Abstract
Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy (BMD). In the last decade several antisense oligonucleotides drugs have been developed to induce a partially functional internally deleted dystrophin, similar to that produced in BMD, and expected to ameliorate the disease course. The pattern of dystrophin expression and functionality in dystrophinopathy patients is variable due to multiple factors, such as molecular functionality of the dystrophin and its distribution. To benchmark the success of therapeutic intervention, a clear understanding of dystrophin expression patterns in dystrophinopathy patients is vital. Recently, several groups have used innovative techniques to quantify dystrophin in muscle biopsies of children but not in patients with milder BMD. This study reports on dystrophin expression using both Western blotting and an automated, high-throughput, image analysis platform in DMD, BMD, and intermediate DMD/BMD skeletal muscle biopsies. Our results found a significant correlation between Western blot and immunofluorescent quantification indicating consistency between the different methodologies. However, we identified significant inter- and intradisease heterogeneity of patterns of dystrophin expression in patients irrespective of the amount detected on blot, due to variability in both fluorescence intensity and dystrophin sarcolemmal circumference coverage. Our data highlight the heterogeneity of the pattern of dystrophin expression in BMD, which will assist the assessment of dystrophin restoration therapies.
Highlights
IntroductionDuchenne and Becker muscular dystrophy (DMD/ BMD) are progressive X-linked neuromuscular disorders that together affect 1 in 3500–5000 newborn males worldwide
We found that there was a significant difference in the percentage of dystrophin-positive fibers between CTRLs versus Duchenne muscular dystrophy (DMD) (p 1⁄4 0.0011), CTRLs versus Intermediate clinical phenotypes (IMD) (p 1⁄4 0.0190) and Becker muscular dystrophy (BMD) versus DMDs (p < 0.0001) and Dystrophin Sarcolemmal Coverage Myofiber sarcolemmal circumference coverage of dystrophin can be visualized as a cumulative frequency graph (Fig. 2d); in this graph, the population showing 0%–25% dystrophin coverage, excluded in the graph 2c, is included
Detailed information on the patterns of dystrophin expression in patients with BMD and IMD is lacking in the recent studies in which novel technologies to measure protein expression and its quantification are used [17, 21], and most recent studies only used Western blot (WB) [9, 22]
Summary
Duchenne and Becker muscular dystrophy (DMD/ BMD) are progressive X-linked neuromuscular disorders that together affect 1 in 3500–5000 newborn males worldwide. J Neuropathol Exp Neurol Volume 00, Number 0, 2021. They are caused by mutations in the DMD gene which lead to absent (DMD) or decreased expression (BMD) of the dystrophin protein and damage and eventually loss of muscle [1]. DMD patients typically live into their 20s. While life expectancy in patients with BMD is closer to that of the general population, a wide variability of severity and outcomes exists. Following the implementation of recent standards of care, DMD males are living into their 30s and in some cases even longer. A more proactive approach to prevent respiratory insufficiency and cardiac failure has improved outcomes in BMD
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