Charcot-Marie-Tooth disease (CMT) due to GJB1 variants (CMTX1) is the second most common form of CMT. It is an X-linked disorder characterized by progressive sensory and motor neuropathy with males affected more severely than females. Many reported GJB1 variants remain classified as variants of uncertain significance (VUS). In this large, international, multicentre study we prospectively collected demographic, clinical and genetic data on patients with CMT associated with GJB1 variants. Pathogenicity for each variant was defined using adapted American College of Medical Genetics criteria. Baseline and longitudinal analyses were conducted to study genotype-phenotype correlations, to calculate longitudinal change using the CMT Examination Score (CMTES), to compare males versus females, and pathogenic/likely pathogenic (P/LP) variants versus VUS. We present 387 patients from 295 families harbouring 154 variants in GJB1. Of these, 319 patients (82.4%) were deemed to have P/LP variants, 65 had VUS (16.8%) and three benign variants (0.8%; excluded from analysis); an increased proportion of patients with P/LP variants compared with using ClinVar's classification (74.6%). Male patients (166/319, 52.0%, P/LP only) were more severely affected at baseline. Baseline measures in patients with P/LP variants and VUS showed no significant differences, and regression analysis suggested the disease groups were near identical at baseline. Genotype-phenotype analysis suggested c.-17G>A produces the most severe phenotype of the five most common variants, and missense variants in the intracellular domain are less severe than other domains. Progression of disease was seen with increasing CMTES over time up to 8 years follow-up. Standard response mean (SRM), a measure of outcome responsiveness, peaked at 3 years with moderate responsiveness [change in CMTES (ΔCMTES) = 1.3 ± 2.6, P = 0.00016, SRM = 0.50]. Males and females progressed similarly up to 8 years, but baseline regression analysis suggested that over a longer period, females progress more slowly. Progression was most pronounced for mild phenotypes (CMTES = 0-7; 3-year ΔCMTES = 2.3 ± 2.5, P = 0.001, SRM = 0.90). Enhanced variant interpretation has yielded an increased proportion of GJB1 variants classified as P/LP and will aid future variant interpretation in this gene. Baseline and longitudinal analysis of this large cohort of CMTX1 patients describes the natural history of the disease including the rate of progression; CMTES showed moderate responsiveness for the whole group at 3 years and higher responsiveness for the mild group at 3, 4 and 5 years. These results have implications for patient selection for upcoming clinical trials.

Using exome sequencing in an individual with Charcot-Marie-Tooth disease (CMT) we have identified a mutation in the X-linked dystrophin-related protein 2 (DRP2) gene. A 60-year-old gentleman presented to our clinic and underwent clinical, electrophysiological and skin biopsy studies. The patient had clinical features of a length dependent sensorimotor neuropathy with an age of onset of 50 years. Neurophysiology revealed prolonged latencies with intermediate conduction velocities but no conduction block or temporal dispersion. A panel of 23 disease causing genes was sequenced and ultimately was uninformative. Whole exome sequencing revealed a stop mutation in DRP2, c.805C>T (Q269*). DRP2 interacts with periaxin and dystroglycan to form the periaxin-DRP2-dystroglycan complex which plays a role in the maintenance of the well-characterized Cajal bands of myelinating Schwann cells. Skin biopsies from our patient revealed a lack of DRP2 in myelinated dermal nerves by immunofluorescence. Furthermore electron microscopy failed to identify Cajal bands in the patient's dermal myelinated axons in keeping with ultrastructural pathology seen in the Drp2 knockout mouse. Both the electrophysiologic and dermal nerve twig pathology support the interpretation that this patient's DRP2 mutation causes characteristic morphological abnormalities recapitulating the Drp2 knockout model and potentially represents a novel genetic cause of CMT.

Charcot-Marie-Tooth disease (CMT) type 2A is a form of peripheral neuropathy, due almost exclusively to dominant mutations in the nuclear gene encoding the mitochondrial protein mitofusin-2 (MFN2). However, there is no understanding of the relationship of clinical phenotype to genotype. MFN2 has two functions: it promotes inter-mitochondrial fusion and mediates endoplasmic reticulum (ER)-mitochondrial tethering at mitochondria-associated ER membranes (MAM). MAM regulates a number of key cellular functions, including lipid and calcium homeostasis, and mitochondrial behavior. To date, no studies have been performed to address whether mutations in MFN2 in CMT2A patient cells affect MAM function, which might provide insight into pathogenesis. Using fibroblasts from three CMT2AMFN2 patients with different mutations in MFN2, we found that some, but not all, examined aspects of ER-mitochondrial connectivity and of MAM function were indeed altered, and correlated with disease severity. Notably, however, respiratory chain function in those cells was unimpaired. Our results suggest that CMT2AMFN2 is a MAM-related disorder but is not a respiratory chain-deficiency disease. The alterations in MAM function described here could also provide insight into the pathogenesis of other forms of CMT.

To extend the phenotypic description of Charcot-Marie-Tooth disease (CMTX1) and to draw new genotype-phenotype relationships.

Mitofusin-2 (MFN2) is one of two ubiquitously expressed homologous proteins in eukaryote cells, playing a critical role in mitochondrial fusion. Mutations in MFN2 (most commonly autosomal dominant) cause Charcot-Marie-Tooth disease type 2A (CMT2A), the commonest axonal form of CMT, with significant allelic heterogeneity. Previous, moderately-sized, cross sectional genotype-phenotype studies of CMT2A have described the phenotypic spectrum of the disease, but longitudinal natural history studies are lacking. In this large multicentre prospective cohort study of 196 patients with dominant and autosomal recessive CMT2A, we present an in-depth genotype-phenotype study of the baseline characteristics of patients with CMT2A and longitudinal data (1-2 years) to describe the natural history. A childhood onset of autosomal dominant CMT2A is the most predictive marker of significant disease severity and is independent of the disease duration. When compared to adult onset autosomal dominant CMT2A, it is associated with significantly higher rates of use of ankle-foot orthoses, full-time use of wheelchair, dexterity difficulties and also has significantly higher CMT Examination Score (CMTESv2) and CMT Neuropathy Score (CMTNSv2) at initial assessment. Analysis of longitudinal data using the CMTESv2 and its Rasch-weighted counterpart, CMTESv2-R, show that over 1 year, the CMTESv2 increases significantly in autosomal dominant CMT2A (mean change 0.84 ± 2.42; two-tailed paired t-test P = 0.039). Furthermore, over 2 years both the CMTESv2 (mean change 0.97 ± 1.77; two-tailed paired t-test P = 0.003) and the CMTESv2-R (mean change 1.21 ± 2.52; two-tailed paired t-test P = 0.009) increase significantly with respective standardized response means of 0.55 and 0.48. In the paediatric CMT2A population (autosomal dominant and autosomal recessive CMT2A grouped together), the CMT Pediatric Scale increases significantly both over 1 year (mean change 2.24 ± 3.09; two-tailed paired t-test P = 0.009) and over 2 years (mean change 4.00 ± 3.79; two-tailed paired t-test P = 0.031) with respective standardized response means of 0.72 and 1.06. This cross-sectional and longitudinal study of the largest CMT2A cohort reported to date provides guidance for variant interpretation, informs prognosis and also provides natural history data that will guide clinical trial design.

We have identified missense mutations at conserved amino acids in the PRPS1 gene on Xq22.3 in two families with a syndromic form of inherited peripheral neuropathy, one of Asian and one of European descent. The disease is inherited in an X-linked recessive manner, and the affected male patients invariably develop sensorineural hearing loss of prelingual type followed by gating disturbance and visual loss. The family of European descent was reported in 1967 as having Rosenberg-Chutorian syndrome, and recently a Korean family with the same symptom triad was identified with a novel disease locus CMTX5 on the chromosome band Xq21.32-q24. PRPS1 (phosphoribosyl pyrophosphate synthetase 1) is an isoform of the PRPS gene family and is ubiquitously expressed in human tissues, including cochlea. The enzyme mediates the biochemical step critical for purine metabolism and nucleotide biosynthesis. The mutations identified were E43D, in patients with Rosenberg-Chutorian syndrome, and M115T, in the Korean patients with CMTX5. We also showed decreased enzyme activity in patients with M115T. PRPS1 is the first CMT gene that encodes a metabolic enzyme, shedding a new light on the understanding of peripheral nerve-specific metabolism and also suggesting the potential of PRPS1 as a target for drugs in prevention and treatment of peripheral neuropathy by antimetabolite therapy.

Charcot-Marie-Tooth disease type 1A (CMT1A) is caused by a uniform 1.5-Mb duplication on chromosome 17p, which includes the PMP22 gene. Patients often present the classic neuropathy phenotype, but also with high clinical variability.

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the β7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on β7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.

Although mutations in more than 90 genes are known to cause CMT, the underlying genetic cause of CMT remains unknown in more than 50% of affected individuals. The discovery of additional genes that harbor CMT2-causing mutations increasingly depends on sharing sequence data on a global level. In this way-by combining data from seven countries on four continents-we were able to define mutations in ATP1A1, which encodes the alpha1 subunit of the Na+,K+-ATPase, as a cause of autosomal-dominant CMT2. Seven missense changes were identified that segregated within individual pedigrees: c.143T>G (p.Leu48Arg), c.1775T>C (p.Ile592Thr), c.1789G>A (p.Ala597Thr), c.1801_1802delinsTT (p.Asp601Phe), c.1798C>G (p.Pro600Ala), c.1798C>A (p.Pro600Thr), and c.2432A>C (p.Asp811Ala). Immunostaining peripheral nerve axons localized ATP1A1 to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures of myelin sheaths. Two-electrode voltage clamp measurements on Xenopus oocytes demonstrated significant reduction in Na+ current activity in some, but not all, ouabain-insensitive ATP1A1 mutants, suggesting a loss-of-function defect of the Na+,K+ pump. Five mutants fall into a remarkably narrow motif within the helical linker region that couples the nucleotide-binding and phosphorylation domains. These findings identify a CMT pathway and a potential target for therapy development in degenerative diseases of peripheral nerve axons.

Mutations in the major peripheral nervous system (PNS) myelin protein, myelin protein zero (MPZ), cause Charcot-Marie-Tooth Disease type 1B (CMT1B), typically thought of as a demyelinating peripheral neuropathy. Certain MPZ mutations, however, cause adult onset neuropathy with minimal demyelination but pronounced axonal degeneration. Mechanism(s) for this phenotype are unknown. We performed an autopsy of a 73-year-old woman with a late-onset neuropathy caused by an H10P MPZ mutation whose nerve conduction studies suggested severe axonal loss but no demyelination. The autopsy demonstrated axonal loss and reorganization of the molecular architecture of the axolemma. Segmental demyelination was negligible. In addition, we identified focal nerve enlargements containing MPZ and ubiquitin either in the inner myelin intralaminar and/or periaxonal space that separates axons from myelinating Schwann cells. Taken together, these data confirmed that a mutation in MPZ can cause axonal neuropathy, in the absence of segmental demyelination, thus uncoupling the two pathological processes. More important, it also provided potential molecular mechanisms as to how the axonal degeneration occurred: either by disruption of glial-axon interaction by protein aggregates or by alterations in the molecular architecture of internodes and paranodes. This report represents the first study in which the molecular basis of axonal degeneration in the late-onset CMT1B has been explored in human tissue.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an untreatable childhood peripheral neuropathy caused by mutations of the mitochondrial fusion protein, mitofusin (MFN) 2. Here, pharmacological activation of endogenous normal mitofusins overcame dominant inhibitory effects of CMT2A mutants in reprogrammed human patient motor neurons, reversing hallmark mitochondrial stasis and fragmentation independent of causal MFN2 mutation. In mice expressing human MFN2 T105M, intermittent mitofusin activation with a small molecule, MiM111, normalized CMT2A neuromuscular dysfunction, reversed pre-treatment axon and skeletal myocyte atrophy, and enhanced axon regrowth by increasing mitochondrial transport within peripheral axons and promoting in vivo mitochondrial localization to neuromuscular junctional synapses. MiM111-treated MFN2 T105M mouse neurons exhibited accelerated primary outgrowth and greater post-axotomy regrowth, linked to enhanced mitochondrial motility. MiM111 is the first pre-clinical candidate for CMT2A.

Mutations in myelin protein zero (MPZ) are generally associated with Charcot-Marie-Tooth type 1B (CMT1B) disease, one of the most common forms of demyelinating neuropathy. Pathogenesis of some MPZ mutants, such as S63del and R98C, involves the misfolding and retention of MPZ in the endoplasmic reticulum (ER) of myelinating Schwann cells. To cope with proteotoxic ER-stress, Schwann cells mount an unfolded protein response (UPR) characterized by activation of the PERK, ATF6 and IRE1α/XBP1 pathways. Previous results showed that targeting the PERK UPR pathway mitigates neuropathy in mouse models of CMT1B; however, the contributions of other UPR pathways in disease pathogenesis remains poorly understood. Here, we probe the importance of the IRE1α/XBP1 signalling during normal myelination and in CMT1B. In response to ER stress, IRE1α is activated to stimulate the non-canonical splicing of Xbp1 mRNA to generate spliced Xbp1 (Xbp1s). This results in the increased expression of the adaptive transcription factor XBP1s, which regulates the expression of genes involved in diverse pathways including ER proteostasis. We generated mouse models where Xbp1 is deleted specifically in Schwann cells, preventing XBP1s activation in these cells. We observed that Xbp1 is dispensable for normal developmental myelination, myelin maintenance and remyelination after injury. However, Xbp1 deletion dramatically worsens the hypomyelination and the electrophysiological and locomotor parameters observed in young and adult CMT1B neuropathic animals. RNAseq analysis suggested that XBP1s exerts its adaptive function in CMT1B mouse models in large part via the induction of ER proteostasis genes. Accordingly, the exacerbation of the neuropathy in Xbp1 deficient mice was accompanied by upregulation of ER-stress pathways and of IRE1-mediated RIDD signaling in Schwann cells, suggesting that the activation of XBP1s via IRE1 plays a critical role in limiting mutant protein toxicity and that this toxicity cannot be compensated by other stress responses. Schwann cell specific overexpression of XBP1s partially re-established Schwann cell proteostasis and attenuated CMT1B severity in both the S63del and R98C mouse models. In addition, the selective, pharmacologic activation of IRE1α/XBP1 signaling ameliorated myelination in S63del dorsal root ganglia explants. Collectively, these data show that XBP1 has an essential adaptive role in different models of proteotoxic CMT1B neuropathy and suggest that activation of the IRE1α/XBP1 pathway may represent a therapeutic avenue in CMT1B and possibly for other neuropathies characterized by UPR activation.

Distal hereditary motor neuropathies comprise a clinically and genetically heterogeneous group of disorders. We recently mapped an X-linked form of this condition to chromosome Xq13.1-q21 in two large unrelated families. The region of genetic linkage included ATP7A, which encodes a copper-transporting P-type ATPase mutated in patients with Menkes disease, a severe infantile-onset neurodegenerative condition. We identified two unique ATP7A missense mutations (p.P1386S and p.T994I) in males with distal motor neuropathy in two families. These molecular alterations impact highly conserved amino acids in the carboxyl half of ATP7A and do not directly involve the copper transporter's known critical functional domains. Studies of p.P1386S revealed normal ATP7A mRNA and protein levels, a defect in ATP7A trafficking, and partial rescue of a S. cerevisiae copper transport knockout. Although ATP7A mutations are typically associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome, we demonstrate here that certain missense mutations at this locus can cause a syndrome restricted to progressive distal motor neuropathy without overt signs of systemic copper deficiency. This previously unrecognized genotype-phenotype correlation suggests an important role of the ATP7A copper transporter in motor-neuron maintenance and function.

Charcot-Marie-Tooth disease type 1A (CMT1A), caused by duplication of the peripheral myelin protein 22 (PMP22) gene, and CMT1B, caused by mutations in myelin protein zero (MPZ) gene, are the two most common forms of demyelinating CMT (CMT1), and no treatments are available for either. Prior studies of the MpzSer63del mouse model of CMT1B have demonstrated that protein misfolding, endoplasmic reticulum (ER) retention and activation of the unfolded protein response (UPR) contributed to the neuropathy. Heterozygous patients with an arginine to cysteine mutation in MPZ (MPZR98C) develop a severe infantile form of CMT1B which is modelled by MpzR98C/ + mice that also show ER stress and an activated UPR. C3-PMP22 mice are considered to effectively model CMT1A. Altered proteostasis, ER stress and activation of the UPR have been demonstrated in mice carrying Pmp22 mutations. To determine whether enabling the ER stress/UPR and readjusting protein homeostasis would effectively treat these models of CMT1B and CMT1A, we administered Sephin1/IFB-088/icerguestat, a UPR modulator which showed efficacy in the MpzS63del model of CMT1B, to heterozygous MpzR98C and C3-PMP22 mice. Mice were analysed by behavioural, neurophysiological, morphological and biochemical measures. Both MpzR98C/ + and C3-PMP22 mice improved in motor function and neurophysiology. Myelination, as demonstrated by g-ratios and myelin thickness, improved in CMT1B and CMT1A mice and markers of UPR activation returned towards wild-type values. Taken together, our results demonstrate the capability of IFB-088 to treat a second mouse model of CMT1B and a mouse model of CMT1A, the most common form of CMT. Given the recent benefits of IFB-088 treatment in amyotrophic lateral sclerosis and multiple sclerosis animal models, these data demonstrate its potential in managing UPR and ER stress for multiple mutations in CMT1 as well as in other neurodegenerative diseases. (Left panel) the accumulation of overexpressed PMP22 or misfolded mutant P0 in the Schwann cell endoplasmic reticulum (ER) leads to overwhelming of the degradative capacity, activation of ER-stress mechanisms, and myelination impairment. (Right panel) by prolonging eIF2α phosphorylation, IFB-088 reduces the amount of newly synthesized proteins entering the ER, allowing the protein quality control systems to better cope with the unfolded/misfolded protein and allowing myelination to progress.

To investigate the effectiveness of targeted next-generation sequencing (NGS) panels in achieving a molecular diagnosis in Charcot-Marie-Tooth disease (CMT) and related disorders in a clinical setting.