Pathogenic effects of a novel heterozygous R350P desmin mutation on the assembly of desmin intermediate filaments in vivo and in vitro

Abstract

Mutations of the human desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities. Here, we report the pathogenic effects of a novel heterozygous R350P desmin missense mutation, which resides in the evolutionary highly conserved coil 2B domain of the alpha-helical coiled-coil desmin rod domain, on the assembly of desmin intermediate filaments (IF) in cultured cells and in vitro. By transfection experiments, we show that R350P desmin is incapable of de novo formation of a desmin IF network in vimentin-free BMGE+H, MCF7 and SW13 cells and that it disrupts the endogenous vimentin cytoskeleton in 3T3 fibroblast cells. Hence, transfected cells displayed abnormal cytoplasmic protein aggregates reminiscent of desmin-positive protein deposits seen in the immunohistochemical and ultrastructural analysis of skeletal muscle derived from the index patient of the affected family. To study the functional effects of the R350P desmin mutation at the protein level, we performed in vitro assembly studies with wild-type (WT) and mutant desmin protein. Our analysis revealed that the in vitro assembly process of R350P desmin is already disturbed at the unit length filament level and that further association reactions generate huge, tightly packed protein aggregates. On assessing the pathogenic effects of R350P desmin in various mixtures with WT desmin, we show that a ratio of 1 : 3 (R350P desmin/WT desmin) is sufficient to effectively block the normal polymerization process of desmin IFs. Our findings indicate that the heterozygous R350P desmin mutation exerts a dominant negative effect on the ordered lateral arrangement of desmin subunits. This disturbance of the lateral packing taking place in the first phase of assembly is ultimately leading to abnormal protein aggregation.

How much mutant protein is needed to cause a protein aggregate myopathy in vivo? Lessons from an exceptional desminopathy

“…In vitro assembly studies on the p.Arg350Pro (R350P) desmin mutant have established a pathogenic ratio of 1:3 of mutant versus wild-type proteins that abolished the normal filament assembly process (Bär et al, 2005a). The present study provides the first insight into the complex pattern of mutant versus wild-type desmin on the mRNA and protein levels in diseased human skeletal muscle.…”

“…1). Standard histology, indirect desmin immunofluorescence staining and ultrastructural analysis were performed as described previously (Bär et al, 2005a). A skeletal muscle biopsy from a previously reported patient with a heterozygous c.735G>C desmin mutation (Vrabie et al, 2005) was included in this study.…”

“…Nucleotide numbering refers to cDNA sequence with +1 corresponding to the A of the ATG start codon. Isolation of DNA from peripheral blood leukocytes and desmin mutation analysis of genomic DNA were performed as described previously (Bär et al, 2005a); sequencing primers are available from the authors upon request. For RT-PCR total RNA was extracted from skeletal muscle samples, cDNA was obtained using p(dN6)-oligomers and M-MLV H(-) reverse transcriptase (Promega) according to the manufactures instructions.…”

“…Insights into the molecular pathogenesis of desminopathies are derived from transfection experiments and in vitro assembly studies which indicate that the majority of desmin rod mutants i) are incapable of forming a de novo desmin intermediate filament network, ii) form abnormal intermediate filament structures, iii) induce a collapse of a pre-existing intermediate filament cytoskeleton, and iv) lead to desmin-positive protein aggregates (Bär et al, 2005a;Bär et al, 2006a;Bär et al, 2005b;Bär et al, 2006b). The ratio of mutant versus wild-type desmin protein is likely to play a critical role in the disease process.…”

156th ENMC International Workshop: Desmin and protein aggregate myopathies, 9–11 November 2007, Naarden, The Netherlands

“…In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named 'scapuloperoneal syndrome type Kaeser'. By genetic analysis of the original kindred, a heterozygous missense mutation of the desmin gene R350P was discovered cosegregating with the disorder, recently described [25]. Examination of 15 patients from five unrelated families harbouring R350P, including the original kindred described by Kaeser, showed large clinical variability, even within the same family, covering scapuloperoneal, limb girdle, and distal phenotypes with variable cardiac or respiratory involvement.…”