The impact of the molecular data on clinical practice in facio-scapulohumeral muscular dystrophy (FSHD)

Abstracts G E N E R A L P O S T E R 2: MUSCULAR DYSTROPHY III AND MYOPATHIES GP2.1 Some difficulties in defining the time when the first affection of mimic muscles appear in patients with autosomal dominant facioscapulolimb muscular dystrophy, type 2 (FSLD2) (or facioscapuloperoneal) V. Kazakov, D. Rudenko

Department of Neurology, Pavlov's State Medical University, St. Petersburg, Russia In FSLD2 studies we found a different degree of weakness of mimic muscles among the patients belonging to the same pedigree. Among 106 from 148 examined patients including 49 with facioscapulo-peroneal phenotypes, slight or minimal weakness of separate mimic muscles or their portions was present. 34 patients had severe asymmetric weakness of orbicularis oris muscle together with different degrees of weakness of some other mimic muscles. However among these patients, having the features of myopathic face, no persons complained of weakness or atrophy of separate mimic muscles or changes of facial expression. These patients as well as their parents and closest relatives did not notice the affection of mimic muscles. The patients who had even the final phenotype of the disease learned about their mimic muscles disturbances for the first time on their examination. The patients considered that in the earlier period of the disease the shoulder girdle muscles were affected. Some of these patients even supposed that weakness of the extensors of the feet was the first symptom of the disease. However a special test questionnaire used in patients and their relatives permitted us to established that in some of these patients, the face was affected simultaneously with shoulder girdle muscles or even earlier. What degree of weakness of some mimic muscles or their portions must be in a patient to suppose their affection? We think if some of our patients were examined by other investigators then the diagnosis would be 'pure' scapuloperoneal dystrophy or even facioscapuloperoneal dystrophy which started in the foot extensors. We suggest that the establishment of the time and degree of affection of separate mimic muscles is a principle and important question.

Keywords: FSHD; FSPD; Mimic muscles GP2.2 MRI to evaluate muscle involvement in patients with autosomal dominant facioscapulolimb muscular dystrophy, type 2 (FSLD2) (or facioscapuloperoneal) D. Rudenko,V.M. Kazakov, L. Tntin, E. Shelkoplyas

Department of Neurology, Parlor's Medical University and Institute of Radiology, St, Petersburg, Russia Thirty-four patients from 12 families and 10 controls were studied. MRI of 20 muscles bilaterally in each patient was performed with a high field unit (Vision 1.5 T, Siemens)and lower field unit (Magneview 0.04 T, Instrumentarium). Axial 10 mm thick sections of the midthigh and ntidlower leg muscles and T1 and T2 weighted images were created with visual and quantitative evaluation. More frequently, the following MRI patterns were found in symptomatic patients (SP): heterogeneity with focal increase of fat within muscles (moth-eaten) and two varieties of homogeneity density with signal intensity, one of which corresponded to clinical muscle weakness (all muscle replaced by fat) and another which did not correspond to clinical muscle weakness. In presymptomatic patients (56%), one could usually notice a homogeneity hyperdensity only in soleus (S) and gastrocnemius (G) muscles without their weakness. In these patients the 'moth-eaten' pattern was never found. In all SP muscle, the density changes appeared earlier than the decrease of muscle volume. When the process extended to the lower limbs the anterior compartment of lower leg muscles was involved at an earlier stage, although the changes of density of S and G muscles without their weakness were evident as well. MRI affection of the posterior group of the thigh muscles appeared later, but quadriceps, gracilis and sartorius muscles were well

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spared. The asymmetry of muscle affections was established without predominant affection of the sides. MRI are useful to obtain information on the distribution and sequence of muscle involvement at different stages of the disease, although no method reveals specific diagnostic details.

Keywords: MRI; Muscle pattern; FSHD GP2.3 High frequency of epilepsy and mental retardation in early onset 4q35facioscapulohumeral muscular dystrophy with huge deletion of KpnI repeats Masanori Funakoshia'b, Kanako Goto a. Kyozo Yonemoto b, Kim Bong Yoon a, Ikuya Nonaka a, Kiichi Arahata '~

~Wational Institute af Neuroscience, NCNP, Tokyo 187, Japan, bDepartment of Rehabilitation Medicine, The Jikei University School of Medicine, Tokyo 105, Japan Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder characterized by progressive weakness and atrophy of the facial, shoulder-girdle and upper arm muscles, with occasional subsequent pelvic-girdle and lower limb involvement. Epilepsy and mental retardation (MR) do not occur frequently in regular 4q35-FSHD, while early onset cases often show MR. We report eighteen 4q35-FSHD patients that were classified as early onset 4q35-FSHD according to the clinical criteria reported by Brouwer, among 155 Japanese 4q35-FSHD patients from 86 families. All of the 155 patients had smaller EcoRI fragments under 35 kb (10-33.5 kb; mean = 18.3 kb, normal 35-kb). The EcoRI fragments of the 86 probands were reduced by 3-kb after Blnl digestion. The early onset patients had significantly smaller EcoRl fragment size (10-17 kb; mean = 12.9 kb) than the later onset group (12-33.5 kb; mean = 18.4 kb) (P < 0.001, t-test). Of the eighteen early onset patients, eight had the smallest EcoRI fragments reported to date (10 kb and 11 kb), which contained only one Kpnl unit (measured by specific quantitative LA-PCR). All of the eight patients were sporadic cases and showed severe clinical phenotype and most had epilepsy and/or MR. The four patients who had both epilepsy and MR showed epileptic EEG records, while no detectable abnormal brain CT/MRI images were found. We concluded that huge deletion within the 4q35-FSHD gene region could produce a severe clinical phenotype which includes central nervous system abnormalities.

Keywords: Facioscapulohumeral muscular dystrophy; Epilepsy; Mental retardation

GP2.4 The impact of the molecular data on clinical practice in facio-scapulohumeral muscular dystrophy (FSHD) Enzo Ricci, Barbara Merico, Giancarlo Deidda, Giuliana Galluzzi, Gabriella Silvestri, Stefania Cacurri, Serenella Servidei, Luciano Felicetti, Pietro Tonali

Institute of Cell Biology CNR, Institute of Neurology, Catholic University, UILDM, Rome, Italy FSHD is an autosomal dominant disease presenting a high degree of variability with regard to age at onset, severity and pattern of muscle involvement, both between and within families. The gene for FSHD has been mapped to the 4q35 subtelomeric region. The distal probe pl3E-11 detects EcoRI fragments ranging between 10 and 27 kb in most FSHD patients, To assess the existence of correlation between fragment size and disease severity, we analysed 142 FSHD patients from 74 families. A severe proximal lower limb involvement was present in 49 patients (35%) among which 18 were wheelchair-bound. A mild form of the disease, restricted to facial and scapular muscles was found in 39 patients (27%). The 4q35 pl3E-11 EcoRI fragment was significantly smaller in the severe group compared with all other patients (P < 0.0001), while the mean age was 37 years in both groups. The 8 patients with very short fragments (less than 14 kb) had a mean age of 20 and a severe

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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