Background: Accurate dystrophin quantitation is essential for evaluating the impact of dystrophin-restoring therapies in Duchenne muscular dystrophy (DMD). Different western blot methodologies (e.g., choice of dystrophin standards, muscle content adjustment, loading controls, and normalization) may lead to variable dystrophin quantification, influencing the assessment of treatment outcomes.
Objective: To describe assays quantifying exon-skipped dystrophin and delandistrogene moxeparvovec micro-dystrophin at baseline and following treatment with dystrophin-restoring therapies, incorporating muscle content adjustment.
Results: For western blots, the normal control (NC) for full-length dystrophin is prepared by pooling healthy muscle biopsy samples to mitigate interindividual variability. Exon-skipped dystrophin is quantified using monoclonal DYS1 antibodies and a 5-point %NC standard curve (0.25%-8.0%). Micro-dystrophin is quantified using monoclonal DYS3 antibodies and a 5-point recombinant micro-dystrophin standard curve (21.9-349.6 fmol/mg protein, equating to 3.4-54.8 %NC). Alpha-actinin serves as an internal protein loading control but is not used for normalization. Because non-muscle tissues contribute to the total protein loaded in a western blot, muscle fiber dystrophin protein may be underestimated. Skeletal muscle content may be quantified by evaluating α-actinin in immunoblots, myosin heavy chain in Coomassie-stained gels, or Masson trichrome (MTri) staining of cryosections. MTri histological staining distinguishes muscle fibers from collagen-containing tissues using Biebrich Scarlet-Acid Fuchsin and Aniline Blue staining, respectively. MTri-stained cryosections cut concurrently with sections used for western blotting undergo color-segmentation-based digital image analysis to determine MTri %muscle. MTri %muscle is the most quantitative method to measure muscle content distribution and is appropriate to adjust the dystrophin protein (%NC) to the proportion of tissue represented by muscle fibers (%muscle).
Conclusions: In conditions with progressive muscle loss and fibrosis like DMD, it is important that biopsy muscle content is precisely measured for reliable dystrophin quantification. MTri staining/analysis represents an appropriate approach to determine muscle content and adjust the amount of dystrophin detected by western blotting.