*3.1. Description of DES Variants and Their Segregation in Families*

Probably disease-causing *DES* variants in heterozygous constitution were identified in six index cases (1.8%). Two missense variants were identified within the non-helical head (amino-teminal) domain of desmin, i.e., in P1 with biventricular form of ACM (NM\_001927.3: c.127A > G; NP\_001918.3: p.(K43E)) and in P2 with DCM (NM\_001927.3: c.170C > T; NP\_001918.3: p.(S57L)) (Figure 1, Tables S2 and S3). Both of them were previously reported in Clinvar database as variants of uncertain significance. In addition, we analyzed the desmin filament formation in transfected HT1080 and in iPSC-derived cardiomyocytes, revealing an abnormal cytoplasmic aggregation in the DES-p.(K43E) variant and known pathogenic DES-p.(R406W) variant (Figure 2). Two novel variants were found in the highly conserved central α-helical rod domain, i.e., in P3 with familial DCM located in the 1B helical domain (NM\_001927.3:c.629C > A; NP\_001918.3: p.(A210D)) and in P4 with familial LVNC in combination with skeletal myopathy located in the 2B helical domain (NM\_001927.3: c.1092G>T; NP\_001918.3: p.(Q364H)) (Figure 1, Tables S2 and S3). The findings in the biopsies are described below. The remaining two probands had the following known *DES* pathogenic variants: P5 with ACM and skeletal myopathy in the 2B helical domain (NM\_001927.3: c.1216C>T; NP\_001918.3: p.(R406W); HGMD database ((http://www.hgmd.cf.ac.uk/ac/index.php) CM000368) [6] and P6 with RCM and skeletal myopathy within the non-helical tail (carboxy-terminal) domain (NM\_001927.3: c.1360C > T; NP\_001918.3: p.(R454W); HGMD CM071700) [26] (Figure 1, Tables S2 and S3). Table S4 contains lists of rare genetic variants of further cardiomyopathy associated genes in all probands (frequency in Exac database less than 0.00001). Just the variant of *MYH7* (NM\_000257.3) c.4679G > C, p.(Arg1560Pro) in proband 4 could be relevant in a patient with LVNC. However, it was not present in other members of the family tested (II 1, 3, 4; III 1, 2) (Figure 1) and did not co-segregate with the phenotype of LVNC. Importantly, any pathogenic variants in mitochondrial proteins coded by nuclear DNA or mitochondrial DNA were not found in these six probands.

Figure 1 illustrates the segregation of *DES* variants in families. Family history or clinical screening revealed a similar cardiac disease in a first-degree relative in P3, P4, and P6 segregating with occurrence of *DES* variants (Figure 1, Table S2). In the father of P2, heterozygous for *DES* p.(S57L) variant, we observed an incomplete penetrance of the disease with atrioventricular block grade I, right bundle branch block, left anterior hemiblock, normal echocardiography, and a mild elevation of creatinine phosphokinase of 6.1 μkat/l (upper limit of normal 2.3 μkat/L) without clinical signs of myopathy. Cases P1 and P5 seemed to be sporadic (segregation assessed in mother and sister of P1, and three siblings of P5).

**Figure 2.** Cell transfection experiments of transfected HT1080 cells and iPSC-derived cardiomyocytes. Mutant and wild-type desmin was expressed with the red fluorescent protein-tag mRuby at the C-terminus (shown in red). Representative confocal images are shown (**A**). In case of HT1080 cells, F-actin was stained using phalloidin-Alexa488 (shown in green) and the nuclei were stained using 4 ,6-diamidin-2-phenylindole (shown in blue). In case of iPSC-cardiomyocytes, the cardiomyocyte marker α-actinin was stained using antibodies (shown in green) and the nuclei were stained with DAPI (shown in blue). Scale bars represent 10 μm. (**B**) Quantification of aggregate formation was performed in three to four independent transfection experiments of HT1080 cells. \* *p* < 0.05 and \*\* *p* < 0.01. The variant DES-p.(Y122C) was used as a positive control forming abnormal cytoplasmic aggregates [20].

#### *3.2. Phenotypes of Desminopathy*

The initial clinical presentation included cardiac arrest due to ventricular tachycardia in the 2nd decennium (P1), complete atrioventricular blockade in the 3rd decennium (P5, P6), and heart failure in the 3rd to 5th decennium (P2, P3, and P4). Skeletal myopathy and dysfunction of bulbar muscles became apparent during the 4th to 6th decennium in cases 4–6 (Table S2 and S3). An unusual clinical presentation had proband 2. A young female presented with acute heart failure, a severe systolic dysfunction of mildly dilated left ventricle, persistent elevation of troponin T (> 10 times the upper limit of normal) (Table S3), and an extensive mid-wall late gadolinium enhancement of the septum and anterior wall of the left ventricle (Figure 3). These findings mimicked inflammatory cardiomyopathy however, there was no sign of inflammation as assessed by endomyocardial biopsy. Inflammation was absent also in her heart explanted during transplantation three years later. The arrhythmogenic left ventricular cardiomyopathy was considered as an alternative diagnosis in P2. However, her electrocardiogram was unremarkable and ventricular extrasystoles were infrequent. Proband 4 presented with a unique phenotype of LVNC. Magnetic resonance imaging (Figure 3) confirmed the diagnosis of LVNC with a percentage of non-compaction within the total left ventricular mass of 43%. Proband 6 was incorrectly diagnosed with mitochondrial disease based on skeletal muscle biopsy performed several years ago. This diagnosis was reclassified to desminopathy after the identification of known pathogenic desmin mutation (p.(R454W)) and morphological analysis of myocardial samples from the explanted heart. Table S3 illustrates additional clinical and laboratory data of the study group including echocardiography. During a median follow-up of 56 months (31–182), five probands (83%) developed end-stage heart failure.

**Figure 3.** Cardiovascular magnetic resonance imaging in patients with left ventricular non-compaction cardiomyopathy (P4) and dilated cardiomyopathy with an extensive late gadolinium enhancement (P2). (**A**,**B**): Four chamber and short axis views of left ventricular non-compaction cardiomyopathy in P4. (**C**,**D**): Two chamber long axis and four chamber views of an extensive late gadolinium enhancement in the ventricular septum and left ventricular anterior wall mimicking inflammatory cardiomyopathy in P2.
