10.5061/DRYAD.NVX0K6DRX
Svaren, John
0000-0003-2963-7921
University of Wisconsin-Madison
Wang, Hongge
Sanofi Translational research
Davison, Matthew
Sanofi Translational research
Shy, Michael
University of Iowa
MicroRNA quantitative RT-PCR analysis of CMT1A Plasma
Dryad
dataset
2021
FOS: Basic medicine
National Center for Advancing Translational Sciences
https://ror.org/04pw6fb54
R21 TR003034
National Institute of Neurological Disorders and Stroke
https://ror.org/01s5ya894
U54 NS065712
Charcot-Marie-Tooth Association
https://ror.org/04k8a6441
2021-05-31T00:00:00Z
2021-05-31T00:00:00Z
en
https://doi.org/10.5281/zenodo.4818612
196553 bytes
4
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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 -80oC. 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 miRCURYTM 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.