BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal disease for which no 
cure is available. Clinical trials have shown to be largely underpowered due to 
inter-individual variability and noisy outcome measures. The availability of 
biomarkers able to anticipate clinical benefit is highly needed to improve 
clinical trial design and facilitate drug development.
METHODS: In this study, we aimed to appraise the value of protein biomarkers to 
predict prognosis and monitor disease progression or treatment outcome in 
patients affected by DMD. We collected clinical data and 303 blood samples from 
157 DMD patients in three clinical centres; 78 patients contributed multiple 
blood samples over time, with a median follow-up time of 2 years. We employed 
linear mixed models to identify biomarkers that are associated with disease 
progression, wheelchair dependency, and treatment with corticosteroids and 
performed survival analysis to find biomarkers whose levels are associated with 
time to loss of ambulation.
RESULTS: Our analysis led to the identification of 21 proteins whose levels 
significantly decrease with age and nine proteins whose levels significantly 
increase. Seven of these proteins are also differentially expressed in 
non-ambulant patients, and three proteins are differentially expressed in 
patients treated with glucocorticosteroids. Treatment with corticosteroids was 
found to partly counteract the effect of disease progression on two biomarkers, 
namely, malate dehydrogenase 2 (MDH2, P = 0.0003) and ankyrin repeat domain 2 (P 
= 0.0005); however, patients treated with corticosteroids experienced a further 
reduction on collagen 1 serum levels (P = 0.0003), especially following 
administration of deflazacort. A time to event analysis allowed to further 
support the use of MDH2 as a prognostic biomarker as it was associated with an 
increased risk of wheelchair dependence (P = 0.0003). The obtained data support 
the prospective evaluation of the identified biomarkers in natural history and 
clinical trials as exploratory biomarkers.
CONCLUSIONS: We identified a number of serum biomarkers associated with disease 
progression, loss of ambulation, and treatment with corticosteroids. The 
identified biomarkers are promising candidate prognostic and surrogate 
biomarkers, which may support drug developers if confirmed in prospective 
studies. The serum levels of MDH2 are of particular interest, as they correlate 
with disease stage and response to treatment with corticosteroids, and are also 
associated with the risk of wheelchair dependency and pulmonary function.