Circulating MyomiRs as Potential Biomarkers to Monitor Response to Nusinersen in Pediatric SMA Patients

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by mutations in survival motor neuron (SMN) 1 gene, resulting in a truncated SMN protein responsible for degeneration of brain stem and spinal motor neurons. The paralogous SMN2 gene partially compensates full-length SMN protein production, mitigating the phenotype. Antisense oligonucleotide nusinersen (Spinraza^®) enhances SMN2 gene expression. SMN is involved in RNA metabolism and biogenesis of microRNA (miRNA), key gene expression modulators, whose dysregulation contributes to neuromuscular diseases. They are stable in body fluids and may reflect distinct pathophysiological states, thus acting as promising biomarkers. Muscle-specific miRNAs (myomiRs) as biomarkers for clinical use in SMA have not been investigated yet. Here, we analyzed the expression of miR-133a, -133b, -206 and -1, in serum of 21 infantile SMA patients at baseline and after 6 months of nusinersen treatment, and correlated molecular data with response to therapy evaluated by the Hammersmith Functional Motor Scale Expanded (HFMSE). Our results demonstrate that myomiR serological levels decrease over disease course upon nusinersen treatment. Notably, miR-133a reduction predicted patients' response to therapy. Our findings identify myomiRs as potential biomarkers to monitor disease progression and therapeutic response in SMA patients.

Keywords: biomarkers; myomiRNAs; nusinersen; spinal muscular atrophy.

Figure 1

MyomiR expression reduction in serum of SMA patients after 6 months of nusinersen treatment. RT-PCR analysis of muscle-specific miRNAs in total RNA extracted from serum of 21 SMA patients demonstrated a significant reduction of miR-133a, -133b and -1 transcriptional levels during nusinersen treatment. A trend in reduction for miR-206 is shown. Each point represents miRNA expression level in serum of a patient at that time-point; trend lines show miRNA changes over time (solid lines). The medians of the myomiR expression levels at baseline and after 6 months of treatment are plotted accordingly for each miRNA (dotted lines). miRNA levels were normalized to miR-16 and expressed as fold changes using the 2^−ΔCt formula. Wilcoxon signed-ranked test, * p < 0.05.

Figure 2

Spearman’s correlations between each myomiR expression in SMA patients during nusinersen treatment. Spearman’s correlation coefficients (r) were used to corroborate relationships between myomiR expression levels in serum of SMA patients at baseline (T0) and 6 months after treatment (T6). Coefficients major than 0.5 indicate a good positive correlation. p values (p < 0.05) and r (r ≥ 0.046) coefficients were assessed by Spearman test.

Figure 3

miR-133a reduction predicts clinical improvement of SMA patients during nusinersen treatment. Logistic regression analysis of miRNA differential expression and clinical response to therapy after 6 months of nusinersen treatment. Each circle represents miRNA expression level in serum of a patient after 6months of treatment. An improvement at the HFMSE score ≥ 3 was considered clinically meaningful (1.0 on y axis). A significant relationship for miR-133a reduction by 6.6 C_t values (inflection point) (A), and a positive trend for miR-133b and -206 are reported (B). * p < 0.05.