AIMS: Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low. METHODS: We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy, intermediate muscular dystrophy and normal control tissue. RESULTS: Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin:spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis. CONCLUSION: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.
AIMS: Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low. METHODS: We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy, intermediate muscular dystrophy and normal control tissue. RESULTS: Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin:spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis. CONCLUSION: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.
Authors: Susan A Elmore; Famke Aeffner; Dinesh S Bangari; Torrie A Crabbs; Stacey Fossey; Shayne C Gad; Wanda M Haschek; Jessica S Hoane; Kyathanahalli Janardhan; Ramesh C Kovi; Gail Pearse; Lyn M Wancket; Erin M Quist Journal: Toxicol Pathol Date: 2017-11-07 Impact factor: 1.902
Authors: Peter P Nghiem; Eric P Hoffman; Priya Mittal; Kristy J Brown; Scott J Schatzberg; Svetlana Ghimbovschi; Zuyi Wang; Joe N Kornegay Journal: Am J Pathol Date: 2013-11 Impact factor: 4.307
Authors: Kristin Wilson; Crystal Faelan; Janet C Patterson-Kane; Daniel G Rudmann; Steven A Moore; Diane Frank; Jay Charleston; Jon Tinsley; G David Young; Anthony J Milici Journal: Toxicol Pathol Date: 2017-10-03 Impact factor: 1.902
Authors: Famke Aeffner; Crystal Faelan; Steven A Moore; Alexander Moody; Joshua C Black; Jay S Charleston; Diane E Frank; Johannes Dworzak; J Kris Piper; Manish Ranjitkar; Kristin Wilson; Suzanne Kanaly; Daniel G Rudmann; Holger Lange; G David Young; Anthony J Milici Journal: Arch Pathol Lab Med Date: 2018-08-31 Impact factor: 5.534