MicroRNAs as Biomarkers of Charcot-Marie-Tooth Disease Type 1A

Abstract

Objective: To determine whether microRNAs (miRs) are elevated in the plasma of individuals with the inherited peripheral neuropathy Charcot-Marie-Tooth disease type 1A (CMT1A), miR profiling was employed to compare control and CMT1A plasma.

Methods: We performed a screen of CMT1A and control plasma samples to identify miRs that are elevated in CMT1A using next-generation sequencing, followed by validation of selected miRs by quantitative PCR, and correlation with protein biomarkers and clinical data: Rasch-modified CMT Examination and Neuropathy Scores, ulnar compound muscle action potentials, and motor nerve conduction velocities.

Results: After an initial pilot screen, a broader screen confirmed elevated levels of several muscle-associated miRNAs (miR1, -133a, -133b, and -206, known as myomiRs) along with a set of miRs that are highly expressed in Schwann cells of peripheral nerve. Comparison to other candidate biomarkers for CMT1A (e.g., neurofilament light) measured on the same sample set shows a comparable elevation of several miRs (e.g., miR133a, -206, -223) and ability to discriminate cases from controls. Neurofilament light levels were most highly correlated with miR133a. In addition, the putative Schwann cell miRs (e.g., miR223, -199a, -328, -409, -431) correlate with the recently described transmembrane protease serine 5 (TMPRSS5) protein biomarker that is most highly expressed in Schwann cells and also elevated in CMT1A plasma.

Conclusions: These studies identify a set of miRs that are candidate biomarkers for clinical trials in CMT1A. Some of the miRs may reflect Schwann cell processes that underlie the pathogenesis of the disease.

Classification of evidence: This study provides Class III evidence that a set of plasma miRs are elevated in patients with CMT1A.

Figure 1. MicroRNA (MiR) Levels in Charcot-Marie-Tooth Disease Type 1A (CMT1A) vs Control Plasma

A total of 21 candidate miRs were analyzed using quantitative real-time PCR platform in a cohort of 109 CMT1A and 52 control plasma samples (table 2). Expression levels of these 21 miRs were normalized to combined levels of 3 reference miRNAs: miR-16, -103, and -30e; negative delta Ct is shown as a scatterplot for control and CMT1A samples. The median and interquartile range are shown.

Figure 2. Receiver Operating Characteristic (ROC) Analysis of Charcot-Marie-Tooth Disease Type 1A (CMT1A) Associated MicroRNAs (MiRs)

ROC plots are shown for selected myomiRs (-206 and -133a) and a Schwann cell–associated miR (-223-3p) in comparison with the transmembrane protease serine 5 (TMPRSS5) and neurofilament light (NfL) levels from the same sample sets. Area under the curve data are shown below the graph.

Figure 3. Correlation Analysis of Plasma MicroRNA (MiR) With Protein Biomarker and Clinical Measurements of Neuropathy

The 2 panels show Pearson correlation analysis of plasma miRs with age, 2 protein biomarkers (neurofilament light [NfL] and transmembrane protease serine 5 [TMPRSS5]), neuropathy scores (Charcot-Marie-Tooth Examination Score [CMTES] and Charcot-Marie-Tooth Neuropathy Score [CMTNS]), and electrophysiologic measures (nerve conduction velocity [NCV] and compound muscle action potential [CMAP]) in (A) control and (B) Charcot-Marie-Tooth disease type 1A (CMT1A) sample sets. Red and blue shading show those correlations that are positive or negative, respectively. Red numbers indicate those correlations where p < 0.05.

Figure 4. Reproducibility and Predicted Targets of Charcot-Marie-Tooth Disease Type 1A (CMT1A)–Associated MicroRNAs (MiRs)

(A) The graph shows miR levels in repeat samples (from 22 CMT1A cases) that had been obtained from patients within 1–3 years from the initial sample collection. The levels of the miRs are shown to compare the miR levels between initial and follow-up samples. (B) Predicted targets of the Schwann cell–associated miRs were analyzed by gene ontology to identify statistically enriched pathways.