Analysis of muscle biopsies from an adult-onset patient D9. (a) H&E stained section of the first biopsy (taken 6 years prior to initiation of ERT) shows vacuolation in ~20-25% fibers (10x). The second biopsy was performed after 6 years of ERT (b-f). (b) H&E staining shows mostly vacuolated fibers (10x) (note, some of the large “holes” are likely freeze artefacts). (c) EM demonstrates the presence of autophagic buildup and “pale” areas (arrows) in the surrounding relatively well preserved fibers (transverse section). Bar: 2 μm. (d-f) Muscle fibers were stained for lysosomal marker LAMP2 (green) and autophagosomal marker LC3 (red). Nuclei are stained with Hoechst (blue). LAMP2/LC3 staining demonstrates prominent autophagic accumulation with inclusions in most fibers; these abnormalities are commonly seen in fibers with mild (d) or no (e and f) lysosomal enlargement. Prominent lysosomal enlargement is seen in occasional fibers (e; arrowheads). Bar: 10 μm.