Cardiomyopathy-Associated Mutations in a Hotspot Region at the C-Terminal Part of Desmin Coil-2 Domain Impair the Intermediate Filament Assembly

Abstract Background The DES gene encodes the intermediate filament protein desmin, which connects different multi-protein complexes like the cardiac desmosomes and is highly important for the structural integrity of cardiomyocytes. Pathogenic DES -mutations cause filament assembly defects leading to cardiomyopathies. However, most DES -variants listed in genetic disease databases are currently classified as variants of unknown significance. Here, we functionally characterized 21 different DES -variants of unknown significance and 18 additional proline variants, localized in a highly conserved stretch at the C-terminus of the desmin coil-2 subdomain. Methods We inserted desmin variants via site-directed-mutagenesis and investigated the filament assembly in transfected cell lines and cardiomyocytes derived from induced pluripotent stem cells by confocal microscopy. In addition, we purified recombinant wild-type and mutant desmin and analyzed the filament formation by atomic force microscopy. Co-expression with wild-type desmin delivered by adeno-associated virus was used to model the heterozygous status of cardiomyopathy patients. Results Twelve DES -variants of unknown significance formed cytoplasmic aggregates, which was likewise verified by atomic force microscopy. Of note, these twelve variants disturb the filament assembly even when co-expressed with wild-type desmin. Using a proline screen, we showed that proline residues localized at nearly each of the positions in this stretch cause filament assembly defects. By modelling the tetrameric structure of desmin, we demonstrated that specific heptad positions as well as positions of intra- and intermolecular ion bridge sites are particularly susceptible mutations that promote desmin aggregation. Conclusion In summary, our study demonstrated that the highly conserved stretch at the C-terminus of the coil-2 subdomain is a hotspot region, where several pathogenic DES -mutations cause an aberrant desmin aggregation. Based on our functional data we suggest to re-classify the aggregate-forming variants as likely-pathogenic mutations rather than variants of unknown significance. Our study may have relevance for the genetic counselling of cardiomyopathy patients with similar DES -variants.