Objective: To determine if microRNA’s (miR) are elevated in the plasma of individuals affected by the inherited peripheral neuropathy Charcot-Marie-Tooth Disease, type 1A (CMT1A), miR profiling was employed to compare control and CMT1A plasma.

Methods: We undertook a screen of CMT1A and control plasma samples to identify miRs that are elevated in CMT1A using a pilot screen of plasma miR by next generation sequencing, followed by validation of selected miRs by quantitative PCR, and correlation with clinical data and protein biomarkers.

Results: After an initial pilot screen, a broader screen confirmed elevated levels of several muscle-associated miRNAs (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 L, NfL) measured on the same sample set shows a comparable elevation of several miRs and ability to discriminate cases from controls. In addition, the putative Schwann cell miRs correlate with a recently described TMPRSS5 biomarker that is also elevated in CMT1A plasma, and this protein is most highly expressed in Schwann cells.

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.

This study is designed to determine if miR’s from muscle and Schwann cells are elevated in plasma of patients with CMT1A. This study provides Class III evidence that a set of plasma miRs are elevated in patients with CMT1A.
Standard Protocol Approvals, Registrations, and Patient Consents

 Subjects with CMT1A were identified and evaluated in the Inherited Neuropathy Consortium (INC) clinic in the Department of Neurology at Iowa and the National Hospital for Neurology and Neurosurgery, London, UK. Institutional Review Board approval was obtained from the University of Iowa. Written informed assent/consent was provided by participants under protocols approved by the ethics board of the NIH Rare Diseases Clinical Research Network (Protocol INC6601) and the National Hospital for Neurology and Neurosurgery Research Ethics Committee/ Central London REC 3 09/H0716/61. All of the patients are followed in the INC natural history studies and most of the patients were enrolled in the recently published natural history study of CMT1A ¹⁶. The sample cohorts include many samples used in a previous study of protein biomarkers ¹⁰.

Subjects were diagnosed with CMT1A on the basis of clinical evidence of sensory and/or motor peripheral neuropathy (including length dependent sensory loss, weakness and atrophy of the distal musculature and decreased deep tendon reflexes), nerve conduction studies, and confirmatory genetic testing for the PMP22 duplication in the subject or affected first degree relatives. Subjects and normal controls provided two 6 ml EDTA containing tubes of blood during their visit for plasma extraction¹⁷ which was performed within 15 minutes of the blood draw and stored in aliquots at -80^oC. All subjects were examined clinically by investigators who were certified by the INC for the proper administration of the CMTNSv2 (CMT neuropathy score), a validated 9 item, 36 composite score based on patients symptoms (3 items), examination findings (4 items) and electrophysiology (2 items)¹⁸. CMTESv2 (CMT examination score) scores were also calculated which included the 7 items of patients’ symptoms and examination findings in the CMTNSv2 but excludes the physiological results. Thus, the CMTES has a maximum score of 28 rather than 36 points ¹⁸. These scores were then subjected to Rasch modification to generate CMTNS-R and CMTES-R   ¹⁹.

The controls were age-matched, with a similar average age (Table 2) and similar male/female distribution. In some cases, controls are unaffected family members of CMT1A patients who have been demonstrated to not have CMT. None of the control subjects have acquired neuropathy.

Nerve Conduction Studies: Ulnar motor conduction velocities (MNCV) were performed by standard techniques ²⁰ with recording over the belly of the Abductor Digiti Minimi (ADM) with stimulation at the wrist and below the elbow. Maximum compound muscle action potential (CMAP) amplitudes were recorded using baseline to peak measurements in mV.

MicroRNA isolation from plasma

MiRs were extracted from plasma samples using miRCURY^TM RNA Isolation Kit - Biofluids (Exiqon, Frederick, MD, catalog #300112; or by Qiagen purification kit (#217204). Thawed samples were centrifuged at 3000 x g for 5min; then 200uL of supernatants were used to isolate RNA using the manufacturer’s protocol; final elution volume is 50ul water.

MicroRNA quantitative RT-PCR on Biomark HD platform

The custom RT (reverse transcriptase) primer pool and preAmp primer pool were made according to the protocol for creating custom RT and preamplification pools using TaqMan® MicroRNA Assays (Life Technologies Corporation, 6055 Sunol Blvd, Pleasanton, CA, 4465407 revision date Feb 2019, Rev D). Ten mL of each individual 5x RT primer of the 24 candidate miRNAs were combined,  and a RT primer pool was made by adding TE buffer (10 mM Tris, 1 mM EDTA) to 1 mL with each primer at a final concentration of 0.05x. Five mL of each 20x TaqMan MicroRNA assay for the 24 candidate miRNAs were combined with 380mL TE buffer, resulting a 500 mL PreAmp primer pool with each assay at a final concentration of 0.2x.

The quantitative PCR procedure was a modification of a Fluidigm protocol (Fluidigm Corporation, 2 Tower Place, Suite 2000, South San Francisco, CA 94080, MicroRNA Real-Time PCR Using Dynamic Array IFCs, PN 100-1616 C1). The 10 µL RT reaction was composed of the following (µL): RT primer pool (0.05x) 4.0, 100mM dNTPs (with dTTP) 0.2, multiScribe reverse transcriptase (50 U/µL) 1.5, 10X reverse transcription buffer 1.0, RNase inhibitor (20 U/µL) 0.13, Nuclease-free water 0.17, total RNA 3.0. The RT reaction was performed in a thermocycler: 16 °C for 2 min, 42 °C for 1 min, and 50 °C for 1 sec, repeat these three steps for 40 cycles; then 85 °C for 5 min; then hold at 4 °C. The 5 µL of preamplification mixture contained the following (µL): TaqMan preAmp master mix (2X) 2.5, PreAmp primer pool 1.0, RT product 1.5. The preamplification reaction was performed using the following condition: 95 °C  10 min, 55 °C for 2 min, 72 °C for 2 min, 18 cycles of (95 °C for 15 sec, 60 °C for 4 min), 99.9 °C for 10 min, then hold at 4 °C. The preamplification products were diluted 10-fold by adding 45 µL of 0.1xTE to 5 µL of preamplification product and used for subsequent real-time quantitative PCR run on the Biomark High Density platform.

The 10X assays were prepared by combining 3 µL of 20x TaqMan miRNA assay (Life Technologies) and 3 µL of 2x Assay loading reagent (Fluidigm 100-7611), resulting in final concentration (at 10x) of primers at 9 µM and probe at 2 µM. Samples were prepared by combining 3 µL of 2x master mix (TaqMan Fast Advanced Master Mix, Life Technologies 4444557), 0.3 µL of 20x GE Sample Loading Reagent (Fluidigm 100-7610), and 2.7 µL of 10x diluted preamplification product. For the Fast TaqMan assays (Biomark High Density only, 100-6174 C1): 3 µL of each assay and 3 µL of each sample were loaded into the respective inlets on the 192.24 IFC. RT-qPCR results, which are expressed as raw Ct values, were normalized to three reference miRNAs miR-16, miR-103, and miR-30e. The relative expression (fold change) was calculated using the 2^−ΔCt method.

Statistical Analysis

Data were analyzed using GraphPad Prism v7.03 (GraphPad, San Diego CA) using unpaired t-tests. Pearson correlation coefficients were calculated for each marker relative to neuropathy score (CMTES-R, CMTES-N, Ulnar CMAP, Ulnar MNCV). The TMPRSS5, NfL, and miRNA-206, -133a and -223-3p ROC (receiver operating characteristic) curves and AUC (area under the curve) calculations were generated using GraphPad Prism v7.03.

The file contains Ct data from qRT-PCR for selected microRNA's in human plasma samples. Three of the miRNA's had been used for normalization purposes, as noted above. Other columns include de-identified patient numbers, diagnosis (CMT1A or control) and the CMTES/CMTNS neuropathy scores and Rasch-modified derivatives. In addition, age, nerve conduction velocity and compound muscle action potentials are provided. Missing values indicate that a given miRNA was not detected in a specific sample.