P.13.12 An objective method for immunofluorescence analysis of dystrophin levels in muscle from DMD patients in clinical studies

Abstract

Duchenne muscular dystrophy (DMD) is characterized by absence or very low (trace) expression of dystrophin at the sarcolemmal membrane of the muscle fibers. In clinical studies aiming to restore dystrophin expression, dystrophin levels are measured in muscle biopsies by immunofluorescence analysis of cross-sections or western blot analysis of total protein extracts. However, appropriate quantification poses a technical challenge as dystrophin levels may be low and/or variable between fibers in the same biopsy. We have developed an immunofluorescence method and automated image analysis that measures the dystrophin intensity per individual fiber in a biopsy. It reproducibly detects even small differences in dystrophin levels. Muscle cross-sections co-stained for dystrophin and spectrin are imaged by confocal microscopy and image analysis is performed using Definiens software. Using a customized algorithm, and the sarcolemmal spectrin signal as a mask, the software automatically segments each image into individual muscle fibers, measures the varying dystrophin intensity per individual fiber, and objectively produces a histogram of the distribution in the fiber population, including revertant fibers. Duplicated analysis of the biopsies on the same or multiple days and by different operators was shown to be reproducible, objective and able to distinguish between different low dystrophin levels in Becker muscular dystrophy and DMD samples. Moreover, in DMD patients treated with antisense oligonucleotides to restore dystrophin expression, comparisons of the dystrophin intensity distribution histograms of pre- and post-treatment muscle biopsies showed increases in dystrophin intensities of entire muscle fiber populations post-treatment.