ABSTRACT
The present work aimed to study dystrophin gene mutations in DMD patients and to investigate some of the biochemical mechanisms that might be involved in the pathogenesis of muscle wasting in this disease. The present study was conducted on 40 male subjects divided into two groups: Group 1: included 30 patients with the firm diagnosis of DMD as confirmed by negative immunohistochemical staining of muscle biopsies for dystrophin protein. Group 2 included 10 healthy male volunteers of matched age. All individuals were subjected to measurements of serum creatine kinase [CK], RANTES, and MDA. A soleus muscle biopsy was taken from every individual and was subjected to measurement of intracellular Ca[2+]concentration [[Ca2+]i], and assays of PLA[2], calpain, calpastatin, and caspase-3 activities in muscle tissue homogenates. DNA was extracted from peripheral blood leukocytes and exons 19, 44, and 50 were amplified by isolated polymerase chain reaction [PCR] technique. The amplified exons were then subjected to agarose gel electrophoresis for detection of exon mutations. The present study showed that 7/30 of the DMD patients had mutations in the form of exon deletions. Exon 50 showed the highest frequency of deletions [4/30]. The study also showed that 5/30 of the patients had deletions of only one exon, whereas 2/30 of them had combined 2 exon deletions. None of the patients had combined 3 exon deletions. The present study also revealed significant increase in serum levels of CK, RANTES, and MDA in DMD patients as compared to the control. It also revealed significant increase in soleus muscle [Ca2+]i], in the activities of PLA[2], calpain, and caspase-3 in muscle tissue homogenates, and in the ratio of calpain/calpastatin activities in the DMD patients as compared to the control group. On the contrary, this study showed a significant decrease in calpastatin activity in DMD patients in comparison to the control group. From the present work it could be concluded that DMD is a complex disease and that dystrophin deficiency is necessary, but not sufficient on its own to fully account for the pathophysiology of the disease which entails the interplay of several factors and mechanisms. Accordingly, secondary modifiers must play an important role in determining the ultimate fate of dystrophin-deficient fibers e.g. cytosolic calcium overload, increased activities of PLA[2] and of the calpain system, and enhanced oxidative stress and apoptosis. Moreover, the inflammatory response within the diseased muscle is one likely candidate among potential disease-modifying factors. Therefore, although there is currently no effective therapy for this fatal muscle disease, various strategies should be developed driven by the increasing understanding of the molecular mechanisms involved in the progression of muscle wasting. These strategies should include the use of membrane stabilizing agents, Ca[2+] channel blockers, PLA[2] inhibitors, calpain inhibitors, anticaspases, and ROS scavengers