Myotonic disorders

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Pla(form-Oral Presentations

Monday, July 8, 2002

Inflammatory Myopathies (15:00-16:30)

15 15:40 CD8CD28 Double-Positive T Cells in Patients with Polymyositis

13 15:00 Beta Chemokines and their Receptors in Inflammatory Myopathies J.L. De Bleecker* t, B. De Paepe t, G. Racz 2, J.-M. S chroeder 3. 1Ghent,

Belgium; 2Szeged, Hungary; 3Aachen, Germany Objective: Further studies on the role of chemoattractant cytokines (chemokines) and their receptors in the initiation and progression of polymyositis (PM), sporadic inclusion body myositis (sIBM) and dermatomyositis (DM). Methods: Various chemokines were visualized in frozen muscle sections using single and multistep immunofluorescence. In protein extracts, density of chemokine receptor subtypes CCR1 to CCR5 was quantitatively analyzed using actin as an internal standard. Results: Increased expression of Monocyte Chemoattractant Protein-1 (MCP-1) was observed in PM and sIBM, localized to actively invading T cells and a subset of macrophages, and to capillaries near endomysial inflammatory foci. In DM, perifascicular and perimysial blood vessels were also strongly positive at sites remote from inflammatory exudates. Only chemokine receptors CCR2A and CCR2B were undetectable in normal muscle, but present in all three inflammatory myopathies. For both CCR4 and CCR5, elevated levels of protein were observed: CCR4:2.53 (DM), 7.00 (sIBM) and 2.74 (PM) times; CCR5:2.36 (DM), 2.20 (sIBM) and 4.42 (PM) times. CCR1 content was reduced in all three inflammatory myopathies. Conclusion: We report the expression of MCP-1 and its receptors CCR2A and CCR2B, and an increased expression of the chemokine receptors CCR4 and CCR5 in each main inflammatory myopathy. This differential expression of beta chemokines and their receptors offers hope for more selective immunotherapy. 14 Polymyositis, A Disease Entity Disputed

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M. de Visser* 1, LM. Bronner 1, M.EG. van der Meulen 2, J.E. Hoogendijk 2 , J.H.J. Wokke 2, H. Burger 2, A.E. Voskuyl I , H.J. Dinant t, W.H.J.P. Linssen t, W.J. van Venrooij 3. 1Amsterdam,

2 Utrecht, 3Nijmegen, The Netherlands Objective: According to the 1975 Bohan and Peter criteria, dermatomyositis is differentiated from polymyositis only by typical skin changes. Recently, histopathological characteristics are found to distinguish polymyositis from dermatomyositis and sporadic inclusion body myositis. Nevertheless, the Bohan and Peter criteria are still widely used. We investigated the applicability of broadly accepted diagnostic features, including histopathology. Methods: Inclusion criteria: 1) previous diagnosis of myositis established in third-line referral center; 2) subacute onset of symmetrical, proximal weakness. Exclusion criteria: 1) sporadic inclusion body myositis, muscular dystrophy, rhabdomyolysis, and exposure to myotoxic drugs; 2) clinical data or muscle biopsy not available. We reviewed the clinical charts and muscle biopsies of all included patients (n=165), and we re-examined 111 patients (67%). Results: The diagnoses at presentation according to pre-defined criteria, were as follows: polymyositis 9 (5%), dermatomyositis 54 (33%), myositis with connective tissue disease 32 (19%), myositis with malignancy 3 (2%), unspecified myositis 38 (23%), possible myositis 29 (18%). At follow-up, 5 of the 9 polymyositis patients had typical inclusion body myositis features. The remaining 4 patients fulfilled criteria for polymyositis, but lacked the assumed typical picture. Conclusions: (1) Polymyositis must be disputed as a specific disease entity. (2) At presentation, 40% of the patients can not be specifically diagnosed.

Ken-ya Murata*, Miwa Takamure, Satoshi Ueno. Nara, Japan Objective: To examine the pathogenic roles of costimulatory molecules in polymyositis (PM), lymphocytes expressing CD4, CD8 and CD28 molecules in patients with PM, with Duchenne muscular dystrophy (DMD), and healthy volunteers were counted using flow cytometry. Methods: All PM patients had proximal muscle weakness associated with electrophysical myopathic changes. Their serum creatine kinase levels were elevated. The patients were receiving neither corticosteroids nor immunosuppressive therapy at the time of the study. Blood samples were obtained from 8 PM patients, 10 DMD patients, and 10 volunteers. FITCconjugated anti-CD4, CD8, and phycoerythrin-conjugated anti-CD28 antibodies were used for single or dual color flow cytometry. Results: There were no statistical differences among the three groups in terms of numbers and frequencies of CD4-positive, CD8-positive, or CD4CD28 double-positive T cells. There was, however, a difference in the frequency of CD8CD28 double-positive T cells observed (4.7 4- 1.9% in PM group vs 16.0 4- 6.9% in the DMD group and 17.8 4- 8.1% in the volunteers, P < 0.01). Conclusion: CD8CD28 double-positive T cells appear to be involved in PM. Understanding the role of these costimulatory molecules in the pathogenesis of PM may lead to improving PM therapy, particularly immunomodulating treatment. 16 16:00 Class I MHC Detection as a Diagnostic Tool in Muscle Biopsies of Patients Suffering from Dermatomyositis D. Figarella-Branger*, M. Civatte, N. Schleinitz, J.R. Harl , J. Pouget, J.F. Pellissier. Marseille, France Objective: To evaluate the diagnostic value of class I MHC detection in muscle biopsies of patients presenting with clinical features of dermatomyositis (DM). Methods: A retrospective study was performed on muscle biopsies in 22 patients presenting with clinical features of DM. Immunohistochemical detection of class I MHC was done in all cases. Results: On pathological features two groups of patients were recorded: group I (14 patients) with typical features of DM and group II (8 patients) almost normal muscle biopsies (no inflammatory exudates, no perifascicular atrophy). Abnormal sarcolemmal class I MHC expression was recorded in all cases. In all muscle biopsies of group I patients, class I MHC expression was observed in almost all fibers but was stronger in perifascicular areas (8 patients) or was restricted to perifascicular atrophic fibers (6 patients). In all muscle biopsies of group II patients only some perifascicular fibers expressed class I MHC. According to Bohan and Peter criteria, patients were classified as definite DM (9 group I and 3 group II patients), probable DM (5 group I and 2 group II patients) possible DM (3 group II patients). Conclusions: Abnormal perifascicular class I MHC expression is of diagnostic value in patients presenting with clinical features of DM especially when muscle biopsy fails to show typical features such as inflammatory infiltrates and/or perifascicular atrophy.

Myotonic Disorders (15:00-16:30) 17 15:00 Modafinil Reduces Somnolence and Enhances Mood in Patients with Myotonic Muscular Dystrophy J.R. MacDonald* 1, j. Hill 2, M.A. Tarnopolsky 3. 1Hamilton, Canada;

2Mississauga, Canada; 3Canada Objective: To evaluate the potential for Modafinil to reduce daytime somnolence in patients with myotonic dystrophy.

Oral Presentations in a Workshop

Monday, July 8, 2002

Methods: Thirty-six patients with myotonic dystrophy were randomized to Modafinil (100 mg X 7 d > 200 mg X 7 d) and placebo for 14 days using double-blind, cross-over design. Before and after each trial, subjects completed handgrip strength testing, spirometry and quality of life measures (RAND). On day 7 and 14 each subject completed a modified Epworth Sleepiness Scale (ESS), the Stanford Sleepiness Scale (SSS), and the Profile of Mood States (POMS). Results: The ESS showed less somnolence on the Modafinil treatment phase (P < 0.05), with similar resuks for the SSS on day 7 of the Modafinil phase (P < 0.05). The POMS showed lesser fatigue/inertia and higher tension/anxiety scores for Modafinil versus placebo (P < 0.05). The RAND showed an increase in energy during the Modafinil phase (P < 0.01). There were no improvements in voluntary or respiratory strength measures. Three patients withdrew due to reported side effects (2 on Modafinil and 1 on placebo). Conclusions: This study confirmed the results of a smaller open preliminary study by showing that Modafinil reduced excessive daytime somnolence in patients with myotonic dystrophy. This study was partially supported by Draxis Pharmaceuticals, Mississauga, Ontario, CANADA. Dr. McDonald was funded by a Premier's Research Excellence Award. 18

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Nuclear Localization of the Myotonic Dystrophy-Associated Six5 Protein using a New Panel of Monoclonal Antibodies Nguyen thi Man * 1, y . c .N. Pham 1, G.E. Morris 1, G.M. Hamilton 2, K.J. Johnson 2. 1Wrexham, United Kingdom; 2Glasgow, United Kingdom

Objectives: Six5 is one member of the Six family of homeoboxcontaining transcription factors involved in eye development. In myotonic dystrophy (DM), an expanded CTG repeat lies within the 5'-UTR of the Six5 gene. Like DM patients, Six5 knockout mice develop cataracts. Six5 protein is produced from three exons, A, B and C. Monoclonal antibodies against two different regions of Six5 are needed for conclusive identification of authentic Six5 and studies of its distribution. Methods: We cloned exons A and B separately into a bacterial expression plasmid and used the recombinant proteins to immunize Balb/c mice for hybridoma production. Transfection of HeLa cells with full-length Six5 in pCDNA4 results in localization to the nucleus and we screened hybridomas for their ability to recognize the nuclear recombinant Six5 in transfected cells. Results: Ten mAbs against exon A and four mAbs against exon B were obtained and cloned twice to homogeneity. Conclusions: Six5 migrates on SDS-PAGE as a 100kD protein, slightly larger than predicted from the amino-acid sequence and recognized by all 14 mAbs. The mAbs also detect endogenous Six5 in nuclei by immunofluorescence microscopy. Six5 was present in all fetal and adult cell types tested. Supported by the Muscular Dystrophy Campaign (UK).

19 Stratagem in vitro for Gene Therapy Directed to Myotonic Dystrophy

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J. Puymirat',~I , M.A. Langlois I , G. Doucet I , L. Timchenko:, J. Rossi 3.

1City of Quebec, Canada; :Houston, USA; SCity of Hope, USA There is evidence showing that the specific targeting of the mutant transcripts could be essential for the development of a gene therapy for DM1. We developed a strategy which utilises the differential subcellular localization of the normal and mutant transcripts, using antisense RNA and ribozymes. Using antisense ODNs, we identified one antisense RNA and three ribozyme cleavage sites in the 3'UTR of the DMPK. We have designed a retrovirus which produces an (CUG)n antisense RNA, under the control of the 5'LTR. We showed that antisense RNA destroy by 80% and 50% the levels of the mutant and normal DMPK transcripts, respectively. This effect is associated with a complete restoration in glucose uptake,

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a complete disappearance of CUG binding activity and an increase in myoblasts differentiation. We designed a vector (pTz) which express the ribozyme under the control of the RNAmeti promoter and this vector was used to transfect human DM myoblasts with 750 repeats. The levels of the normal and mutant DMPK mRNAs were decreased by 50 and 63, respectively. These results indicate that antisense RNA and ribozymes can preferentially affect the expression of the mutant transcripts and therefore could be useful for the development of a gene therapy for DM. 20

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Progressions of both (CTG)n Expansions and Muscular Disability Rating Scale (MDRS) are Age Dependent in Myotonic Dystrophy Type 1 (DM1) Masanobu Kinoshita* 1, Toshiyuki Ohtake 2, Tetsuo Komori 2, Tadaaki Maedal, Tadasuke Nagano 1, Masaru Kikkawa 1, Satoko Nakamura 1, Akihiko Matsuda l , Tetsuya Mitarai l , Kazuhiko Hirose 3. 1Fourth

Department of lnternal Medicine, Saitama Medical Centeg Saitama Medical School, Saitama, Japan; 2Department of Neurology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan; 31nternal Medicine, Towa Hospital, Tokyo, Japan Background: The mutation responsible for DM 1 is an unstable expansion of a (CTG),, in the 3 t untranslated region of a gene encoding DMPK. The somatic instability of (CTG),, expansions has been reported. We therefore investigate whether the progressions of (CTG),, expansion in leukocytes and MDRS might be age dependent. Object and Methods: We examined both the (CTG),, length in two peripheral blood samples and the values of MDRS in 10 patients with DM1 (Male 4, Female 6) over a period of time ranging from 3 year to 7 years (mean4-SD 4.54-1.2 years). The patient's initial length varied from approximately 60-70 to 1670 repeats. To determine the length, Southern blot analysis was performed using the method described previously (Muscle Nerve 19: 240, 1996). The MDRS described by Mathieu et al. were used to estimate the extent of muscular involvement. Results: In 9 cases, the lengths have expansions with an average size of 1674-97 repeats and the values of MDRS were elevated with an average values of 1.04-0. In one case with approximately 60-70 repeat, when compared with the lengths in leukocytes and the values of MDRS 5 years previously, the both were the same value. Conclusion: Age-dependent (CTG) ,~ expansion and MDRS elevation in DM 1 patients with large expansion may be more dominant than in patients with small expansions. MONDAY, JULY 8, 2002

Oral Presentations in a Workshop WS-1 Studies on the Expression of Obscurin; A Giant Modular Protein Associated with Myofibrils S.H. Laval*, L.V.B. Anderson, H.J. Blair, J. Moss, K.M.D. Bushby.

Newcastle upon Tyne, UK Objective: Obscurin is new member of the titin and Dbl families of myosin light chain kinases (MLCK) that is thought to orchestrate the assembly of myofibrils in striated muscle. Regulation of obscurin gene expression is complex and multiple alternatively spliced products, translational start sites and stop codons have been identified. Methods: To elucidate obscurin expression we have performed Northern analysis and developed monoclonal antibodies to two different epitopes using peptides from the deduced sequence. Results: The gene appears to encode a large ~20kb transcript that is most abundant in skeletal muscle. Smaller transcripts of between 9.5 and 15kb were detected in brain; but not in heart, kidney, liver, spleen, lung, thymus, uterus or ovary. Two alternative 3' ends were identified, one of which encodes serine-threonine kinase domains similar to those of other