Fig 2: Exon 75s created by cryptic splice site activation. A. Schematic illustration of part of the genomic structure of the DMD gene. Authentic splicing proceeds from exon 74 to exon 75 (diagonal line), whereas novel alternative splicing proceeds from exon 74 to the cryptic splice acceptor site (bold diagonal line). Boxes and lines indicate exons and introns, respectively. Partial nucleotide sequences of exons and introns are shown as upper- and lower-case letters, respectively. The branch point sequence is underlined. Numbers under the schema indicate probability scores for splice acceptor sites and branch points. B. Nucleotide sequence of exon 75s (top) and its translated amino acid sequence (bottom). Codon 42 of exon 75s is a stop codon.

Fig 3: Overview of Dmd gene mutations in IF rats. (A) Sequence of the Dmd gene of IF rats. Arrows indicate the location of primers used in PCR. (B) Sequence of mutated Dmd mRNA in IF rats. Arrows indicate the location of primers used. (C) Genomic PCR results for the Dmd gene in wild-type (WT) and IF rats. Note the absence of PCR product in IF when primers #1R or #2F, the corresponding sequences of which are lacking in the Dmd gene, were used. Also note the absence of PCR product in wild type when the primer set #1F and #2R was used owing to the region being too large to amplify. (D) RT-PCR results for Dmd cDNA in wild-type and IF rats. Note the absence of PCR product in IF when primer #1F, the corresponding sequence of which is lacking in Dmd mRNA, was used. (E) The molecular structure of full-length dp427 (upper) and truncated dp427 (below). ABD1, actin-binding domain 1; CH, calponin homology domain; CR, Cysteine-rich domain; C-term, C-terminal domain; H, Hinge; R, spectrin-like repeat. The black arrowhead indicates the mutation site.

Fig 4: Results of the analysis of the DMDMex dataset Principal component analysis of the DMDMex RNA‐seq counts.Volcano plot for the F test on differential expression between healthy individuals and DMD patients.Scatter plot showing the group effect at baseline (x‐axis) and the effect of the interaction between age and group (y‐axis). Black dots represent non‐differentially expressed genes, while blue dots represent differentially expressed genes.Venn diagram showing the overlap between the genes identified as differentially expressed in DMD patients in Wong et al (2009), in Liu et al (2015) and in this study.Volcano plot for the F test on differential expression between treated and untreated DMD patients.Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al (2015).Scatter plot showing genes for which a significant effect of treatment with steroids. The group effects at baseline are plotted on the x‐axis, while the effects of steroids are plotted on the y‐axis.Heatmap obtained by Ingenuity Pathway Analysis comparing the pathways affected in DMD patients to the pathways affected at different time points in mouse.Correlation circle representing the correlation between physical tests and their first two principal components.Scatter plot showing the relationship between the first principal component of body measurements and the expression levels of LAPTM4B.

Fig 5: Progressive exacerbation of muscular dystrophy in DMD rats. (a) Representative images of 6-month-old WT (left panel) and DMD rats (right panel). (b) Body weight comparison of WT and DMD rats of 1–9 months of age (n = 6). (c) Quantification of maximum muscle strength by grip test at the indicated ages of WT and DMD rats (2 months: WT: n = 7, DMD: n = 6, 4 months: WT: n = 7, DMD: n = 7, 6 months: WT: n = 8, DMD: n = 12, 8 months: WT: n = 4, DMD: n = 7, 10 months: WT: n = 11, DMD: n = 21). (d) Representative HE stains of TA muscle sections from 6-month-old WT rats and 1- to 10-month-old DMD rats. Scale bar = 100 μm. The following symbols, ψ, *, #, and ζ, indicate inflammatory cell infiltration, necrotic myofibres, fibrosis, and adipogenesis, respectively. (e) Immunoblotting analysis of perilipin expression in WT and DMD rats. Full-length blots are presented in Supplementary Figure 6a. (f) Quantification of perilipin protein expression (n = 6, each). (g) Representative Masson Trichrome stains of TA muscle sections from 1- to 10-month-old WT and DMD rats. Scale bar = 100 μm. (h) Quantification of Masson Trichrome staining positive area per section (n = 6, each). (i) Immunohistochemical analysis of eMHC in TA muscle sections from WT and DMD rats. Scale bar = 100 μm. (j) Quantification of eMHC positive fibres per section (n = 6, each). (k,l) Quantification of (k) Pax7+ cells and (l) MyoD+ cells of skeletal muscle primary cells from WT and DMD rats (n = 6, each). Data are expressed as mean ± SEM except for (b), which is expressed as mean ± SD, and were compared by Tukey Kramer’s test. Different letters indicate statistically significant differences (p < 0.05). Progressive decrease of both Pax7+ and MyoD+ cells was observed in DMD rats. Significant decrease of the number of MyoD+ cells was observed in DMD rats from 1 month compared to WT, while from 3 months about the number of Pax7+ cells. For (c), (h), (k) and (l), the result of statistical comparison only between the genotypes at each indicated ages was displayed. When a significant age-related difference was observed by the Tukey–Kramer’s test, the † mark was added beside the legend of the graph. *p < 0.05. **p < 0.01. ***p < 0.001. N.D. not detected.