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.