Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials.

Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels.

Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable.

Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.

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This proves that the lack of trust by the FDA on dystrophin meaures is unfounded.  The FDA has what it need to make a decision on Eteplirsen.

A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

I also think this proves that the FDA has all they need Jason.

Thank you for posting the article here Jon Moulton. I had posted it on Facebook in answer to a discussion about the problems with dystrophin quantitation and the anouncement that Sarepta would now delay filing with the FDA but the discussion disappeared and no one commented on it while it was still up, as far as I had witnessed anyway.

As a scientist, what do you think about Prosensa's article Jon Moulton? Is it a sensitive, reproducible and objective method(s)? Could it be used to convince the FDA that drytrophin quantitation can be used for filing and approving new drugs for DMD now ? Or has it confused the FDA about dystrophin? Just trying to wrap my head around it all. Thanks.


Hi Andrea,

Ultimately the FDA will decide what level of precision and accuracy are acceptable in an assay.  I have not seen them specify levels of precision and accuracy that they would accept.  Without a target, when methods are published  the FDA is free to accept or reject them.  If the FDA specifies what they will accept, it becomes an engineering problem to meet the specifications.  Without a specification, the decision to accept or reject is political.

Yes, well it's the politics that is killing me, figuratively, and my son literally. 

Thanks Jon.

Sarepta is using a similar Immunofluorescence Analysis already.
Here is a slide from a sarepta  Credit Suisse Healthcare Conference presentation.

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