Pathological And Molecular Biological Studies Of Limb-girdle Muscular Dystrophy Type 2A

Objective: Limb-girdle muscular dystrophy is a group of hereditary diseases progressed slowly, with proximal muscle weakness and atrophy as their major clinical expressions, and the major pathological appearance is muscle fibers degenerating, necrotic and regenerating. It belongs to the progressive muscular dystrophy, and according to the inherited pattern and pathogenic gene, it can be classified into autosomal dominant inheritance(LGMD1A~1G), and autosomal recessive inheritance (LGMD2A~2L). The pathogenic gene of LGMD2A is CAPN3, which encodes calpain-3. The mutation of CAPN3 caused the protein expressed abnormal, followed with clinical and pathological expressions. With skeletal muscle biopsy, histochemical stains, immunohistochemical stains and western blotting, in order to investigated the pathological and molecular biological characters, also the potential pathogenesis, meanwhile to study the relations between calpain-3 and other PMD related proteins.Methods: Select biopsied skeletal muscle specimens of 10 cases with LGMD2A, frozen serial sections in 7μm or 10μm.1 Histochemical stains: including Hematoxylin and Eosin(HE), Modified Gomori trichrome(MGT), Nicotinamide Adenine Dinucleotide Tetra-zolium Reductase(NADH-TR), cytochrome c oxidase(CCO), succinate dehydrogenase(SDH), AMP deaminase, Acid phosphatase, Adenosine triphosphatase (ATPase), periodic acid schiff(PAS), Oil red O and Sudan blue.2 Immunohistochemical stains(2 cases with Duchenne muscular dystrophy and 2 cases with LGMD2B as control): anti-dystrophin-N, -C , -R , sarcoglycan-α,-β,-γ,-δ,dysferlin , caveolin-3 monoclone antibodies immunohisto- chemical stains.3 Western blotting: western blotting analysis labeled with anti-calpain-3 and caveolin-3 monoclone antibodies.Results:1 Pathological characters of histochemical and enzymological stains: fiber size variation, muscle fibers degenerating, necrotic and regenerating presented in a variable extent, and connective tissue elements increased. NADH-TR, CCO and SDH stains, the activities showed focal decrease in many fibers, and lobulated fibers can be observed. The activity of AMP is normal. Acid phosphotase staining, the activity is increased in degenerating fibers and inflammatory cells, with red staining. ATPase reacted sections, the random checkerboard distribution ofⅠandⅡtypes are preserved, occasionally with some neurogenic pathological changes, such as muscle fiber predominance and group atrophy. Muscle fiber glycogen contents presents normal in PAS. Oil red O and Sudan blue stains, lipid contents increased in some fibers.2 Pathological characters of immunohistochemical stains: anti-dystrophin-N, -C, -R, sarcoglycan-α,-β,-γ,-δ, dysferlin, caveolin-3 monoclone antibodies staining, 10 cases with LGMD2A, the corresponding proteins expressed normal with a dark brown colour; Duchenne muscular dystrophy(DMD) patients, dystrophin-N, -C, -R showed complete deficient on the sarcolemma, and sarcoglycan-α,-β,-γ,-δexpressed weakly, while dysferlin and caveolin-3 were normal; LGMD2B, dysferlin was absent on sarcolemma and in the sarcoplasm, but the other proteins expressed normal.3 Western blotting: compared with the control, the band at 94kd(calpain-3) is completely absent in 10 cases, and additional band at 30kd expressed weakly. The band at 22kd(caveolin-3) protein expressed normal in all of the 10 cases.Conclusions:1 Major clinical symptoms of patients with LGMD2A: consistent with common clinical appearances of LGMD, without special features. Onset at childhood or adulthood, often with a positive family history. First onset at the proximal limb muscles with weakness and atrophy, and always involved lower limb. The pathogenetic condition progressed slowly, and CK increased slightly to markedly. EMG showed myogenic change.2 Biopsied skeletal muscle, histochemical stains: muscular dystrophy pathological change. Muscle fibers degenerating, necrotic and regenerating in variable extents. The splitting fibers can be observed in the chronic cases. The connective tissue elements increased. NADH-TR, CCO and SDH stains, the activity of enzymes showed abnormal, such as lobulated fibers.3 Immunohistochemical stains: anti-dystrophin-N,-C,-R ,sarcoglycan,dysferlin and caveolin-3 monoclonal antibody stains, the expression of corresponding proteins are normal, and the diagnosis of Duchenne muscular dystrophy, Becker muscular dystrophy, LGMD2C~2F, LGMD2B and LGMD1C should be excluded. Suggesting that the calpain-3 had no relationship with the proteins mentioned above in the 10 cases.4 Western blotting: the band corresponding to calpain-3 protein(94kd) showed completely absent in the 10 patients with LGMD2A. LGMD2A can be final diagnosed when the calpain-3 is completely absent. Western blotting is an effective method to diagnose LGMD2A.5 The pathogenesy of LGMD2A may include: structure or function defection of calpain-3; abnormal of binding between calpain-3 and titin; apoptosis of skeletal muscle cell.