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Stéphane Blot
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8982-0317
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Background
Canine models of Duchenne muscular dystrophy (DMD) are a valuable tool to evaluate potential therapies because they faithfully reproduce the human disease. Several cases of dystrophinopathies have been described in canines, but the GRMD (Golden Retriever Muscular Dystrophy) model remains the most used in preclinical studies. Here we report a new spontaneous dystrophinopathy in a Labrador retriever strain, named LRMD (Labrador Retriever Muscular Dystrophy).
Methods
A colony of LRMD dogs was established from spontaneous cases. Fourteen LRMD dogs were followed-up and compared to the GRMD standard using several functional tests. The disease causing mutation was studied by several molecular techniques and identified using RNA-sequencing.
Results
The main clinical features of the GRMD disease were found in LRMD dogs; the functional tests provided data roughly overlapping with those measured in GRMD dogs, with similar inter-individual heterogeneity. The LRMD causal mutation was shown to be a 2.2‑Mb inversion disrupting the DMD gene within intron 20 and involving the TMEM47 gene. In skeletal muscle, the Dp71 isoform was ectopically expressed, probably as a consequence of the mutation. We found no evidence of polymorphism in either of the two described modifier genes LTBP4 and Jagged1. No differences were found in Pitpna mRNA expression levels that would explain the inter-individual variability.
Conclusions
This study provides a full comparative description of a new spontaneous canine model of dystrophinopathy, found to be phenotypically equivalent to the GRMD model. We report a novel large DNA mutation within the DMD gene and provide evidence that LRMD is a relevant model to pinpoint additional DMD modifier genes.
Keywords
Dog, canine, Neuromuscular disorders, animal model, DMD, LRMD, Dystrophin, Inversion, Dp71
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- TableS1primers.docx
- TableS2DMDdogmodelsv4.xlsx
- FigureS1.tif
Pedigree of the LRMD colony. The colony was founded by crossing an affected male with his carrier sister. Ten subsequent litters were obtained, and a total of 14 LRMD dogs survived the neonatal period. The colony presented high levels of inbreeding (consanguinity coefficients min 25 % max 41 %).
- FigureS2.tif
Phenotype in the neonatal period: fulminating form and growth retardation. A: Serial sections of muscle tissue sampled on a deceased LRMD neonate stained with H&E (left) and Alizarin red S (right)., Necrosis and calcium overload were seen in the diaphragm, and to a lesser extent, in the tongue and the sartorius cranialis muscle. Other muscles, such as the triceps brachii, the tibialis cranialis or the biceps femoris, were relatively spared by this rhabdomyolysis process. The results observed are consistent with the muscle lesions observed in the GRMD neonatal fulminating form. B: Whole section view of a diaphragm biopsy taken from a deceased LRMD neonate. The strong staining with Alizarin red S indicates that there is a significant calcium overload in this muscle. C: Kaplan-Meier survival curve during the neonatal period (from birth to 2 months of age) showing that half of the LRMD dogs died within their first days of life due to neonatal fulminating forms. Only 47 % of the LRMD dogs survived to weaning (2 months of age). D: Weight curves during the neonatal period (from birth to weaning, 2 months of age) from 5 different litters, showing growth retardation in most LRMD dogs (in black) relative to healthy littermates (in grey). E: Picture of a 15-week-old LRMD dog (LRMD7, on the right) compared to a healthy littermate (carrier female) illustrating the difference in size F: Picture of a one month-old LRMD dog (LRMD13, on the right) compared to a healthy male littermate.
- FigureS3.tif
Histological findings in LRMD skeletal muscles. A: evolution of the muscle pathology with age. H&E stained biopsies x20. Illustration of the aspect of the biceps femoris at 5 different ages: 2 months, 4 months, 1 year, 2 years and 6 years. A significant number of necrosis-regeneration lesions are noted at early stages; these lesions are associated with inflammatory foci and sporadic calcifications. With time endomysial fibrosis and adiposis dominate the pathological context. B: illustration of all the elementary lesions found in LRMD muscles. Entire section and details of an extensor carpi radialis biopsy taken at the age of 4 months (LRMD7). This biopsy had an elevated pathological index (62.5 %). Abbreviations: BF: biceps femoris.
- FigureS4.tif
Immunohistochemical characterization of the expressed dystrophins in LRMD muscles. Serial sections from a biceps femoris (LRMD3), immunohistochemistry using the following antibodies: A: Dys 2 (dystrophin, C-terminal part), B: bDG (beta-dystroglycan), C : MANEX 1A (dystrophin, N-terminal part), D : MANEX 1011C (dystrophin, exons 10-11), E: Dys 1 (dystrophin, central rod domain repeats 8-10), F: MANDYS 107 (dystrophin, central rod domain repeat 15). Most of the myofibres show a marked immunoreactivity with the Dys2 (C-term) antibody, associated with a beta-dystroglycan relocalization. Some of the Dys 2 negative myofibres (asterisks) were positive for the antibodies specific for the N-terminal part of the protein (MANEX 1A, MANEX 1011C). No immunoreactivity was seen in any case when using antibodies specific for the central rod domain.
- FigureS5.tif
Correlation between Dp71 expression and histological lesions In 28 biopsies from 6 muscles sampled from 8 different LRMD dogs the proportion of Dys2+ fibres was quantified and compared to the pathological index on H&E stained serial sections. The correlation was not significant (Pearson’s R= -0.32; p =0.069).
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CURRENT STATUS: Published
View the published article at Skeletal Muscle.
Version 2
Posted 26 Jun, 2020
No community comments so far
Editorial decision: Accept
On 08 Jul, 2020
Review #1 received
Received 05 Jul, 2020
Reviewer #2 agreed
On 01 Jul, 2020
Review #2 received
Received 01 Jul, 2020
Reviewer #1 agreed
On 29 Jun, 2020
Reviewers invited
Invitations sent on 28 Jun, 2020
Editor assigned
On 25 Jun, 2020
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On 24 Jun, 2020
Editor invited
On 24 Jun, 2020
No comments provided
Editorial decision: Major revision
On 27 Mar, 2020
Review #1 received
Received 26 Mar, 2020
Review #2 received
Received 16 Mar, 2020
Reviewer #2 agreed
On 05 Mar, 2020
Reviewer #1 agreed
On 04 Mar, 2020
Editor assigned
On 02 Mar, 2020
Reviewers invited
Invitations sent on 02 Mar, 2020
Submission checks complete
On 01 Mar, 2020
Editor invited
On 01 Mar, 2020
First submitted
On 27 Feb, 2020
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Subject Areas
Animal Science
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A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
See the published version of this preprint at Skeletal Muscle.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Stéphane Blot
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8982-0317
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Skeletal Muscle.
Version 2
Posted 26 Jun, 2020
No community comments so far
Editorial decision: Accept
On 08 Jul, 2020
Review #1 received
Received 05 Jul, 2020
Reviewer #2 agreed
On 01 Jul, 2020
Review #2 received
Received 01 Jul, 2020
Reviewer #1 agreed
On 29 Jun, 2020
Reviewers invited
Invitations sent on 28 Jun, 2020
Editor assigned
On 25 Jun, 2020
Submission checks complete
On 24 Jun, 2020
Editor invited
On 24 Jun, 2020
No comments provided
Editorial decision: Major revision
On 27 Mar, 2020
Review #1 received
Received 26 Mar, 2020
Review #2 received
Received 16 Mar, 2020
Reviewer #2 agreed
On 05 Mar, 2020
Reviewer #1 agreed
On 04 Mar, 2020
Editor assigned
On 02 Mar, 2020
Reviewers invited
Invitations sent on 02 Mar, 2020
Submission checks complete
On 01 Mar, 2020
Editor invited
On 01 Mar, 2020
First submitted
On 27 Feb, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Animal Science
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Skeletal Muscle.
Background
Canine models of Duchenne muscular dystrophy (DMD) are a valuable tool to evaluate potential therapies because they faithfully reproduce the human disease. Several cases of dystrophinopathies have been described in canines, but the GRMD (Golden Retriever Muscular Dystrophy) model remains the most used in preclinical studies. Here we report a new spontaneous dystrophinopathy in a Labrador retriever strain, named LRMD (Labrador Retriever Muscular Dystrophy).
Methods
A colony of LRMD dogs was established from spontaneous cases. Fourteen LRMD dogs were followed-up and compared to the GRMD standard using several functional tests. The disease causing mutation was studied by several molecular techniques and identified using RNA-sequencing.
Results
The main clinical features of the GRMD disease were found in LRMD dogs; the functional tests provided data roughly overlapping with those measured in GRMD dogs, with similar inter-individual heterogeneity. The LRMD causal mutation was shown to be a 2.2‑Mb inversion disrupting the DMD gene within intron 20 and involving the TMEM47 gene. In skeletal muscle, the Dp71 isoform was ectopically expressed, probably as a consequence of the mutation. We found no evidence of polymorphism in either of the two described modifier genes LTBP4 and Jagged1. No differences were found in Pitpna mRNA expression levels that would explain the inter-individual variability.
Conclusions
This study provides a full comparative description of a new spontaneous canine model of dystrophinopathy, found to be phenotypically equivalent to the GRMD model. We report a novel large DNA mutation within the DMD gene and provide evidence that LRMD is a relevant model to pinpoint additional DMD modifier genes.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- TableS1primers.docx
- TableS2DMDdogmodelsv4.xlsx
- FigureS1.tif
Pedigree of the LRMD colony. The colony was founded by crossing an affected male with his carrier sister. Ten subsequent litters were obtained, and a total of 14 LRMD dogs survived the neonatal period. The colony presented high levels of inbreeding (consanguinity coefficients min 25 % max 41 %).
- FigureS2.tif
Phenotype in the neonatal period: fulminating form and growth retardation. A: Serial sections of muscle tissue sampled on a deceased LRMD neonate stained with H&E (left) and Alizarin red S (right)., Necrosis and calcium overload were seen in the diaphragm, and to a lesser extent, in the tongue and the sartorius cranialis muscle. Other muscles, such as the triceps brachii, the tibialis cranialis or the biceps femoris, were relatively spared by this rhabdomyolysis process. The results observed are consistent with the muscle lesions observed in the GRMD neonatal fulminating form. B: Whole section view of a diaphragm biopsy taken from a deceased LRMD neonate. The strong staining with Alizarin red S indicates that there is a significant calcium overload in this muscle. C: Kaplan-Meier survival curve during the neonatal period (from birth to 2 months of age) showing that half of the LRMD dogs died within their first days of life due to neonatal fulminating forms. Only 47 % of the LRMD dogs survived to weaning (2 months of age). D: Weight curves during the neonatal period (from birth to weaning, 2 months of age) from 5 different litters, showing growth retardation in most LRMD dogs (in black) relative to healthy littermates (in grey). E: Picture of a 15-week-old LRMD dog (LRMD7, on the right) compared to a healthy littermate (carrier female) illustrating the difference in size F: Picture of a one month-old LRMD dog (LRMD13, on the right) compared to a healthy male littermate.
- FigureS3.tif
Histological findings in LRMD skeletal muscles. A: evolution of the muscle pathology with age. H&E stained biopsies x20. Illustration of the aspect of the biceps femoris at 5 different ages: 2 months, 4 months, 1 year, 2 years and 6 years. A significant number of necrosis-regeneration lesions are noted at early stages; these lesions are associated with inflammatory foci and sporadic calcifications. With time endomysial fibrosis and adiposis dominate the pathological context. B: illustration of all the elementary lesions found in LRMD muscles. Entire section and details of an extensor carpi radialis biopsy taken at the age of 4 months (LRMD7). This biopsy had an elevated pathological index (62.5 %). Abbreviations: BF: biceps femoris.
- FigureS4.tif
Immunohistochemical characterization of the expressed dystrophins in LRMD muscles. Serial sections from a biceps femoris (LRMD3), immunohistochemistry using the following antibodies: A: Dys 2 (dystrophin, C-terminal part), B: bDG (beta-dystroglycan), C : MANEX 1A (dystrophin, N-terminal part), D : MANEX 1011C (dystrophin, exons 10-11), E: Dys 1 (dystrophin, central rod domain repeats 8-10), F: MANDYS 107 (dystrophin, central rod domain repeat 15). Most of the myofibres show a marked immunoreactivity with the Dys2 (C-term) antibody, associated with a beta-dystroglycan relocalization. Some of the Dys 2 negative myofibres (asterisks) were positive for the antibodies specific for the N-terminal part of the protein (MANEX 1A, MANEX 1011C). No immunoreactivity was seen in any case when using antibodies specific for the central rod domain.
- FigureS5.tif
Correlation between Dp71 expression and histological lesions In 28 biopsies from 6 muscles sampled from 8 different LRMD dogs the proportion of Dys2+ fibres was quantified and compared to the pathological index on H&E stained serial sections. The correlation was not significant (Pearson’s R= -0.32; p =0.069).
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