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.