The integrated stress response contributes to tRNA synthetase-associated peripheral neuropathy.

Dominant mutations in ubiquitously expressed transfer RNA (tRNA) synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth (CMT) disease. Genetic evidence in mouse and Drosophila models suggests a gain-of-function mechanism. In this study, we used in vivo, cell type­specific transcriptional and translational profiling to show that mutant tRNA synthetases activate the integrated stress response (ISR) through the sensor kinase GCN2 (general control nonderepressible 2). The chronic activation of the ISR contributed to the pathophysiology, and genetic deletion or pharmacological inhibition of Gcn2 alleviated the peripheral neuropathy. The activation of GCN2 suggests that the aberrant activity of the mutant tRNA synthetases is still related to translation and that inhibiting GCN2 or the ISR may represent a therapeutic strategy in CMT.

Are we prepared for clinical trials in Charcot-Marie-Tooth disease?

There has been considerable progress in developing treatments for Charcot-Marie-Tooth disease with a number of therapies either completing or nearing clinical trials. In the case of CMT1A, the commonest subtype of CMT, there have been more than five randomised, double blind placebo-controlled trials. Although these trials were negative for the primary outcome measure, considerable lessons have been learnt leading to the collection of large prospective natural history data sets with which to inform future trial design as well as the development of new and sensitive outcome measures. In this review we summarise the difficulties of conducting clinical trials in a slowly progressive disease such as CMT1A and the requirement for sensitive, reproducible and clinically relevant outcome measures. We summarise the current array of CMT specific outcome measures subdivided into clinical outcome measures, functional outcome measures, patient reported outcome measures, biomarkers of disease burden and treatment specific biomarkers of target engagement. Although there is now an array of CMT specific outcome measures, which collectively incorporate clinically relevant, sensitive and reproducible outputs, a single outcome measure incorporating all three qualities remains elusive.

Mutation update for myelin protein zero-related neuropathies and the increasing role of variants causing a late-onset phenotype.

Mutations of myelin protein zero gene (MPZ) are found in 5% of Charcot-Marie-Tooth patients. In 2004, Shy et al. identified two main phenotypes associated with them: an early-onset subtype with mainly demyelinating features and a late-onset subgroup with prominent axonal impairment. We evaluated whether novel MPZ mutations described in literature during the last 14 years could still fit with this classification. We collected and revised reports of 69 novel MPZ mutations. Almost 90% of them could be alternatively classified as responsible for: (a) an early-onset phenotype, with first limitations starting before 3 years (2.5 ± 0.50 years), motor milestones delays, frequently severe course and upper limb MNCVs below 15 m/s; (b) late-onset neuropathy, with mean age of onset of 42.8 ± 1.5 years and mean upper limbs motor nerve conduction velocities (MNCVs) of 47.2 ± 1.4 m/s; (c) a phenotype more similar to typical CMT1A neuropathy, with onset during the 2nd decade, MNCV in the range of 15-30 m/s and slowly progressive course. The present work confirms that P0-related neuropathies may be separated into two main distinct phenotypes, while a third, relatively small, group comprehend patients carrying MPZ mutations and a childhood-onset disease, substantiating the subdivision into three groups proposed by Sanmaneechai et al. (Brain 138:3180-3192, 2015). Interestingly, during the last years, an increasing number of novel MPZ mutations causing a late-onset phenotype has been described, highlighting the clinical relevance of late-onset P0 neuropathies. Since the family history for neuropathy is often uncertain, due to the late disease onset, the number of patients carrying this genotype is probably underestimated.

CMT subtypes and disease burden in patients enrolled in the Inherited Neuropathies Consortium natural history study: a cross-sectional analysis.

BACKGROUND: The international Inherited Neuropathy Consortium (INC) was created with the goal of obtaining much needed natural history data for patients with Charcot-Marie-Tooth (CMT) disease. We analysed clinical and genetic data from patients in the INC to determine the distribution of CMT subtypes and the clinical impairment associated with them. METHODS: We analysed data from 1652 patients evaluated at 13 INC centres. The distribution of CMT subtypes and pathogenic genetic mutations were determined. The disease burden of all the mutations was assessed by the CMT Neuropathy Score (CMTNS) and CMT Examination Score (CMTES). RESULTS: 997 of the 1652 patients (60.4%) received a genetic diagnosis. The most common CMT subtypes were CMT1A/PMP22 duplication, CMT1X/GJB1 mutation, CMT2A/MFN2 mutation, CMT1B/MPZ mutation, and hereditary neuropathy with liability to pressure palsy/PMP22 deletion. These five subtypes of CMT accounted for 89.2% of all genetically confirmed mutations. Mean CMTNS for some but not all subtypes were similar to those previously reported. CONCLUSIONS: Our findings confirm that large numbers of patients with a representative variety of CMT subtypes have been enrolled and that the frequency of achieving a molecular diagnosis and distribution of the CMT subtypes reflects those previously reported. Measures of severity are similar, though not identical, to results from smaller series. This study confirms that it is possible to assess patients in a uniform way between international centres, which is critical for the planned natural history study and future clinical trials. These data will provide a representative baseline for longitudinal studies of CMT. CLINICAL TRIAL REGISTRATION: ID number NCT01193075.

Mutations in the tail domain of DYNC1H1 cause dominant spinal muscular atrophy.

OBJECTIVE: To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600). METHODS: Target exon capture and next generation sequencing was used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed. RESULTS: Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state-a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function. CONCLUSIONS: We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein (DYNC1H1) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance.

Strategy for genetic testing in Charcot-Marie-disease.

BACKGROUND: Charcot Marie Tooth disease (CMT) affects one in 2500 people. Genetic testing is often pursued for family planning purposes, natural history studies and for entry into clinical trials. However, identifying the genetic cause of CMT can be expensive and confusing to patients and physicians due to locus heterogeneity. METHODS: We analyzed data from more than 1000 of our patients to identify distinguishing features in various subtypes of CMT. Data from clinical phenotypes, neurophysiology, family history, and prevalence was combined to create algorithms that can be used to direct genetic testing for patients with CMT. FINDINGS: The largest group of patients in our clinic have slow motor nerve conduction velocities (MNCV) in the upper extremities. Approximately 88% of patients in this group have CMT1A. Those who had intermediate MNCV had primarily CMT1X (52.8%) or CMT1B (27.8%). Patients with very slow MNCV and delayed walking were very likely to have CMT1A (68%) or CMT1B (32%). No patients with CMT1B and very slow MNCV walked before 15 months of age. Patients with CMT2A form our largest group of patients with axonal forms of CMT. INTERPRETATION: Combining features of the phenotypic and physiology groups allowed us to identify patients who were highly likely to have specific subtypes of CMT. Based on these results, we created a series of algorithms to guide testing. A more detailed review of this data is published in Annals of Neurology (1).

MFN2 mutations cause severe phenotypes in most patients with CMT2A.

BACKGROUND: Charcot-Marie-Tooth disease type 2A (CMT2A), the most common form of CMT2, is caused by mutations in the mitofusin 2 gene (MFN2), a nuclear encoded gene essential for mitochondrial fusion and tethering the endoplasmic reticulum to mitochondria. Published CMT2A phenotypes have differed widely in severity. METHODS: To determine the prevalence and phenotypes of CMT2A within our clinics we performed genetic testing on 99 patients with CMT2 evaluated at Wayne State University in Detroit and on 27 patients with CMT2 evaluated in the National Hospital for Neurology and Neurosurgery in London. We then preformed a cross-sectional analysis on our patients with CMT2A. RESULTS: Twenty-one percent of patients had MFN2 mutations. Most of 27 patients evaluated with CMT2A had an earlier onset and more severe impairment than patients without CMT2A. CMT2A accounted for 91% of all our severely impaired patients with CMT2 but only 11% of mildly or moderately impaired patients. Twenty-three of 27 patients with CMT2A were nonambulatory prior to age 20 whereas just one of 78 non-CMT2A patients was nonambulatory after this age. Eleven patients with CMT2A had a pure motor neuropathy while another 5 also had profound proprioception loss. MFN2 mutations were in the GTPase domain, the coiled-coil domains, or the highly conserved R3 domain of the protein. CONCLUSIONS: We find MFN2 mutations particularly likely to cause severe neuropathy that may be primarily motor or motor accompanied by prominent proprioception loss. Disruption of functional domains of the protein was particularly likely to cause neuropathy.

Determinants of reduced health-related quality of life in pediatric inherited neuropathies.

OBJECTIVE: We have shown that health-related quality of life (QOL) in children with inherited neuropathies (Charcot-Marie-Tooth disease [CMT]) is significantly reduced compared to population norms, thus establishing its utility as an outcome measure in therapeutic trials. However, the Australian ascorbic acid trial in children with CMT type 1A (CMT1A) identified no change in QOL scores despite a trend toward improvement in nerve conduction velocities in the treated group. The objective of this study was to identify clinical, electrophysiologic, and functional correlates of QOL in children with CMT1A, to guide future investigations of strategies to improve QOL and reduce disability in these patients. METHODS: In this cross-sectional study, a series of multivariate regression models were developed to determine whether QOL scores could be explained by demographic and symptom data, standardized measures of gross motor function, foot/ankle and hand/finger involvement, electrophysiology, and gait characteristics in 70 children aged 5-16 years with CMT1A. RESULTS: Independent determinants of reduced QOL in children with CMT1A, from strongest to weakest, were leg cramps, hand tremor, short step length, reduced long jump distance, ankle inflexibility, poor agility and endurance, advancing age, and foot drop. Many of the standardized clinical and electrophysiologic measures used as endpoints in clinical trials of CMT correlated poorly with QOL. CONCLUSION: QOL is negatively affected by CMT1A in children. Multivariate modeling suggests that interventions designed to improve leg cramps, tremor, agility, endurance, and ankle flexibility might have a substantial effect on QOL in children with CMT1A.

Diagnosis and new treatments in genetic neuropathies.

The genetic neuropathies are a clinically and genetically heterogeneous group of diseases of which the most common types are Charcot-Marie-Tooth disease (CMT), the hereditary sensory and autonomic neuropathies and the distal hereditary motor neuropathies. More than 30 causative genes have been described, making an accurate genetic diagnosis increasingly possible. Although no specific therapies are yet available, research into their pathogenesis has revolutionised our understanding of the peripheral nervous system and allowed the development of rational approaches to therapy. The first therapeutic trials in CMT are currently underway. This review will suggest an approach to the diagnosis of these disorders and provide an update on new therapies.

X-linked distal hereditary motor neuropathy maps to the DSMAX locus on chromosome Xq13.1-q21.

OBJECTIVE: To clinically characterize and map the gene locus in a three-generation family with an X-linked adult-onset distal hereditary motor neuropathy. METHODS: Microsatellite markers spanning the juvenile distal spinal muscular atrophy (DSMAX) locus were genotyped and analyzed using genetic linkage analysis. The promoter, untranslated and coding region of the gap junction beta1 (GJB1) gene was sequenced. Nine positional candidate genes were screened for disease mutations using high-resolution melt (HRM) analysis. RESULTS: The family showed significant linkage to markers on chromosome Xq13.1-q21. Haplotype construction revealed a disease-associated haplotype between the markers DXS991 and DX5990. Sequence analysis excluded pathogenic changes in the coding and promoter regions of the GJB1 gene. Additional fine mapping in the family refined the DSMAX locus to a 1.44-cM interval between DXS8046 and DXS8114. HRM analysis did not identify disease-associated mutations in the coding region of nine candidate genes. CONCLUSION: We have identified a family with adult-onset distal hereditary motor neuropathy that refines the locus reported for juvenile distal spinal muscular atrophy (DSMAX) on chromosome Xq13.1-q21. Exclusion of mutations in the coding and regulatory region of the GJB1 gene eliminated the CMTX1 locus as a cause of disease in this family. Nine positional candidate genes in the refined interval underwent mutation analysis and were eliminated as the pathogenic cause of DSMAX in this family. The syndrome in this family may be allelic to the juvenile distal spinal muscular atrophy first reported at this locus.

Different clinical and magnetic resonance imaging features between Charcot-Marie-Tooth disease type 1A and 2A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the more frequent cause of demyelinating CMT, and CMT2A is the most common cause of axonal CMT. We conducted a magnetic resonance imaging (MRI) study on 39 CMT1A and 21 CMT2A patients to compare their neuroimaging patterns and correlate with clinical features. CMT1A patients showed selective fatty infiltration with a preference for anterior and lateral compartment muscles, whereas CMT2A patients showed a preference for superficial posterior compartment muscles. Early-onset CMT2A patients showed more severe leg fatty atrophy than late-onset CMT2A patients. In late-onset CMT2A, soleus muscle was the earliest, and most severely affected than the other leg muscles. Selective involvement of intrinsic foot muscles is a characteristic pattern of minimal CMT1A and CMT2A. Our MRI study demonstrates different patterns of fatty infiltration involving superficial posterior compartment muscles in CMT2A (partial T-type), and peroneal nerve innervated muscles in CMT1A (P-type).

Neuropathy progression in Charcot-Marie-Tooth disease type 1A.

OBJECTIVE: To determine the rate of disease progression in Charcot-Marie-Tooth disease type 1A (CMT1A). BACKGROUND: CMT1A is the most common inherited peripheral neuropathy, affecting approximately 1:5,000 people irrespective of ethnic background or gender. There is no cure for CMT1A. Clinical trials are being initiated that use the CMT Neuropathy Score (CMTNS), a composite score based on patient symptoms, signs, and neurophysiologic abnormalities, as the primary outcome variable. The sensitivity of the CMTNS or any other score to change over time, as a measure of CMT1A progression, has yet to be determined. METHODS: We determined the CMTNS as well as the Neuropathy Impairment Score (NIS) on 72 patients followed for up to 8 years. The rate of disease progression was evaluated for the CMTNS and NIS using mixed effects linear regression models, adjusting for age and gender. RESULTS: Both CMTNS and NIS showed changes over time. The CMTNS increased an average of 0.686 points per year (95% CI 0.461 to 0.911, p

CMT1X phenotypes represent loss of GJB1 gene function.

OBJECTIVE: To investigate possible genotype-phenotype correlations and to evaluate the natural history of patients with Charcot-Marie-Tooth disease type 1X (CMT1X). BACKGROUND: CMT1X is caused by over 260 distinct mutations in the gap junction beta 1 (GJB1) gene, located on the X chromosome, which encodes the gap junction protein connexin 32 (Cx32). The natural history of CMT1X is poorly understood, and it remains unknown whether particular mutations cause more severe neuropathies through abnormal gain-of-function mechanisms. METHODS: We evaluated 73 male patients with CMT1X, who each have 1 of 28 different GJB1 mutations predicted to affect nearly all domains of Cx32. Disability was evaluated quantitatively by the CMT Neuropathy Score (CMTNS) as well as by the CMT Symptom Score (CMTSS) and the CMT Examination Score (CMTES), which are both based on the CMTNS. Patients were also evaluated by neurophysiology. RESULTS: In all patients, disability increased with age, and the degree of disability was comparable with that observed in patients with a documented GJB1 deletion. Disability correlated with a loss of motor units as assessed by motor unit number estimates. CONCLUSIONS: Taken together, these data suggest that most GJB1 mutations cause neuropathy by a loss of normal connexin 32 function. Therefore, treatment of male patients with Charcot-Marie-Tooth disease type 1X may prove amenable to gene replacement strategies.

Skin biopsies demonstrate MPZ splicing abnormalities in Charcot-Marie-Tooth neuropathy 1B.

OBJECTIVE: To demonstrate that intronic mutations in the myelin protein zero (MPZ) cause Charcot-Marie-Tooth neuropathy 1B (CMT1B) by disrupting MPZ splicing. METHODS: We report a family with a T>G transversion at the invariant + 2 position in intron 4 of MPZ (c.614 + 2T>G) that abolishes 5' donor site recognition and is predicted to alter MPZ splicing. We obtained detailed clinical and neurophysiologic analysis of the family. We performed skin biopsies to investigate splicing abnormalities, MPZ protein levels, and localization in myelinated nerves. RESULTS: Patients developed a late onset neuropathy with minimally slow nerve conduction velocities. Skin biopsies confirmed the predicted skipping of exon 4 and downstream frameshift of the mutant MPZ. Quantitative immuno-EM demonstrated normal nerve MPZ levels, suggesting that the mutant MPZ was transported to compact myelin. CONCLUSIONS: Intronic mutations cause CMT1B by disrupting splicing and certain MPZ mutations may cause neuropathy by interacting with the wild type MPZ in the extracellular space of compact myelin.

Reliability and validity of the CMT neuropathy score as a measure of disability.

OBJECTIVE: To determine the validity and reliability of the Charcot-Marie-Tooth disease (CMT) neuropathy score (CMTNS) in patients with inherited neuropathy. BACKGROUND: Natural history studies and potential treatment trials for patients with various forms of CMT are limited by the lack of quantitative methodologies to monitor disease progression. Most cases of CMT can be considered length-dependent axonal neuropathies because disability for even the demyelinating forms correlates with length-dependent axonal degeneration. The total neuropathy score (TNS) is a validated composite measure of disability in length-dependent axonal neuropathies but is weighted toward predominantly sensory neuropathies. Thus, the authors have devised a CMTNS, modified from the TNS, to provide a single measure to quantify CMT disability. METHODS: The authors measured inter- and intrainvestigator reliability of the CMTNS and performed a validation of the score with the Neuropathy Impairment Score (NIS), patient self-assessment scores, an ambulation index, and other measures of disability. RESULTS: Inter- and intrainvestigator reliability was more than 95% in the 60 patients evaluated. Patients could be divided into mild (CMTNS, < or =10), moderate (CMTNS, 11 to 20), and severe (CMTNS, > or =21) categories and demonstrated excellent correlations among all measures of disability. CONCLUSION: The Charcot-Marie-Tooth disease (CMT) neuropathy score is a validated measure of length-dependent axonal and demyelinating CMT disability and can be investigated as an end point for longitudinal studies and clinical trials of CMT.

Quantitative sensory testing: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology.

OBJECTIVE: This assessment evaluates the clinical utility, efficacy, and safety of quantitative sensory testing (QST). METHODS: By searching MEDLINE, Current Contents, and their personal files, the authors identified 350 articles. Selected articles utilized computer operated threshold systems, manually operated threshold systems, and electrical threshold devices. The authors evaluated the use of normal values and the degree of reproducibility between the same and different systems. Articles were rated using a standard classification of evidence scheme. RESULTS: Because of differences between systems, normal values from one system cannot be transposed to others. Reproducibility of results was also an important concern, and there is no consensus on how it should be defined. The authors identified no adequately powered class I studies demonstrating the effectiveness of QST in evaluating any particular disorder. A number of class II and III studies demonstrated that QST is probably or possibly useful in identifying small or large fiber sensory abnormalities in patients with diabetic neuropathy, small fiber neuropathies, uremic neuropathies, and demyelinating neuropathy. CONCLUSIONS: QST is a potentially useful tool for measuring sensory impairment for clinical and research studies. However, QST results should not be the sole criteria used to diagnose pathology. Because malingering and other nonorganic factors can influence the test results, QST is not currently useful for the purpose of resolving medicolegal matters. Well-designed studies comparing different QST devices and methodologies are needed and should include patients with abnormalities detected solely by QST.

Mutations in the neurofilament light chain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease.

Neurofilament light chain polypeptide (NEFL) is one of the most abundant cytoskeletal components of the neuron. Mutations in the NEFL gene were recently reported as a cause for autosomal dominant Charcot-Marie-Tooth type 2E (CMT2E) linked to chromosome 8p21. In order to investigate the frequency and phenotypic consequences of NEFL mutations, we screened 323 patients with CMT or related peripheral neuropathies. We detected six disease associated missense mutations and one 3-bp in-frame deletion clustered in functionally defined domains of the NEFL protein. Patients have an early onset and often a severe clinical phenotype. Electrophysiological examination shows moderately to severely slowed nerve conduction velocities. We report the first nerve biopsy of a CMT patient with a de novo missense mutation in NEFL, and found an axonal pathology with axonal regeneration clusters and onion bulb formations. Our findings provide further evidence that the clinical variation observed in CMT depends on the gene mutated and the specific type of mutation, and we also suggest that NEFL mutations need to be considered in the molecular evaluation of patients with sporadic or dominantly inherited CMT.

Protein zero is necessary for E-cadherin-mediated adherens junction formation in Schwann cells.

Protein Zero (P0), the major structural protein in the peripheral nervous system (PNS) myelin, acts as a homotypic adhesion molecule and is thought to mediate compaction of adjacent wraps of myelin membrane. E-Cadherin, a calcium-dependent adhesion molecule, is also expressed in myelinating Schwann cells in the PNS and is involved in forming adherens junctions between adjacent loops of membrane at the paranode. To determine the relationship, if any, between P0-mediated and cadherin-mediated adhesion during myelination, we investigated the expression of E-cadherin and its binding partner, beta-catenin, in sciatic nerve of mice lacking P0 (P0(-/-)). We find that in P0(-/-) peripheral myelin neither E-cadherin nor beta-catenin are localized to paranodes, but are instead found in small puncta throughout the Schwann cell. In addition, only occasional, often rudimentary, adherens junctions are formed. Analysis of E-cadherin and beta-catenin expression during nerve development demonstrates that E-cadherin and beta-catenin are localized to the paranodal region after the onset of myelin compaction. Interestingly, axoglial junction formation is normal in P0(-/-) nerve. Taken together, these data demonstrate that P0 is necessary for the formation of adherens junctions but not axoglial junctions in myelinating Schwann cells.