Figure 3. Histopathology.

Histopathology of patient II-1 TA biopsy (A–E, G–K, M–N, P) shows myopathic changes: fiber size variation and numerous internal nuclei, some with central pallor in H&E (A, arrowheads) and rimmed vacuoles in Herovici (B, arrowheads). Subsarcolemmal TNPO3 accumulation is observed in TNPO3 IHC staining (C, arrowheads). A fiber with small cytoplasmic bodies is seen in Gomori trichrome staining (D, black arrowhead) and a fiber with myofibrillar pathology (D, white arrowhead). In a serial section, mitochondrial NADH staining reveals an uneven staining pattern in central parts of the muscle fibers (E). Gomori trichrome of patient I-3 TA biopsy (F) shows a ragged red fiber (black arrowhead) and myofibrillar pathology (white arrowhead). Large (arrowhead) and small myotilin accumulations are observed in IHC staining (G). In IF double staining, p62 (green) and TDP-43 (red) colocalize in inclusion bodies (H, arrowheads) in the rimmed vacuolar fiber. The mitochondrial CHCHD10 shows subsarcolemmal accumulation in several muscle fibers (I). IF double staining of desmin (green) and alpha-B-crystallin (red) shows both cytoplasmic and subsarcolemmal overexpression and colocalization (J, arrowheads), as does desmin (green) and tropomyosin (red) in K. Notably, desmin (green) and tropomyosin (red) show overexpression in patient III-1 muscle at only age 16 months (L). In confocal microscopy, RBM4 (green) is often excluded from the nuclei (M, arrowheads) in the patient biopsies, whereas SRRM2 (green) in N shows strictly nuclear localization in the patient, a pattern similar to control muscles. Confocal analysis of TNPO3 shows normal nuclear localization in patient III-1 (O), but often perinuclear accumulation of TNPO3 in adult patient biopsy (P, arrowheads), which shows as a nuclear rim. (A–L), scale bar = 100 μm; (M–P), scale bar = 50 μm. IF = immunofluorescent; IHC = immunohistochemistry; TA = tibialis anterior; TNPO3 = transportin-3.