Molecular signatures of inherited and acquired sporadic late onset nemaline myopathies

Figures

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  Fig. 1. Quantitative myopathological analysis of inherited and sporadic late-onset nemaline myopathies (A) Unbiased quantitative myopathological analysis was performed by systematic random sampling of digitized images of muscle sections. The distribution of nemaline rods in muscle fibers was categorized as (B) subsarcolemmal aggregates, (C) central aggregates, or (D) diffuse. (E) In some inherited nemaline myopathy samples, > 90% of fibers contained rods. (F) Atrophic fibers filled with rods, (G) necrotic fibers (*), and increased endomysial connective tissue (arrow) were seen almost exclusively in sporadic late-onset nemaline myopathy. (H) In inherited nemaline myopathy (iNM), fibers containing nemaline rods were more frequent than in sporadic late onset nemaline myopathy (SLONM). Bars represent mean ± standard deviation. (I) The diameters of fibers containing rods were larger in iNM than in SLONM. Lines represent the mean of each group. (J) In iNM, rods were most often found in aggregates, whereas they were diffusely distributed in SLONM. (K) Necrotic fibers, regenerating fibers, atrophic fibers filled with rods, and increased endomysial connective tissue were all more common in SLONM.…

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  Fig. 2. Proteomic analysis of inherited and sporadic late-onset nemaline myopathies after laser capture microdissection (A) Nemaline rod and rod-free areas from sporadic late onset nemaline myopathy (SLONM) and inherited nemaline myopathy (iNM) samples were subjected to laser capture microdissection and subsequently processed for proteomic analysis. (B) Volcano plots demonstrate differentially expressed proteins between rod and rod-free areas, as well as between SLONM and iNM samples. The left panel depicts protein abundance in nemaline rod areas relative to rod-free areas across all samples, whereas the center and right panels depict protein abundance in SLONM relative to iNM in rod and rod-free areas, respectively. (C) Comparison of the rod areas of SLONM and iNM showed enrichment of targets corresponding to several immune, structural, metabolic, and cellular pathways

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  Fig. 3. Transcriptomic analysis of inherited and sporadic late-onset nemaline myopathies (A) Bulk RNA sequencing was performed from sporadic late onset nemaline myopathy (SLONM) and inherited nemaline myopathy (iNM) muscle samples. (B) Transcriptomic analysis demonstrated 32 differentially expressed genes between the two disorders. (C) A heat map demonstrates the differential transcriptomic profiles of iNM and SLONM patient samples. (D) Principal component analysis showed differential clustering of iNM and SLONM transcriptomic profiles. (E) Ingenuity Pathway Analysis identified several pathways differentially expressed between the two disorders, the top 10 of which are illustrated here. (F) Differentially expressed genes in these pathways included several sarcomeric proteins, some of which were represented in multiple pathways. Bars represent mean ± SEM

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  Fig. 4. Analysis of immunoglobulin expression and autoimmune markers in inherited and sporadic late-onset nemaline myopathies (A) Proteomic analysis showed an increased abundance of IgG heavy chains and both kappa and lambda light chains in nemaline rod areas in sporadic late-onset nemaline myopathy (SLONM) relative to inherited nemaline myopathies (iNM). There was also a trend towards an increased abundance of immunoglobulin chains in the nemaline rod areas of SLONM relative to rod-free areas. Striped bars indicate immunoglobulin chains that were only detected in one of the groups of samples compared *p < 0.05. (B) Immunofluorescent staining demonstrated accumulation of kappa light chains in atrophic fibers in 38% of SLONM biopsies (bottom row), but not in iNM (top row). Scale bar = 400 µm. (C) Immunoglobulin genes were also differentially expressed between SLONM and iNM. (D) Immunostaining for Major Histocompatibility Antigen-I (MHC-I) showed reactivity in SLONM biopsies, occurring predominantly in atrophic fibers (first panel, SLONM with monoclonal gammopathy; second panel, SLONM without monoclonal gammopathy). Biopsies from patients with inherited nemaline myopathy showed no MHC-I…