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How much mutant protein is needed to cause a protein aggregate myopathy in vivo? Lessons from an exceptional
Christoph S Clemen1, Dirk Fischer, Jens Reimann
1Institute of Biochemistry I, Medical Faculty, University of Cologne, Germany.
Abstract:
Myofibrillar myopathies are caused by mutations in desmin, alphaB-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 disease-causing 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 intra- and 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 wild-type 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.
Insights
This study identifies a truncated desmin protein as the cause of a rare myofibrillar myopathy. Protein levels vary significantly within muscle, complicating the determination of disease-causing thresholds.
Area of Science:
- Genetics
- Molecular Biology
- Neurology
Background:
- Myofibrillar myopathies (MFMs) are a group of inherited muscle disorders.
- Mutations in genes like desmin cause MFMs, often resulting in heterozygous single amino acid substitutions or small in-frame deletions.
- Standard immunodetection techniques struggle to differentiate mutant from wild-type proteins in human skeletal muscle.
Purpose of the Study:
- To investigate the molecular basis of an exceptional desminopathy.
- To identify the specific disease-causing desmin mutation and its pathogenic mechanism.
- To analyze the expression levels and distribution of mutant desmin in affected muscle tissue.
Main Methods:
- Genetic analysis of patients with desminopathy.
- Immunoblotting to detect desmin protein variants in skeletal muscle.
- RT-PCR to identify desmin mRNA species.
- Semiquantitative RT-PCR and protein quantification to assess mutant desmin levels.
- In vitro desmin assembly studies.
Main Results:
- A heterozygous c.735G>C mutation in desmin was identified in affected patients.
- Skeletal muscle samples revealed full-length desmin and a truncated 50 kDa variant.
- RT-PCR detected mRNA for wild-type desmin and two mutant forms: p.Glu245Asp and p.Asp214_Glu245del.
- The truncated p.Asp214_Glu245del desmin was identified as the disease-causing mutant.
- Truncated desmin mRNA ranged from 24% to 37%, but protein levels varied widely (5% to 43%) within and between muscle biopsies.
Conclusions:
- The truncated p.Asp214_Glu245del desmin protein is pathogenic in myofibrillar myopathy.
- High intra- and interindividual variability in mutant desmin protein levels complicates defining in vivo pathogenic thresholds.
- The complex distribution of mutant protein reflects local expression and turnover differences within myonuclear domains.
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