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Impaired contractile properties in single muscle fibres isolated from patients affected by FSHD
S16
CIS8
CP1
Impaired contractile properties in single muscle fibres isolated from patients affected by FSHD
ANALYSIS
R. Bottinelli ~, R. Tupler2, I.Vos ~, L. Barbierato 2, M.C.Zanardi ~, M. Canepari ~, M.A.Pellegrino I, A.Berardinelli3, G.Lanzi~, C.Reggiani~ Istituto di Fisiologia umana~, Biologia Generale e Genetica:, Fondazione C.Mondino I.R.C.C.S), Universita' di Pavia, Pavia, Italy In muscles of FSHD patients contractile strength decreases without significant degenerative processes and without signs of denervation. This may suggest that individual fibres might loose their contractile ability. To test this hypothesis and contribute to the understanding of the pathological phenotype at cellular level, single fibres isolated from bioptic samples of Deltoid of FSHD patients (n=4) and control healthy subjects (n=2) were compared. Histological examination of the samples did not show any major alteration, except a size heterogeneity in both slow and fast fibres size. Contractile properties of various fibre types were investigated using short (1.5-2 ram) segments activated in Ca2+ buffered solutions at 12°C, 2.5 ~tm sarcomere length. Both isometric tension and maximum shortening velocity were significantly reduced in slow fibres from FSHD patients compared to those of controls. Fast (IIA) fibres were not significantly affected. Calcium sensitivity of both fibre types was similar in control and FSHD fibres.The results support the hypothesis that the decrease of contractile strength is present also at individual fibre level at least in slow fibres.
CO1 MONOCLONAL
OF THE EMERIN GENE I N FAMILIAL AND SPORADIC CASES OF EMERY-DREIFUSS MUSCULAR DYSTROPHY S. Llcnse 1, N. Romem 2, JC. B,'u'bot 1, S. Bione 3, D. Toniolo 3, J.C. Kaplan 1, D. Rtcan 1 1: Lab. Biochim & Gdndt. Mol. and INSERM 129, CHU Cochin, Paris; 2: Hop. Robert Debrt;, Paris; 3: 1st. Genet Biochim., Pavia Nineteen iodependent mutations have already been described in the ubiquitously expressed 2100 bp long emerin gene of patients with a familial form of X-linked Emery-Dreifuss muscular dystrophy (Xq28). None has been described in sporadic cases so far. We present our results obtained in 2 unrelated families with Xlinked EDMD and in 10 sporadic cases of alleged EDMD. In 3 patients of a s,'une family we fimnd, by direct sequencing of RTPCR ,'unplificd lymphoblastoid cell RNA , a 4 bp deletion (nt 675-679) in the terminal part of the coding sequence (exon 6) inlroducing a premature stop codon expected to result in a 19 aminoacid truncated protein. Direct genetic counselling could be provided to two at risk maternal aunts who did not carry the deletion. In one sporadic case the ,'unplified RT-PCR products were normal in length and in sequence. We are currently searching for mutations, both in mRNA and in genomic sequences, in another famili,'d case and in 9 sporadic cases of EDMD.
CO2 ANTIBODIES
AGAINST
EMERIN, FOR THE DIAGNOSIS OF EMERYDREIFUSS M U S C U L A R D Y S T R O P H Y . Sushila Manilal, Nguyen thi Man, Caroline Sewry 1, and Glenn E. Morris. MRIC Biotechnotogy Group, N.E. Wales Institute, Deeside, Clwyd, CH5 4BR, U.K. 1Dept. of Paediatrics and Neonatal Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0NN, U. K. Mutations in the STA gene encoding the protein, emerin have recently been shown to be responsible for the X-linked form of Emery-Dreifnss muscular dystrophy (EDMD). Although the gene is highly expressed in skeletal muscles and heart, STA has been shown to be ubiquitously expressed in most tissues. Emerin is a serine-rich protein having a hydrophilic N-terminns and a short hydrophobic C-region. We have expressed the hydrophilic region of emerin as a recombinant protein by PCR cloning from total human muscle eDNA and used this to produce a panel of 12 mAbs against emerin. The mAbs, which recognise several different epitopes, detected a protein band on Western blots of normal human muscle which was completely absent from muscle of EDMD patient. The mAbs were also used to demonstrate the absence of emerin in the EDMD patient by immunohistochemistry and to determine the localisation of emerin in normal tissues. This work was support by a Research Development (DevR) award Higher Education Funding Council (Wales).
Evidence of genetic heterogeneity in facioscapulohumeral muscular dystrophy Italian families. R. Tupler 1, L. Barbierato l, M. Memmi1, A. Berardinelh 2, G. Piccolo 2, R. Bottinelli 3 L. Palmucci4, A. Ferlini 5, D. De Grandis5, I. Marchi6, A. Ottolini 7, C. Reggiani3, , P. Maraschio. 1 IBiologia Generate e Genetica Medica, 2IRCCS "C.Mondino"and 31stituto di Fisiologia Umana, University of Pavia; 4Centre per le malattie neuromuscolari "P. Peirolo", University of Turin; 5Divisione di Neurologla, Arcispedale Sant'Anna, Fen'am; 6Istituto Nazionale per la Ricerca sul Cancro, Sassari; 7Istituto Angelo Custode, Predore, Bergamo. Facioseapuiohumeral muscular dystrophy (FSHD) is a progressive disease, tranmaitt~ in an autosomal dominant fashion, although isolated and autosomal recessive cases has been described. FSHD has been mapped by linkage analysis in chromosome 4q35-qter region. Subsequently, the isolation of the pl3E-ll probe (D4F104S1), which detects EcoRI fragments containing taademly repeated KpnI units (D4Z4) located in the 4q subtelomeric heterochromatin, led to the suggestion that the rearrangements revealed by pl3E-11 in sporadic and familial FSHD cases may be the underlying defect. Other studies on extended FSHD podegrees failed to show an association with chromosome 4q35 genetic marker and indicated FSHD to be a heterogeneous disorder. Here we describe the genetic haplotyping of 11 Italian families. In most of the affected subjects an EcoRI fragment smaller than 30 Kb was revealed. One family, in which pl 3E-11 EcoRI rearranged fragments were present in both healthy and affected individuals, has been excluded from linkage to the chromosome 4q35 region. Genetic heterogeneity in Italian families and the role of D4Z4 rearrangements are discussed.
CIS8
CP1
Impaired contractile properties in single muscle fibres isolated from patients affected by FSHD
ANALYSIS
R. Bottinelli ~, R. Tupler2, I.Vos ~, L. Barbierato 2, M.C.Zanardi ~, M. Canepari ~, M.A.Pellegrino I, A.Berardinelli3, G.Lanzi~, C.Reggiani~ Istituto di Fisiologia umana~, Biologia Generale e Genetica:, Fondazione C.Mondino I.R.C.C.S), Universita' di Pavia, Pavia, Italy In muscles of FSHD patients contractile strength decreases without significant degenerative processes and without signs of denervation. This may suggest that individual fibres might loose their contractile ability. To test this hypothesis and contribute to the understanding of the pathological phenotype at cellular level, single fibres isolated from bioptic samples of Deltoid of FSHD patients (n=4) and control healthy subjects (n=2) were compared. Histological examination of the samples did not show any major alteration, except a size heterogeneity in both slow and fast fibres size. Contractile properties of various fibre types were investigated using short (1.5-2 ram) segments activated in Ca2+ buffered solutions at 12°C, 2.5 ~tm sarcomere length. Both isometric tension and maximum shortening velocity were significantly reduced in slow fibres from FSHD patients compared to those of controls. Fast (IIA) fibres were not significantly affected. Calcium sensitivity of both fibre types was similar in control and FSHD fibres.The results support the hypothesis that the decrease of contractile strength is present also at individual fibre level at least in slow fibres.
CO1 MONOCLONAL
OF THE EMERIN GENE I N FAMILIAL AND SPORADIC CASES OF EMERY-DREIFUSS MUSCULAR DYSTROPHY S. Llcnse 1, N. Romem 2, JC. B,'u'bot 1, S. Bione 3, D. Toniolo 3, J.C. Kaplan 1, D. Rtcan 1 1: Lab. Biochim & Gdndt. Mol. and INSERM 129, CHU Cochin, Paris; 2: Hop. Robert Debrt;, Paris; 3: 1st. Genet Biochim., Pavia Nineteen iodependent mutations have already been described in the ubiquitously expressed 2100 bp long emerin gene of patients with a familial form of X-linked Emery-Dreifuss muscular dystrophy (Xq28). None has been described in sporadic cases so far. We present our results obtained in 2 unrelated families with Xlinked EDMD and in 10 sporadic cases of alleged EDMD. In 3 patients of a s,'une family we fimnd, by direct sequencing of RTPCR ,'unplificd lymphoblastoid cell RNA , a 4 bp deletion (nt 675-679) in the terminal part of the coding sequence (exon 6) inlroducing a premature stop codon expected to result in a 19 aminoacid truncated protein. Direct genetic counselling could be provided to two at risk maternal aunts who did not carry the deletion. In one sporadic case the ,'unplified RT-PCR products were normal in length and in sequence. We are currently searching for mutations, both in mRNA and in genomic sequences, in another famili,'d case and in 9 sporadic cases of EDMD.
CO2 ANTIBODIES
AGAINST
EMERIN, FOR THE DIAGNOSIS OF EMERYDREIFUSS M U S C U L A R D Y S T R O P H Y . Sushila Manilal, Nguyen thi Man, Caroline Sewry 1, and Glenn E. Morris. MRIC Biotechnotogy Group, N.E. Wales Institute, Deeside, Clwyd, CH5 4BR, U.K. 1Dept. of Paediatrics and Neonatal Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0NN, U. K. Mutations in the STA gene encoding the protein, emerin have recently been shown to be responsible for the X-linked form of Emery-Dreifnss muscular dystrophy (EDMD). Although the gene is highly expressed in skeletal muscles and heart, STA has been shown to be ubiquitously expressed in most tissues. Emerin is a serine-rich protein having a hydrophilic N-terminns and a short hydrophobic C-region. We have expressed the hydrophilic region of emerin as a recombinant protein by PCR cloning from total human muscle eDNA and used this to produce a panel of 12 mAbs against emerin. The mAbs, which recognise several different epitopes, detected a protein band on Western blots of normal human muscle which was completely absent from muscle of EDMD patient. The mAbs were also used to demonstrate the absence of emerin in the EDMD patient by immunohistochemistry and to determine the localisation of emerin in normal tissues. This work was support by a Research Development (DevR) award Higher Education Funding Council (Wales).
Evidence of genetic heterogeneity in facioscapulohumeral muscular dystrophy Italian families. R. Tupler 1, L. Barbierato l, M. Memmi1, A. Berardinelh 2, G. Piccolo 2, R. Bottinelli 3 L. Palmucci4, A. Ferlini 5, D. De Grandis5, I. Marchi6, A. Ottolini 7, C. Reggiani3, , P. Maraschio. 1 IBiologia Generate e Genetica Medica, 2IRCCS "C.Mondino"and 31stituto di Fisiologia Umana, University of Pavia; 4Centre per le malattie neuromuscolari "P. Peirolo", University of Turin; 5Divisione di Neurologla, Arcispedale Sant'Anna, Fen'am; 6Istituto Nazionale per la Ricerca sul Cancro, Sassari; 7Istituto Angelo Custode, Predore, Bergamo. Facioseapuiohumeral muscular dystrophy (FSHD) is a progressive disease, tranmaitt~ in an autosomal dominant fashion, although isolated and autosomal recessive cases has been described. FSHD has been mapped by linkage analysis in chromosome 4q35-qter region. Subsequently, the isolation of the pl3E-ll probe (D4F104S1), which detects EcoRI fragments containing taademly repeated KpnI units (D4Z4) located in the 4q subtelomeric heterochromatin, led to the suggestion that the rearrangements revealed by pl3E-11 in sporadic and familial FSHD cases may be the underlying defect. Other studies on extended FSHD podegrees failed to show an association with chromosome 4q35 genetic marker and indicated FSHD to be a heterogeneous disorder. Here we describe the genetic haplotyping of 11 Italian families. In most of the affected subjects an EcoRI fragment smaller than 30 Kb was revealed. One family, in which pl 3E-11 EcoRI rearranged fragments were present in both healthy and affected individuals, has been excluded from linkage to the chromosome 4q35 region. Genetic heterogeneity in Italian families and the role of D4Z4 rearrangements are discussed.