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.