Homozygous COQ7 mutation: a new cause of potentially treatable distal hereditary motor neuropathy.

Distal hereditary motor neuropathy represents a group of motor inherited neuropathies leading to distal weakness. We report a family of two brothers and a sister affected by distal hereditary motor neuropathy in whom a homozygous variant c.3G>T (p.1Met?) was identified in the COQ7 gene. This gene encodes a protein required for coenzyme Q10 biosynthesis, a component of the respiratory chain in mitochondria. Mutations of COQ7 were previously associated with severe multi-organ disorders characterized by early childhood onset and developmental delay. Using patient blood samples and fibroblasts derived from a skin biopsy, we investigated the pathogenicity of the variant of unknown significance c.3G>T (p.1Met?) in the COQ7 gene and the effect of coenzyme Q10 supplementation in vitro. We showed that this variation leads to a severe decrease in COQ7 protein levels in the patient's fibroblasts, resulting in a decrease in coenzyme Q10 production and in the accumulation of 6-demethoxycoenzyme Q10, the COQ7 substrate. Interestingly, such accumulation was also found in the patient's plasma. Normal coenzyme Q10 and 6-demethoxycoenzyme Q10 levels were restored in vitro by using the coenzyme Q10 precursor 2,4-dihydroxybenzoic acid, thus bypassing the COQ7 requirement. Coenzyme Q10 biosynthesis deficiency is known to impair the mitochondrial respiratory chain. Seahorse experiments showed that the patient's cells mainly rely on glycolysis to maintain sufficient ATP production. Consistently, the replacement of glucose by galactose in the culture medium of these cells reduced their proliferation rate. Interestingly, normal proliferation was restored by coenzyme Q10 supplementation of the culture medium, suggesting a therapeutic avenue for these patients. Altogether, we have identified the first example of recessive distal hereditary motor neuropathy caused by a homozygous variation in the COQ7 gene, which should thus be included in the gene panels used to diagnose peripheral inherited neuropathies. Furthermore, 6-demethoxycoenzyme Q10 accumulation in the blood can be used to confirm the pathogenic nature of the mutation. Finally, supplementation with coenzyme Q10 or derivatives should be considered to prevent the progression of COQ7-related peripheral inherited neuropathy in diagnosed patients.

Repurposing pentamidine using hyaluronic acid-based nanocarriers for skeletal muscle treatment in myotonic dystrophy.

In a context of drug repurposing, pentamidine (PTM), an FDA-approved antiparasitic drug, has been proposed to reverse the splicing defects associated in myotonic dystrophy type 1 (DM1). However, clinical use of PTM is hinder by substantial toxicity, leading to find alternative delivery strategies. In this work we proposed hyaluronic acid-based nanoparticles as a novel encapsulation strategy to efficiently deliver PTM to skeletal muscles cells. In vitro studies on C2C12 myoblasts and myotubes showed an efficient nanoparticles' internalization with minimal toxicity. More interestingly, our findings evidenced for the first time the endosomal escape of hyaluronic acid-based nanocarriers. Ex vivo studies showed an efficient nanoparticles' internalization within skeletal muscle fibers. Finally, the therapeutic efficacy of PTM-loaded nanosystems to reduce the number of nuclear foci has been demonstrated in a novel DM1 in vitro model. So far, current data demonstrated the potency of hyaluronic acid-based nanosystems as efficient nanocarrier for delivering PTM into skeletal muscle and mitigate DM1 pathology.

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons.

Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

Expanding the phenotypic variability of MORC2 gene mutations: From Charcot-Marie-Tooth disease to late-onset pure motor neuropathy.

MORC2 gene encodes a ubiquitously expressed nuclear protein involved in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous mutations in MORC2 gene have been associated with a spectrum of disorders affecting the peripheral nervous system such as Charcot-Marie-Tooth (CMT2Z), spinal muscular atrophy-like with or without cerebellar involvement, and a developmental syndrome associated with impaired growth, craniofacial dysmorphism and axonal neuropathy (DIGFAN syndrome). Such variability in clinical manifestations associated with the increasing number of variants of unknown significance detected by next-generation sequencing constitutes a serious diagnostic challenge. Here we report the characterization of an in vitro model to evaluate the pathogenicity of variants of unknown significance based on MORC2 overexpression in a neuroblastoma cell line SH-EP or cortical neurons. Likewise, we show that MORC2 mutants affect survival and trigger apoptosis over time in SH-EP cell line. Furthermore, overexpression in primary cortical neurons increases apoptotic cell death and decreases neurite outgrowth. Altogether, these approaches establish the pathogenicity of two new variants p.Gly444Arg and p.His446Gln in three patients from two families. These new mutations in MORC2 gene are associated with autosomal dominant CMT and with adult late onset proximal motor neuropathy, further increasing the spectrum of clinical manifestations associated with MORC2 mutations.

MiR-422a promotes loco-regional recurrence by targeting NT5E/CD73 in head and neck squamous cell carcinoma.

At the time of diagnosis, 60% of patients with head and neck squamous cell carcinoma (HNSCC) present tumors in an advanced stage (III-IV) of disease and 80% will relapse within the first two years post-treatment, due to their frequent radio(chemo)resistance. To identify new molecular targets and companion biomarkers, we have investigated the miRNome of 75 stage III-IV oropharynx tumors without relapse (R) or with loco-regional relapse (non-responder, NR) within two years post-treatment. Interestingly, miR-422a was significantly downregulated in NR tumors, in agreement with the increase in cell proliferation and adhesion induced by miR-422a inhibition in vitro. Furthermore, we identified CD73/NT5E oncogene as target of miR-422a. Indeed, modulation of the endogenous level of miR-422a inversely influences the expression and the enzymatic activity of CD73. Moreover, knocking down CD73 mimics the effects of miR-422a upregulation. Importantly, in tumors, miR-422a and CD73 expression levels are inversely correlated, and both are predictive of relapse free survival - especially considering loco(regional) recurrence - in vitro two independent cohorts of advanced oropharynx or HNSCC (N=255) tumors. In all, we reported, for the first time, that MiR-422a and its target CD73 are involved in early loco(regional) recurrence of HNSCC tumors and are new targets for personalized medicine.

Telomere profiling: toward glioblastoma personalized medicine.

Despite a standard of care combining surgery, radiotherapy (RT), and temozolomide chemotherapy, the average overall survival (OS) of glioblastoma patients is only 15 months, and even far lower when the patient cannot benefit from this combination. Therefore, there is a strong need for new treatments, such as new irradiation techniques. Against this background, carbon ion hadrontherapy, a new kind of irradiation, leads to a greater biological response of the tumor, while minimizing adverse effects on healthy tissues in comparison with RT. As carbon ion hadrontherapy is restricted to RT-resistant patients, photon irradiation resistance biomarkers are needed. Long telomeres and high telomerase activity have been widely associated with photon radioresistance in other cancers. Moreover, telomere protection, telomere function, and telomere length (TL) also depend on the shelterin protein complex (TRF1, TRF2, TPP1, POT1, TIN2, and hRAP1). We thus decided to evaluate an enlarged telomeric status (TL, telomerase catalytic subunit, and the shelterin component expression level) as a potential radioresistance biomarker in vitro using cellular models and ex vivo using patient tumor biopsies. In addition, nothing was known about the role of telomeres in carbon ion response. We thus evaluated telomeric status after both types of irradiation. We report here a significant correlation between TL and the basal POT1 expression level and photon radioresistance, in vitro, and a significant increase in the OS of patients with long telomeres or a high POT1 level, in vivo. POT1 expression was predictive of patient response irrespective of the TL. Strikingly, these correlations were lost, in vitro, when considering carbon irradiation. We thus propose (1) a model of the implications of telomeric damage in the cell response to both types of irradiation and (2) assessment of the POT1 expression level and TL using patient tumor biopsies to identify radioresistant patients who could benefit from carbon hadrontherapy.