Abstract
Clinical trials using strategies aimed at inducing dystrophin expression in Duchenne muscular dystrophy (DMD) are underway or at advanced planning stage, including splice switching antisense oligonucleotides (AON), drugs to induce read-through of nonsense mutations and viral mediated gene therapy. In all these strategies, different dystrophin proteins, often internally deleted, are produced, similar to those found in patients with the milder DMD allelic variant, Becker muscular dystrophy (BMD). The primary biological endpoint of these trials is to induce functional dystrophin expression. A reliable and reproducible method for quantification of dystrophin protein expression at the sarcolemma is crucial to monitor the biochemical outcome of such treatments. We developed a new high throughput semi quantitative fluorescent immunofluorescence method for quantifying dystrophin expression in transverse sections of skeletal muscle. This technique is completely operator independent as it based on an automated scanning system and an image processing script developed with Definiens software. We applied this new acquisition-analysis method to quantify dystrophin and sarcolemma-related proteins using paediatric control muscles from cases without a neuromuscular disorder as well as DMD and BMD samples. The image analysis script was instructed to recognize myofibres immunostained for spectrin or laminin while dystrophin was quantified in each identified myofibre (from 2,000 to over 20,000 fibres, depending on the size of the biopsy). We were able to simultaneously extrapolate relevant parameters such as mean sarcolemmal dystrophin, mean spectrin and laminin intensity, fibre area and diameter. In this way we assessed dystrophin production in each muscle fibre in samples of DMD, BMD and controls. This new method allows the unbiased quantification of dystrophin in every myofibre within a transverse muscle section and will be of help for translational research projects as a biological outcome in clinical trials in DMD and BMD.