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A novel high-throughput immunofluorescence analysis method for quantifying dystrophin intensity in entire transverse sections of Duchenne muscular dystrophy muscle biopsy samples

A novel high-throughput immunofluorescence analysis method for quantifying dystrophin intensity in entire transverse sections of Duchenne muscular dystrophy muscle biopsy samples
A novel high-throughput immunofluorescence analysis method for quantifying dystrophin intensity in entire transverse sections of Duchenne muscular dystrophy muscle biopsy samples

Clinical trials using strategies aimed at inducing dystrophin expression in Duchenne muscular dystrophy (DMD) are underway or at advanced planning stage, including splice switching antisense oligonucleotides (AON), drugs to induce read-through of nonsense mutations and viral mediated gene therapy. In all these strategies, different dystrophin proteins, often internally deleted, are produced, similar to those found in patients with the milder DMD allelic variant, Becker muscular dystrophy (BMD). The primary biological endpoint of these trials is to induce functional dystrophin expression. A reliable and reproducible method for quantification of dystrophin protein expression at the sarcolemma is crucial to monitor the biochemical outcome of such treatments. We developed a new high throughput semi quantitative fluorescent immunofluorescence method for quantifying dystrophin expression in transverse sections of skeletal muscle. This technique is completely operator independent as it based on an automated scanning system and an image processing script developed with Definiens software. We applied this new acquisition-analysis method to quantify dystrophin and sarcolemma-related proteins using paediatric control muscles from cases without a neuromuscular disorder as well as DMD and BMD samples. The image analysis script was instructed to recognize myofibres immunostained for spectrin or laminin while dystrophin was quantified in each identified myofibre (from 2,000 to over 20,000 fibres, depending on the size of the biopsy). We were able to simultaneously extrapolate relevant parameters such as mean sarcolemmal dystrophin, mean spectrin and laminin intensity, fibre area and diameter. In this way we assessed dystrophin production in each muscle fibre in samples of DMD, BMD and controls. This new method allows the unbiased quantification of dystrophin in every myofibre within a transverse muscle section and will be of help for translational research projects as a biological outcome in clinical trials in DMD and BMD.

Adolescent, Biopsy, Child, Child, Preschool, Clinical Trials as Topic, Dystrophin, Fluorescent Antibody Technique, Genetic Therapy, High-Throughput Screening Assays, Humans, Image Processing, Computer-Assisted, Laminin, Muscle Fibers, Skeletal, Muscular Dystrophy, Duchenne, Oligonucleotides, Antisense, Sarcolemma, Software, Spectrin, Journal Article, Research Support, Non-U.S. Gov't
1932-6203
1-21
Sardone, Valentina
a98ee6c8-bfeb-4793-87f9-be6a34adbd50
Ellis, Matthew
afbca752-ced4-40dd-b0af-d9ecffbd5b63
Torelli, Silvia
602488be-46e8-424f-975a-0134a7715e27
Feng, Lucy
00f5d331-6b81-40e8-92f8-fa6a15992d9f
Chambers, Darren
ff78a735-65b4-4aff-8b89-4308011dcc40
Eastwood, Deborah
dc1140b3-10d4-4a7c-bf1e-66f94e6a9e06
Sewry, Caroline
63dd53e7-0f24-45b8-a634-e3a1f339c47f
Phadke, Rahul
ddd1d98b-41ac-456b-bb1b-34a895c30e3b
Morgan, Jennifer E
784c5ac9-07ea-4193-a35f-f6a4201d7501
Muntoni, Francesco
24b9757d-8a5b-4532-94f6-6d25ba539ee7
Sardone, Valentina
a98ee6c8-bfeb-4793-87f9-be6a34adbd50
Ellis, Matthew
afbca752-ced4-40dd-b0af-d9ecffbd5b63
Torelli, Silvia
602488be-46e8-424f-975a-0134a7715e27
Feng, Lucy
00f5d331-6b81-40e8-92f8-fa6a15992d9f
Chambers, Darren
ff78a735-65b4-4aff-8b89-4308011dcc40
Eastwood, Deborah
dc1140b3-10d4-4a7c-bf1e-66f94e6a9e06
Sewry, Caroline
63dd53e7-0f24-45b8-a634-e3a1f339c47f
Phadke, Rahul
ddd1d98b-41ac-456b-bb1b-34a895c30e3b
Morgan, Jennifer E
784c5ac9-07ea-4193-a35f-f6a4201d7501
Muntoni, Francesco
24b9757d-8a5b-4532-94f6-6d25ba539ee7

Sardone, Valentina, Ellis, Matthew, Torelli, Silvia, Feng, Lucy, Chambers, Darren, Eastwood, Deborah, Sewry, Caroline, Phadke, Rahul, Morgan, Jennifer E and Muntoni, Francesco (2018) A novel high-throughput immunofluorescence analysis method for quantifying dystrophin intensity in entire transverse sections of Duchenne muscular dystrophy muscle biopsy samples. PLoS ONE, 13 (3), 1-21, [e0194540]. (doi:10.1371/journal.pone.0194540).

Record type: Article

Abstract

Clinical trials using strategies aimed at inducing dystrophin expression in Duchenne muscular dystrophy (DMD) are underway or at advanced planning stage, including splice switching antisense oligonucleotides (AON), drugs to induce read-through of nonsense mutations and viral mediated gene therapy. In all these strategies, different dystrophin proteins, often internally deleted, are produced, similar to those found in patients with the milder DMD allelic variant, Becker muscular dystrophy (BMD). The primary biological endpoint of these trials is to induce functional dystrophin expression. A reliable and reproducible method for quantification of dystrophin protein expression at the sarcolemma is crucial to monitor the biochemical outcome of such treatments. We developed a new high throughput semi quantitative fluorescent immunofluorescence method for quantifying dystrophin expression in transverse sections of skeletal muscle. This technique is completely operator independent as it based on an automated scanning system and an image processing script developed with Definiens software. We applied this new acquisition-analysis method to quantify dystrophin and sarcolemma-related proteins using paediatric control muscles from cases without a neuromuscular disorder as well as DMD and BMD samples. The image analysis script was instructed to recognize myofibres immunostained for spectrin or laminin while dystrophin was quantified in each identified myofibre (from 2,000 to over 20,000 fibres, depending on the size of the biopsy). We were able to simultaneously extrapolate relevant parameters such as mean sarcolemmal dystrophin, mean spectrin and laminin intensity, fibre area and diameter. In this way we assessed dystrophin production in each muscle fibre in samples of DMD, BMD and controls. This new method allows the unbiased quantification of dystrophin in every myofibre within a transverse muscle section and will be of help for translational research projects as a biological outcome in clinical trials in DMD and BMD.

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More information

Accepted/In Press date: 5 March 2018
Published date: 26 March 2018
Keywords: Adolescent, Biopsy, Child, Child, Preschool, Clinical Trials as Topic, Dystrophin, Fluorescent Antibody Technique, Genetic Therapy, High-Throughput Screening Assays, Humans, Image Processing, Computer-Assisted, Laminin, Muscle Fibers, Skeletal, Muscular Dystrophy, Duchenne, Oligonucleotides, Antisense, Sarcolemma, Software, Spectrin, Journal Article, Research Support, Non-U.S. Gov't

Identifiers

Local EPrints ID: 428175
URI: http://eprints.soton.ac.uk/id/eprint/428175
ISSN: 1932-6203
PURE UUID: 735d089a-47a6-4c52-99dc-f98db6342d37

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Date deposited: 13 Feb 2019 17:30
Last modified: 16 Mar 2024 00:08

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Contributors

Author: Valentina Sardone
Author: Matthew Ellis
Author: Silvia Torelli
Author: Lucy Feng
Author: Darren Chambers
Author: Deborah Eastwood
Author: Caroline Sewry
Author: Rahul Phadke
Author: Jennifer E Morgan
Author: Francesco Muntoni

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