Levels of dystrophin expression reported in patients with Becker muscular dystrophy (BMD) vary dramatically due to multiple factors, including the natural variation in actual dystrophin levels across patients, use of different detection methods, and application of different reference standards. Dystrophin levels in baseline Becker muscular dystrophy biopsies Lastly, dystrophin is found in low abundance in healthy muscle tissue and at even lower abundance in diseased muscle, which pushes the limits of sensitivity for various assay designs and detection methods. Further, some DMD biopsies contain high fat and fibrotic tissue, which could impact the accuracy of dystrophin quantitation. The large size of the dystrophin protein and its susceptibility to proteolysis pose challenges with extracting the protein, and there can be further difficulties associated with electrophoretic separation on a sodium dodecyl sulfate polyacrylamide gel for accurate, albeit relative, quantification. An added complication stems from the inability to utilize a single healthy standard across sites because these biopsies are typically small in size. The issue with this practice is that all reports of dystrophin amounts, even with “quantitative methods”, are relative in nature because dystrophin quantity in muscle biopsies from healthy controls varies between individuals. For example, to construct a 50% standard, equal protein amounts of healthy lysate and DMD lysate are combined. A standard curve is typically setup such that healthy control lysate is spiked into DMD lysate to create a standard curve with a range of dystrophin amounts relative to the healthy control which is set at 100%.
The use of healthy controls as the standard for quantifying dystrophin has led to the practice of reporting dystrophin amounts in terms of percent dystrophin found in healthy control (e.g., percentage of healthy control where the control is assumed to be 100%). Thus, extracts of healthy control muscle must be used to generate a standard curve. A dystrophin protein standard does not exist at this time because large scale recombinant expression of the complete 427 kD protein has not been possible. 3 Western Blot is a method to detect and quantify proteins by transferring (blotting) proteins separated by electrophoresis from a gel to a membrane. Quantification of dystrophin protein by western blot involves many potential challenges that should be carefully considered when developing a method for use in clinical study. This report presents a review of the previously reported dystrophin amounts in muscular dystrophy biopsies, along with an analysis of critical variables impacting dystrophin quantitation. Eteplirsen (Exondys 51™ Sarepta Therapeutics, Inc., Cambridge, MA) received accelerated approval from the US Food and Drug Administration (FDA) based on an increase in dystrophin in some treated patients, as assessed by western blot, 1,2 highlighting the importance of dystrophin quantification as an essential component of biochemical efficacy for dystrophin-restoring therapies. Currently, multiple therapeutic agents that aim to restore dystrophin expression are being evaluated in clinical trials ( identifiers: NCT02310906, NCT02740972, NCT03368742, NCT03508947, NCT03769116, and NCT03362502). Keywordsĭuchenne muscular dystrophy, western blot methods, dystrophin western blot, muscular dystrophy biopsy, dystrophin quantification Article:ĭuchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that disrupt the production of functional dystrophin protein, resulting in progressive muscle damage and loss of contractile function.
A review of the dystrophin western blot data on Duchenne and Becker muscular dystrophy biopsies is conducted, along with a thorough investigation of methodologies to quantify dystrophin. A few key changes to standards and data analysis parameters can result in a low level of dystrophin (20% of the levels reported in healthy human muscle. Nonoptimized western blot methods can reflect inaccurate results, especially in the quantitation of low dystrophin levels. The approval, along with the initiation of clinical trials evaluating other dystrophin-restoring therapies, highlights the importance of accurate dystrophin quantitation. The Duchenne muscular dystrophy community has recently seen the first approved therapy for the restoration of dystrophin, based on its ability to increase levels of dystrophin protein, as determined by western blot.
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