*3.3. Morphology of Desminopathy in Myocardial and Skeletal Muscle Samples*

An immunohistochemical examination of myocardial samples in P1–P3 and P6 showed a diffuse alteration of desmin distribution in cardiomyocytes with a formation of desmin aggregates revealing strong immunoreactivity in the cytoplasm (shown in P1, P3; Figure 4A,E). Electron microscopy of cardiomyocytes in P1, P2, and P6 revealed myofibrillar disruption, streaming Z bands, and deposits of dense, amorphous granulofilamentous material of variable size and shape (shown in P1, P2; Figure 4B,C). In addition, we constructed a set of expression plasmids for the six *DES* missense variants and transfected HT1080 as a cell model without endogenous desmin expression and iPSC-derived cardiomyocytes. These experiments revealed a severe intermediate filament formation defect for *DES*-p.(K43E) and *DES*-p.(R406W) underlining their pathogenicity. Furthermore, electron microscopy of cardiac tissue demonstrated in P2, P4, and P6 focally increased the number of mitochondria, often in clusters, with loss of mitochondrial spatial organization (P2; Figure 4D). Importantly, desmin aggregates were absent in myocardial samples of P4 both at immunohistochemical and ultrastructural analysis. An expression of desmin in myocardium (P2, P4, and P6) was also assessed by Western blot analysis. There was an obvious reduction of the signal in P4 (Figure 1B).

Samples of the skeletal muscle (P4–P5 m. soleus, P4 intercostal muscle, P6 m. deltoideus) showed different findings in P4 and P6 as compared with P5. The morphological analysis in P4 and P6 detected only mild myopathic changes. The light microscopy with hematoxylin-eosin staining showed a marked variability in fiber size and increased number of internal nuclei (P4; Figure 4F). No inclusions were observed by light microscopy. Similarly, desmin immunohistochemistry did not reveal any protein aggregates in the sarcoplasma of P4 and P6 (P4; Figure 4G). In the NADH and SDH reactions, many fibers did not possess the characteristic checkerboard pattern, and in a proportion of fibers there was increased oxidative activity at the periphery of the muscle fibers, indicating the pathological accumulation of mitochondria (P4; Figure 4H). However, no typical ragged red fibers were observed. The distribution of COX reactivity was altered similarly to a NADH/SDH pattern with very few COX-negative fibers present (P4; Figure 4I). On the other hand, the muscle biopsy in P5 showed severe myopathic changes with a large amount of fibro-fatty tissue in the interstitium of the muscle. Desmin immunohistochemistry confirmed in P5 a diffuse alteration of desmin distribution with a formation of desmin aggregates in the cytoplasm of muscle fibers.

An ultrastructural analysis of skeletal muscle biopsies revealed a focally increased number of mitochondria, often in clusters, with an altered distribution in P4 and P6 (P4; Figure 4J) however, no ultrastructural abnormality in mitochondria morphology was observed. Typical deposits of dense granulofilamentous material were absent in P4 and were not observed in P6 at the first reading. Thus the first description of the skeletal muscle biopsy in P6 led to the diagnosis of mitochondrial myopathy. Nevertheless, the second reading of the skeletal muscle biopsy performed with the knowledge of the results of genetic tests and abnormal immunostaining of desmin in myocardium discovered a focus of dense amorphous material in a single fiber at electron microscopy (not shown).

**Figure 4.** Illustration of histopathology, immunohistochemistry, and electron microscopy in individuals with the novel desmin variants. (**A**): Desmin immunohistochemistry (left ventricular myocardium, explanted heart, P1) documenting a diffuse alteration of desmin distribution with a formation of desmin aggregates revealing strong immunoreactivity in the cytoplasm. Original magnification ×400. (**B**): Electron

microscopy (left ventricular myocardium, explanted heart, P1) detects amorphous granulofilamentous material in the cytoplasm of cardiomyocytes compatible with desmin aggregates. Original magnification ×10,000. (**C**,**D**): Electron microscopy (left ventricular myocardium, explanted heart, P2). (**C**): Pathological dense granulofilamentous inclusions in the cytoplasm of cardiomyocyte. Original magnification ×12,000. (**D**): Increased number of mitochondria in cardiomyocyte, often in clusters, with altered distribution. Original magnification ×8000. (**E**): Desmin immunohistochemistry (right ventricular myocardium, endomyocardial biopsy, P3) revealed an abnormal staining of cardiomyocytes with a formation of desmin positive aggregates. Original magnification ×400. (**F**–**J**) Diagnostic skeletal muscle biopsy specimens, Soleus muscle, P4, original magnification ×400. (**F**): By light microscopy with hematoxyllin-eosin, there was a marked variability in fiber size, absent inclusions, and increased number of internal nuclei. (**G**): Desmin immunohistochemistry did not reveal any protein aggregates in the sarcoplasma. (**H**): Nicotinamide adenine dinucleotide (NADH) and succinate dehydrogenase (SDH) immunohistochemistry identified few muscle fibers with increased oxidative activity at their periphery, indicating the pathological accumulation of mitochondria. However, no typical ragged red fibers were observed. (**I**): Very few COX-negative fibers were also present. (**J**): Electron-microscopic analysis revealed increased number of mitochondria, often in clusters, with altered distribution. No accumulation of intermediate filaments was observed. Original magnification ×6000.

#### *3.4. Indications for the Pathogenicity of the Novel Desmin Variants*

A typical myocardial histopathology and ultrastructure with pathological desmin-immunoreactive aggregates strongly supported the pathogenicity of desmin variants p.(K43E), p.(S57L), and p.(A210D). In addition, the desmin filament formation experiments in transfected HT1080 and in iPSC-derived cardiomyocytes revealed an abnormal cytoplasmic aggregation of DES-p.(K43E). On the other hand, the pathogenicity of the novel desmin variant p.(Q364H) is supported mainly by decreased myocardial desmin expression and co-segregation of the above desmin variant in the family in the absence of other segregating cardiomyopathy-related genes as assessed by WES in the proband.
