cases) decreases sharply with the number of repeats. Since a high frequency of de novo mutation is correlated with low fitness in any dominant disease, it is possible to calculate the mean fitness of a given repeat number. Severe and very mild disease courses were observed in the same families and a Bayesian estimation of mutation penetrance suggested that patients-only studies overestimate FSHD severity in 1-3 repeats mutation carriers. Large deletions are however unambiguously associated with a higher risk of a severe course as shown by the proportion of de novo mutations and somatic mosaic cases. When both parents of isolated cases with a 2534 kb fragment (6 to 10 D4Z4 units) have been studied, one of the parent has always been found to be an asymptomatic cartier showing a reduced penetrance of these mutations. The possibility that any 9-10 units fragment is involved in the pathophysiology with a low penetrance should be considered.

hypothesised in which the hydrophobic COOH-terminal domain is inserted in the inner nuclear membrane and the hydrophilic NH2-terminal domain is associated with the nuclear lamina. We investigated the possible locafisation of emerin in the nuclear envelope and in the in situ nuclear matrix preparations of cell monolayers (SAOS, MG 63, Hela cells) by means of immunolabelling with a polyclonal anti-emerin antibody. We found emerin at the nuclear periphery in all cultured cells examined. The positive labelling for emerin remained through the matrix extraction procedure and it was still clearly visible after the last step (high salt extraction). This finding is consistent with the biochemical data reported about an emerin association with the nuclear fraction from muscle and brain, and fits with a model in which the binding with the nuclear matrix is stable. The detergent solubilisation step did not affect the emerin detection, suggesting that the COOH-terminal hydrophobic domain is not responsible for maintaining the perinuclear localisation. The hypotheses about an emerin role in association with the peripheral nuclear matrix are confirmed by these immunocytochemical findings which suggest a possible emerin involvement in some of the nuclear matrix known functions.

Keywords: Emery-Dreifuss muscular dystrophy; Emerin GP2.7 Clinical and genetic analyses of Emery-Dreifuss muscular dystrophy and rigid spine syndrome Shinichiro Kuboa'b, Kim Bong Yoon~, Toshifumi Tsukaharaa, Kiichi Arahata~

"National Institute of Neuroscience, NCNP, ToIcvo 187, Japan, bTokyo Medical College, To~'o 160, Japan Emery-Dreifuss muscular dystrophy(EDMD) shows clinical similarities with rigid spine syndrome (RSS) because of the joint contractures and slowly progressive wasting and weakness in muscle. Clinically, EDMD can be differentiated from RSS by the presence of intra-atrial conduction block which is often life-threatening, and therefore, precise diagnosis is essential. To detect mutations in the STA gene (responsible for X-linked EDMD) among patients having a clinical diagnosis of RSS, if any, we examined six cases with RSS and a case with EDMD. Based on the STA gene sequence, eight PCR primer pairs (designed by Nigro et al., 1995) were used to amplify the entire coding sequence of emerin together with the exon-intron junctions. When SSCP showed any mobility shift, we sequenced the corresponding PCR fragments. We found two novel mutations in the STA gene. In a patient with EDMD, an acceptor-site mutation of the intron 5 (A1506 to G) was detected. In one of the six RSS patients, 1 base pair deletion was detected in exon 1 (GI41). Both mutations were expected to produce truncated proteins lacking the C-terminus, and emerin was not detected in these patients. The remaining five patients with RSS showed no mutation in the STA gene. in conclusion, RSS includes a group of forme t'ruste X-linked EDMD.

Keywords: Emery-Dreifuss muscular dystrophy; Rigid spine syndrome; Keywords: FSHD; Penetrance; Mutation

Genetic diagnosis

GP2.6 Immunocytochemical detection of emerin within the nuclear matrix

GP2.8 Early onset X-linked Emery-Dreifuss muscular dystrophy resembling limb-girdle muscular dystrophy

S. Squarzonia, P. Sabatelli a, A. Ognibeneb, D. Tonioloc, L. Cartegni c, F. Cobianchi c, S. Petrini a, L. Merlini d, N.M. Maraldi ~'b

"lst. Citomorfologia N.P. CNR c/o IOR, Bologna, bLab. Biologia Cellulare e Microscopia Elettronica IOR, Bologna, "Ist. Genetica Biochimica Evoluzionistica CNR, Pavia, dLab. Patologia Neuromuscolare IOR, Bologna, Italy Emerin is a protein that is altered or missing in the X-linked form of Emery-Dreifuss muscular dystrophy. Emerin was localised at the nuclear rim in different normal tissues, such as skeletal, cardiac and smooth muscle etc.; it was also present in the nuclear fraction from human skeletal muscle and rabbit brain. Emerin shares some homology with thymopoietins and the nuclear lamina-associated protein 2. A model has been

C.A. Sewrya'b, E.J. Lichtarowicz-Krynska~, S.B. Manila] c, D. Recan a, J. Taylor~, S. Llense a, J-C. Kaplan~, V. Dubowitza, G.E. Morrisc, F. Muntoni a

aNeuromuscular Unit and bMRC Muscle Cell Biology Group, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, UK, ~'Biotechnology Group, NEWI, Plas Coch, Wrexham, LLll 2AW, UK, aLaboratoire de Biochimie et G~ndtique Moleculaire, Htpital Cochin 123 Boulevard de Port-Royal, 75104 Paris, France Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked neuromuscular disorder caused by defects in the STA gene located on Xp28. The gene codes for a nuclear protein called emerin. Affected patients usually

Abstracts present in early adolescence with scapulo-peroneal muscle weakness and wasting, contractures of the Tendo Achilles and elbow flexors, and spinal rigidity. We report a case of EDMD with an unusually early presentation at the age of 2.5 years with pelvic weakness and tightness of the left Tendo Achilles, but no other contractures. Serum creatine kinase was elevated (1994 IU/I; normal < 200 IU/I); muscle biopsy showed marked dystrophic changes and a possible diagnosis of Duchenne or Becker, or a limb-girdle muscular dystrophy were considered. However, expression of dystrophin and ee-sarcoglycan, were normal, and no abnormalities in the DMD gene were found. Six years later clinical examination of the proband's maternal cousin, aged 14 years, showed typical features of EDMD. This was confirmed in both affected boys by the absence of emerin in muscle and leukocytes, and identification of a novel mutation in exon 4 of the STA gene. Carrier status in both mothers was also confirmed by mutational and protein analysis. Emery-Dreifuss muscular dystrophy should therefore be considered in all cases of unexplained early onset muscular dystrophy, even in the absence of the typical clinical features.

Keywords: Emery-Dreifuss; Muscular dystrophy; Emerin GP2.9 X-linked Emery-Dreifuss muscular dystrophy: molecular diagnosis by protein analysis and use of the skin biopsy in female carriers J. Colomer a, J. Pradas b, M. Guitet a, J. Vila ~, J. Corbera ~, S. Manilal d, G.E. Morris d aServei de Neurologia, bServei d'Anatomia Patologica, ~Hospital de Sam Pau, Barcelona, Spain, aMR1C Biochemistry Group, N.E. Wales Institute, Wales, UK

Introduction. Emery-Dreifuss muscular dystrophy (EDMD), is characterised by cardiac conduction defect, muscular contractures and a slow progression usually inherited as a X-linked recessive form. Recently the gene responsible for the illness has been known as STA and is located in the Xq28 region. The deficient product of the gene is the emerin, a protein component of nuclear membrane and ubiquitous in different tissues. We report the findings of two brothers and their obligate carrier mother affected from this type of dystrophy. Patients and methods. The initial symptoms of the older brother, now 12 years old, was weakness in bending the arms and toe walking. The weakness spread slowly to the rest of the muscles developing contraetures at the elbows and in the Achilles tendon. The CK was 20× normal values. The ECG showed a migrated pacemaker. A muscular biopsy was performed on his younger brother now 6 that follow a very similar disease course. The stains for the emerin with a monoclonal NCL-emerin antibody did not show emerin. A G994>A deletion in the STA gene was detected. A skin biopsy was performed in the mother and tested with monoclonal NCL-emerin. About 50% of nuclei were emerin positive. Conclusion. The EDMD has a specific phenotype. The lack of emerin in the nuclear membrane and the presence of any mutation at the STA gene allow us to make the diagnosis. The ubiquity of the protein enables us to perform the diagnosis with a skin biopsy and at the same time to establish a carrier status. Keywords: Emery-Dreifuss muscular dystrophy; Emerin; Skin biopsy GP2.10 An early-onset autosomal recessive muscular dystrophy with distal wasting and rigidity of the spine F. Muntoni ~, J, Taylor~, V. Dubowitz ~, C.A. Sewry ~'b "Department of Paediatrics & Neonatal Medicine, t'MRC Muscle Cell Biology, Group, Hammersmith Hospital, London, UK We report five cases (3 males and 2 females) from 5 unrelated Cancasian families who presented in the first few years of life with symptoms of progressive muscle weakness affecting not only the proximal limb girdles, but also the distal muscles of the lower limbs and the neck flexors. All children had a similar clinical phenotype characterised by selective

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weakness and wasting of the deltoids, biceps, neck flexors and distal leg muscles, followed shortly after by a severe rigidity of the spine and achilles tendon contractures. Intellectual function was normal. The age of onset was in the first 2 years of life in four children and in-utero in one. Motor milestones were normal in 4, and all cases achieved independent ambulation. The increasing spinal rigidity in conjunction with the marked neck weakness caused all 5 children to adopt a forward stooping posture with a hyper-extended neck, both when sitting and walking. Serum CK levels were markedly elevated in all (5-10 times the upper limit of normal). Muscle biopsies showed similar dystrophic changes and normal expression of dystrophin, sarcoglycans, laminin chains and emerin. All children followed a similar progressive clinical course and lost ambulation before the age of 8 years due to a combination of weakness and contractures. Three of these children have significantly impaired respiratory function and one has developed severe nocturnal hypoventilation. Cardiac function is normal. These cases appear to represent a severe, progressive, autosomal recessive muscular dystrophy, charactefised by early onset distal wasting, contractures, rigidity of the spine, and respiratory failure. While the clinical phenotype has some resemblance to a severe EmeryDreifuss dystrophy, there was no cardiac involvement and emerin was normal.

Keywords: Rigid spine; Distal myopathy; Autosomal recessive GP2.11 A family with severe pseudo-dominant Emery-Dreifuss muscular dystrophy due to emerin deficiency D. Rdcan ~, S, Llense a, J.-C. Barbot a, F. Leturcq", N. Deburgrave a, J.-M. Dupontb, P. Warrot a, C. Giraudet a, F. Fraisse a, D. Amsallemc, J. Bensaid d, J.A. Urtizberea ~, G.E. Morris f J.-C. Kaplan"

~%aboratoire de Biochimie et Gdngtique Moleculaire, HOpital Cochin, F75014 Paris, J'Laboratoire de Cytogdndtique, H@ital Cochin F-75104 Paris, "Service de POdiatric, CHU St-Jacques, F-25030 Besanfon, riService de cardiologie, CHU Dupuytren, F-87402 Limoges, eDirection des Affaires Mddicales, AFM, F-91002 Evo:, France, fBiotechnology Group, NEW1, Plas Coch, Wrexham, LLI1 2AW, UK We present here a family that had been previously reported as a dominant form of Emery-Dreifuss muscular dystrophy (EDMD) because four patients (two males and two females) were affected in four generations. They exibited predominant cardiac symptoms (permanent auricular paralysis) requiring a pace-maker at age 14 in a male, 23 in his mother, and 42 in his maternal grand-mother, with atypical skeletal muscle involvement. Actually, the segregation in this family was compatible with X-linkage. The availability of anti-emerin antibodies helped in clarifying the issue. Emerin was found to be absent on Western blots of fibroblasts and lymphoblastoid cultured cells (LCL) from the youngest affected male. Emerin was only barely visible in LCL from his severely affected mother. The gene defect in this family is a deletion removing the distal part of the emerin (STA) gene. We found no X-autosome translocation in the manifesting female, however a skewed X chromosome inactivation was ascertained in her LCL by methylation sensitive restriction enzyme digestion of the androgen receptor (AR) locus. This family is intriguing because of the unusual severity of the cardiac involvement both in males and in females. We are currently investigating the 3' limit of the deletion. This observation examplifies the importance of analysing the emerin protein in suspected cases.

Keywords: Emery-Dreifuss muscular dystrophy; Emerin; Auricular paralysis

GP2.12 Comparison of three functional assessment scales in neuromuscular diseases Ayse Karaduman a, Ctineyt Akg61 a, 0znur Tuncaa, 0zgen Arasa, Yavuz Yakut a, Haluk Topaloglu b