Proteomic profiling of Duchenne muscular dystrophy : protein patterns and candidate markers of disease

Duchenne muscular dystrophy (DMD) caused by mutations in the dystrophin gene is a severe chronic muscle-wasting disease leading to early loss of ambulation in patients and to death by the third decade. Other muscular dystrophies exist including amongst others DMD’s milder allelic form Becker Muscular Dystrophy and the heterogeneous group of limb-girdle muscular dystrophies differ in age of onset, severity, and affected proteins.
In diagnosing muscular dystrophies, the assessment of multiple proteins in a muscle biopsy by immunohistochemical methods is considered the gold standard, as the identification of the underlying mutation is not always feasible or sufficient. The reproducibility and sensitivity of reverse protein arrays and their excellent correlation with immunohistochemistry and immunoblotting combined with minimal sample and antibody consumption make them an ideal approach for the assessment of muscular expression of multiple proteins in small biopsies. We have evaluated a set of antibodies currently used in standard diagnostic processes for muscular dystrophies on human muscle tissue and cultured primary human myotubes and show high correlations with Western blot data and reproducible significant differences in dystrophin, sarcoglycan, and dystroglycan expression between control and patient samples. This suggests that reverse protein arrays can quantitatively measure muscle proteins in as little as 10mg muscle tissue. This technology could be of interest not only in diagnostic processes, but especially for protein quantification of multiple, follow-up biopsies during clinical trials in upcoming therapy approaches such as exon-skipping, when protein expression in muscle is considered an important outcome measure or biomarker.
Despite the precise and extensive knowledge about the dystrophin gene and its protein, precise molecular and cellular events that eventually lead to muscle fiber degeneration in DMD are poorly understood. Downstream pathogenic events in metabolic pathways and cellular signalling that are key factors causing the ultimate degeneration of muscle fibers in DMD and reflecting disease state can be elucidated using mass spectrometry-based proteomics experiment. Proteomic profiling of DMD muscle tissue and comparing the resulting pattern to other muscular dystrophies has revealed a drastic increase in the muscle-specific member of the small heat shock protein family HSPb2 and, to a lesser extent, a decrease in GPD1L that was not seen in other muscular dystrophies. We are currently implementing a set of experiments to validate HSPb2 as disease marker for duchenne muscular dystrophy in cultured primary myotubes from DMD patients and, if applicable, in serum from DMD patients. HSPb2 is a promising candidate that could be applied as signature molecule as part of a protein panel that can be used to assessed disease state in DMD or therapeutic effects of novel drugs or treatments.