The aim of this work is to contribute to the characterisation of new or as yet unidentified neuromuscular diseases (in collaboration with : P. Guicheney, M. Bitoun, G. Bonne, D. Hantaï, A. Ferreiro, F. Leturcq, P. Richard, J. Lunardi, N. Monnier, J. Laporte, V. Biancalana, A. Oldfors, F. Muntoni, N. Clarke, N. Laing, C.Wallgren-Pettersson, AL Taratuto etc) The goal of this work is to contribute at the characterisation of new or not yet identified neuromuscular diseases (in collaborations).
To be mentioned here:
- The first protocol of gene therapy done in humans. This phase I essay of clinical research, without any direct benefit for the patients, has been performed using a plasmid with total cDNA of dystrophin in patients with disease of Duchenne or Becker. The principal investigators were Pr Michel Fardeau (Institut de myologie) and Pr Serge Herson (Internal medicine CHUPS). This essay has been performed in collaboration with the Transgene collaboration.
- The immunocytochemical, histo-enzymological and ultrastructural analyses of abnormalities associated with congenital myopathies, in particular core myopathies (Central Core Disease (CCD), Multi-minicore), correlated with molecular studies, allows to bring precisions at the clinical entities which are not yet well defined or differentiated. In the domain of congenital myopathies with cores, we have also contributed to the clinical and morphological characterisation of patients with CCD linked or not-linked with the gene RYR1.
- In the domain of congenital myopathies, we have also contributed at the study of nemalin myopathies (NM) with the identification of one of the first children with a severe form of NM, which was associated at a mutation stop of the gene ACTA1 and at the study of myopathies with congenital fiber type disproportion (CFTD) linked at the gene TPM3.
- Thanks to the immunocytochemical study with antibodies against alpha- and beta-dystroglycan, collagen VI and perlecan, which we have systematically introduced in our laboratory, it has been possible to perform a reclassification of multiple muscular dystrophies of at that time unknown origin. In this domain, we have contributed to the identification of congenital muscular dystrophy associated at collagen 6, as well as to the study of muscular dystrophies with deficiency in alpha-dystroglycan, linked or not-linked at the gene FKRP.
- A first clinical and morphological analysis of a large series of patients with a congenital myopathy of the centronuclear type (CNM) has been performed. This work has contributed in an important way to linkage analyses on the autosomal dominant form of CNM and to the identification of the causal gene DNM2 that encodes Dynamine 2. Next, we have described the selectivity of the muscular affection in patients with a DNM2 mutation by a clinical and muscle imaging study. Later, we have identified the severe clinical and sporadic forms associated with DNM2. Next, we concentrated our interest on the histopathological analysis of patients in whom a mutation in DNM2 had been excluded. These patients make up a heterogeneous group with as common characteristic a variable number of fibres with internalized nuclei. This work has led to a classification in several subgroups of patients based on the morphological aspects observed at the muscular biopsy, as well as the mode of transmission and the clinical presentation.
- We have also identified a group of CNM patients that presented a curious morphological feature characterized by the presence of a variable number of fibers « en collerettes » or “dark necklace” fibres (DNF). These dark necklaces are situated a few micrometers of the sarcolemma and have been observed in the two types of fibres. The dark necklaces are colored by HE, GT, PAS, NADH-TR, SDH and COX, but they are not stained by myofibrillar ATPase. Some nuclei are aligned with the dark necklace. The ultrastructural analysis showed that the dark necklace consists of myofibrils with a small diameter, which are surrounded by mitochondria and sarcotubular structures. The immunohistochemical analysis has shown an intense staining with the anti-SERCA1 and SERCA2 antibodies, bot not with the other proteins of the sarcoplasmic reticulum (calsequestrin, the ryanodin receptor, triadin), and the T-tubule (dihydropyridin receptor-alpha1subunit). We have observed a moderate reaction with the anti-desmin and alphaB-crystallin antibodies, but less with anti-myotilin. Clinically, it is sporadic cases, with symptoms that started in the first decennium of their lives, with a proximal weakness of the lower limbs and a slowly progressive evolution. The genetic analysis has permetted to exclude mutations in the genes DNM2, hJumpy and BIN. The analysis of the gene MTM is currently performed. The mechanism of this defect in the organisation of the sarcoplasm still has to be elucidated.
- We are studying the clinical, immunohistochemical and ultrastructural characteristics in patients with myofibrillar myopathy (MFM). The ultrastructural findings in 19 patients with different genetically-proven MFMs (9 desmin, 5 alpha-B-crystallin, 3 ZASP, 2 myotilin) were analyzed. In one patient with a ZASP mutation, we additionally performed an immuno-electron microscopic (EM) study, using antibodies against desmin, alpha-B-crystallin, ZASP and myotilin. The ultrastructural findings in desminopathies and alpha-B-crystallinopathies were very similar and consisted of electrondense granulofilamentous accumulations and sandwich formations. They differed in the presence of early apoptotic nuclear changes in alpha-B-crystallinopathies. ZASPopathies were characterized by filamentous bundles (labeled with the myotilin antibody on immunoEM), and floccular accumulations of thin filamentous material. Tubulofilamentous inclusions in sarcoplasm and myonuclei in combination with filamentous bundles were characteristic for myotilinopathies. We conclude from this study that MFMs ultrastructural findings can direct diagnostic efforts towards the causal gene mutated, and that EM should be included in the diagnostic workup of MFMs. The immunohistochemical study involves 15 patients with a genetically-proven MFM (7 desmin, 3 alpha-B-crystallin, 3 ZASP, 2 myotilin) in whom we used 23 antibodies including several novel antibodies against distinct Z-disc and M-line proteins. This study is performed in collaboration with Dieter O. Fürst and Peter F.M. van der Ven of the Institute for Cell Biology, Department of Molecular Cell Biology, University of Bonn, Bonn (Germany) and with Rudolf Kley of the Neurologische Univ.-Klinik Bergmannsheil, Ruhr-Universität Bochum, Bochum (Germany). The results of this study are currently being analyzed.