Report of a TREAT-NMD/World Duchenne Organisation Meeting on Dystrophin Quantification Methodology

Abstract

Representatives of academia, patient organisations, industry and the United States Food and Drug Administration attended a workshop on dystrophin quantification methodology. The aims of the workshop were to provide an overview of methods used to quantify dystrophin levels in human skeletal muscle and their applicability to clinical trial samples, outline the gaps with regards to validating the methods for robust clinical applications prior to regulatory agency review, and to align future efforts towards further optimizing these methods. The workshop facilitated a constructive but also critical discussion on the potential and limitations of techniques currently used in the field of translational research (western blot and immunofluorescence analysis) and emerging techniques (mass spectrometry and capillary western immunoassay). Notably, all participants reported variation in dystrophin levels between muscle biopsies from different healthy individuals and agreed on the need for a common reference sample.

Challenges of Interpreting Dystrophin Content by Western Blot

“…The inability of LC-MS to resolve differences in dystrophin <5% has also been reported by others. 3 As is the case with any dystrophin quantification method, a dystrophin LC-MS method should be examined for the ability to resolve changes in dystrophin in the range that is reported.…”

“…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. 3 Western Blot is a method to detect and quantify proteins by transferring (blotting) proteins separated by electrophoresis from a gel to a membrane. 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.…”

A high–throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies

“…Irrespective of the individual therapeutic approach, immunohistochemical evaluation and quantification of sarcolemmal dystrophin is a key pathological outcome measure. To evaluate the molecular efficacy and success of DMD clinical trials, robust, reliable and objective methodology for dystrophin quantification must be utilised [3,6,35]. Furthermore, there is a growing interest to not only localise and quantify the level of restored dystrophin in myofibres of post-treatment biopsies, but also to correlate its levels to key DAP interactions, to demonstrate surrogate molecular functionality [14,29,30].…”

“…In addition, many scanners may not be calibrated in the same way as traditional fluorescent microscopes to account for day to day variability. It is therefore essential for automated image analysis systems and digital scripts to be suitably optimised and validated for use in a clinical trial environment [3]. Furthermore, images must be of a consistent quality having been immunostained or histochemically stained and acquired via highly reproducible methods [1,3,4,35,38].…”

Phenotype predictions for exon deletions/duplications: A user guide for professionals and clinicians using Becker and Duchenne muscular dystrophy as examples

“…Relevant positive and negative controls should be included. DMD, Duchenne muscular dystrophy; RT-PCR, Reverse transcription polymerase chain reaction When dystrophin is present at less than 3% of normal levels, generally patients will develop DMD, while patients with levels of 10% or more of (altered) dystrophin generally will develop BMD (Aartsma-Rus et al, 2019). However, it is not known how much dystrophin is needed, and this probably depends on variables like which internally deleted protein is produced (different dystrophins will have different functionalities), the amount of dystrophin production, the number of dystrophin-positive cells, and other factors like genetic modifiers.…”