CMT Type Research Articles

Phenotypic variability of TRPV4 related neuropathies

Mutations in the transient receptor potential vanilloid 4 (TRPV4) gene have been associated with autosomal dominant skeletal dysplasias and peripheral nervous system syndromes (PNSS). PNSS include Charcot–Marie–Tooth disease (CMT) type 2C, congenital spinal muscular atrophy and arthrogryposis and scapuloperoneal spinal muscular atrophy. We report the clinical, electrophysiological and muscle biopsy findings in two unrelated patients with two novel heterozygous missense mutations in the TRPV4 gene. Whole exome sequencing was carried out on genomic DNA using Illumina TruseqTM 62Mb exome capture. Patient 1 harbours a de novo c.805C > T (p.Arg269Cys) mutation. Clinically, this patient shows signs of both scapuloperoneal spinal muscular atrophy and skeletal dysplasia. Patient 2 harbours a novel c.184G > A (p.Asp62Asn) mutation. While the clinical phenotype is compatible with CMT type 2C with the patient's muscle harbours basophilic inclusions. Mutations in the TRPV4 gene have a broad phenotypic variability and disease severity and may share a similar pathogenic mechanism with Heat Shock Protein related neuropathies.

Ser135Phe mutation in HSPB1 (HSP27) from Charcot–Marie–Tooth disease type 2F families

Charcot–Marie–Tooth disease (CMT) is a group of clinically and genetically heterogeneous peripheral neuropathies. We identified two axonal CMT type 2F (CMT2F) families presented with distally predominant weakness in upper and lower extremities with sensory involvement. This study identified a c.404C>T (p.Ser135Phe) mutation in HSPB1 gene as the underlying cause of the both families by applying of whole exome sequencing. The p.Ser135Phe mutation was completely cosegregated with the affected members in the both families, and it was not found in 300 healthy controls. This mutation has been previously reported as the causes of CMT2F or hereditary motor neuropathy 2B (dHMN2B). The mutation was located in the highly conserved alpha-crystallin domain, and several in silico analyses also predicted that the mutation is likely to be pathogenic. HSPB1 encodes heat shock protein 27 (HSP27) which belongs to the superfamily of small stress induced proteins. These results suggest that the HSPB1 mutation is underlying cause of CMT2F phenotype shown in the present families. We believe that this study will be useful for the molecular diagnosis of peripheral neuropathies.

65. Peripheral nerve ultrasonography: Clinical use

Charcot–Marie–Tooth disease (CMT) is an heterogeneous group of hereditary neuropathies characterized by weakness and atrophy of the limbs, loss of sensation and absent or reduced tendon reflexes. Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) causes progressive or recurrent weakness and numbness of the limbs. Both CMT and CIDP share similar nerve conduction study abnormalities (reduced amplitude of sensory and motor potentials, and reduced conduction velocity). The aim of our study was to compare the ultrasound (US) data recorded in genetic neuropathy (CMT) and inflammatory neuropathy (CIDP). We enrolled 10 patients with CMT type 1A and 10 patients with CIDP. In this study we compared left and right median nerve cross-sectional area (CSA) and echogenicity nerve in the forearm, between two groups of patients to evaluate a possible difference. Three US classes were identified based on CSA and echogenicity. For all patient included neurophysiological assessment was used to estimate a possible correlation between the nerve conduction study variables and US data. US of the nerve can be a tool of significant value in the differential diagnosis in different neuropathies.

Characterization of the mitofusin 2 R94W mutation in a knock-in mouse model.

Charcot-Marie-Tooth disease (CMT) comprises a group of heterogeneous peripheral axonopathies affecting 1 in 2,500 individuals. As mutations in several genes cause axonal degeneration in CMT type 2, mutations in mitofusin 2 (MFN2) account for approximately 90% of the most severe cases, making it the most common cause of inherited peripheral axonal degeneration. MFN2 is an integral mitochondrial outer membrane protein that plays a major role in mitochondrial fusion and motility; yet the mechanism by which dominant mutations in this protein lead to neurodegeneration is still not fully understood. Furthermore, future pre-clinical drug trials will be in need of validated rodent models. We have generated a Mfn2 knock-in mouse model expressing Mfn2(R94W), which was originally identified in CMT patients. We have performed behavioral, morphological, and biochemical studies to investigate the consequences of this mutation. Homozygous inheritance leads to premature death at P1, as well as mitochondrial dysfunction, including increased mitochondrial fragmentation in mouse embryonic fibroblasts and decreased ATP levels in newborn brains. Mfn2(R94W) heterozygous mice show histopathology and age-dependent open-field test abnormalities, which support a mild peripheral neuropathy. Although behavior does not mimic the severity of the human disease phenotype, this mouse can provide useful tissues for studying molecular pathways associated with MFN2 point mutations.

A novel LITAF/SIMPLE mutation within a family with a demyelinating form of Charcot–Marie–Tooth disease

Charcot–Marie–Tooth disease type 1 (CMT1) is a common disorder of the peripheral nervous system. The underlying genetic cause is highly heterogeneous, and mutations in SIMPLE (small integral membrane protein of lysosome/late endosome) represent a rare cause of CMT type 1, named CMT1C. Herein, we report the clinical, electrophysiological, and neuropathological findings of an Italian CMT1 family with a novel SIMPLE missense mutation. The family exhibited electrophysiological signs of demyelination, predominantly affecting the lower limbs, with conduction blocks, and a wide variability of age of onset among the members. Molecular analysis identified the novel heterozygous missense mutation p.Pro135Arg in SIMPLE which co-segregated with the disease within the pedigree. In conclusion, our findings confirm that the genetic analysis of LITAF/SIMPLE should be considered for the diagnostic flow-chart of CMT1 patient, especially when nerve conduction studies show the presence of conduction blocks.

Evolution of Drug Resistance: Insight on TEM β-Lactamases Structure and Activity and β-Lactam Antibiotics

Since the discovery of the first penicillin bacterial resistance to β-lactam antibiotics has spread and evolved promoting new resistances to pathogens. The most common mechanism of resistance is the production of β-lactamases that have spread thorough nature and evolve to complex phenotypes like CMT type enzymes. New antibiotics have been introduced in clinical practice, and therefore it becomes necessary a concise summary about their molecular targets, specific use and other properties. β-lactamases are still a major medical concern and they have been extensively studied and described in the scientific literature. Several authors agree that Glu166 should be the general base and Ser70 should perform the nucleophilic attack to the carbon of the carbonyl group of the β-lactam ring. Nevertheless there still is controversy on their catalytic mechanism. TEMs evolve at incredible pace presenting more complex phenotypes due to their tolerance to mutations. These mutations lead to an increasing need of novel, stronger and more specific and stable antibiotics. The present review summarizes key structural, molecular and functional aspects of ESBL, IRT and CMT TEM β-lactamases properties and up to date diagrams of the TEM variants with defined phenotype. The activity and structural characteristics of several available TEMs in the NCBI-PDB are presented, as well as the relation of the various mutated residues and their specific properties and some previously proposed catalytic mechanisms.

Clinical-sonographic and neurophysiological comparisons in hereditary motor and sensory neuropathy

The article presents the results of ultrasound studies in patients with hereditary motor and sensory neuropathy, or Charcot-Marie-Tooth disease (CMT). Based on standard electroneuromiography, the patients were divided into two groups with demyelinating (n=25) or axonal (n=31) process, CMT1 and CMT2 respectively. Demyelinating forms were characterized by specific prolonged sonographic changes with uniform thickening of all studied nerves and abnormalities of their cable structure. In axonal forms the character of structural changes was not as severe and specific as in CMT1, but symmetrical prolongation of the nerve structure was seen in this type of CMT as well. The description of genetically confirmed familial cases of HMSN type 1X in male relatives is presented, which demonstrates modern potential of unltrasound studies in diagnosing the involvement of peripheral nerves in patients from a heterogeneous group of hereditary neuropahies.

Neuropathic pain model of peripheral neuropathies mediated by mutations of glycyl-tRNA synthetase.

Charcot-Marie-Tooth disease (CMT) is the most common inherited motor and sensory neuropathy. Previous studies have found that, according to CMT patients, neuropathic pain is an occasional symptom of CMT. However, neuropathic pain is not considered to be a significant symptom associated with CMT and, as a result, no studies have investigated the pathophysiology underlying neuropathic pain in this disorder. Thus, the first animal model of neuropathic pain was developed by our laboratory using an adenovirus vector system to study neuropathic pain in CMT. To this end, glycyl-tRNA synthetase (GARS) fusion proteins with a FLAG-tag (wild type [WT], L129P and G240R mutants) were expressed in spinal cord and dorsal root ganglion (DRG) neurons using adenovirus vectors. It is known that GARS mutants induce GARS axonopathies, including CMT type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V). Additionally, the morphological phenotypes of neuropathic pain in this animal model of GARS-induced pain were assessed using several possible markers of pain (Iba1, pERK1/2) or a marker of injured neurons (ATF3). These results suggest that this animal model of CMT using an adenovirus may provide information regarding CMT as well as a useful strategy for the treatment of neuropathic pain.Graphical

Novel C12orf65 mutations in patients with axonal neuropathy and optic atrophy

ObjectiveCharcot-Marie Tooth disease (CMT) forms a clinically and genetically heterogeneous group of disorders. Although a number of disease genes have been identified for CMT, the gene discovery for some complex...

Coexistence of peripheral myelin protein 22 and dystrophin mutations in a chinese boy

We describe a 10-year-old Chinese boy with features of Charcot-Marie-Tooth disease (CMT) and Duchenne muscular dystrophy (DMD). Case report. Weakness and mild sensory loss in the distal extremities, pes cavus, and nerve conduction findings suggested demyelinating neuropathy, while moderate calf pseudohypertrophy, proximal muscle weakness, a myopathic pattern on electromyography, and deficiency of dystrophin immunohistochemical staining on muscle biopsy indicated DMD. Genetic testing revealed a large deletion spanning exon 50 in the gene coding for dystrophin and duplications in the gene coding for peripheral myelin protein 22. This is an interesting and very rare case of CMT type 1A comorbid with DMD. This results in an unusual phenotype and rapid deterioration of motor function. Usage of both target region capture and next generation sequencing is a powerful tool for predicting precisely the range of the large DNA fragment deletion in DMD.

Hereditary motor and sensory neuropathy (HMSN) type X1 in an Argentinean family reveals independent GJB1/Cx32 mutations at the identical nucleotide position

X-linked Charcot–Marie-Tooth disease (CMT Type X1, OMIM: 302800) represents a frequent cause of hereditary peripheral motor and sensory neuropathies and is associated with mutations in GJB1 encoding the gap junction beta 1 protein connexin 32 (Cx32). Studying an Argentinean family of Italian origin with seven affected males in three generations exhibiting clinical signs of CMT, eight obligate female carriers were identified genealogically. DNA sequencing of exon 2 and adjacent regions of the GJB1 gene in two symptomatic males whose respective maternal grandfathers, both affected, were brothers, revealed mutations in GJB1/Cx32. Surprisingly, each of the two affected patients had a different mutation in hemizygous state at the same nucleotide position: c.383C>T (p.S128L) and c.383C>A (p.S128X). In both cases, the identified mutation was present in heterozygous state in the corresponding maternal genomic DNA. Furthermore, X-chromosomal microsatellite analysis showed identical marker alleles in both patients. Together with the genealogical information, these molecular data imply that a primarily mutated allele mutated for a second time. In conclusion, two different mutations at the same nucleotide position in this Argentinean family represent a finding with a very low probability of occurrence.

Parental mosaicism of a novel PMP22 mutation with a minimal neuropathic phenotype

Genetic germinal and somatic mosaicisms of dominant Charcot-Marie-Tooth disease (CMT) mutations are rarely reported and/or recognized. We describe a novel heterozygous p.Trp39Cys missense mutation in the extracellular domain of the peripheral myelin protein 22 (PMP22) associated with an early-onset demyelinating CMT type 1 E (CMT1E) in two siblings born from asymptomatic non-consanguineous parents. The 29-year-old mother, harboring approximately 20% of the mutant PMP22 allele in blood, had minor signs of distal polyneuropathy (pes cavus, decreased ankle jerk reflexes and vibration sense in legs) and slight reduction of sural nerve action potentials (SNAPs). Authors suggest that mutations of CMT-related genes which originate in post-zygotic stages may be associated with mild phenotypes of peripheral neuropathy.

D.P.11 Dual pathology identified by next generation sequencing in siblings with peripheral neuropathy, ataxia and retinal dystrophy

Two children, born to healthy consanguineous South Asian parents, presented with recurrent aspiration in infancy, peripheral neuropathy, ataxia and retinal dystrophy. The older child had pyrexias and pneumonia in infancy due to aspiration, requiring gastrostomy and fundoplication. Nystagmus was noted aged 8months with subsequent visual impairment and signs of rod-cone retinal dystrophy on ERG. By 18months she had severe motor delay, generalised hypotonia and absent lower limb reflexes. EMG and NCVs indicated a severe demyelinating neuropathy. By 5years she was markedly ataxic and had developed a progressive scoliosis. Cranial MRI demonstrated delayed myelination but no cerebellar abnormalities. No mutations were identified in the PMP22, MPZ or GDAP1 genes and transferrin IEF, ATM, aprataxin, senataxin and vitamin E levels were normal. Muscle biopsy showed some reduction of activity of respiratory chain complexes 4 and 5 but no underlying mitochondrial abnormality was identified. The younger sibling had a similar presentation but had silent aspiration which did not require surgery and had no scoliosis at age 7. Autozygosity mapping identified seven regions of autozygosity >2Mb, on chromosomes 5, 8, 11, 12(x2), 13 and 22. Using exome capture Agent SureSelect all exon kit on an ABI SOLiD 4.0 platform, seven homozygous variants were identified within these regions. One was a c.627T>G; p.Phe209Leu mutation in the PDE6A gene, known to cause Retinitis Pigmentosa 43. Another was a novel c.1585C>T; p.Arg529Cys variant in the SH3TC2 gene, which is associated with CMT type 4C. Both mutations were confirmed by Sanger sequencing. These genes are separated by only 800kb and are therefore unlikely to segregate independently. Attempts at reaching a clinical diagnosis in these siblings had been directed towards identifying a single underlying disorder. The molecular findings indicated that they had dual pathology, illustrating the diagnostic power of new genetic technologies.

Identification of Drug Modulators Targeting Gene-Dosage Disease CMT1A

The structural integrity of myelin formed by Schwann cells in the peripheral nervous system (PNS) is required for proper nerve conduction and is dependent on adequate expression of myelin genes including peripheral myelin protein 22 (PMP22). Consequently, excess PMP22 resulting from its genetic duplication and overexpression has been directly associated with the peripheral neuropathy called Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent type of CMT. Here, in an attempt to identify transcriptional inhibitors with therapeutic value toward CMT1A, we developed a cross-validating pair of orthogonal reporter assays, firefly luciferase (FLuc) and β-lactamase (βLac), capable of recapitulating PMP22 expression, utilizing the intronic regulatory element of the human PMP22 gene. Each compound from a collection of approximately 3,000 approved drugs was tested at multiple titration points to achieve a pharmacological end point in a 1536-well plate quantitative high-throughput screen (qHTS) format. In conjunction with an independent counter-screen for cytotoxicity, the design of our orthogonal screen platform effectively contributed to selection and prioritization of active compounds, among which three drugs (fenretinide, olvanil, and bortezomib) exhibited marked reduction of endogenous Pmp22 mRNA and protein. Overall, the findings of this study provide a strategic approach to assay development for gene-dosage diseases such as CMT1A.

Validation of the Charcot–Marie–Tooth disease pediatric scale as an outcome measure of disability

Charcot-Marie-Tooth disease (CMT) is a common heritable peripheral neuropathy. There is no treatment for any form of CMT, although clinical trials are increasingly occurring. Patients usually develop symptoms during the first 2 decades of life, but there are no established outcome measures of disease severity or response to treatment. We identified a set of items that represent a range of impairment levels and conducted a series of validation studies to build a patient-centered multi-item rating scale of disability for children with CMT. As part of the Inherited Neuropathies Consortium, patients aged 3 to 20 years with a variety of CMT types were recruited from the USA, United Kingdom, Italy, and Australia. Initial development stages involved definition of the construct, item pool generation, peer review, and pilot testing. Based on data from 172 patients, a series of validation studies were conducted, including item and factor analysis, reliability testing, Rasch modeling, and sensitivity analysis. Seven areas for measurement were identified (strength, dexterity, sensation, gait, balance, power, endurance), and a psychometrically robust 11-item scale was constructed (CMT Pediatric Scale [CMTPedS]). Rasch analysis supported the viability of the CMTPedS as a unidimensional measure of disability in children with CMT. It showed good overall model fit, no evidence of misfitting items, and no person misfit, and it was well targeted for children with CMT. The CMTPedS is a well-tolerated outcome measure that can be completed in 25 minutes. It is a reliable, valid, and sensitive global measure of disability for children with CMT from the age of 3 years.

Genetic disruption of Pten in a novel mouse model of tomaculous neuropathy

‘Tomacula’ and myelin outfoldings are striking neuropathological features of a diverse group of inherited demyelinating neuropathies. Whereas the underlying genetic defects are well known, the molecular mechanisms of tomacula formation have remained obscure. We hypothesized that they are caused by uncontrolled, excessive myelin membrane growth, a process, which is regulated in normal development by neuregulin-1/ErbB2, PI3 Kinase signalling and ERK/MAPK signalling. Here, we demonstrate by targeted disruption of Pten in Schwann cells that hyperactivation of the endogenous PI3 Kinase pathway causes focal hypermyelination, myelin outfoldings and tomacula, even when induced in adult animals by tamoxifen, and is associated with progressive peripheral neuropathy. Activated AKT kinase is associated with PtdIns(3,4,5)P3 at paranodal loops and Schmidt–Lanterman incisures. This striking myelin pathology, with features of human CMT type 4B1 and HNPP, is dependent on AKT/mTOR signalling, as evidenced by a significant amelioration of the pathology in mice treated with rapamycin. We suggest that regions of non-compact myelin are under lifelong protection by PTEN against abnormal membrane outgrowth, and that dysregulated phosphoinositide levels play a critical role in the pathology of tomaculous neuropathies.

Successful Alternative Treatment Containing Vindesine for Acute Lymphoblastic Leukemia With Charcot-Marie-Tooth Disease

Peripheral neuropathy is a well-known side effect of vincristine (VCR), a microtubule inhibitor commonly used to treat malignancies. Severe neurological adverse events can occur in patients with Charcot-Marie-Tooth disease (CMT) treated with VCR. Vindesine is also a microtubule inhibitor, which, like VCR, is widely used to treat malignancies. The case of an 11-year-old female patient with CMT type 1A who developed severe peripheral neuropathy induced by VCR given for her acute lymphoblastic leukemia is reported. Alternative treatment containing vindesine instead of VCR led to a successful outcome without a relapse of leukemia or neurological worsening of CMT.

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.

Mutation analysis of PMP22 in Slovak patients with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies

Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies are the most commonly inherited neurological disorders in humans, characterized by clinical and genetic heterogeneity. The most prevalent clinical entities belonging to this group of disorders are CMT type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). CMT1A and HNPP are predominantly caused by a 1.5 Mb duplication and deletion in the chromosomal region 17p11.2, respectively, and less frequently by other mutations in the peripheral myelin protein 22 (PMP22) gene. Despite being relatively common diseases, they haven't been previously studied in the Slovak population. Therefore, the aim of this study was to identify the spectrum and frequency of PMP22 mutations in the Slovak population by screening 119 families with CMT and 2 families with HNPP for causative mutations in this gene. The copy number determination of PMP22 resulted in the detection of CMT1A duplication in 40 families and the detection of HNPP deletion in 7 families, 6 of which were originally diagnosed as CMT. Consequent mutation screening of families without duplication or deletion using dHPLC and sequencing identified 6 single base changes (3 unpublished to date), from which only c.327C>A (Cys109X) present in one family was provably causative. These results confirm the leading role of PMP22 mutation analysis in the differential diagnosis of CMT and show that the spectrum and frequency of PMP22 mutations in the Slovak population is comparable to that seen in the global population.

Genetics of Neuropathies

Charcot-Marie-Tooth disease (CMT) encompasses the heritable motor and sensory neuropathies that comprise most of the inherited peripheral neuropathies. Due to the great genetic heterogeneity of the condition, it can be difficult to determine what type of CMT a person has. The major phenotypic features of the CMT subtypes are delineated, as well as recommendations for focused genetic testing.