| Objective:Genetically Limb girdle muscular dystrophy (LGMD) is a group of highly heterogeneous neuromuscular disorders, which predominantly involved in proximal limb muscles. Up to date, there are at least 7 autosomal dominant (LGMD1A LGMD1G) and 12 autosomal recessive(LGMD2A-LGMD2L) forms which had been identified for their responsible genes and the products. LGMD was first named by Walton and Nattrass in 1954, a group of genetic muscle diseases that was first classified to distinguish it from DMD/BMD and facioscapulohumeral muscular dystrophy. Clinical signs of LGMD were characterized by progressive weakness and atrophy of limb girdle muscular and progresses gradually. These are nonspecific. The analyses of gene mutation and protein expression was essential means to diagnosis LGMD. The gene responsible for LGMD2A has been localized to chromosome 15q15.1-q15.3. It encodes calpain-3, the skeletal muscle-specific member of the calpains. Mutations in the dysferlin gene can cause muscular dystrophies called dysferlinopathy. To clarify the frequency and clinicopathological features of LGMD2A in Chinese, we performed protein analyses of caveoiln-3,calpain-3,dysferlin,sarcoglycan in 37 Limb girdle muscular dystrophy (LGMD).Material and methods:We examined a total of 37 LGMD patients'muscles obtained from the peripheral nerve and muscular disease institute of shandong qilu hospital, including 7 LGMD2B patients clinically and histochemically diagnosed. As a control we selected 5 normal muscle specimens confirmed by pathologic profiles. The biopsied skeletal muscle specimens were flash-frozen in isopentane chilled with liquid nitrogen, and then were made into 4μm section. We used antibodies for dystrophin C-terminal,dystrophin Rod,dystrophin N-terminal,caveolin-3,α-,β-,γ-,δ-sarcoglycan and dysferlin (Novocastra Laboratories) for IHC. Expression of calpain-3 and dysferlin protein was observed using Western blotting (WB).Result:5 cases with no or only trace expression of calpain-3 protein were diagnosed as calpainopathy (LGMD2A) by Western blot analysis. The expression of dystrophin, caveolin-3,α-,β-,γ-andδ-sarcoglycan protein were normally staining of 5 LGMD2A and 7 LGMD2B patients'specimens by IHC.2 of 5 LGMD2A patients had reduced staining of dysferlin by IHC. Altogether 8 patients showed deficient in dysferlin staining, WB was performed using the total protein extracting from the muscle tissue.6 had an dysferlin expression above 30% of the normal. Calpain-3 expression of 2 LGMD2B and 23 of 30 LGMD with excluding LGMD2B showed varied reduced level in WB. They were excluced from primary LGMD2B and LGMD2A.2 patients had a normal calpain-3 expression.Conclusion:1.5 LGMD2A patients was about 13.5% of the LGMD(5/37), less than the situation in Japanese (about 26%).2. We had used two type of immunologic skills in the reseach, IHC and WB.We apply calpain-3 expression by WB to remedy that calpian-3was not used by IHC.5 cases with no or only trace expression of calpain-3 protein were diagnosed as calpainopathy (LGMD2A) by Western blot analysis.2 of 5 LGMD2A patients had reduced staining of dysferlin by IHC,but dysferlin expression above 15% of the normal by WB. Respecting to the reliability of WB in testing the protein deficiency,We suggest that at first an IHC should be performed in diagnosis of dysferlinopathy, and WB would be necessary for the uncertain cases. 3. Pathologically, there was marked variation in fibre size and most small fibres were round. Some necrotic and regenerating fibers were seen. Fibres with centrally placed nuclei can be found frequently. It has no specificity. These will make it difficult in differential diagnosis during LGMDs. A molecular biologic method will be necessary in this situation. Labelling the dystrophin C-terminal,dystrophin Rod,dystrophin N-terminal,caveolin-3,calpain-3,α-,β-,γ-,δ-sarcoglycan and dysferlin around the muscle fiber using IHC, or testing the expression level of related protein in muscle tissue with WB would solve the problem. |
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