Material and methods
Patients and mutation
analysis
Clinical evaluation and biological samples were obtained after informed
written consent and were secured in accordance with the protocol
approved by national ethic committees. Routine biological analyses were
performed in the three patients, as well as nerve conduction studies
(NCS) and electromyogram (EMG) studies.
Patient from family 2 performed whole-body muscle magnetic resonance
imaging (MRI) and was screened for mutations in the SMN1, HSPB1,
HSPB3 and HSPB8 genes. Additionally, whole-exome sequencing was
performed. Exome enrichment was done with the SeqCap EZ MedExome Target
Enrichment Kit, and sequencing was done using the NextSeq 500 sequencing
system following the manufacturers’ instructions.
Patient II4 from family 2 performed a next-generation sequencing-based
gene panel that includes 76 genes. A search for mutations using Sanger
sequencing of the codified regions of the MORC2 gene was performed in
patient III5.
Plasmids
MORC2-flag fusion protein was realized by Genescript by cloning MORC2
ORF cDNA (NM_001303256.1) in pcDNA3.1-C-DYK plasmid. Mutagenesis was
done by site-directed mutagenesis following the manufacturer
recommendation (QuikChange II Site-Directed Mutagenesis Kit – Agilent)
and clones were validated by sequencing. MORC2-flag variants were
subcloned in pCAGIG vector (addgene # plasmid 11159 ; (Matsuda and
Cepko, 2004)). MORC2-flag (3kb) variants were amplified by high fidelity
PCR (Q5, New England BioLabs) and cloned using XhoI / NotI. Clones were
validated by sequencing the full coding sequence.
Cell culture
Human neuroblastoma cell line SH-EP (RRID:CVCL_0524 ; an epitheloid
subclone of the human NB line SK-N-SH) was used in this study. Cells
were defrosted and cultured in a modified DMEM culture medium (DMEM
medium with 10% Fetal Bovine Serum and 1% penicillin/streptomycin) and
were transfected using JetPRIME following manufacturer recommendations
(Polyplus transfection). Cortical neuron culture was done as described
previously in Jacquier et al (Jacquier et al., 2009). Briefly, embryonic
mice at E15.5 days of development were sacrificed. The motor cortex was
dissected and trypsinized. Cortical neurons were then transfected by
electroporation using a cuvette electroporator (BTX ECM830) with the
following parameters 220 volts, 5 ms pulse length, 3 times at 1-second
interval. Electroporated neurons were plated in standard conditions
(neurobasal plus medium + B27 plus + 2% horse serum + 1% penicillin /
streptomycin).
Western Blot
SH-EP were plated in 6 well plate and transfected with the different
plasmids using the JetPrime reagent (Polyplus transfection). Twenty-four
hours later, cells were washed in PBS and extracted directly in 200 µL
1X Laemmli buffer (50 mM Tris HCl pH 6.8, 10% glycerol, 100 mM DTT, 2%
SDS, bromophenol blue) supplemented with 20U of Benzonase (Merck
Millipore) per wells to digest the DNA. After 15 min incubation at room
temperature, totals extracts are boiled and loaded in an 8% SDS-PAGE,
transferred on nitrocellulose membrane, blotted with the indicated
antibodies, and revealed with the ECL clarity (BioRad).
Immuno fluorescent staining and
quantification
Cells line or cortical neuron culture were fixed using 4% formaldehyde
for 10 min at RT. Then, cells were washed in PBS and put 1 hour in
blocking buffer (PBS with 4% BSA, 0.3% Triton x100, 0.1M glycine)
before primary antibody incubation overnight at 4°c. After three PBS
washes, secondary antibodies were incubated in a blocking buffer for 1
hour at RT, washed and mounted. The following antibodies were used.
Mouse anti GFP at 1/1000 (Santa Cruz, sc-9996); Rabbit anti-Cleaved
caspase-3 Asp175 at 1/400 (Cell signaling technology, #9661); Rabbit
anti FLAG M2 at 1/1000 (Cell signaling technology, #14793).
Images acquisitions were done using EVOS M5000 microscope (ThermoFisher
Scientific) or LSM800 confocal microscope (Zeiss). Images were analyzed
using ImageJ software or Metamorph software. In SH-EP experiments,
quantification of GFP positive cells were done on at least 80 images
from at least 4 independent experiments. To overcome difference in
transfection efficiency between conditions, survival was normalized by
day 1. To pool the data between independent experiments, survival was
expressed in percentage of the control. Caspase 3 activation
quantification was established by the percentage of caspase 3 activated
positive cell in GFP positive cells expressing MORC2 and normalized at
100% for the control. In cortical neuron experiments, quantification of
the soma number was done on confocal images using Metamorph software on
at least 120 fields per condition from 4 independent experiments. The
mean number of cortical neurons per field was normalized to the value at
day 1 and compared to the WT condition. The percentage of
activated-caspase3 positive neurons in electroporated neurons (i.e. GFP
positive) was quantified on 80 fields per condition from 4 independent
experiments and normalized to the value obtained with WT MORC2. Neurite
length for each neurons was automatically measured in confocal images
using the specific plugin “neurite outgrowth” of the Metamorph
software. Quantification was performed on thirty fields per experiment
in 4 independent experiments at day 5.
Statistical analysis
Each experiment was independently repeated at least three times. Data
were analyzed with Excel (Microsoft) or Prism 5 (GraphPad Software Inc).
Data from several groups showing normality and equal variance were
analyzed with one-way ANOVA followed by Dunnett’s multiple comparison
test. Otherwise, data that do not show a Gaussian distribution were
analyzed with Kruskal-Wallis nonparametric test followed by Dunns post
hoc test. Asterisks describes values levels of statistical significance
as following: Non significant ns : p<0.05 ; Significant *
p<0.01 ; Very significant ** p<0.001 ; Extremely
significant *** p<0.0001.
Ethical approval
All procedures performed in studies involving human participants were in
accordance with the ethical standards of the institutional and/or
national research committee and with the 1964 Helsinki declaration and
its later amendments or comparable ethical standards. Informed consent
was obtained from all individual participants included in the study. All
procedures performed in studies involving animals were in accordance
with the ethical standards of the institution or practice at which the
studies were conducted.