DOI: 10.1161/circgen.125.005653 ISSN: 2574-8300
Cardiomyopathy-Associated Mutations in a Hotspot Region at the C-Terminal Part of Desmin Coil-2 Domain Impair the Intermediate Filament Assembly
Jonas Reckmann, Hendrik Milting, Sabrina Voß, Marco T. Radukic, Franziska Klag, Franziska Flottmann, Alexander Lütkemeyer, Joline Groß, Anna Gärtner, Sandra Landwehr, Dario Anselmetti, Annika Hoyer, Kristian M. Müller, Jan Gummert, Volker Walhorn, Andreas Brodehl
BACKGROUND:
The
DES
gene encodes the IF (intermediate filament) protein desmin, which connects different multiprotein complexes, such as 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 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. Coexpression 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 were likewise verified by atomic force microscopy. Of note, these 12 variants disturb the filament assembly even when coexpressed 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 modeling the tetrameric structure of desmin, we demonstrated that specific heptad positions, as well as positions of intramolecular and intermolecular ion bridge sites, are particularly susceptible to mutations that promote desmin aggregation.
CONCLUSIONS:
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 molecular data, we suggest reclassifying the aggregate-forming variants as likely pathogenic mutations rather than variants of unknown significance. Our study may have relevance for the genetic counseling of cardiomyopathy patients with similar
DES
variants.