Pezzani , Raffaele (2008) Studio molecolare dei difetti di glicosilazione dell'alfa distroglicano in pazienti affetti da distrofia muscolare congenita e distrofia muscolare dei cingoli. [Ph.D. thesis]
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Muscular dystrophies are a group of diseases genotypically and clinically heterogeneous, characterized by progressive muscular weakness.
The histopathologic profile can vary from mild to severe myopathy, with degeneration, regeneration and adipose-fiber substitution.
Muscular dystrophy etiopathogenesis involves basal membrane elements and muscle fiber cytoskeleton alterations. A prominent role is played by the dystrophin glycoprotein complex (DGC), which provides a strong stabilization link between the intracellular cytoskeleton and extracellular matrix. The DGC is composed by dystrophin, sarcoglycans, distrobrevins, sintrophins, sarcospan and dystroglycan. Dystroglycan is a glycoprotein, encoded by one gene DAG1, that is post-translationally cleaved into 2 subunits, ?-dystroglycan and ?-dystroglycan. The dystroglycan ensure connection and stability and is expressed in a large variety of tissue, including skeletal and cardiac muscle, central and peripheral nervous system tissue and epithelia.
?-DG is a highly glycosylated peripheral membrane protein, which binds to several molecules (laminin, agrin, perlecan, neurexin, biglycan) and tightly interacts with the extracellular portion of ?-DG. ?-DG has the ability to connect to the cytoplasmic domain of dystrophin, to caveolin-3 and other proteins implicated in signal trasduction. The predicted molecular weight of ?-dystroglycan is about 72 kDa, but can vary due to glycosylation. In fact ?-dystroglycan has an apparent molecular weight of 156 kDa in skeletal muscle.
So far no pathogenic mutations have been identified in genes encoding for dystroglycan. It is clear that the pathogenesis of in muscular dystrophy involves post-translational processing events that are important for the interaction of dystroglycan with its ligands. In particular ?-dystroglycan glycosylation has a critical role in maintaining integrity of extracellurar matrix proteins.
Alterations in ?-dystroglycan glycosylation lead to a particular group of recessive autosomic muscular dystrophy called dystroglycanopathies.
The dystroglycanopathies show strong phenotypic heterogeneity: in fact at the most severe end of the clinical spectrum are Walker-Warburg Syndrome (WWS), Muscle-Eye-Brain (MEB), Fukuyama congenital muscular dystrophy (FCMD), MDC1C. At the mild end there is Limb-Girdle muscular dystrophy (LGMD). Genetically, dystroglycanopathies are caused by mutations in 6 known genes: POMT1, POMT2, POMGnT1, Fukutin, LARGE, FKRP. All these genes encode for putative or known glycosyltransferases.
This present work intends to characterize a large group of patients affected by congenital or limb-girdle muscular dystrophy with unknown etiopathogenesis, trying to extend genotype and phenotype correlations. In fact a precise molecular diagnosis will provide an essential basis for further studies leading to identification of new therapeutic strategies.
Patient groups are sorted from the muscular biopsy database “Centro delle Malattie Neuromuscolari”, Department of Neuroscience, University of Padova, which comprises over 8000 samples. In order to be selected, each patient must meet one of these criteria: a) be affected by congenital muscular dystrophy or limb-girdle muscular dystrophy with unknown etiopathogenesis, b) have floating CK values, weakness during fever or steroid responsiveness. 234 patients were selected for screening.
Immunohistochemistry was performed on 234 muscular biopsies. Cryosections were incubated with an antibody directed to the ?-dystroglycan epitope (IIH6C4 - Upstate Biotechnology).
Immunoblot was performed on 94 samples, with reduced ?-dystroglycan glycosylation in immunohistochemistry. The analysis shows exact congruence between immunohistochemistry and immunoblot data.
Of 94 analyzed patients, 26 were idenfied as having pathogenic mutations. 73% of patients showed reduced ?-dystroglycan glycosylation, while 27% of patients exhibited total absence of ?-dystroglycan glycosylation (in immunohistochemistry and immunoblot). The data set obtained by mutation analysis lead to a general mutation rate of 28% (26 patients out of 94 patients with ?-dystroglycan deficit) and can be subdivided as a) 24% if mutation rate is examined inside partially deficit patient group, or in b) 46% if mutation rate is considered inside totally deficit patient group.
In conclusion, in the last few years it has become more and more evident that ?-dystroglycan play a pivotal role in the dystrophin glycoprotein complex (DGC), permitting connection and assuring stability between the intracellular cytoskeleton and extracellular matrix. Moreover a better understanding of ?-dystroglycan O-glycosylation could lead to the development of new therapeutic approaches. In fact, post-translational processes that alter dystroglycan glycosylation are the basis of muscular dystrophy pathogenesis, principally dystroglycanopathy. The mutation rate found in our patients (with ?-dystroglycan deficit) is about 26%, which an incidence of 50% in patients with total absence of ?-dystroglycan. This work confirms high genotypic and phenotypic heterogeneity of dystroglycanopathy and the wide phenotypic spectrum related to these disorders.
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