Combining Protein Expression and Molecular Data Improves Mutation Characterization of Dystrophinopathies

Figures

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  Figure 1. Workflow chart for all patients referring to us. All patients were first immunohistochemical analyzed using a panel of antibodies for muscle proteins such as dystrophin, utrophin, α, β, γ, sarcoglycans caveolin-3, dysferlin, merosin, and nNOS to establish the primary protein deficiency. Based on the results obtained by immunohistochemical staining, the patients were classified into four groups: (a) patients with a deficiency of dystrophin, (b) patients with a deficiency of sarcoglycans, (c) patients with a deficiency of caveolin 3, and (d) patients with all proteins present (data not shown). Patients from the group (a), (b), and (d) were further analyzed by multiplex WB to confirm the immunohistochemical result and to identify modification of calpain-3 for the others. A total of 40 unrelated male patients identified with a deficiency of dystrophin were recruited for this study. These patients were further tested for a complete molecular characterization of the DMD gene mutations.

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  Figure 2. Immunofluorescent staining of dystrophin in normal control muscle and twin sisters (#F1 and #F2). (A) Immunostaining for dystrophin with antibodies against all three dystrophin domains for twin sisters revealed a difference between them. The first sister (F1) shows a reduction/ discontinuous signal for dystrophin and overexpression of utrophin from NMJ to the whole sarcolemma while the other sister (F2) presented only a slight reduction of signal for dystrophin. Scale bar 50, μm. (B) Dystrophin WB analysis of muscle biopsy sample from both sisters revealed the same difference between sisters with reduced intensity of bands for dystrophin with (a) antibody directed against the dystrophin rod domain (DYS1) and (b) antibody directed against the dystrophin C-terminal (DYS2) for sister F1 and an almost normal expression for sister F2 compared with control muscle sample. (C) HRM analysis revealed the same sequence variant for both sisters in exon 16 in the DMD gene (red line). Also, patient #18 was identified with a variation in exon 16 (blue line) compared with normal (Gray lines). (D) A section of the sequencing electropherogram for one twin compared with control.

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  Figure 3. The spectrum of mutations identified in the DMD gene in the group of 40 unrelated Romanian patients.

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  Figure 4. Frequency of mutation per exons in the DMD gene. The exons 48 and 49 are the most involved in mutations in the DMD gene in our study lot.

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  Figure 5. Schematic representation of the distribution pattern of mutations along the DMD gene.