The primary molecular endpoint for many Duchenne muscular dystrophy (DMD) clinical trials is the induction, or increase in production, of dystrophin protein in striated muscle. For accurate endpoint analysis, it is essential to have reliable, robust and objective quantification methodologies capable of detecting subtle changes in dystrophin expression. In this work, we present further development and optimisation of an automated, digital, high-throughput script for quantitative analysis of multiplexed immunofluorescent (IF) whole slide images (WSI) of dystrophin, dystrophin associated proteins (DAPs) and regenerating myofibres (fetal/developmental myosin-positive) in transverse sections of DMD, Becker muscular dystrophy (BMD) and control skeletal muscle biopsies. The script enables extensive automated assessment of myofibre morphometrics, protein quantification by fluorescence intensity and sarcolemmal circumference coverage, colocalisation data for dystrophin and DAPs and regeneration at the single myofibre and whole section level. Analysis revealed significant variation in dystrophin intensity, percentage coverage and amounts of DAPs between differing DMD and BMD samples. Accurate identification of dystrophin via a novel background subtraction method allowed differential assessment of DAP fluorescence intensity within dystrophin positive compared to dystrophin negative sarcolemma regions. This enabled surrogate quantification of molecular functionality of dystrophin in the assembly of the DAP complex. Overall, the digital script is capable of multiparametric and unbiased analysis of markers of myofibre regeneration and dystrophin in relation to key DAPs and enabled better characterisation of the heterogeneity in dystrophin expression patterns seen in BMD and DMD alongside the surrogate assessment of molecular functionality of dystrophin. Both these aspects will be of significant relevance to ongoing and future DMD and other muscular dystrophies clinical trials to help benchmark therapeutic efficacy.

中文翻译：

---

高通量数字脚本，用于多重免疫荧光分析和定量分析肌营养不良症中的肌膜蛋白和肌膜蛋白。

许多Duchenne肌营养不良症（DMD）临床试验的主要分子终点是横纹肌中肌营养不良蛋白的诱导或产量增加。对于准确的终点分析，至关重要的是要有可靠，可靠且客观的定量方法，能够检测肌营养不良蛋白表达的细微变化。在这项工作中，我们提出了自动化，数字化，高通量脚本的进一步开发和优化，用于定量分析肌营养不良蛋白，肌营养不良蛋白相关蛋白（DAP）的多重免疫荧光（IF）全玻片图像（WSI）和再生肌纤维（胎儿/ DMD，贝克尔肌营养不良症（BMD）和对照骨骼肌活检标本的横切面中出现发育性肌球蛋白阳性）。该脚本可对肌纤维形态进行广泛的自动评估，通过荧光强度和肌膜周覆盖率，肌营养不良蛋白和DAP的共定位数据以及单个肌纤维和整个切片水平的再生对蛋白质进行定量。分析显示，在不同的DMD和BMD样品之间，肌营养不良蛋白强度，DAP覆盖率和DAP含量之间存在显着差异。通过一种新型的背景扣除法对肌营养不良蛋白进行准确鉴定，与肌营养不良蛋白阴性肌膜区相比，可以在肌营养不良蛋白阳性患者中对DAP荧光强度进行差异评估。这可以替代DAP复合物组装过程中肌营养不良蛋白的分子功能的替代量化。总体，该数字脚本能够对与关键DAP相关的肌纤维再生和肌营养不良的标志物进行多参数无偏分析，并能够更好地表征BMD和DMD中肌营养不良蛋白表达模式的异质性，同时对肌营养不良蛋白的分子功能进行替代评估。这两个方面都将与正在进行的和将来的DMD和其他肌营养不良症的临床试验具有重要的关联，以帮助确定治疗效果。