Cysteinyl-tRNA Synthetase Mutations Cause a Multi-System, Recessive Disease That Includes Microcephaly, Developmental Delay, and Brittle Hair and Nails.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.

Revisiting the pathogenic mechanism of the GJB1 5' UTR c.-103C > T mutation causing CMTX1.

The second most common form of Charcot-Marie-Tooth neuropathy (CMT), X-linked CMT type X1 (CMTX1), is caused by coding and non-coding mutations in the gap junction beta 1 (GJB1) gene. The non-coding GJB1 c.-103C > T mutation (NM_000166.5) has been reported to cause CMTX1 in multiple families. This study assessed the internal ribosomal entry site (IRES) activity previously reported for the rat Gjb1 P2 5' untranslated region (UTR). Using a bicistronic assay and transfecting RT4 Schwann cells, IRES activity of the human GJB1 P2 5' UTR was compared to the GJB1 P2 5' UTR containing either the c.-103C > T mutation or the non-pathogenic c.-102G > A variant. No differences in GJB1 P2 5' UTR IRES activity were observed between the negative control, the wild-type P2 5' UTR, the c.-103C > T 5' UTR or the c.-102G > A 5' UTR, irrespective of the GJB1 intron being present (p = .429 with intron, and p = .865 without). A theoretical c.-131A > G variant was predicted to result in the same RNA secondary structure as the GJB1 c.-103C > T P2 5' UTR. However, no significant difference was observed between expression from the wild-type GJB1 P2 5' UTR and the GJB1 c.-131A > G variant (p = .688). Deletion of the conserved region surrounding the c.-103C > T mutation (c.-108_-103del) resulted in significantly higher expression than the c.-103C > T mutation alone (p = .019), suggesting that the conserved c.-108_-103 region was not essential for translation. The reporter assays in this study do not recapitulate the previously reported GJB1 IRES activity and suggest an alternate pathogenic mechanism for the c.-103C > T CMTX1 non-coding mutation.

Farnesol Ameliorates Demyelinating Phenotype in a Cellular and Animal Model of Charcot-Marie-Tooth Disease Type 1A.

Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disease affecting the peripheral nervous system that is caused by either the demyelination of Schwann cells or degeneration of the peripheral axon. Currently, there are no treatment options to improve the degeneration of peripheral nerves in CMT patients. In this research, we assessed the potency of farnesol for improving the demyelinating phenotype using an animal model of CMT type 1A. In vitro treatment with farnesol facilitated myelin gene expression and ameliorated the myelination defect caused by PMP22 overexpression, the major causative gene in CMT. In vivo administration of farnesol enhanced the peripheral neuropathic phenotype, as shown by rotarod performance in a mouse model of CMT1A. Electrophysiologically, farnesol-administered CMT1A mice exhibited increased motor nerve conduction velocity and compound muscle action potential compared with control mice. The number and diameter of myelinated axons were also increased by farnesol treatment. The expression level of myelin protein zero (MPZ) was increased, while that of the demyelination marker, neural cell adhesion molecule (NCAM), was reduced by farnesol administration. These data imply that farnesol is efficacious in ameliorating the demyelinating phenotype of CMT, and further elucidation of the underlying mechanisms of farnesol's effect on myelination might provide a potent therapeutic strategy for the demyelinating type of CMT.

Enhanced axonal neuregulin-1 type-III signaling ameliorates neurophysiology and hypomyelination in a Charcot-Marie-Tooth type 1B mouse model.

Charcot-Marie-Tooth (CMT) neuropathies are a group of genetic disorders that affect the peripheral nervous system with heterogeneous pathogenesis and no available treatment. Axonal neuregulin 1 type III (Nrg1TIII) drives peripheral nerve myelination by activating downstream signaling pathways such as PI3K/Akt and MAPK/Erk that converge on master transcriptional regulators of myelin genes, such as Krox20. We reasoned that modulating Nrg1TIII activity may constitute a general therapeutic strategy to treat CMTs that are characterized by reduced levels of myelination. Here we show that genetic overexpression of Nrg1TIII ameliorates neurophysiological and morphological parameters in a mouse model of demyelinating CMT1B, without exacerbating the toxic gain-of-function that underlies the neuropathy. Intriguingly, the mechanism appears not to be related to Krox20 or myelin gene upregulation, but rather to a beneficial rebalancing in the stoichiometry of myelin lipids and proteins. Finally, we provide proof of principle that stimulating Nrg1TIII signaling, by pharmacological suppression of the Nrg1TIII inhibitor tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17), also ameliorates the neuropathy. Thus, modulation of Nrg1TIII by TACE/ADAM17 inhibition may represent a general treatment for hypomyelinating neuropathies.

A Deregulated Stress Response Underlies Distinct INF2-Associated Disease Profiles.

BACKGROUND: Monogenic diseases provide favorable opportunities to elucidate the molecular mechanisms of disease progression and improve medical diagnostics. However, the complex interplay between genetic and environmental factors in disease etiologies makes it difficult to discern the mechanistic links between different alleles of a single locus and their associated pathophysiologies. Inverted formin 2 (INF2), an actin regulator, mediates a stress response-calcium mediated actin reset, or CaAR-that reorganizes the actin cytoskeleton of mammalian cells in response to calcium influx. It has been linked to the podocytic kidney disease focal segemental glomerulosclerosis (FSGS), as well as to cases of the neurologic disorder Charcot-Marie-Tooth disease that are accompanied by nephropathy, mostly FSGS. METHODS: We used a combination of quantitative live cell imaging and validation in primary patient cells and Drosophila nephrocytes to systematically characterize a large panel of >50 autosomal dominant INF2 mutants that have been reported to cause either FSGS alone or with Charcot-Marie-Tooth disease. RESULTS: We found that INF2 mutations lead to deregulated activation of formin and a constitutive stress response in cultured cells, primary patient cells, and Drosophila nephrocytes. We were able to clearly distinguish between INF2 mutations that were linked exclusively to FSGS from those that caused a combination of FSGS and Charcot-Marie-Tooth disease. Furthermore, we were able to identify distinct subsets of INF2 variants that exhibit varying degrees of activation. CONCLUSIONS: Our results suggest that CaAR can be used as a sensitive assay for INF2 function and for robust evaluation of diseased-linked variants of formin. More broadly, these findings indicate that cellular profiling of disease-associated mutations has potential to contribute substantially to sequence-based phenotype predictions.

Charcot neuroarthropathy of the knee due to idiopathic sensory peripheral neuropathy.

BACKGROUND: Charcot neuroarthropathy is a systemic disease that generates pathological changes in the musculoskeletal system, causing instability, dislocations, and deformities. Charcot neuroarthropathy of the knee, due to either diabetes mellitus or syringomyelia, is anecdotally reported with the epidemic of the diseases. However, idiopathic sensory peripheral neuropathy can inflict osteoarticular structures directly, inducing a dysfunctional Charcot neuroarthropathy. An early diagnosis and effective relief of the symptomatic deformity is essential for the treatment. CASE PRESENTATION: We report the case of a patient with idiopathic sensory peripheral neuropathy who presented with a swelling right knee, as well as distorted and painless gait disorder, diagnosed as Charcot neuroarthropathy of the knee. Partial weight bearing with a hinged knee brace was used to correct the abnormal alignment and gait posture, and bisphosphonates were prescribed to decrease pathological bone resorption. Although the alignment and Knee Society Score got a gradual deterioration, the combination of orthosis and pharmacy could alleviate the symptom to a certain extent. CONCLUSION: The diagnosis of Charcot neuroarthropathy of the knee is rare that requiring early diagnosis. The presence of features, including painlessness, numbness, and deformed arthropathy following chronic-onset algesthesia loss should be taken carefully.

Charcot-Marie-Tooth: From Molecules to Therapy.

Charcot-Marie-Tooth (CMT) is the most prevalent category of inherited neuropathy. The most common inheritance pattern is autosomal dominant, though there also are X-linked and autosomal recessive subtypes. In addition to a variety of inheritance patterns, there are a myriad of genes associated with CMT, reflecting the heterogeneity of this disorder. Next generation sequencing (NGS) has expanded and simplified the diagnostic yield of genes/molecules underlying and/or associated with CMT, which is of paramount importance in providing a substrate for current and future targeted disease-modifying treatment options. Considerable research attention for disease-modifying therapy has been geared towards the most commonly encountered genetic mutations (PMP22, GJB1, MPZ, and MFN2). In this review, we highlight the clinical background, molecular understanding, and therapeutic investigations of these CMT subtypes, while also discussing therapeutic research pertinent to the remaining less common CMT subtypes.

Calcium Deregulation and Mitochondrial Bioenergetics in GDAP1-Related CMT Disease.

The pathology of Charcot-Marie-Tooth (CMT), a disease arising from mutations in different genes, has been associated with an impairment of mitochondrial dynamics and axonal biology of mitochondria. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause several forms of CMT neuropathy, but the pathogenic mechanisms involved remain unclear. GDAP1 is an outer mitochondrial membrane protein highly expressed in neurons. It has been proposed to play a role in different aspects of mitochondrial physiology, including mitochondrial dynamics, oxidative stress processes, and mitochondrial transport along the axons. Disruption of the mitochondrial network in a neuroblastoma model of GDAP1-related CMT has been shown to decrease Ca2+ entry through the store-operated calcium entry (SOCE), which caused a failure in stimulation of mitochondrial respiration. In this review, we summarize the different functions proposed for GDAP1 and focus on the consequences for Ca2+ homeostasis and mitochondrial energy production linked to CMT disease caused by different GDAP1 mutations.

[Research progress in the mouse models of Charcot-Marie-Tooth disease type 2 (CMT2)].

Charcot-Marie-Tooth disease (CMT) is a kind of common hereditary motor and sensory neuropathies with a global prevalence of about 1 in 2500. Clinically, CMT can be divided into two main types: a demyelinating type (CMT1, CMT3, CMT4 and CMTX1) and an axonal type (CMT2). Up to now, about 17 unique genes related to CMT2 have been mapped and cloned. However, the pathogenesis of these disease-causing genes is still unknown. The mouse models have been playing an important role in understanding the molecular mechanism of CMT2. Recently, near 10 transgenic, knock-in and knock-out mouse models of CMT2 have been generated. In this review, we briefly introduce the construction strategy of the CMT2 mouse models, summarize the research progress of the CMT2 mouse models, and analyze in detail a few typical mouse models of CMT2.

A compound heterozygous mutation in HADHB gene causes an axonal Charcot-Marie-tooth disease.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a heterogeneous disorder of the peripheral nervous system. So far, mutations in hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), beta subunit (HADHB) gene exhibit three distinctive phenotypes: severe neonatal presentation with cardiomyopathy, hepatic form with recurrent hypoketotic hypoglycemia, and later-onset axonal sensory neuropathy with episodic myoglobinuria. METHODS: To identify the causative and characterize clinical features of a Korean family with motor and sensory neuropathies, whole exome study (WES), histopathologic study of distal sural nerve, and lower limb MRIs were performed. RESULTS: WES revealed that a compound heterozygous mutation in HADHB is the causative of the present patients. The patients exhibited an early-onset axonal sensorimotor neuropathy without episodic myoglobinuria, and showed typical clinical and electrophysiological features of CMT including predominant distal muscle weakness and atrophy. Histopathologic findings of sural nerve were compatible with an axonal CMT neuropathy. Furthermore, they didn't exhibit any other symptoms of the previously reported HADHB patients. CONCLUSIONS: These data implicate that mutation in HADHB gene can also cause early-onset axonal CMT instead of typical manifestations in mitochondrial trifunctional protein (MTP) deficiency. Therefore, this study is the first report of a new subtype of autosomal recessive axonal CMT by a compound heterozygous mutation in HADHB, and will expand the clinical and genetic spectrum of HADHB.

Myotubularin phosphoinositide phosphatases in human diseases.

The level and turnover of phosphoinositides (PIs) are tightly controlled by a large set of PI-specific enzymes (PI kinases and phosphatases). Mammalian PI phosphatases are conserved through evolution and among this large family the dual-specificity phosphatase (PTP/DSP) are metal-independent enzymes displaying the amino acid signature Cys-X5-Arg-Thr/Ser (CX5RT/S) in their active site. Such catalytic site characterizes the myotubularin 3-phosphatases that dephosphorylate PtdIns3P and PtdIns(3,5)P2 and produce PtdIns5P. Substrates of myotubularins have been implicated in endocytosis and membrane trafficking while PtdIns5P may have a role in signal transduction. As a paradox, 6 of the 14 members of the myotubularin family lack enzymatic activity and are considered as dead phosphatases. Several myotubularins have been genetically linked to human diseases: MTM1 is mutated in the congenital myopathy X-linked centronuclear or myotubular myopathy (XLCNM) and MTMR14 (JUMPY) has been linked to an autosomal form of such disease, while MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) neuropathies. Furthermore, recent evidences from genetic association studies revealed that several other myotubularins could be associated to chronic disorders such as cancer and obesity, highlighting their importance for human health. Here, we discuss cellular and physiological roles of myotubularins and their implication in human diseases, and we present potential pathological mechanisms affecting specific tissues in myotubularin-associated diseases.

Recurrence of acute Charcot neuropathic osteoarthropathy after conservative treatment.

BACKGROUND: Charcot neuropathic osteoarthropathy (CN) is a chronic, progressive-destructive process affecting the feet of patients with sensory neuropathy. Data on CN recurrence are underrepresented in the literature. The aim of the present study was to evaluate the rate of CN recurrence after its treatment and to find predisposing factors. METHODS: Fifty-two patients (age 59 ± 11 years, 16 female) with acute CN with 57 affected feet were enrolled. Comorbidities, localization, and stage of disease at first diagnosis as well as ulcerations, need for surgery, noncompliance, and subsequent treatment (orthopedic footwear or orthotic treatment) during the course of therapy were recorded. During follow-up, the incidence of recurrence of CN was observed. Mean follow-up was 47 ± 40 months. RESULTS: Diabetes was the most common reason for sensory neuropathy (79%). Recurrence of CN was seen in 13 feet (23%) with an interval of 27 ± 31 months (range, 3-102 months) after the end of initial immobilization. Patients with recurrence were immobilized for a shorter period of time and had a more advanced stage of CN at time of first diagnosis. Predictors of recurrence were noncompliance (odds ratio 19.7; confidence interval, 4.1-94.4; P < .001) and obesity (odds ratio 6.4; confidence interval, 1.6-25.9; P = .06). CONCLUSIONS: Recurrence of osteoarthropathic activity is a possible complication after conservative treatment of CN. Obesity and noncompliance are strong predictors for the recurrence of CN. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Charcot-Marie-Tooth type 4B is caused by mutations in the gene encoding myotubularin-related protein-2.

A gene mutated in Charcot-Marie-Tooth disease type 4B (CMT4B), an autosomal recessive demyelinating neuropathy with myelin outfoldings, has been mapped on chromosome 11q22. Using a positional-cloning strategy, we identified in unrelated CMT4B patients mutations occurring in the gene MTMR2, encoding myotubularin-related protein-2, a dual specificity phosphatase (DSP).

MSC p43 required for axonal development in motor neurons.

Neuron connectivity and correct neural function largely depend on axonal integrity. Neurofilaments (NFs) constitute the main cytoskeletal network maintaining the structural integrity of neurons and exhibit dynamic changes during axonal and dendritic growth. However, the mechanisms underlying axonal development and maintenance remain poorly understood. Here, we identify that multisynthetase complex p43 (MSC p43) is essential for NF assembly and axon maintenance. The MSC p43 protein was predominantly expressed in central neurons and interacted with NF light subunit in vivo. Mice lacking MSC p43 exhibited axon degeneration in motor neurons, defective neuromuscular junctions, muscular atrophy, and motor dysfunction. Furthermore, MSC p43 depletion in mice caused disorganization of the axonal NF network. Mechanistically, MSC p43 is required for maintaining normal phosphorylation levels of NFs. Thus, MSC p43 is indispensable in maintaining axonal integrity. Its dysfunction may underlie the NF disorganization and axon degeneration associated with motor neuron degenerative diseases.

Dynamin 2 in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is an inherited neuronal disorder, and is induced by mutations of various genes associated with intracellular membrane traffic and cytoskeleton. A large GTPase, dynamin, which is known as a fission protein for endocytic vesicles, was identified as a gene responsible for dominant-intermediate CMT type 2B (DI-CMT2B). Of these mutants, the PH domain, which is required for interaction with phosphoinositides, was mutated in several families. Interestingly, the expression of a deletion mutant, 551Δ3, did not impair endocytosis, but induced abnormal accumulation of microtubules. Recent evidence has shown that dynamin 2 regulates the dynamic instability of microtubules, and 551Δ3 lacks this function. We propose a model for the regulation of the dynamic instability of microtubules by dynamin 2 and discuss the relationship between dynamin 2 and CMT.

[Hereditary sensory and motor neuropathy and hereditary sensory and autonomic neuropathies: recent advances]. / Nouveautés dans les maladies de Charcot-Marie-Tooth et les neuropathies sensitives et dysautonomiques héréditaires.

This review summarizes the recent genetic advances in hereditary sensorimotor neuropathy also called Charcot-Marie-Tooth disease. The different new genes discovered in 2010 and their underlying phenotypes will be presented.

Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease.

Mutations in GDAP1 lead to severe forms of the peripheral motor and sensory neuropathy, Charcot-Marie-Tooth disease (CMT), which is characterized by heterogeneous phenotypes, including pronounced axonal damage and demyelination. We show that neurons and Schwann cells express ganglioside-induced differentiation associated protein 1 (GDAP1), which suggest that both cell types may contribute to the mixed features of the disease. GDAP1 is located in the mitochondrial outer membrane and regulates the mitochondrial network. Overexpression of GDAP1 induces fragmentation of mitochondria without inducing apoptosis, affecting overall mitochondrial activity, or interfering with mitochondrial fusion. The mitochondrial fusion proteins, mitofusin 1 and 2 and Drp1(K38A), can counterbalance the GDAP1-dependent fission. GDAP1-specific knockdown by RNA interference results in a tubular mitochondrial morphology. GDAP1 truncations that are found in patients who have CMT are not targeted to mitochondria and have lost mitochondrial fragmentation activity. The latter activity also is reduced strongly for disease-associated GDAP1 point mutations. Our data indicate that an exquisitely tight control of mitochondrial dynamics, regulated by GDAP1, is crucial for the proper function of myelinated peripheral nerves.

Hereditary predominantly motor neuropathies.

PURPOSE OF REVIEW: We review recent advances in Charcot-Marie-Tooth disease (CMT), the most frequent inherited neuromuscular disorder. RECENT FINDINGS: During the last year further progresses have occurred in this field and concerned identification of novel mutations in recently identified genes, allowing better definition of associated phenotypes; increased knowledge on pathophysiologic mechanisms of the different CMT types, with the contribution of cellular and animal model studies; studies on the natural history of CMT and attempts at developing appropriate outcome measures to assess disease course and intervention efficacy; trials with ascorbic acid in CMT type 1A; and studies on new possible therapeutic strategies. SUMMARY: Such advances have implications on clinical management of CMT and are modifying the clinical approach to CMT, by improving diagnostic tools, allowing better definition of prognosis, and increasing the hope for future effective treatments. Research on CMT is important as is shedding light on important pathways that regulates the normal function of axonal transport, vesicular trafficking, and also revealing new aspects of intracellular organelles' function and interactions.

The role of mitochondrial network dynamics in the pathogenesis of Charcot-Marie-Tooth disease.

Mitochondrial dysfunction plays a relevant role in the pathogenesis of neurological and neuromuscular diseases. Mitochondria may be involved as a primary defect of either the mtDNA or nuclear genome encoded subunits of the respiratory chain. These organelles have also been directly involved in the pathogenesis of Mendelian neurodegenerative disorders caused by mutations in nuclear-encoded proteins targeted to mitochondria, such as Friedreich ataxia, hereditary spastic paraplegia, or some monogenic forms of Parkinson disease. In addition, mitochondria also participate in the pathogenic mechanisms affecting neurodegenerative disorders such Huntington disease or amyotrophic lateral sclerosis. Cell death in neurodegeneration associated with neurological diseases usually occurs by apoptosis being the most common route the intrinsic mitochondria pathway. Along with regulation of apoptosis, mitochondria also modulate cell pathogenesis by means of energy production, reactive oxygen species (ROS) generation, and calcium buffering. Mitochondria form dynamic tubular networks that continually change their shape and move throughout the cell. Here we review the critical role of mitochondria in monogenic neuromuscular disorders, especially inherited peripheral neuropathies caused by abnormal mitochondrial network dynamics. In yeast, at least three proteins are required for mitochondrial fusion, Fzo1, Ugo1 and Mgm1. The human counterparts of Fzo1p and Mgm1p, MFN1/MFN2 and OPA1 respectively, are related to human disease. Mutations in the MFN2 gene cause the most frequent form of autosomal dominant axonal Charcot-Marie-Tooth disease, CMT2A. Mutations in OPA1 cause autosomal dominant optic atrophy (ADOA). For the opposite process of mitochondrial fission, four proteins are at least involved in yeast. Very recently a mutation in the DRP1 gene (the human homologue of yeast Dnm1) has been reported in an infant with a syndrome with encephalopathy, optic atrophy and lactic acidosis. GDAP1 has been recently related to the mitochondrial fission in mammalian cells and, interestingly, mutations in the GDAP1 gene are the cause of the most common form of autosomal recessive CMT, either axonal or demyelinating. These and other disorders are the most recent instances of disease related with mitochondrial abnormal motility, fusion and fission. We propose that the pathomechanisms underlying these disorders also include a complex relationship between mitochondrial dynamics and transport across the axon.