Discussion
Here we report the use of in vitro models to assess the
pathogenicity of variants of unknown significance in the MORC2gene. To this end, we have used the human neuroblastoma cell line SH-EP
which is a proliferative and easy to transfect cell line, and the
primary cortical neurons which are abundant and easy to electroporate.
We found that in SH-EP line the overexpression of a pathogenic MORC2
mutant significantly decreased survival and increased apoptosis. The
same effect was observed in primary cortical neurons in which we found
that neurite outgrowth was also significantly reduced.
A previous study performed in rat sensory neurons showed that the pS87L
mutation in MORC2 which is associated with a severe SMA like phenotype
caused axonal swelling (Sancho et al., 2019). To date, only one in
vitro cell model has been developed to establish the pathogenicity ofde novo variants of the MORC2 gene (Guillen Sacoto et al.,
2020). This model is based on the ability of MORC2 to regulate the
activity of the HUSH complex involved in epigenetic silencing (Liu et
al., 2017; Tchasovnikarova et al., 2017). This approach was performed on
a population of FACS-isolated HeLa cells that displayed epigenetic
repression of an integrated GFP reporter by the HUSH complex. The
knockout of the MORC2 gene de-repressed the GFP reporter gene and
the ability of transfected MORC2 constructs to restore repression GFP
repression by the HUSH complex was evaluated by FACS (Tchasovnikarova et
al., 2017; Douse et al., 2018; Guillen Sacoto et al., 2020). Altogether,
these studies provided a precise evaluation of the regulatory function
of MORC2 mutants in the HUSH complex but did not take into account
possible other biological functions and revealed the necessity to
develop simple in vitro models applicable to the analysis of many
variants in the context of hospital facilities to help clinicians to
quickly investigate new variants and establish molecular diagnosis.
We have identified two different mutations in the MORC2 gene in
patients from two unrelated families. Patients from the first family
presented a form of young adult-onset, autosomal dominant axonal
neuropathy with progressive weakness and mild sensory impairment. The
overall clinical manifestations of the present patients resemble those
in the original paper by Sevilla et al. describing the p.R252W and
p.S87L mutations, but with a less prominent sensory loss at neurological
examination and a slightly later age of onset. Some patients have been
identified with early, SMA-like disease onset and primary involvement of
the proximal muscles (Albulym et al., 2016; Laššuthová et al., 2016;
Schottmann et al., 2016; Sevilla et al., 2016). Patients we identified
in the second family present with late-onset distal SMA clinical
features without any sign of sensory nerve involvement. Although the
involvement of the sensory nerves was an initial and prominent feature,
especially in patients with SMA-like phenotypes (Sevilla et al., 2016),
some studies reported minor or even none sensory complaints (Albulym et
al., 2016; Laššuthová et al., 2016; Schottmann et al., 2016). The main
clinical characteristic of MORC2 mutated patients is the
spreading of muscle weakness to proximal muscles. It is observed in the
majority of patients and leads to severe weakness and walking
difficulties (Sevilla et al., 2016; Semplicini et al., 2017). In some
patients, the clinical phenotype tends to be more complex since it may
include symptoms of the central nervous system (CNS), which comprise
pyramidal signs, neurodevelopmental disorders with growth retardation
and cerebellar atrophy (Albulym et al., 2016; Schottmann et al., 2016;
Guillen Sacoto et al., 2020). Altogether, MORC2 is associated
with a broad genetic and phenotypic variability that is challenging for
neurologists. Interestingly, apart from SMN only a few genes can
cause SMA-like phenotypes in adults: TRPV4, CHCHD10; BICD2,
DYNHC1H1, SETX, VRK1, VAPB and ASAH1 genes have been reported (Peeters
et al., 2014). Our study identifies a new variant of MORC2 associated
with a SMA-like phenotype in adults, thus increasing the number of
patients with such phenotype. This suggests that MORC2 should be
included not only in the panel of genes involved in CMT but also in
searches for genes causing proximal SMA. To some extend, considering the
broad range of phenotypes associated with MORC2 mutations, MORC2 should
also be considered for other panels such as spinocerebellar ataxia, or
neurodevelopmental disorders.