Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies

Fig. 1. Overview of the ACTA1 mutations. A Schematic representation of the seven ACTA1 exons and position of the mutations in the ten novel patients (bold) and in the previously reported cases (light). B Resolved 3D protein structure of an αskm-actin monomer with position of the missense mutations highlighted in red

Fig. 2. Muscle histology. Gomori trichrome staining on transverse muscle sections evidenced fibre size variability throughout the samples. The images also show an increased interstitial connective tissue in patients 4, 5, 7, 10, and 13 (B, C, E, H, I), abundant cytoplasmic rods in patients 2, 6, 8, 9, and 13 (A, D, F, G, I), cytoplasmic bodies (arrows) in patients 4, 5, 8, and 9 (B, C, F, G), major accumulations of thin filaments in patient 13 (I), as well as intranuclear rods in patient 7 (arrow, highlighted in the inset). In patient 10 (H), no rods or cytoplasmic bodies were observed

Fig. 3. Cytoplasmic and intranuclear rods by electron microscopy. Ultrastructural investigations of the muscle biopsy specimens illustrate cytoplasmic rods with the typical lattice structure in patients 4, 6, and 9 (D, E, F), thin filaments emanating from thickened Z-lines in patients 6 and 9 (arrow) (E, F), numerous intranuclear rods in patients 7 (arrow) and 12 (A, B, C), accumulations of thin filaments in patients 7 (arrow) and 13 (H, I), and major myofibre disorganization in patients 4, 7, and 9 (D, G, H). Note the square shape of the intranuclear rods in patient 12 (B, C)

Fig. 4. Cytoplasmic bodies by electron microscopy. Cytoplasmic bodies showing the characteristic halo of radiating filaments as illustrated on muscle sections from patients 4, 5, 8, and 9 (arrows, A–D). The patients also displayed fibre size variability, myofibre disorganization and endomysial fibrosis. The enlargement of the perinuclear space is indicated in patient 9 (arrow, D)

Fig. 5. Ultrastructure of the nuclei. Electron microscopy detected nuclear anomalies in the muscle samples including intranuclear rods in patients 5 and 7 (B, C), and especially a significant enlargement of the perinuclear space scaling up to 165 nm in patient 5 (B), 195 nm in patient 1 (A), 217 nm in patient 4 (E), 230 nm in patient 11 (F), 390 nm in patient 7 (D), and 1230 nm in patient 6 (C) as indicated by the red bars. Patient 1 additionally displayed dense heterochromatic areas (A)

Fig. 6. Ultrastructural endplates and membrane anomalies. Electron microscopy uncovered an abnormal organization of the skeletal muscle basal membrane in atrophic fibres showing ripples of excessive membrane projecting into the interstitium in patients 8 and 11 (arrows, B, D), and depleted postsynaptic membrane fold of the neuromuscular junctions in patients 5 and 11 (A, C, arrows)

Fig. 7. Immunofluorescence of nuclei. Representative images of muscle samples showing an abnormal nuclear shape in patients 6 and 7 compared with the age-matched control as illustrated by the lamin A/C, Nesprin-1, and Nesprin-2 signals. Laminin indicates the basal lamina. The images correspond to a single z-plane after super-resolution of confocal acquisition

Fig. 8. Cardiac α-actin immunofluorescence. (A) Cardiac α-actin signals (Magenta) on muscle sections from patients and age-matched controls. Laminin appears in Cyan. Scale bar:100 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu $$\end{document}μm (B) Normal distribution of each fibre intensity for cardiac actin demonstrates an increased cardiac α-actin level in patients 6 and 7 compared with the other patients and the controls. The number of analysed fibres is indicated for each patient. (C) Western blot on muscle extracts shows intense cardiac α-actin bands in patients 7 and 8, and a lower expression in patient 2 and in the controls. Ponceau red served as loading control