MicroRNAs as serum biomarkers in Becker muscular dystrophy

Abstract

Becker muscular dystrophy (BMD) is an X-linked neuromuscular disorder due to mutation in the DMD gene, encoding dystrophin. Despite a wide clinical variability, BMD is characterized by progressive muscle degeneration and proximal muscle weakness. Interestingly, a dysregulated expression of muscle-specific microRNAs (miRNAs), called myomirs, has been found in patients affected with muscular dystrophies, although few studies have been conducted in BMD. We analysed the serum expression levels of a subset of myomirs in a cohort of 29 ambulant individuals affected by BMD and further classified according to the degree of alterations at muscle biopsy and in 11 age-matched healthy controls. We found a significant upregulation of serum miR-1, miR-133a, miR-133b and miR-206 in our cohort of BMD patients, supporting the role of these miRNAs in the pathophysiology of the disease, and we identified serum cut-off levels discriminating patients from healthy controls, confiming the potential of circulating miRNAs as promising noninvasive biomarkers. Moreover, serum levels of miR-133b were found to be associated with fibrosis at muscle biopsy and with patients' motor performances, suggesting that miR-133b might be a useful prognostic marker for BMD patients. Taken together, our data showed that these serum myomirs may represent an effective tool that may support stratification of BMD patients, providing the opportunity of both monitoring disease progression and assessing the treatment efficacy in the context of clinical trials.

Keywords: BMD; Becker muscular dystrophy; biomarkers; miR-133b; miRNA; serum; skeletal muscle.

FIGURE 1

Muscle biopsy. Representative H&E pictures of the normal muscle (A) and in BMD patients. (B) Mild cases showed limited alterations in fibre size, a mild fibrosis, and a few central nuclei. (C) Moderate cases showed an increase in fibre size variability and in fibrotic tissue. (D) Severe cases showed an important presence of fibroadipose tissue, a significant variation in fibre size, with both highly atrophic and hypertrophic fibres and an increase in nuclear centralization

FIGURE 2

Distribution of serum miRNA in BMD patients and healthy controls. (A) Serum miR‐1 levels were higher in BMD than in healthy controls (p = 0.0024). Scatter dot plot values represent means and standard error of the mean (SEM). (B) Serum miR‐31 levels did not significantly differ between BMD and controls (p = 0.0822). (C) Serum miR‐133a levels were higher in BMD than in healthy controls (p < 0.0001). (D) Serum miR‐133b levels were higher in BMD than in healthy controls (p = 0.0002). (E) Serum miR‐206 levels were higher in BMD than in healthy controls (p < 0.0001)

FIGURE 3

ROC analysis. Accuracy of serum miR‐1 (A), miR‐133a (B), miR‐133b (C) and miR‐206 (D) for the diagnosis of BMD vs healthy controls

FIGURE 4

Correlation analysis. (A) miR‐1 and miR‐133a were positively correlated (r = 0.858, p < 0.0001). (B) miR‐1 and miR‐133b were positively correlated (r = 0.743, p < 0.0001). (C) miR‐1 and miR‐206 were positively correlated (r = 0.56, p < 0.003). (D) miR‐133b and miR‐133a were positively correlated (r = 0.901, p < 0.0001). (E) miR‐133b and miR‐206 were positively correlated (r = 0.677, p = 0.0001). (F) miR‐206 and miR‐133a were positively correlated (r = 0.635, p < 0.0004). Spearman's correlation coefficient was used