RICERCA ITALIANA     

Molecular pathogenesis and clinical evolution in limb-girdle muscular dystrophies

Università degli Studi di Padova

Abstract

We propose an innovative research on limb-girdle muscular dystrophies (LGMD) that will go beyond their molecular diagnosis to include pathogenetic mechanisms that might lead to new pharmacological or gene replacemnet strategies. A group of clinical centers with a specific interest in the research area of neuromuscular disorders will cooperate to organize a large clinico-molecular network to study molecular pathogenesis and phenotype correlations in LGMD.
The term LGMD covers a large spectrum of inherited muscle disorders characterized by progressive muscle weakness and wasting with autosomal recessive or dominant inheritance in which the primary defect might be either due to a specific muscle protein defect or to a genetic mutation (table 1). These muscle disorders might be associated with a variable degree of clinical severity and sometimes with cardiac, respiratory or PNS involvement. Cognitive involvement and quality of life have not been so far investigated and this will be part of our project. Our cooperative group will utilize the facility of a tissue bank located in Padova, that includes over 6000 biopsies collected over 30 years and collects DNA, cell lines of most recognized neuromuscular disorders, so far identified. Furthemore a collection of neuromuscular cases including several LGMD biopsies which are available in the Unit of Messina and Padova, and over 100 molecularly defined cases have been collected by the Unit of Pisa. The cooperation between these different centers will allow to reach the following objectives:
1. investigate molecular defects that cause limb-girdle muscular dystrophy in which several genes have been identified in order to study their molecular pathophysiology (LGMD 2A-2I, 1B-1C);
2. identify new clinical phenotypes in limb-girdle dystrophies and correlate them with a corresponding muscle specific defect;
3. study gene expression by microchips analysis and RT-PCR in biopsies already obtained from limb-girdle muscular dystrophy 1B, 1C, 2A, 2I, correlate changes found by muscular magnetic resonance and perform aerobic muscle exercise in selected case in order to correlate muscle atrophy and fatigue with extramuscular features (respiratory insufficiency, cardiac changes, cognitive fetaures and peripheral nervous system involvement) in one of the largest series of myopathic patients at european level.
A definite goal will be to create a common network of excellence as a result of our cooperation, for clinico- molecular analysis of muscle patients. Patients will also benefit both in term of diagnosis and of genetic and secondary prevention (i.e. cardiopathy: in FKRP-patients, or laminopathies), while this research project might shed light on possible future therapeutic strategies, since we will investigate different molecular pathogenetic mechanisms.
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Principal Investigator

Corrado Angelini Università degli Studi di PADOVA

Research Objectives

A clinical network has been formed by a group of clinical centers with a specific interest in muscle disorders that will cooperate in order to study clinical phenotype, molecular mechanisms and genetic mutations in limb-girdle muscular dystrophies. These myopathies include various clinical phenotypes characterized by progressive muscle weakness and wasting but they also present frequent involvement of heart and skeletal muscle. Our collaborative groups will utilize both a Telethon neuromuscular tissue bank in Padova and the large collections of muscle biopsies available in the other centers (Messina, Pisa e Torino). A first goal is to take advantage of this collaborative network for research purposes and implement collaboration on molecular analysis between the various centers, another goal is to share common clinical and MRI protocols to delineate clinical phenotypes. The collaboration between the various centers will pursue the following objectives:
1. clarify molecular defects and genetic mutations that cause limb-girdle muscular dystrophy with autosomal dominant or autosomal recessive inheritance in which several genes have been identified (units of Padova, Torino and Messina). In particular we will further investigate DNA to identify pathogenetic mutations in over 100 cases with calpain defect, over 50 cases with alpha-sarcoglycanopathy, 20 cases with alpha-dystroglycan defect, 20 cases of suspected caveolinopathy and 10 cases with suspected lamin A/C mutations.
2. identify new clinical phenotypes and correlate them with a corresponding muscle specific protein defect (units of Padova and Pisa). The coexistence of proximal/distal phenotype or only hyperCKemia has been described in the dysferlinopathy and caveolinopathy, while neurogenic and myogenic features might be associated to lamin A/C defects, the hyperCKemias represent an interesting phenotype that deserves further investigation and presents genetic heterogeneity.
3. study by the new technique of MRI imaging the pathophysiologic and pathogenetic mechanisms so to delineate evolution and progression at the basis of limb-girdle muscular dystrophies(Unit of Padova and Pisa).
4. Costruction of a limb-girdle muscular dystrophy integrated database available by internet but anounimuized. During the project we will produce a large amount of data. These will concern both clinical data collected on LGMD patients and pathophysiological data obtained by MRI imaging from several muscles. We want to collect all these data in a common database that will be planned in the course of yearly meetings of our research units of the collaborative group.
All these objectives will allow to create a cooperative clinico-molecular network and might help in establishing a better collaboration for clinico-molecular research, in the interest of muscle patients, that will also benefit from this cooperation, both from a more defined molecular diagnosis at the present time, while in the future this research network might generate new data, that might be useful for therapeutic strategies. <<<

Timescale

24 months

National and international background

During the past 15 years an increasing number of genes have been identified that cause different forms of muscular dystrophy (Fig. 1)(1-10)and numerous protein and loci have been found to characterize LGMD (10-12). In the current project we will particularly study a) recessive (2A-2I) and dominant limb-girdle dystrophies (1B, 1C); b) proximal and distal myopathies due to lamin A/C defect. In our cooperative group a common scientific basis of clinical and molecular protocols is available, and we plan to further implement it: we have already established a western blot protocol (2) to molecularly identify protein defects in recessive cases of limb-girdle muscular dystrophy. A number of investigations have led to a profound change in the classification of different types of limb-girdle muscular dystrophy with a new focus on the molecular genetic analysis rather than on clinical symptoms only. It is therefore advisable, once the primary defect has been established to study clinical phenotypes in order to investigate the marked phenotypical heterogeneity in patients with these muscular dystrophies and establish if the clinical phenotype is due to environmental (i.e. diet, exercise) or post-translational factors that might modify gene expressions; for this objective we will utilize the new microchips technology. Gene expression profiling by microarray is a powerful technique to explore pathogenetic mechanisms in limb-girdle muscular dystrophy, as we already did in dysferlinopathy(3). This technique was utilized by various researchers in neurological diseases (4), in inflammatory muscle diseases (5) or in Duchenne muscular dystrophy (6-8), gene expression profiling reveals a series of transcripts related to both necrosis and apoptosis, it documents both increased and differential inflammatory response and muscle regeneration. This new technology allows to identify overexpressed genes related to necrosis, regeneration, inflammatory response and other underexpressed genes (i.e. sarcomeric proteins). It is not well known if the molecular profiles of muscle tissues in muscular dystrophy patients are distinct and represent molecular signatures from which diagnostic insight might follow for each type of dystrophy. One of the major hurdles will be to dissect which gene expression profiles are common to the various types of muscular dystrophies i.e.features of degeneration, regeneration, inflammatory process, from gene expression profiling related to pathophysiologic mechanisms characteristic for each form, to this purpose "hierarchical clustering" analysis might be used. A possible key is to analyze the results of this molecular technology in each experiment is to go back to clinico- pathological changes, were all gene expression can be queried in a single patient. In a recent paper (10) our group by microarray investigations in collaboration with Dr. Hoffman has proposed a new pathogenetic mechanism involving disruption of Rb-MyoD pathway in muscle regeneration in laminopathies. Furthermore specific RNA changes can be further validated by RT-PCR technique that we recently acquired. We want to investigate in a detailed way the differential clinical features of LGMD, to do this we propose an innovative clinico-radiological score and we want to test several muscle groups by the validate MRC scale. The use of MRI and specific clinical protocol will allow to better define the cause of muscle fatigue and weakness in these myopathies. Further we plan to investigate both cognitive features and quality of life in LGMD, since both these features have been so far neglected (13). A significant problem which remains in our understanding in the pathogenesis in the non-sarcoglycan LGMD (14) is the variability of presentation of severity event in a same kindred. Strategies for this purpose are planned.
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