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

Abstract

Myofibrillar myopathies are caused by mutations in desmin, αB-crystallin, myotilin, ZASP, and filamin C genes. Since the vast majority of myofibrillar myopathy causing mutations are heterozygous single amino acid substitutions or small in-frame deletions, the pathogenic role of mutant versus wild-type protein cannot be assessed in human skeletal muscle by standard immunodetection techniques. We report on an exceptional desminopathy due to a heterozygous c.735G>C mutation. Immunoblotting detected full-length 53 kDa desmin and a truncated 50 kDa variant in skeletal muscle from three affected patients of two different families. RT-PCR identified three desmin mRNA species encoding for wild-type and two mutant proteins, p.Glu245Asp and p.Asp214_Glu245del. Since previous functional studies on the p.Glu245Asp mutant showed biological properties identical to wild-type desmin, the truncated p.Asp214_Glu245del desmin is the diseasecausing mutant. Semiquantitative RT-PCR established a fraction of the truncated desmin mRNA species in a range from 24% to 37%. Initial quantification of corresponding desmin proteins in the muscle biopsy of the index patient of one family indicated a fraction of only 10% of the truncated species. However, serial analyses of different sections from each muscle biopsy revealed a high intraand interindividual variability of the truncated desmin protein level within a range from 5% to 43%. Desmin assembly studies in vitro have established clear-cut pathogenic ratios of mutant versus wildtype proteins. However, our findings point out a far more complex situation in human skeletal muscle. The heterogeneously distributed mutation load within and between individual specimens, which reflects local differences in the expression and/or turnover of the mutant protein in different areas containing multiple myonuclear domains, renders it impossible to define an exact pathogenic threshold of a specific mutant in vivo.

The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments

“…The presence of cytoplamic aggregates is a key feature in human desminopathy and recently it was reported that the aggregates contain both mutant and WT desmin (Clemen et al, 2009). Similarly, the aggregates formed as a result of expressing desmin K190A, although not as prevalent, resembled the aggregates observed in desminopathy muscle biopsies, which contain desmin and other sarcomere-associated proteins (Schroder et al, 2007).…”

Mutant desmin substantially perturbs mitochondrial morphology, function and maintenance in skeletal muscle tissue

“…These deletions were observed in one previously reported patient harboring the heterozygous E245D/D214_E245del (c.735G>C) desmin mutation (Fig. 6a, fourth column), who underwent muscle biopsy at the age of 38 years [14], and a second patient with a R350P (c.1049G>C) desmin mutation. Here, it is noteworthy that the R350P patient with the large-scale mtDNA deletions (father; 52 years of age at biopsy; Fig.…”

“…In our electron microscopic analysis of skeletal muscle tissue from desminopathy patients, we discovered that in the case of the heterozygous c.735G>C desmin mutation that leads to the expression of two mutant desmin proteins, E245D and D214_E245del [14], areas with a subsarcolemmal accumulation of mitochondria (Fig. 3a, ma) did prominently localize in close association with pathological protein aggregates composed of granulofilamentous material (Fig.…”

“…3Electron microscopic visualization of mitochondrial pathology in human and murine desminopathies. a Granulofilamentous material (gfm), subsarcolemmal accumulation of mitochondria (ma), and multiple enlarged mitochondria ( arrowheads ) in skeletal muscle tissue from a German patient harboring a heterozygous E245D/D214_E245del (c.735G>C) desmin mutation [14]. b Upper panel normal mitochondria regularly positioned adjacent to myofibrillar Z-discs in murine wild-type (WT) soleus muscle.…”

“…Tissue samples derived from diagnostic vastus lateralis muscle biopsies of two patients from a previously reported family with a heterozygous c.1049G>C (R350P) desmin mutation [3] and of one previously reported patient harboring the heterozygous c.735G>C desmin mutation leading to the expression of two mutant desmin protein species (E245D and D214_E245del) [14] were obtained from the Department of Neurology, University Hospital Bergmannsheil, Ruhr-University, Bochum, Germany and the Department of Neurology, University Hospital Bonn, Germany, respectively. Human desmin reference sequences: NM_001927.3; P17661.…”