*2.2. Genetic Analysis and Detection of Variants*

To detect causal genetic variants, WES was performed according to internationally accepted guidelines [18]. Full technical details are provided in the Supplementary Materials. The criteria for classifying variants as putative disease-causing variants included their rare occurrence (≤0.05% among control samples), changes in predicted amino acid sequences, conservation across different species (http://www.ncbi.nlm.nih.gov/BLAST/), segregation within the family, and previously reported pathogenicity in databases.

Exons with identified variants of the *DES* gene were PCR amplified (Table S1) from genomic DNA of all available individuals from the analyzed families and sequenced using the version 3.1 Dye Terminator cycle sequencing kit with electrophoresis on an ABI 3500XL Avant Genetic Analyzer (both ThermoFisher Scientific; Waltham, MA, USA). Data were analyzed using Sequencing Analysis software version 6.0 (both ThermoFisher Scientific; USA) and the segregation of the candidate *DES* variants with the phenotype was evaluated.

## *2.3. In Vitro Analysis of DES Variants*

As many but not all pathogenic *DES* variants cause an abnormal cytoplasmic desmin aggregation, we constructed for the identified*DES* variants expression plasmids by site-directed mutagenesis (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. Desmin encoding parts of all plasmids were verified by Sanger sequencing (Macrogen, Amsterdam, Netherlands). The plasmid pmRuby-N1-DES and pmRuby-N1-DES-p.(Y122C) have been previously described [19,20]. Previously reported variant DES-p.(Y122C) was used as a positive control forming abnormal cytoplasmic aggregates [20]. HT1080 cells, which do not express endogenous desmin and cardiomyocytes derived from human induced pluripotent stem cells (iPSC) (NP00040-8) were transfected using Lipofectamin 3000 (ThermoFisher Scientific) or nucleofection using the 4D Nucleofector (Lonza, Cologne, Germany) in combination with the P3 Primary Cell 4D Nucleofector Kit according to the manufacturer's instructions. The differentiation of hiPSCs has been previously described [21]. Transfected HT1080 cells were fixed using 4% paraformaldehyde, permeabilized using 0.05% Triton X100, and stained with phalloidin conjugated with Alexa-488. Transfected hiPSC-derived cardiomyocytes were stained with primary antibodies against the Z-band protein α-actinin as a cardiomyocytes specific marker (Sigma-Aldrich, Missouri, MO, USA, #A7732) in combination with secondary antibodies conjugated to Alexa-488 (ThermoFisher). Confocal microscopy was performed as previously described [22].

## *2.4. Statistical Analysis of Aggregate Formation*

A total of 3 to 4 independent transfection experiments were analyzed by counting the number of aggregate forming cells. Non-parametric Kruskal–Wallis for multiple comparison was performed using GraphPad Prism version 8.3.0 for Windows (GraphPad Software, San Diego, CA, USA). *p*-values <0.05 were considered as significant.
