Defining Haplosufficiency in Autosomal Recessive Limb-Girdle Muscular Dystrophy Using Molecular Markers in Disease Carriers

Figures

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  Figure 1. LGMDR1 Carriers Have Reduced CAPN3 Gene/Protein Expression (A) Venn-diagram highlighting number of DEGs in common between 3 contrasts: controls vs carriers, controls vs LGMDR1, and carriers vs LGMDR1. (B) Heatmap ranking the top 100 DEGs comparing controls vs LGMDR1. DEGs are defined as genes with adj. p value <0.05 and log2FoldChange >1. (C) CAPN3 normalized counts gleaned from RNA-seq differential expression analysis. (D) Immunoblot depicting full-length (94 kDa) and autolysis (55–60 kDa) CAPN3 products. (E) Quantification of relative total calpain-3 expression (94 kDa +55–60 kDa) for each sample across 3 technical replicates. Protein quantification was normalized to total protein yielded from stain-free protein blot. Ordinary one-way ANOVA with Tukey correction. *p < 0.05, **p < 0.01, **p < 0.001. All other comparisons are not statistically significant. LGMD = limb-girdle muscular dystrophy.

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  Figure 2. LGMDR9 Carriers Have FKRP Gene/Protein Expression Similar to Controls (A) Venn-diagram highlighting number of DEGs in common between 3 contrasts: controls vs carriers, controls vs LGMDR9, and carriers vs LGMDR9. (B) Heatmap ranking the top 100 DEGs comparing controls vs LGMDR9. DEGs are defined as genes with adj. p value <0.05 and log2FoldChange >1. (C) FKRP normalized counts gleaned from RNA-seq analysis. (D and E) Immunoblotting detecting FKRP protein. FKRP protein quantification was normalized to total protein yielded from stain-free protein blot. Ordinary one-way ANOVA with Tukey correction. *p < 0.05, **p < 0.01, **p < 0.001. All other comparisons are not statistically significant. LGMD = limb-girdle muscular dystrophy.

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  Figure 3. LGMDR9 Carriers Exhibit Decreased Matriglycan and ∼50% Laminin-Binding Ability (A–D) Immunoblot of core dystroglycan (AF6868 antibody) displaying higher (150 kDa) and lower (100 kDa) MW α-dystroglycan as well as β -dystroglycan. Total core a-dystroglycan represents 100 + 150 kDa bands. (E and F) IIH6 banding determining extent of matriglycan expression. (G and H) Laminin-overlay assay displaying sample ability to bind to laminin antigen (Engelbreth-Holm-Swarm). All sample measurements are normalized to total protein yielded from stain-free protein blot. Ordinary one-way ANOVA with Tukey's correction. *p < 0.05, **p < 0.01, **p < 0.001. All other comparisons are not significant. LGMD = limb-girdle muscular dystrophy.

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  Figure 4. LGMDR3 Carriers Exhibit α and β Sarcoglycan Levels Similar to Controls (A) Venn-diagram highlighting number of DEGs in common between 3 contrasts: controls vs carriers, controls vs LGMDR3, and carriers vs LGMDR3. (B) Heatmap ranking the top 100 DEGs comparing controls vs LGMDR3. DEGs are defined as genes with adj. p value <0.05 and log2FoldChange >1. (C) SGCA normalized counts gleaned from RNA-seq analysis. (D–F) Immunoblot depicting SGCA, SGCB expression averaged for each sample across 3 technical replicates. All sample measurements are normalized to total protein yielded from stain-free protein blot. Ordinary one-way ANOVA with Tukey correction. *p < 0.05, **p < 0.01, **p < 0.001. All other comparisons unless indicated on plot are not significant. LGMD = limb-girdle muscular dystrophy.