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A high-throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies

A high-throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies
A high-throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies

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.

2051-5960
Scaglioni, Dominic
01dca81f-d72e-4f12-b5c0-8d7ba8aecaf6
Ellis, Matthew
afbca752-ced4-40dd-b0af-d9ecffbd5b63
Catapano, Francesco
6be7d3ce-6254-479e-a4dd-859ce92d4385
Torelli, Silvia
602488be-46e8-424f-975a-0134a7715e27
Chambers, Darren
ff78a735-65b4-4aff-8b89-4308011dcc40
Feng, Lucy
00f5d331-6b81-40e8-92f8-fa6a15992d9f
Sewry, Caroline
63dd53e7-0f24-45b8-a634-e3a1f339c47f
Morgan, Jennifer
10085cd7-1de2-4671-87b5-d61221898925
Muntoni, Francesco
24b9757d-8a5b-4532-94f6-6d25ba539ee7
Phadke, Rahul
ddd1d98b-41ac-456b-bb1b-34a895c30e3b
Scaglioni, Dominic
01dca81f-d72e-4f12-b5c0-8d7ba8aecaf6
Ellis, Matthew
afbca752-ced4-40dd-b0af-d9ecffbd5b63
Catapano, Francesco
6be7d3ce-6254-479e-a4dd-859ce92d4385
Torelli, Silvia
602488be-46e8-424f-975a-0134a7715e27
Chambers, Darren
ff78a735-65b4-4aff-8b89-4308011dcc40
Feng, Lucy
00f5d331-6b81-40e8-92f8-fa6a15992d9f
Sewry, Caroline
63dd53e7-0f24-45b8-a634-e3a1f339c47f
Morgan, Jennifer
10085cd7-1de2-4671-87b5-d61221898925
Muntoni, Francesco
24b9757d-8a5b-4532-94f6-6d25ba539ee7
Phadke, Rahul
ddd1d98b-41ac-456b-bb1b-34a895c30e3b

Scaglioni, Dominic, Ellis, Matthew, Catapano, Francesco, Torelli, Silvia, Chambers, Darren, Feng, Lucy, Sewry, Caroline, Morgan, Jennifer, Muntoni, Francesco and Phadke, Rahul (2020) A high-throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies. Acta Neuropathologica Communications, 8 (1), [53]. (doi:10.1186/s40478-020-00918-5).

Record type: Article

Abstract

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.

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More information

Accepted/In Press date: 16 March 2020
Published date: 17 April 2020

Identifiers

Local EPrints ID: 446222
URI: http://eprints.soton.ac.uk/id/eprint/446222
ISSN: 2051-5960
PURE UUID: 0dbb264a-ec7f-476e-af7a-3845ba987a03

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Date deposited: 29 Jan 2021 17:30
Last modified: 16 Mar 2024 10:34

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Contributors

Author: Dominic Scaglioni
Author: Matthew Ellis
Author: Francesco Catapano
Author: Silvia Torelli
Author: Darren Chambers
Author: Lucy Feng
Author: Caroline Sewry
Author: Jennifer Morgan
Author: Francesco Muntoni
Author: Rahul Phadke

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