CMT Type Research Articles

Sensory-Motor Neuropathy in Mfn2 T105M Knock-in Mice and Its Reversal by a Novel Piperine-Derived Mitofusin Activator.

Mitochondrial dysfunction is a hallmark of many genetic neurodegenerative diseases, but therapeutic options to reverse mitochondrial dysfunction are limited. While recent studies support the possibility of improving mitochondrial fusion/fission dynamics and motility to correct mitochondrial dysfunction and resulting neurodegeneration in Charcot-Marie-Tooth disease (CMT) and other neuropathies, the clinical utility of reported compounds and relevance of preclinical models are uncertain. Here, we describe motor and sensory neuron dysfunction characteristic of clinical CMT type 2 A in a CRISPR/Casp-engineered Mfn2 Thr105Met (T105M) mutant knock-in mouse. We further demonstrate that daily oral treatment with a novel mitofusin activator derived from the natural product piperine can reverse these neurologic phenotypes. Piperine derivative 8015 promoted mitochondrial fusion and motility in Mfn2-deficient cells in a mitofusin-dependent manner and reversed mitochondrial dysfunction in cultured fibroblasts and reprogrammed motor neurons from a human CMT2A patient carrying the MFN2 T105M mutation. Like previous mitofusin activators, 8015 exhibited stereospecific functionality, but the more active stereoisomer, 8015-P2, is unique in that it has subnanomolar potency and undergoes entero-hepatic recirculation which extends its in vivo half-life. Daily administration of 8015-P2 to Mfn2 T105M knock-in mice for 6 weeks normalized neuromuscular and sensory dysfunction and corrected histological/ultrastructural neurodegeneration and neurogenic myoatrophy. These studies describe a more clinically relevant mouse model of CMT2A and an improved mitofusin activator derived from piperine. We posit that 8015-P2 and other piperine derivatives may benefit CMT2A or other neurodegenerative conditions wherein mitochondrial dysdynamism plays a contributory role. SIGNIFICANCE STATEMENT: Mitochondrial dysfunction is widespread and broadly contributory in neurodegeneration, but difficult to target therapeutically. Here, we describe 8015-P2, a new small molecule mitofusin activator with ∼10-fold greater potency and improved in vivo pharmacokinetics versus comparators, and demonstrate its rapid reversal of sensory and motor neuron dysfunction in an Mfn2 T105M knock-in mouse model of Charcot-Marie-Tooth disease type 2 A. These findings further support the therapeutic approach of targeting mitochondrial dysdynamism in neurodegeneration.

Charcot-Marie-Tooth Type 2 Disease and Relapsing Remitting Multiple Sclerosis Coexistence

Multiple sclerosis (MS) and Charcot-Marie-Tooth disease (CMT) association has been reported, namely in CMTX and CMT1A. A 37-year-old woman with CMT type 2 (MFN2 mutation) developed subacute brainstem syndrome (left internuclear ophthalmoplegia, facial palsy and ataxia). Brain magnetic resonance imaging revealed infratentorial, periventricular and subcortical lesions, typical of MS (positive CSF oligoclonal bands, negative anti-aquaporin 4 and anti-MOG antibodies). Patient underwent natalizumab, reaching NEDA-3 – no evidence of disease activity - (3-years follow-up). Patients with MFN2 mutations may disclose optic atrophy and periventricular white matter T2 signal changes mimicking MS. Notwithstanding, this is, probably, the first report of proven concomitance between both conditions.

Cochlear implantation in patients with Charcot-Marie-Tooth disease: two cases with a review of the literature.

To report two cases of bilateral cochlear implantation (CI) in Charcot-Marie-Tooth disease (CMT) patients with novel mutations. Furthermore, we conducted a detailed literature review on the profile and outcomes of CI in this uncommon clinical circumstance. Case 1 involved a 25-year-old woman who was referred for sudden hearing loss (HL) in her left ear and had a 7-year history of HL in her right ear. She was diagnosed with CMT type 1 with a thymidine phosphorylase gene mutation. CI was performed on her left side because her hearing gradually worsened to deafness in both ears. At 3months post-operation, her speech discrimination score without lip-reading improved from 0 to 100%. She underwent a second CI on her right ear 6months after her first CI. Two years from her first operation, the speech discrimination score was 100%. Case 2 received her first CI on her right ear at the age of nine for her bilateral HL. She was diagnosed with CMT type 2 with a Twinkle mitochondrial DNA helicase gene mutation. Preoperatively, the speech discrimination score in both ear-aided conditions was 70%. At the 7-year post-operation follow-up, the speech discrimination score was 76%. A second CI was performed due to decreasing hearing ability in her left ear. The speech discrimination score showed 100% at 7months after the second CI. CI is an effective hearing rehabilitation option for CMT patients with severe-to-profound SNHL. Neuro-otologists should consider CI as a treatment option, even though hearing loss in CMT is associated with auditory neuropathy spectrum disease (ANSD).

Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

SORDD: mutation frequency and phenotype in predominantly axonal Charcot-Marie-Tooth disease of undefined genetic cause

Pathogenic, biallelic variants in SORD were identified in 2020 as a novel cause for autosomal-recessive Charcot-Marie-Tooth disease (CMT) type 2, an inherited neuropathy. SORD codes for the enzyme sorbitol dehydrogenase. Loss of this enzyme‘s activity leads to an increase of sorbitol in serum. We retrospectively screened 166 patients with axonal neuropathy (predominantly CMT type 2, but including intermediate form of CMT and distal hereditary motor neuropathy (dHMN)) without identified genetic etiology for SORD mutations at a single large German neuromuscular center. Clinical and electrophysiology exam findings were analyzed for genotype–phenotype correlation. Five patients of the total cohort of 166 patients harbored pathogenic variants in SORD (3%). The homozygous frameshift variant c.757delG (p.Ala253Glnfs*27) was the most common (4/5). One additional case carried this variant on one allele only and an additional pathogenic missense variant c.458C > A (p.Ala153Asp) on the other allele. Age of onset ranged from early infancy to mid-twenties, and phenotypes comprised axonal CMT (4) and dHMN (1). Our findings strengthen the importance of screening for pathogenic variants in SORD, especially in patients with genetically unconfirmed axonal neuropathy, especially CMT type 2 and dHMN.

Home-based multi-sensory and proximal strengthening program to improve balance in Charcot-Marie-Tooth disease Type 1A: A proof of concept study.

People with Charcot-Marie-Tooth Disease (CMT) frequently report problems with balance, which lead to an increased risk of falls. Evidence is emerging of training interventions to improve balance for people with CMT, but to date all have relied on clinic-based treatment and equipment. This proof-of-concept study explored whether a multi-modal program of proprioceptive rehabilitation and strength training can be delivered at home, to improve balance performance in people with CMT Type 1A. Fourteen participants with CMT Type 1A were recruited into this randomized, two-arm study. Baseline assessments included measures of disease severity, posturography, physical function, and patient-reported outcome measurements. All participants received one falls education session. Participants were randomized to either 12 weeks of balance training or 12 weeks of usual activities. The intervention comprised a home-based, multi-sensory balance training and proximal strengthening program, supported by three home visits from a physiotherapist. Thirteen participants completed the study. The intervention was successfully implemented and well tolerated, with high participation levels. Functional measures of balance and walking showed strong effect sizes in favor of the training group. Posturography testing demonstrated moderate improvements in postural stability favoring the intervention group. Inconsistent changes were seen in lower limb strength measures. The intervention was feasible to implement and safe, with some evidence of improvement in balance performance. This supports future studies to expand this intervention to larger trials of pragmatic, home-delivered programs through current community rehabilitation services and supported self-management pathways.

Preclinical Efficacy of Peripheral Nerve Regeneration by Schwann Cell-like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells in C22 Mice.

Charcot-Marie-Tooth disease (CMT) is a hereditary disease with heterogeneous phenotypes and genetic causes. CMT type 1A (CMT1A) is a type of disease affecting the peripheral nerves and is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene. Human tonsil-derived mesenchymal stem cells (TMSCs) are useful for stem cell therapy in various diseases and can be differentiated into Schwann cell-like cells (TMSC-SCs). We investigated the potential of TMSC-SCs called neuronal regeneration-promoting cells (NRPCs) for peripheral nerve and muscle regeneration in C22 mice, a model for CMT1A. We transplanted NRPCs manufactured in a good manufacturing practice facility into the bilateral thigh muscles of C22 mice and performed behavior and nerve conduction tests and histological and ultrastructural analyses. Significantly, the motor function was much improved, the ratio of myelinated axons was increased, and the G-ratio was reduced by the transplantation of NRPCs. The sciatic nerve and gastrocnemius muscle regeneration of C22 mice following the transplantation of NRPCs downregulated PMP22 overexpression, which was observed in a dose-dependent manner. These results suggest that NRPCs are feasible for clinical research for the treatment of CMT1A patients. Research applying NRPCs to other peripheral nerve diseases is also needed.

Experience with the management of 2599 cases of congenital muscular torticollis and a multicenter epidemiological investigation in 17 hospitals in China

BackgroundCongenital muscular torticollis (CMT) is a common musculoskeletal disease affecting infants and young children. If CMT is not treated correctly and timely, it can lead to limited head and neck movements, head and neck deviation, and abnormal posture. In order to improve patients' symptoms and alleviate the negative impact of the disease on their lives, we are committed to exploring the treatment of CMT.MethodsThe general clinical and ultrasonographic data of 2599 children with CMT who received standardized treatment at Shenzhen Children’s Hospital from 2004 to 2020 were retrospectively reviewed. According to given treatment, children with CMT were divided into the physiotherapy group, physiotherapy combined with glucocorticoid treatment group, and surgical treatment group. We divided children with CMT into local mass, uniform thickening, and atrophy according to ultrasound features. General clinical information, treatment, and ultrasound examination data in each group were compared. Additionally, electronic medical records of 2344 patients admitted due to CMT in 17 tertiary children’s hospitals of China’s Futang Research Center of Pediatric Development (FRCPD) from 2015 to 2019 were retrospectively analyzed. Data on sex, age, year of admission and discharge, and treatment costs during hospitalization were extracted from the first medical record pages according to the ICD codes. The data were assessed for normality using the Kolmogorov–Smirnov test. Depending on the data distribution, they were analyzed using parametric tests, such as the t-test, or non-parametric tests. Qualitative data are expressed as percentages (%) and analyzed using the chi-square or Fisher’s exact probability test, with α = 0.05 as the test level. P < 0.05 was considered to be indicative of a statistically significant difference.ResultsThree types of CMT were defined based on sternocleidomastoid muscle ultrasound examination characteristics: local mass, uniform thickening, and atrophy. Age at first diagnosis was 69.21 ± 108.41 days in local mass type group, 216.85 ± 324.09 days in uniform thickening group, and 417.88 ± 739.05 days in atrophy- type group; while age at first physiotherapy use was 94.06 ± 206.49 days, 255.00 ± 430.62 days, 540.92 ± 1059.29 respectively. The children included in local mass type group have shown a high success rate of conservative treatment, with a rate of 7.5% of children underwent surgery. Age at first diagnosis was 112.44 ± 224.12 days in the physiotherapy group, 115.87 ± 144.86 days in the physiotherapy combined with glucocorticoid treatment subgroup, whereas the age at first physiotherapy use was 137.38 ± 312.11 and 196.91 ± 344.26 days respectively. In the observation period (2015–2019) the mean age at surgery for CMT in 17 tertiary children’s hospitals of the FRCPD was 50 months. Overall, 663 children with CMT were 1–2 years of age, accounting for the largest proportion (28.3%). Followed by 417 individuals (17.8%) were 7–14 years of age, indicating that there are still more children with CMT receiving surgical treatment later.ConclusionsEarly diagnosis and treatment are essential to improve the conservative treatment success rate and achieve good prognosis in children with CMT. Our team’s concept for treating CMT is as follows: after diagnosing the children, we will adopt the standardized protocol of treatment, with physiotherapy combined with the injection of glucocorticoid drugs and SCM release surgery, when needed. This program has a high conservative treatment success rate and may facilitate the achievement of better prognosis and reduced teratogenicity rate.

Genetic study of inherited peripheral neuropathy disease (Charcot–Marie Tooth) disease

Background Charcot-Marie-Tooth disease (CMT) is the most common hereditary neuromuscular disorder. Charcot-Marie-Tooth disease can be caused by mutations in many different genes. These genes provide instructions for making proteins involved in the functioning of the peripheral nerves of the feet, legs, and hands. Peripheral myelin protein 22 (PMP22) related neuropathies account for over 50% of inherited peripheral neuropathies. Agene copy variation results in CMT1A (duplication of 17p11.2.p12) and hereditary neuropathy with liability to pressure palsies (HNPP; single deletion). Point mutation comprise both phenotypes. The underlying pathological mechanisms are incompletely understood and biallilic mutations of PMP22 are very rare. Objective To study clinical characteristics of a cohort of Egyptian patients with CMT; frequency of PMP22 gene duplication and deletion by MLPA and molecular diagnosis. Methodology We applied our study on 97 Egyptian patients from 70 unrelated families with suspected CMT disease, patients were referred from different provinces to Ain Shams neuromuscular outpatient clinic. After an informed and written consent, all patients were examined neurologically and systemically. Diagnosis was based on clinical symptoms, electromyography and genetic study. Results Clinical classification of our patients revealed high incidence rate of AR CMT type patients (43.3%) and sporadic cases (39.3%), while AD CMT type represents (17.5%), the cause of the high rate of negative family history of same disease affection may be for many reasons mild subclinical expression in other family members, autosomal recessive inheritance, or a de novo heterozygous pathogenic variant in a gene associated with autosomal dominant inheritance. Neurophysiological classification according to median NCV shows that 53 patients (51.5%) have axonal sensory-motor affection with median NCV &amp;lt;38 m/s, 26 patients (26.8%) have demyelinating CMT type, and 19 patients (19.6%) have intermediate type (mixed axonal and demyelinating affection with median NCV range between 35 m/s to 45 m/s. Thus the commonest form is CMT 2 (axonal AR type) followed by CMT 4 (demyelinating AR type) with lower incidence of CMT 1 (demyelinating AD type). Conclusion from our study we concluded that (AR CMT type) is not uncommon in Egypt because of the high prevalence of consanguinity, and that PMP22 duplication is not a common cause of Egyptian inherited neuropathy disease.

Myelin protein zero mutation-related hereditary neuropathies: Neuropathological insight from a new nerve biopsy cohort.

Myelin protein zero (MPZ/P0) is a major structural protein of peripheral nerve myelin. Disease-associated variants in the MPZ gene cause a wide phenotypic spectrum of inherited peripheral neuropathies. Previous nerve biopsy studies showed evidence for subtype-specific morphological features. Here, we aimed at enhancing the understanding of these subtype-specific features and pathophysiological aspects of MPZ neuropathies. We examined archival material from two Central European centers and systematically determined genetic, clinical, and neuropathological features of 21 patients with MPZ mutations compared to 16 controls. Cases were grouped based on nerve conduction data into congenital hypomyelinating neuropathy (CHN; n = 2), demyelinating Charcot-Marie-Tooth (CMT type 1; n = 11), intermediate (CMTi; n = 3), and axonal CMT (type 2; n = 5). Six cases had combined muscle and nerve biopsies and one underwent autopsy. We detected four MPZ gene variants not previously described in patients with neuropathy. Light and electron microscopy of nerve biopsies confirmed fewer myelinated fibers, more onion bulbs and reduced regeneration in demyelinating CMT1 compared to CMT2/CMTi. In addition, we observed significantly more denervated Schwann cells, more collagen pockets, fewer unmyelinated axons per Schwann cell unit and a higher density of Schwann cell nuclei in CMT1 compared to CMT2/CMTi. CHN was characterized by basal lamina onion bulb formation, a further increase in Schwann cell density and hypomyelination. Most late onset axonal neuropathy patients showed microangiopathy. In the autopsy case, we observed prominent neuromatous hyperinnervation of the spinal meninges. In four of the six muscle biopsies, we found marked structural mitochondrial abnormalities. These results show that MPZ alterations not only affect myelinated nerve fibers, leading to either primarily demyelinating or axonal changes, but also affect non-myelinated nerve fibers. The autopsy case offers insight into spinal nerve root pathology in MPZ neuropathy. Finally, our data suggest a peculiar association of MPZ mutations with mitochondrial alterations in muscle.

Characteristic 3D foot motion patterns during gait of patients with Charcot-Marie-Tooth identified by cluster analysis

BackgroundCMT is a clinically and genetically heterogenous disease with varying degrees of progression. Different foot deformities, gait and movement patterns are observed. In order to achieve an improved, targeted treatment strategy, the participants are divided into characteristic groups using a mathematical cluster analysis based on the data from the three-dimensional foot kinematics during walking. MethodsOutpatients from age 5–64 years (N = 33 participants, 62 feet) with a proven CMT type 1 (N = 16, 31 feet) or CMT without any further type assignment (N = 17, 31 feet) were retrospectively analyzed. After a standard clinical examination, participants underwent 3D gait analysis using the Oxford Foot Model. To classify the movement patterns, a k-means cluster analysis was calculated based on the principal component analysis (PCA) of the foot kinematics data. Gait parameters, clinical parameters and X-ray data were statistically tested. ResultsThe cluster analysis divided the gait data of the participants into two groups. Cluster 1 (N = 21 participants, 34 feet) showed increased dorsiflexion of the hindfoot and increased plantarflexion of the forefoot with cavus position in the sagittal plane, a hindfoot inversion and forefoot pronation with hindfoot varus in the frontal plane and in the transversal plane a forefoot adduction. Cluster 2 (N = 17 participants, 28 feet) deviated significantly from the norm mainly in the frontal plane and were characterized by a strong eversion of the hindfoot with a supination in the forefoot. DiscussionBased on the findings, the resultant clusters can be interpreted as cavovarus feet (cluster 1) and pes valgus (cluster 2). The most reliable variables in the 3D gait analysis to classify CMT feet with regard to significance are the ones in the frontal plane.This subdivision of participants goes hand in hand with the various necessary guidelines for orthopedic treatment.

Boosting peripheral BDNF rescues impaired in vivo axonal transport in CMT2D mice.

Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterizing Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor tropomyosin receptor kinase receptor B (TrkB), underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice displayed early and persistent disturbances in axonal transport of neurotrophin-containing signaling endosomes in vivo. We discovered that brain-derived neurotrophic factor (BDNF)/TrkB impairments correlated with transport disruption and overall CMT2D neuropathology and that inhibition of this pathway at the nerve-muscle interface perturbed endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restored physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.

Natural history of ankle function during gait in youth with Charcot-Marie-Tooth disease types 1 and 2

BackgroundCharcot-Marie-Tooth disease (CMT) can cause progressive muscle weakness and contracture, leading to gait abnormalities such as increased and delayed peak ankle dorsiflexion and reduced ankle power generation in terminal stance. Understanding strength loss on ankle function during gait is important for interpreting treatment outcomes and evaluating new therapies designed to improve gait. Research questionDo ankle kinematics and kinetics vary as a function of age, disease progression with associated loss of muscle strength and CMT type in youth with CMT types 1 and 2? MethodsA prospective convenience sample of 45 participants with CMT1 and 2, ages 7–22 years, underwent comprehensive gait analysis. Seventeen patients underwent repeat analyses totaling 67 tests. Generalized mixed effects linear modeling was used to compare CMT1 versus CMT2 and to examine the effects of age on ankle strength, range of motion, kinematics, and kinetics within each CMT type. ResultsPlantarflexor and dorsiflexor strength were less in CMT2 compared with CMT1 (p ≤ 0.05), while peak dorsiflexion in terminal stance (TST) was greater (p = 0.02). Peak plantarflexion moment and power generation were also less in CMT2 (p ≤ 0.02). In CMT1, peak dorsiflexion in TST increased with age through 13 years (p = 0.004); then plateaued in the normal range (p = 0.73). Peak ankle angle in mid-swing was closely related to the angle in TST (p < 0.001) following a similar pattern with age. In CMT2, no significant associations were observed between age, peak dorsiflexion in TST, and peak ankle angle in mid-swing (p ≥ 0.19). There were no consistent trends with age for individual patients with repeat tests. SignificanceThe heterogeneity of joint level impairments and gait kinematics and kinetics point to the importance of having an in-depth understanding of gait at the individual patient level using comprehensive gait analysis including valid and reliable strength measures.

Clinical phenotypic diversity of NOTCH2NLC-related disease in the largest case series of inherited peripheral neuropathy in Japan

BackgroundNOTCH2NLC GGC repeat expansions have been associated with various neurogenerative disorders, including neuronal intranuclear inclusion disease and inherited peripheral neuropathies (IPNs). However, only a few NOTCH2NLC-related disease studies in IPN...

Gene Repair of iPSC Line with GARS (G294R) Mutation of CMT2D Disease by CRISPR/Cas9.

Charcot-Marie-Tooth disease (CMT) severely affects patient activity, and may cause disability. However, no clinical treatment is available to reverse the disease course. The combination of CRISPR/Cas9 and iPSCs may have therapeutic potential against nervous diseases, such as CMT. In the present study, the skin fibroblasts of CMT type 2D (CMT2D) patients with the c.880G>A heterozygous nucleotide mutation in the GARS gene were reprogrammed into iPSCs using three plasmids (pCXLE-hSK, pCXLE-hUL and pCXLE-hOCT3/4-shp5-F). Then, CRISPR/Cas9 technology was used to repair the mutated gene sites at the iPSC level. An iPSC line derived from the GARS (G294R) family with fibular atrophy was successfully induced, and the mutated gene loci were repaired at the iPSC level using CRISPR/Cas9 technology. These findings lay the foundation for future research on drug screening and cell therapy. iPSCs can differentiate into different cell types, and originate from autologous cells. Therefore, they are promising for the development of autologous cell therapies for degenerative diseases. The combination of CRISPR/Cas9 and iPSCs may open a new avenue for the treatment of nervous diseases, such as CMT.

The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy

BackgroundClinically, Charcot-Marie-Tooth disease (CMT)-associated muscle atrophy still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT type 4F (CMT4F) by destroying the myelin sheath form, which may be related to the inhibitory role of Ezrin in the self-association of L-periaxin. However, it is still unknown whether L-periaxin and Ezrin are independently or interactively involved in the process of muscle atrophy by affecting the function of muscle satellite cells.MethodA gastrocnemius muscle atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells were treated with adenovirus-mediated overexpression or knockdown of Ezrin. Then, overexpression of L-periaxin and NFATc1/c2 or knockdown of L-periaxin and NFATc3/c4 mediated by adenovirus vectors were used to confirm their role in Ezrin-mediated myoblast differentiation, myotube formation and gastrocnemius muscle repair in a peroneal nerve injury model. RNA-seq, real-time PCR, immunofluorescence staining and Western blot were used in the above observation.ResultsFor the first time, instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/fusion in vitro. In vivo transduction of adenovirus vectors carrying Ezrin, but not Periaxin, into the gastrocnemius muscle in a peroneal nerve injury model increased the numbers of muscle myosin heavy chain (MyHC) I and II type myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of overexpressed Ezrin combined with incubation of knockdown L-periaxin within the injured peroneal nerve or injection of knockdown L-periaxin into peroneal nerve-injured gastrocnemius muscle not only increased the number of muscle fibers but also recovered their size to a relatively normal level in vivo. Overexpression of Ezrin promoted myoblast differentiation/fusion, inducing increased MyHC-I+ and MyHC-II + muscle fiber specialization, and the specific effects could be enhanced by the addition of adenovirus vectors for knockdown of L-periaxin by shRNA. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by knockdown of Ezrin by shRNA in vitro but decreased myotube length and size. Mechanistically, overexpressing Ezrin did not alter protein kinase A gamma catalytic subunit (PKA-γ cat), protein kinase A I alpha regulatory subunit (PKA reg Iα) or PKA reg Iβ levels but increased PKA-α cat and PKA reg II α levels, leading to a decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the effects of overexpressing-Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by an increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, overexpressing Ezrin enhanced type I muscle fiber specialization, accompanied by an increase in NFATc2/c3 levels and a decrease in NFATc1 levels. Furthermore, overexpressing NFATc2 or knocking down NFATc3 reversed the inhibitory effects of Ezrin knockdown on myoblast differentiation/fusion.ConclusionsThe spatiotemporal pattern of Ezrin/Periaxin expression was involved in the control of myoblast differentiation/fusion, myotube length and size, and myofiber specialization, which was related to the activated PKA-NFAT-MEF2C signaling pathway, providing a novel L-Periaxin/Ezrin joint strategy for the treatment of muscle atrophy induced by nerve injury, especially in CMT4F.

Novel Variants in MPV17, PRX, GJB1, and SACS Cause Charcot-Marie-Tooth and Spastic Ataxia of Charlevoix-Saguenay Type Diseases.

Charcot-Marie-Tooth disease (CMT) and autosomal recessive spastic ataxia of Charlevoix-Saguenay type (ARSACS) are large heterogeneous groups of sensory, neurological genetic disorders characterized by sensory neuropathies, muscular atrophies, abnormal sensory conduction velocities, and ataxia. CMT2EE (OMIM: 618400) is caused by mutations in MPV17 (OMIM: 137960), CMT4F (OMIM: 614895) is caused by PRX (OMIM: 605725), CMTX1 (OMIM: 302800) is caused by mutations in GJB1 (OMIM: 304040), and ARSACS (OMIM: 270550) is caused by mutations in SACS (OMIM: 604490). In this study, we enrolled four families: DG-01, BD-06, MR-01, and ICP-RD11, with 16 affected individuals, for clinical and molecular diagnoses. One patient from each family was analyzed for whole exome sequencing and Sanger sequencing was done for the rest of the family members. Affected individuals of families BD-06 and MR-01 show complete CMT phenotypes and family ICP-RD11 shows ARSACS type. Family DG-01 shows complete phenotypes for both CMT and ARSACS types. The affected individuals have walking difficulties, ataxia, distal limb weakness, axonal sensorimotor neuropathies, delayed motor development, pes cavus, and speech articulations with minor variations. The WES analysis in an indexed patient of family DG-01 identified two novel variants: c.83G>T (p.Gly28Val) in MPV17 and c.4934G>C (p.Arg1645Pro) in SACS. In family ICP-RD11, a recurrent mutation that causes ARSACS, c.262C>T (p.Arg88Ter) in SACS, was identified. Another novel variant, c.231C>A (p.Arg77Ter) in PRX, which causes CMT4F, was identified in family BD-06. In family MR-01, a hemizygous missense variant c.61G>C (p.Gly21Arg) in GJB1 was identified in the indexed patient. To the best of our knowledge, there are very few reports on MPV17, SACS, PRX, and GJB1 causing CMT and ARSACS phenotypes in the Pakistani population. Our study cohort suggests that whole exome sequencing can be a useful tool in diagnosing complex multigenic and phenotypically overlapping genetic disorders such as Charcot-Marie-Tooth disease (CMT) and spastic ataxia of Charlevoix-Saguenay type.

MPZ-, GDAP1-, and NEFL-Related Charcot-Marie-Tooth Disease with Diverse Clinical and Electrophysiological Phenotypes

Charcot-Marie-Tooth disease (CMT) is a spectrum of clinically and genetically heterogeneous peripheral neuropathies. CMT can be classified into demyelinating, intermediate, or axonal neuropathy based on clinical, histopathological, and electrophysiological findings. Approximately 140 genes have been reported to be associated with CMT. Mutations in the myelin protein zero (MPZ), ganglioside-induced differentiation related protein 1 (GDAP1), and neurofilament light-chain polypeptide gene (NEFL) genes have been reported to cause all three types of CMT, which is noteworthy because most CMT-related genes cause a single type of neuropathy (either demyelinating or axonal). In contrast, it remains unclear why these genes cause several types of CMT. CMT is presently incurable; however, ongoing attempts to treat CMT with various drugs and dietary supplements have increased the importance of an exact genetic diagnosis for precision medicine. Therefore, it is important to identify the causative mutations and compare the associated clinical characteristics. Taken together, a comparison of causative mutations and clinical features of patients with MPZ, GDAP1, and NEFL mutations will be the first step in understanding how different types of CMT are caused, and will enable a molecular genetic diagnosis. In this review, we describe the clinical, electrophysiological, and genetic characteristics of MPZ-, GDAP1-, and NEFL-related CMT.

I.10 Gene therapy and other novel treatment approaches for CMT

There is still no effective drug treatment available for Charcot-Marie-Tooth disease (CMT). Current management relies on rehabilitation therapy, surgery for skeletal deformities, and symptomatic treatment. The challenge is to find disease-modifying therapies. Several approaches, including gene silencing (by means of ASO, siRNA, CRISPR-Cas9 editing) to counteract the <i>PMP22</i> gene overexpression in the most frequent CMT1A type are under investigation. PXT3003 is the compound in the most advanced phase for CMT1A, as a second phase-III trial is ongoing. Gene therapy to substitute defective genes (particularly in recessive forms associated with loss-of-function mutations) or insert novel ones (e.g., <i>NT3</i> gene) are being developed and tested in animal models. Novel treatment approaches are also aimed at developing compounds acting on pathways important for different CMT types. Modulation of the Neuregulin pathway determining myelin thickness is promising for both hypo-demyelinating and hypermyelinating neuropathies; intervention on Unfolded Protein Response seems effective for rescuing misfolded myelin proteins such as P0 in CMT1B. HDAC6 inhibitors improved axonal transport and ameliorated phenotypes in different CMT models. Other potential therapeutic strategies include targeting macrophages, lipid metabolism, and Nav1.8 sodium channel in demyelinating CMT and the P2X7 receptor, which regulates calcium influx into Schwann cells, in CMT1A. Further approaches are aimed at correcting metabolic abnormalities, including the accumulation of sorbitol caused by biallelic mutations in the sorbitol dehydrogenase (SORD) gene and of neurotoxic glycosphingolipids in HSN1.

P.220 Neuropsychological functioning in CMT type 2Z: A case report of language deficits masquerading as intellectual disability

CMT type 2Z is an increasingly studied form of CMT caused by mutation in the microrchidia family CW-type zinc finger 2 gene (<i>MORC2</i>). Intellectual disability is commonly reported in the small available literature in individuals with CMT2Z; however, neuropsychological data is lacking in this population, and especially in children. Here we present a case report of an 8-year-old boy with genetically confirmed CMT2Z due to a <i>de novo</i> pathogenic variant in the <i>MORC2</i> gene. This boy was referred for comprehensive neuropsychological evaluation by his neuromuscular multidisciplinary team. Results revealed a pattern of strengths and weaknesses consistent with parent report of developmental, language, and academic difficulties. However, overall thinking skills were highly variable, with Average visual and visuospatial skills in contrast to impaired verbal, working memory, and processing speed skills (WISC-V FSIQ=70, GAI=81; VCI=65, VSI=100, FRI=97, WMI=69, PSI=49). Level of independence day-to-day as rated by his mother fell in the impaired range (Vineland-3 ABC=67). Academic achievement was also impaired across skills (WIAT-III Early Reading Skills SS=40, Single Word Reading SS=52, Alphabet Writing Fluency SS=72, Spelling SS=51, Math Problem Solving SS=51, Numerical Operations SS=68). No clinically significant concerns were endorsed by his mother in terms of social-emotional functioning (i.e., externalizing, internalizing, or behavioral symptoms). Diagnostically, this child presents with a complex picture. Impairments in adaptive functioning raise concern for intellectual disability; however, given this child's uneven intellectual profile, with Average visual/visuospatial skills compared to impaired verbal skills, and impairments in virtually all other receptive and expressive language skills measured on exam, observed qualitatively, and reported by his mother, the most appropriate diagnosis is a language disorder. Children with language disorders have persistent difficulties in use of language due to deficits in production including reduced vocabulary, limited sentence structure, and impairments in discourse. Children with language disorders also have difficulties learning adaptive skills and with academic learning, which is also consistent with this child's profile. More research is needed, but the neurocognitive profile of children with CMT2Z is likely more complex than currently reported.