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Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies

AIMS: 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 Duch...

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Autores principales: Vetter, Tatyana A., Nicolau, Stefan, Bradley, Adrienne J., Frair, Emma C., Flanigan, Kevin M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9184255/
https://www.ncbi.nlm.nih.gov/pubmed/34847621
http://dx.doi.org/10.1111/nan.12785
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author Vetter, Tatyana A.
Nicolau, Stefan
Bradley, Adrienne J.
Frair, Emma C.
Flanigan, Kevin M.
author_facet Vetter, Tatyana A.
Nicolau, Stefan
Bradley, Adrienne J.
Frair, Emma C.
Flanigan, Kevin M.
author_sort Vetter, Tatyana A.
collection PubMed
description AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
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spelling pubmed-91842552022-10-14 Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies Vetter, Tatyana A. Nicolau, Stefan Bradley, Adrienne J. Frair, Emma C. Flanigan, Kevin M. Neuropathol Appl Neurobiol Original Articles AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. John Wiley and Sons Inc. 2021-12-28 2022-04 /pmc/articles/PMC9184255/ /pubmed/34847621 http://dx.doi.org/10.1111/nan.12785 Text en © 2021 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Original Articles
Vetter, Tatyana A.
Nicolau, Stefan
Bradley, Adrienne J.
Frair, Emma C.
Flanigan, Kevin M.
Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies
title Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies
title_full Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies
title_fullStr Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies
title_full_unstemmed Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies
title_short Automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies
title_sort automated immunofluorescence analysis for sensitive and precise dystrophin quantification in muscle biopsies
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9184255/
https://www.ncbi.nlm.nih.gov/pubmed/34847621
http://dx.doi.org/10.1111/nan.12785
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