Functional recovery of a novel knockin mouse model of dysferlinopathy by readthrough of nonsense mutation

Figures

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  Figure 1. Structure of dqxDysf−/− knockin mice The nonsense mutation c.2494C>T, its exon, and each 300 bp of flanking introns from human dysferlin sequence were introduced to the homologous region of mouse dysferlin. The chromatogram reads the sequence around the introduced nonsense mutation (shaded in blue). Reference human and mouse sequences are aligned below the chromatogram.

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  Figure 2. Histopathology and physiology of untreated 30-week-old dqxDysf−/− mice (A) Hematoxylin and eosin staining (top row, original magnification ×400) and immunofluorescence of dysferlin (bottom row, TA; third row, quadriceps; green, dysferlin; blue, DAPI; magnification ×400) of C57BL/6 (left column) and untreated dqxDysf−/− (right column) mice. (B) Grip strength. (C) Latency to fall from rotarod. (D) Running distance on treadmill. (E) Specific tetanic tension. (F) Cross-sectional area of EDL muscles. (G) Absolute force on tetanic contraction. (H) Eccentric force measurements. (B–H) n = 4–5 for each group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared with C57BL/6; error bar, standard error.

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  Figure 3. Dose response of dqxDysf−/− mice to 2 weeks of ataluren treatment (A) Western blot of dysferlin and quantification (∗∗∗p < 0.001 between C57BL/6 and untreated dqxDysf−/−; ##p < 0.01 between untreated dqxDysf−/− and dqxDysf−/− treated with 0.9 mg/mL ataluren; error bar, standard error; n = 3–4 for each group). (B) Grip strength. (C) Latency to fall from rotarod. (D) Running distance on treadmill. (B–D) Blank bar, before treatment at the age of 9 weeks; solid bar, after 2 weeks of ataluren treatment (∗p < 0.05, ∗∗∗p < 0.001; error bar, standard error; n = 6–7 for each group). (E) Eccentric contraction force in percentage of the initial force (open circle, untreated C57BL/6; open square, C57BL/6 with ataluren treatment; solid diamond, untreated dqxDysf−/−, n = 10; solid square, dqxDysf−/− treated with 0.3 mg/mL ataluren, n = 5; solid triangle, dqxDysf−/− treated with 0.9 mg/mL ataluren, n = 11; solid circle, dqxDysf−/− treated with 3.0 mg/mL ataluren, n = 5; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with untreated dqxDysf−/− mice; error bar, standard error).

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  Figure 4. Serial response of dqxDysf−/− and A/J mice treated with 0.9 mg/mL oral ataluren (A) Western blot of dysferlin and quantification (∗∗∗p < 0.0001 between C57BL/6 and untreated dqxDysf−/−; #p < 0.05, ##p < 0.01 compared with untreated dqxDysf−/−). (B) Grip strength (n = 3–8 for each group). (C) Latency to fall from rotarod (n = 3–6 for each group). (D) Running distance on treadmill (n = 3–5 for each group). (B–D) Serial measurement of physical function from 0 to 8 weeks of ataluren treatment (blank bar, untreated dqxDysf−/−; solid bar, dqxDysf−/− treated with 0.9 mg/mL ataluren; gray bar, A/J mice treated with 0.9 mg/mL ataluren; ∗p < 0.05, ∗∗p < 0.01 compared with the baseline of each mouse group; error bar, standard error). (E) Eccentric contraction force of dqxDysf−/− after ataluren treatment, in percentage of the initial force (diamond, baseline; square, 1 week of treatment; triangle, 2 weeks of treatment; circle, 4 weeks of treatment; asterisk, 8 weeks of treatment; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with untreated dqxDysf−/− mice; error bar, standard error).

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  Figure 5. Histopathology of dqxDysf−/− mice and expression of dysferlin transcript after 2 weeks of 0.9 mg/mL ataluren treatment (A) Hematoxylin and eosin staining (top row, original magnification ×400) and immunofluorescence of dysferlin (middle row; green, dysferlin; blue, DAPI; original magnification ×400) of TA muscle from C57BL/6 (left column), untreated dqxDysf−/− (middle column), and ataluren-treated dqxDysf−/− (right column) mice. (B) Proportion of IgG-positive muscle fibers was more frequent in untreated dqxDysf−/− mice but reduced after ataluren treatment (∗∗p < 0.01 between C57BL/6 and dqxDysf−/− mice; #p < 0.05 between treated and untreated dqxDysf−/− mice; error bar, standard error; n = 5 for C57BL/6, n = 3 for each treated and untreated dqxDysf−/− group). (C) Fibers with central nucleus were more frequent in dqxDysf−/− mice (∗p < 0.05 compared with C57BL/6) regardless of ataluren treatment (error bar, standard error; n = 5–7 for each group). (D) The expression of dysferlin transcript is reduced in the untreated mouse (∗p < 0.05), which was reverted insignificantly by ataluren treatment (error bar, standard error; n = 4–5 for each group). (E) Serum creatine kinase activity of…