miRNAs as serum biomarkers for Duchenne muscular dystrophy

Abstract

Dystrophin absence in Duchenne muscular dystrophy (DMD) causes severe muscle degeneration. We describe that, as consequence of fibre damage, specific muscle-miRNAs are released in to the bloodstream of DMD patients and their levels correlate with the severity of the disease. The same miRNAs are abundant also in the blood of mdx mice and recover to wild-type levels in animals 'cured' through exon skipping. Even though creatine kinase (CK) blood levels have been utilized as diagnostic markers of several neuromuscular diseases, including DMD, we demonstrate that they correlate less well with the disease severity. Although the analysis of a larger number of patients should allow to obtain more refined correlations with the different stages of disease progression, we propose that miR-1, miR-133, and miR-206 are new and valuable biomarkers for the diagnosis of DMD and possibly also for monitoring the outcomes of therapeutic interventions in humans. Despite many different DMD therapeutic approaches are now entering clinical trials, a unifying method for assessing the benefit of different treatments is still lacking.

Figure 1. Differential amounts of muscle miRNAs in the sera of healthy versus dystrophic children

1. Box plots comparing microRNA levels in the sera of 7 healthy, 26 Duchenne (DMD), and 5 Becker (BMD) patients (patient information is listed in supplementary Table 1). All data were normalized for three spiked miRNAs (ath-miR-159a, cel-lin-2, and cel-lin-4). miR-1, miR-206, and miR-133 values are shown as copy number per ml of serum. Control miR-223 levels are also shown as fold change values. All values are shown in logarithmic scale. p-values are derived from the comparison of miRNA levels in DMD or BMD versus healthy patients.

2. ROC curves regarding diagnostic power to distinguish healthy from dystrophic cases. Area under the curve (AUC) values are also shown.

Figure 2. Correlation between clinical assessment and serum miRNAs

1. Case-by-case comparison of miRNA levels and CK values with NSAA in 10 DMD children between 3 and 6 years of age (patient information is listed in supplementary Table 1). In each graph, miRNA levels are shown as white dots (copy numbers/ml of serum), NSAA scores as black dots, and CK values (units/ml of serum) as diamonds; regression lines are also displayed. Patients DMD 1–3, due to their age (3 years), have a non-collaborative behaviour to perform the entire NSAA; however, all of them had normal muscle strength at neurological assessment and they are indeed indicated with maximum NSAA score according to normal ambulation. For the outlier patient DMD10, different scales were used (graphs on the right).

2. Spearman analysis correlating miRNA or CK levels with age or NSAA scores in DMD children. In the two tables, Spearman coefficients (R) and p-values (p) are displayed.

Figure 3. Muscle miRNAs (dystromiR) as diagnostic markers for DMD gene therapy

1. Schematic representation of the exon skipping strategy in mdx mice (Denti et al, 2006a).

2. Western blot for dystrophin (DYS) and actinin (ACTN) perfomed with 50 µg of muscle extracts (GAS, gastrocnemius; HEA, heart; DIA, diaphragm) of WT, mdx and AAV-U1#23 treated mdx (AAV#23). In control lane (wild-type; WT), 5 µg of WT protein extract were mixed with 45 µg of mdx proteins in order to avoid signal saturation.

3. Mdx gastrocnemius sections from untreated (mock) or exon skipping-treated (AAV#23) animals were analysed by Hematoxylin/Eosin staining. Original magnification 20×. Scale bar: 100 µm.

4. Box plots comparing microRNA and CK levels in the serum of WT, mdx, and AAV#23-treated mdx mice. miRNA values were normalized for three spiked miRNAs (ath-miR-159a, cel-lin-2, and cel-lin-4). All data are shown as fold change values with respect to WT set to a value of 1. p-values are derived from the comparison between miRNA or CK levels in mdx versus WT animals.