Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies.
Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies.
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DOI：
10.1111/nan.12785
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发表时间：
2022-04
影响因子：
5
通讯作者：
Flanigan, Kevin M.
中科院分区：
文献类型：
--
作者：
Vetter, Tatyana A.;Nicolau, Stefan;Bradley, Adrienne J.;Frair, Emma C.;Flanigan, Kevin M.
关键词：
肌营养不良蛋白是DMD基因的蛋白质产物，通过在收缩和舒张期间稳定肌膜而在肌肉完整性中起关键作用。DMD基因易受各种突变的影响，这些突变可能导致蛋白质的完全丢失、耗尽或截短，从而导致杜氏和贝克肌营养不良症。精确和可重复的肌营养不良蛋白定量在表征DMD突变和评估通过基因疗法诱导肌营养不良蛋白的结果方面至关重要。免疫荧光显微镜对低水平的蛋白质表达具有高灵敏度，沿着定位确认，使其成为定量肌营养不良蛋白表达测定的关键组成部分。我们已经开发了一种自动化和公正的方法，用于精确定量肌肉切片中的肌营养不良蛋白免疫荧光。该方法使用抗肌萎缩蛋白和血影蛋白染色的全组织切片的显微镜图像来测量抗肌萎缩蛋白强度和每条肌纤维肌膜处抗肌萎缩蛋白阳性覆盖的比例。为确保客观性，肌营养不良蛋白和血影蛋白的阈值是根据每个组织切片内的非肌膜信号强度凭经验得出的。此外，该方法易于适用于测量纤维形态和其他组织标记物。我们的方法证明了这种定量方法在肌营养不良蛋白病患者和健康对照样品中广泛的肌营养不良蛋白表达中的灵敏度和重现性，具有较高的操作员间一致性。随着恢复营养不良肌肉中肌营养不良蛋白表达的努力将新的潜在疗法带入临床试验，这种方法代表了有效和精确分析肌营养不良蛋白和反映治疗效果的其他肌肉标志物的有价值的工具。描述了一种新开发的用于定量肌肉中抗肌萎缩蛋白阳性和强度的自动免疫荧光分析方法，并用于评估15个抗肌萎缩蛋白病样本和6个健康对照中的抗肌萎缩蛋白表达。结果为连续组织切片之间和不同操作者之间的良好灵敏度和良好再现性提供了证据。这种可访问的，强大的，高通量的肌营养不良蛋白定量方法代表了肌营养不良蛋白病的临床前和临床研究的一个有价值的新工具。
Dystrophin, the protein product of the DMD gene, plays a critical role in muscle integrity by stabilising the sarcolemma during contraction and relaxation. The DMD gene is vulnerable to a variety of mutations that may cause complete loss, depletion or truncation of the protein, leading to Duchenne and Becker muscular dystrophies. Precise and reproducible dystrophin quantification is essential in characterising DMD mutations and evaluating the outcome of efforts to induce dystrophin through gene therapies. Immunofluorescence microscopy offers high sensitivity to low levels of protein expression along with confirmation of localisation, making it a critical component of quantitative dystrophin expression assays. We have developed an automated and unbiased approach for precise quantification of dystrophin immunofluorescence in muscle sections. This methodology uses microscope images of whole‐tissue sections stained for dystrophin and spectrin to measure dystrophin intensity and the proportion of dystrophin‐positive coverage at the sarcolemma of each muscle fibre. To ensure objectivity, the thresholds for dystrophin and spectrin are derived empirically from non‐sarcolemmal signal intensity within each tissue section. Furthermore, this approach is readily adaptable for measuring fibre morphology and other tissue markers. Our method demonstrates the sensitivity and reproducibility of this quantification approach across a wide range of dystrophin expression in both dystrophinopathy patient and healthy control samples, with high inter‐operator concordance. As efforts to restore dystrophin expression in dystrophic muscle bring new potential therapies into clinical trials, this methodology represents a valuable tool for efficient and precise analysis of dystrophin and other muscle markers that reflect treatment efficacy. A newly‐developed automated immunofluorescence analysis method for quantifying dystrophin positivity and intensity in muscle is described and used to assess dystrophin expression in fifteen dystrophinopathy samples and six healthy controls. The results provide evidence for good sensitivity and excellent reproducibility between serial tissue sections and between different operators. This accessible, robust, and high‐throughput dystrophin quantification method represents a valuable new tool for preclinical and clinical studies in dystrophinopathies.
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影响因子：
11.2
作者：
Flanigan, Kevin M.;Ceco, Ermelinda;Lamar, Kay-Marie;Kaminoh, Yuuki;Dunn, Diane M.;Mendell, Jerry R.;King, Wendy M.;Pestronk, Alan;Florence, Julaine M.;Mathews, Katherine D.;Finkel, Richard S.;Swoboda, Kathryn J.;Gappmaier, Eduard;Howard, Michael T.;Day, John W.;McDonald, Craig;McNally, Elizabeth M.;Weiss, Robert B.
通讯作者：
Weiss, Robert B.
影响因子：
2.8
作者：
Waldrop, Megan A.;Gumienny, Felecia;El Husayni, Saleh;Frank, Diane E.;Weiss, Robert B.;Flanigan, Kevin M.
通讯作者：
Flanigan, Kevin M.
影响因子：
7.1
作者：
Scaglioni, Dominic;Ellis, Matthew;Phadke, Rahul
通讯作者：
Phadke, Rahul
影响因子：
5
作者：
Taylor, L. E.;Kaminoh, Y. J.;Flanigan, K. M.
通讯作者：
Flanigan, K. M.
影响因子：
4.6
作者：
Aeffner, Famke;Faelan, Crystal;Milici, Anthony, I
通讯作者：
Milici, Anthony, I