Family 2
Subject II2 is the only known affected member of a Portuguese
non-consanguineous family (Figure 1). She had no history of physical
development delay. She reported cramps in the lower limbs and a tremor
of the upper extremities in early adulthood. At 30 years, she displayed
muscular weakness with walking impairment and difficulties climbing
stairs. Her father also experienced cramps but there was no familial
history otherwise and no consanguinity. The disease slowly progressed,
and at age 44, her clinical examination revealed distal weakness in the
upper limbs (wrist extensor and interosseous scored as 4/5 on MRC scale)
and a proximal deficit in the lower limbs (quadriceps, psoas, and
hamstring muscles as 4/5 on MRC scale), with pes cavus . Jerk
reflexes were all present except at the ankle. No pyramidal signs or
cranial nerve impairments were observed. Electroneuromyography showed
neurogenic features, in the forearms, and both proximal and distal in
the lower limbs, with normal motor and sensory conduction (Table 1). CK
level was mildly elevated: 258 UI/L (Normal < 200 UI/L). A
whole-body muscle MRI was performed at 46 years of age, showing a
severe, bilateral, and symmetric fibro-fatty substitution more apparent
in muscles of the calf. In the thighs and pelvis, there was a mild fatty
replacement of posterior thigh musculature and glutei (Fig1 B). In the
upper limbs, there was also a mild wasting of biceps brachialis, and
deltoids.
Molecular findings and bioinformatics
analysis
Whole-exome sequencing was performed for both families. The two affected
individuals of the first family bear the same heterozygous mutation
c.1330G>A (p.Gly444Arg) in the MORC2 gene
(NM_001303256.2). Patient II2 from family 2 bears the heterozygous
mutation c.1338C>A (p.His446Gln) in the MORC2 gene.
Sequencing of the MORC2 gene in her mother and sister’s DNA (both
asymptomatic) revealed no mutation. Her father was deceased hence no DNA
was available.
MORC2 protein (NP_001290185) contains several predicted domains (Figure
2A). Interestingly, all known mutations affect the ATPase domain or its
transducer S5 domain (Douse et al., 2018). Both mutations described here
affect conserved amino acids in the S5 domain at positions p444 and p446
(Figure 2B). Neighboring mutations in the S5 domain are p.C407Y (Ando et
al., 2017), p.T424R (Schottmann et al., 2016; Zanni et al., 2017),
p.A431V (Ando et al., 2017), p.D466N (Semplicini et al., 2017).
Interestingly, mutation p.G444R has been reported by Albulym, et al
(Albulym et al., 2016) as a likely pathogenic variant without familial
or functional evidence.
Bioinformatic prediction by HumVAr (PolyPhen-2) identified mutations
p.G444R and p.H446Q as probably pathogenic with a score of 0.945 and
0.878 on a 0 to 1 scale, with a sensitivity of 0.65 or 0.71 and a
specificity of 0.91 or 0.89, respectively. Likewise, the mutation taster
software found that both mutations were pathogenic and were neither
found in gnomAD (The Genome Aggregation Database) nor 1000G.
Interestingly, the crystal structure of MORC2 was solved (Douse et al.,
2018) allowing to map the mutations (Figure 2C) and predicting their
impact on the 3D structure of the protein. This allowed the HOPE
software to predict that the mutations were structurally damaging.
Indeed, the arginine mutant residue in position 444 is bigger and
introduces a charge in a buried residue which probably perturbs protein
folding. The glutamine mutant residue at position 446 breaks a hydrogen
bond between Histidine 444 and Alanine 442 residue and therefore
destabilizes the alpha helix.
Protein construct and subcellular
localization
A flag tag (also called DYK) (Figure sup1A) was fused to the C-terminal
extremity of MORC2 and an internal ribosome entry site (IRES) and eGFP
were added downstream (pCAGIG plasmid, addgene #11159). To check the
subcellular localization of the MORC2-flag fusion protein, SH-EP
neuroblastoma cells were transfected (Reddy et al., 1991) and murine
primary cortical neurons were electroporated. Two days after
transfection, the cells were fixed and MORC2 was stained with the
anti-Flag antibody. Nuclei were counterstained with DAPI (Figure sup1B).
In both cell types, MORC2 was detected in the nucleus.
Site-directed mutagenesis was done on pCAGIG MORC2-Flag to reproduce the
mutations identified in the patients. As positive pathogenic controls
mutations were introduced at the following positions of the MORC2 coding
sequence (NM_001303256): c.754 C>T leading to MORC2
p.R252W related to CMT2 (Sevilla et al., 2016), c.1271 C>G
leading to MORC2 p.T424R related to SMA-like (Schottmann et al., 2016;
Zanni et al., 2017). Similarly, both variants of unknown significance
were generated by mutagenesis at positions c.1330G>A
leading to MORC2 p.G444R and c.1338C>A leading to p.H446Q.
By convention, these variants will be designated in the text by their
position (ie: p.G444R as p444). To evaluate the expression of the
protein, the same amount of each plasmid was transfected in SH-EP and
total proteins were solubilized in Laemmli buffer 24 hours later and
analyzed by western Western Blot (Figure sup1B). Ponceau Red staining
revealed the total protein loaded whereas anti-MORC2 or anti-Flag
antibodies were used to detect MORC2. Altogerther, WT and mutant MORC2
proteins were expressed at the same level. Furthermore,
immunofluorescence and confocal microscopy detected no difference in the
subcellular localization of WT or MORC2 mutants (data not shown).
MORC2 mutants alter SH-EP survival and trigger
apoptosis
To determine the impact of MORC2 variants on SH-EP survival over
time, transfected cells (GFP positive) were analysed after 1, and 3 days
in culture (Figure 3A). The cell number was attributed a value of 100 %
at day 1. To evaluate survival, eGFP positive cells were counted at days
3 and normalized to the number of cells at day 1. At day 3, all mutants
caused a decrease in survival compared to control, including the
variants p444 (84%) and p446 (82%) of unknown significance (Figure
3B).
Activation of the caspase 3 by proteolytic cleavage is a landmark of
apoptosis-mediated cell death. To evaluate if reduced survival induced
by MORC2 mutants involved the induction of apoptosis, activated caspase
3 was detected by immunofluorescence at days 3 of culture after
transfection. Quantification of activated caspase 3 positive cells at
day 3 revealed a significant increase in all MORC2 mutants: p.252
(110%), p424 (127%) or MORC2 p444 (124%), p446 (130%) compared to WT
MORC2 (100%). Altogether, these results suggest that MORC2variants decrease cell survival through apoptosis.
Mutant MORC2 affect cortical neuron
survival
Since mutations in MORC2 are associated with neurodegenerative
disorders, we tested their effect in primary cells more relevant for to
the pathophysiology of MORC2-related disorders. Cortical neurons of the
motor cortex, also called upper motor neurons, are easy to purify in
large quantities and can be transfected by electroporation. To determine
the impact of MORC2 mutants on the survival, cortical neurons were
electroporated with WT, p424, p444 or p446 MORC2 constructs immediately
after dissociation and analysed at days 1, 2 and 5 (Figure 4A). Cortical
neuron counting overtime showed that a decrease in survival was already
observed at day 2 with the pathogenic mutation (p424, 87%) and with the
p444 (84%) and p446 (77%) variants compared to WT MORC2. Similar
results were observed at days 5 after electroporation with the
pathogenic mutant p424 (60.34%) and the two variants of unknown
significance p444 (67.19%) and p446 (47.97%) compared to the WT
(100%), suggesting that these variant are pathogenic.
Since we observed increased apoptosis in SH-EP cell expressing the MORC2
mutants, we investigated whether it was also the case in motor neurons.
Activated-caspase 3 staining was performed 2 days after electroporation
(Figure 4C). The percentage of activated-caspase3 positive neurons in
electroporated neurons (i.e. GFP positive) indicated that after 2 days,
apoptosis was increased in MORC2 p424 (199 %), p444 (239 %), p446 (235
%) compared to WT MORC2 (100%) electroporated neurons.
MORC2 affect cortical neuron neurite
outgrowth
Axonal forms of CMT and SMA affect axonal growth or maintenance in
patients. To investigate the impact of MORC2 mutations on the axonal
compartment, we evaluated neurite outgrowth in neurons electroporated
with the eGFP reporter gene (Figure 4C). Box plot of the results showed
a significant reduction of neurite outgrowth in neurons expressing p424
(mean: 87.96), p444 (53.37) and p446 (66.43) compared to neurons
expressing WT MORC2 (107.85). Altogether, these results show that MORC2
mutants interfere with neurite outgrowth, suggesting that they cause
axonal stress. This supports the conclusion drawn from survival and
apoptosis experiments performed in SH-EP and motor neurons that
indicated that the p444 and p446 variants of unknown significance are
probably pathogenic.