Introduction
MORC2 gene encodes a member of the Microrchidia (MORC) protein
superfamily well conserved among higher Eukaryotes (Inoue, 1999).MORC2 gene is ubiquitously expressed, with the highest expression
in testis, ovary and brain. RT-PCR ELISA shows moderate expression in
lungs, kidneys, liver, and heart, and an even lower expression in
skeletal muscle, pancreas, and spleen (Nagase et al., 1998).
Transcriptomic analysis in mice indicates that Morc2 expression
undergoes spatiotemporal regulation in the brain with a peak expression
at the earlier developmental stages followed by a progressive decrease
during aging suggesting an important role in the development of the
nervous systems (Sancho et al., 2019). MORC2 protein was initially shown
to regulate transcriptional repression, invasiveness, and lipogenesis in
cancer cells (Shao et al., 2010; Sánchez-Solana et al., 2014; Liao et
al., 2017). MORC2 was then shown to relax chromatin and facilitate DNA
double-strand break repair via a DNA-dependent ATPase activity (Li et
al., 2012). More recently, MORC2 was shown to be involved in epigenetic
silencing by the human silencing hub (HUSH) complex (Tchasovnikarova et
al., 2017; Douse et al., 2018).
Heterozygous mutations in MORC2 gene are associated with a
spectrum of disorders affecting the peripheral nervous system. Most
clinical manifestations of MORC2 mutations are associated with an
axonal form of Charcot-Marie-Tooth disease (CMT2Z; OMIM: 616688)
(Albulym et al., 2016; Laššuthová et al., 2016; Sevilla et al., 2016;
Zhao et al., 2016; Semplicini et al., 2017). The main clinical features
of the disease encompass slowly progressive distal weakness, muscle
atrophy associated with sensory impairment, typically occurring during
childhood or adolescence. However, some phenotypic variability exists
among MORC2 mutated CMT patients, some of them exhibiting hearing
loss (Albulym et al., 2016; Sevilla et al., 2016), pyramidal signs and
seizure (Albulym et al., 2016). Occasionally, MORC2 mutations
produce early-onset spinal muscular atrophy-like (SMA-like) phenotypes
characterized by proximal muscle and atrophy without sensory loss with
or without diaphragmatic palsy (Schottmann et al., 2016; Zanni et al.,
2017), microcephaly (Sevilla et al., 2016; Zanni et al., 2017), or
cerebellar atrophy (Schottmann et al., 2016). Recently, 20 individuals
with heterozygous MORC2 mutations were shown to develop a
neurodevelopmental syndrome associated with intellectual disability,
growth retardation, facial dysmorphism, and axonal neuropathy (DIGFAN;
OMIM: 619090) (Guillen Sacoto et al., 2020). The pathophysiological
mechanisms underlying such phenotypic variability cannot be correlated
to specific mutated amino acids and remain unclear. Furthermore, with
the increasing number of variants of unknown significance identified by
NGS, clinicians need to develop a functional assay to decipher between
pathological variants and polymorphism.
Here, to evaluate the pathogenicity of MORC2 variants of unknown
significance, we have developed an in vitro system based on the
overexpression of wild-type (WT) or mutant MORC2 proteins in a
neuroblastoma cell line SH-EP and in primary cortical neurons. By
quantifying survival and apoptosis over time, we show significant
differences between WT and well-characterized MORC2 mutants which allow
to predict the pathogenicity of two new variants c.1330G>A
(p.G444R) and c.1338C>A (p.H446Q) of the MORC2 gene
respectively associated with an autosomal dominant form of CMT and with
an adult late onset SMA-like phenotype.