case_images: PMC11702382_01
case_images: Molecular characterization and muscle imaging phenotype. (a) Pedigree of the family. Clinically affected patients are shown in black. Only the proband and his brother and sister could be tested for MYOT gene analysis. (b) Muscle MRI. In the proband (patient 1), severe fatty replacement is present in small glutei in the pelvis. In the thigh, fatty replacement shows a patchy asymmetrical distribution in posterior more than in anterior compartments, with severe involvement of adductor muscles, more patchy and asymmetrical involvement in biceps femoris, semimembranosus and semitendinosus. In the legs, soleus, medial gastrocnemius and peroneal muscles are involved. In the brother (patient 2), fatty replacement is present in small glutei bilaterally and middle and large gluteus on the left side. In the thigh patchy involvement of the left vastus lateralis and adductor muscles is present, while in the legs both soleus and to a lesser extent gastrocnemius are affected. (c) IGV screenshot of the proband's long-read sequencing results of the duplicated region, including the entirety of MYOT as well as part of PKD2L2. The phased (haplotyped) alleles are shown in light blue and pink. The reads supporting the duplication (4 out of 20 and 4 out of 17 reads spanning the upstream and downstream breakpoints of the duplication, respectively) are shown in linked mode at the top of the alignment view. These supporting reads are characterized by their stretches of "rainbow" bases (arrows), which indicate that the reads, for those parts, map to another location in the genome than expected. By investigation of the sequences represented by these reads, the duplication was concluded to be in tandem. (d) Western blot analysis of MYOT protein expression of total muscle lysate of patient 1 compared to a pool of healthy controls. Myosin stained by Coomassie is the loading control. The patient and control pool were run on the same gel, the image was cropped for greater clarity.
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case_images: PMC11702382_01_ENE-32-e70029-g001.jpg
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case_images: His deceased father presented a similar gait impairment since the age of 70 (Figure 1a). Muscle MRI shows fatty replacement of small glutei and both thigh and leg muscles, with greater involvement of posterior than anterior compartments and with prominent bilateral involvement of soleus muscles in both patients (Figure 1b). This pattern is overall similar in both patients, but more limited and more asymmetrical in the older subjectively asymptomatic brother (Figure 1b). In order to define the orientation of the duplication, long-read PacBio HiFi sequencing was performed proving the duplication to be in tandem (Figure 1c) and further refining the breakpoints (chr5: 137,832,296-137,897,203dup). Compared to a pooled group of healthy controls, MYOT expression seemed to increase (Figure 1d).
case_images: Myopathological findings. (a) Light microscopy. Hematoxylin and eosin (H&E) staining shows increased fiber size variability with many hypertrophic fibers and some atrophic fibers, sometimes with an angular appearance. Some central nuclei are present and connective tissue content is increased. Cytosolic vacuoles, mostly non-rimmed are indicated by black arrows. Basophilic material is evident in several fibers (white arrows). Similar findings are evident with Gomori trichrome staining (GT). NADH-tetrazolium histoenzymatic reaction (NADH) shows abnormal myofibrillar network organization in both cases, with areas of variable size lacking reaction with core-like lesions, and focal areas corresponding to the amorphous material indicated above showing intense reaction. Staining with anti-myotilin antibody shows intense reaction of the protein deposits above described (MYOT). Strong reaction with anti-p62 antibody in some muscle fibers. (b) Quantification of atrophic and hypertrophic muscle fibers. Atrophy factor is 274 for case 1 (n = 164 fibers analyzed) and 172 for case 2 (n = 533 fibers analyzed); hypertrophy factor was abnormally increased to 1884 for case 1 and 536 for case 2.
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case_images: PMC11702382_01_ENE-32-e70029-g003.jpg
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case_images: Light microscopy examination of the muscle biopsy of both patients showed the presence of central nuclei in some fibers, increased fiber size variability with the coexistence of hypertrophic muscle fibers and atrophic fibers, increased collagen tissue, myofibrillar disarray, large cytoplasmic rimmed and non-rimmed vacuoles, and amorphous protein aggregates (Figure 2a). Protein aggregates were weakly positive for Desmin but strongly reactive with anti-Myotilin antibody (Figure 2a). Moreover, protein aggregates showed p62 (Figure 2a) and partially by anti-TDP43 antibody immunoreactivity. Both hypotrophic and hypertrophic fibers were present in both cases; however, hypertrophic fibers were more frequent than atrophic fibers (Figure 2b).
case_images: Electron microscopy. Destruction of myofibrillar organization with accumulation of material derived from Z-disk (left). Severe autophagic abnormalities and accumulation of degraded cytosolic material (right).
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case_images: Electron microscopy examination showed areas of focal myofibrillar disarray, signs of lysosomal/autophagic abnormalities including rimmed vacuoles (Figure 3) and lipofuscin, and occasional tubular aggregates.