Dystromirs as serum biomarkers for monitoring the disease severity in Duchenne muscular Dystrophy

Abstract

Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls. We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials. Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.

Figure 1. Serum levels of the analysed dystromirs in DMD, BMD, UCMD and controls.

Absolute quantification of miR-1, miR-206, miR-31, miR-133a and miR-133b in serum samples of 44 DMD, 5 BMD, 16 UCMD patients and 14 healthy controls. The data are presented in a logarithmic scale as miRNA copy number per ml normalized to the spiked-in C. elegance miRNAs (cel-miR-54, cel-miR-39, cel-miR-238). P-values derived from t-test are presented with *, **, *** and correspond to p <0.05, p<0.01 and p<0.001 respectively.

Figure 2. Serum levels of the analysed dystromirs and functional ability of DMD patients.

A. MiR-1, miR-206, miR-31, miR-133a and miR-133b in ambulant and non-ambulant DMD patients. B. Correlation analysis between the level of the dystromirs and the NSAA scores. The data are presented in a logarithmic scale as miRNA copy number per ml normalized to the spiked-in C. elegance miRNAs (cel-miR-54, cel-miR-39, cel-miR-238). P-values derived from t-test are presented with **, *** and correspond to p<0.01 and p<0.001 respectively.

Figure 3. Serum level of the analysed dystromirs in DMD patients according to clinical variables.

Level of miR-1, miR-206, miR-31, miR-133a and miR-133b in serum samples of DMD patients: A. without the need of scoliosis surgery and with scoliosis surgery performed or recommended; B. not on glucocorticoid treatment, on intermittent or on daily steroid regimen; C. without cardiomyopathy and with cardiac abnormality. The data are presented in a logarithmic scale as miRNA copy number per ml normalized to the spiked-in C. elegance miRNAs (cel-miR-54, cel-miR-39, cel-miR-238). P-values derived from t-test are presented with *, ** and correspond to p <0.05 and p<0.01 respectively.

Figure 4. Correlation analysis between the level of the analysed dystromirs in DMD and the FVC values.

Linear regression analysis between the levels of miR-1, miR-206, miR-31, miR-133a and miR-133b in serum samples and FVC scores in DMD patients showed correlation between the level of miR-1 (p = 0.0034, r² = 0.34) and miR-133b (p = 0.0035, r² = 0.34) with the FVC scores. The data are presented as miRNA copy number per ml normalized to the spiked-in C. elegance miRNA s (cel-miR-54, cel-miR-39, cel-miR-238). Regression lines are also presented.

Figure 5. Age profile of miR-1, miR-206, miR-31, miR-133a and miR-133b in serum samples.

Level of miR-1, miR-206, miR-31, miR-133a and miR-133b in serum samples according to the age of the: A. DMD patients; B. UCMD patients; C. healthy controls. The data are presented as miRNA copy number per ml normalized to the spiked-in C. elegance miRNAs (cel-miR-54, cel-miR-39, cel-miR-238). Regression line is also presented.

Figure 6. miRNAs as biomarkers for the outcome of therapeutic interventions: Systemic eteplirsen treatment trial.

Absolute quantification of miR-1, miR-206, miR-31, miR-133a and miR-133b in serum of: A. DMD patients before treatment with eteplirsen (pre-treatment), after treatment (post-treatment) and controls; B. low responders, responders and good responders; C. patients who received low dose and patients with high dose of eteplirsen. The data are presented in a logarithmic scale as miRNA copy number per ml normalized to the spiked-in C. elegance miRNAs (cel-miR-54, cel-miR-39, cel-miR-238). P-values derived from two-tailed t-test are presented with *, *** and correspond to p <0.05 and p<0.001 respectively.

Figure 7. miRNAs as biomarkers for the outcome of therapeutic interventions: Intramuscular eteplirsen treatment trial.

MiR-1, miR-206, miR-31, miR-133a and miR-133b expression in: A. untreated and treated muscle biopsy samples and controls; B. patients who received low dose and patients with high dose of eteplirsen. The data are presented in a logarithmic scale as relative fold change with respect to the control samples and normalized to U6 snRNA expression. P-values derived from two-tailed t-test are presented with *** and correspond to p<0.001.