Diagnosis of muscular dystrophies is currently based on invasive methods 
requiring muscle biopsies or blood tests. The aim of the present study was to 
identify urinary biomarkers as a diagnostic tool for muscular dystrophies. Here, 
the urinary proteomes of Duchenne muscular dystrophy (DMD) patients and healthy 
donors were compared with a bottom-up proteomic approach. Label-free analysis of 
more than 1100 identified proteins revealed that 32 of them were differentially 
expressed between healthy controls and DMD patients. Among these 32 proteins, 
titin showed the highest fold change between healthy subjects and DMD patients. 
Interestingly, most of the sequenced peptides belong to the N-terminal and 
C-terminal parts of titin, and the presence of the corresponding fragments in 
the urine of DMD patients was confirmed by Western blot analysis. Analysis of a 
large cohort of DMD patients and age-matched controls (a total of 104 
individuals aged from 3 to 20 years) confirmed presence of the N-ter fragment in 
all but two patients. In two DMD patients aged 16 and 20 years this fragment was 
undetectable and two healthy controls of 16 and 19 years with serum CK >800 IU/L 
demonstrated a low level of the fragment. N- and C-terminal titin fragments were 
also detected in urine from patients with other muscular dystrophies such as 
Becker muscular dystrophy and Limb-girdle muscular dystrophy (type 1D, 2D and 
2J) but not in neurogenic spinal muscular atrophy. They were also present in 
urine of dystrophin-deficient animal models (GRMD dogs and mdx mice). Titin is 
the first urinary biomarker that offers the possibility to develop a simple, 
non-invasive and easy-to-use test for pre-screening of muscular dystrophies, and 
may also prove to be useful for the non-invasive follow up of DMD patients under 
treatment.