The Dysferlin Expression In Muscular Dystrophy And Miyoshi Myopathy

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 had been identified for their responsible genes and the products. Distal myopathy is a group of primary myopathy, characterized by the weakness and waste in calf muscles and distal anterior compartment myopathy. Genetically and pathologically it is classified for Nonaka distal myopathy(DMRV), Welander distal myopathy, Miyoshi myopathy(MM), oculopharyngeal distal myopathy(OPDM). They are highly heterogeneous in the involvement of muscle group and their progression. Mutations in the dysferlin gene can cause muscular dystrophies called dysferlinopathy, which include limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). To clarify the frequency and clinicopathological features of dysferlinopathy in Chinese, we performed protein analyses of dysferlin in 98 progressive muscular dystrophy(PMD). Material and methods:We examined a total of 98 PMD patients' muscles obtained from the peripheral nerve and muscular disease institute of shandong qilu hospital, including 56 LGMD, 6 MM, 14 BMD and 22 DMD patients clinically and histochemically diagnosed. Expression of dysferlin protein was observed using immunohistochemistry (IHC) and Western blotting (WB). As a contral 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 dysferlin, a-sarcglycan, dystrophin C-terminal, dystrophin Rod and dystrophin N-terminal (Novocastra Laboratories) for IHC. For the patients showed deficiency in dysferlin staining, we performed WB using the total protein extracting from the muscle tissue. Result:Altogether 43 dystrophin, 16 dysferlin and 9α—sarcoglycan deficiency were found in the group by IHC. There were 20 absence, 23 defect in dystrophin staining, 5 absence and 11 defect in dysferlin staining , 3 absence and 6 defect in α—sarcoglycan staining. 5 had a deficiency both in dystrophin and α—sarcoglycan staining, in the 5 patients 2 were dystrophin absent with α — sarcoglycan defect, 1α —sarcoglycan absent with dystrophin defect, 2 absent in both α—sarcoglycan and dystrophin staining and 1 defect in the two protein. 1 showed deficient both in dysferlin and α— sarcoglycan staining. For the 16 patients showed deficient in dysferlin staining, WB was performed using the total protein extracting from the muscle tissue. 5 showed that the protein was absent completely, coincident with IHC. 3 had an dysferlin expression below 15% of the normal. The 8 patients had a normal expression both in a— sarcoglycan and dystrophin , they were diagnosed as primary dysferlinopathy according to the diagnostic criteria of ENMC. The other 8 patients who showed a dysferlin deficiency in IHC identified an expression over 30% of the normal in WB, they were excluced from primary dysferlinopathy. Conclusion:1. 3 had a predominant involvement in their lower proximal muscles in the 8 dysferlinopathy patients, about 7% of the LGMD(3/44), less than the situation in Japanese ( about 18%) , but it is higher than the average level (about 1%) in the world. There were 5 MM in the 8 dysferlinopathy patients, about 83%(5/6) of the distal myopathy. This means that MM would take a more important role in the phenotype of dysferlinopathy in our country.2. We had used two type of immunologic skills in the reseach, IHC and WB. We found that the patients who had a dysferlin absence in IHC would had the same result in WB, the both skills showed a high coincidence in the situation. But in the test for dysferlin defect they could have a conspicuous difference. 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 cases3. The BMD patients have a relatively mild phenotype of dystrophinopathy. They may have a different clinical manifestation inter- and intra-familial, and overlapping with LGMD in clinical manifestation. And it has no specificity in histology. These will make it difficult in differential diagnosis between the BMD and LGMD. A molecular biologic method will be necessary in this situation. Labelling the dysferlin or dystrophin around the muscle fiber using IHC, or testing the expression level of related protein in muscle tissue with WB would solve the problem.4. More often than usual in other PMD, we could find the inflammatory cell infiltration in dysferlinopathy's histological specimens. In these 8 dysferlinopathy, 3 were found the inflammatory cell infiltration in their muscle biopsy, taking about 37.5% of the 8 patients. The inflammatory cell scatters in the extracellular matrix, or invading degenerating fibers, or locating around the blood vessel. Muscle inflammation has made it difficult to distinguish the dysferlinopathy from polymyositis (PM) patients, the later is characteristic of muscle inflammation and degeneration. Furthermore they share the similar onset age and clinical manifestation. Labeling the dysferlin around the muscle fiber in IHC or test the protein level with WB would be an effective method in distinguishing the twins.