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With therapeutic trials on the horizon for Charcot-Marie-Tooth type 1A (CMT1A), reliable, valid, and responsive clinical outcome assessments and biomarkers are essential. Accelerate Clinical Trials in CMT (ACT-CMT) is an international study designed to address important gaps in CMT1A clinical trial readiness including the lack of a validated, responsive functional outcome measure for adults, and a lack of validated biomarkers for multicenter application in clinical trials in CMT1A. The primary aims of ACT-CMT include validation of the Charcot-Marie-Tooth Functional Outcome Measure, magnetic resonance imaging of intramuscular fat accumulation as a lower limb motor biomarker, and in-vivo reflectance confocal microscopy of Meissner corpuscle sensory receptor density, a sensory biomarker. Initial studies have indicated that these measures are feasible, reliable and valid. A large prospective, multi-site study is necessary to fully validate and examine the responsiveness of these outcome measures in relation to existing outcomes for use in future clinical trials involving individuals with CMT1A. Two hundred 15 adults with CMT1A are being recruited to participate in this prospective, international, multi-center study. Serial assessments, up to 3 years, are performed and include the CMT-FOM, CMT Exam Score-Rasch, Overall Neuropathy Limitations Scale, CMT-Health Index, as well as nerve conduction studies, and magnetic resonance imaging and Meissner corpuscle biomarkers. Correlations using baseline data will be examined for validity. Longitudinal analyses will document the changes in function, intramuscular fat accumulation, Meissner corpuscle sensory receptor density. Lastly, we will use anchor-based and other statistical methods to determine the minimally clinically important change for these clinical outcome assessments and biomarkers in CMT1A. Reliable, and responsive clinical outcome assessments of function and disease progression biomarkers are urgently needed for application in early and late phase clinical trials in CMT1A. The ACT-CMT study protocol will address this need through the prospective, longitudinal, multicenter examination in unprecedented detail of novel and existing clinical outcome assessments and motor and sensory biomarkers, and enhance international clinical trial infrastructure, training and preparedness for future therapeutic trials in CMT and related neuropathies.

Objective Experimental therapies under development for Friedreich’s Ataxia (FRDA) require validated biomarkers. In‐vivo reflectance confocal microscopy (RCM) of skin is a noninvasive way to quantify Meissner’s corpuscle (MC) density and has emerged as a sensitive measure of sensory polyneuropathies. We conducted a prospective, cross‐sectional study evaluating RCM of MCs and conventional peripheral nerve measures as candidate peripheral nerve markers in FRDA. Methods Sixteen individuals with FRDA and 16 age‐ and gender‐matched controls underwent RCM of MC density and morphology, skin biopsies for epidermal nerve fiber density (ENFD), nerve conduction studies (NCS), and quantitative sensory testing (QST) including touch, vibration, and cooling thresholds. Results MC densities were measurable in all participants with FRDA, and were lower at digit V (hand), thenar eminence, and arch (foot) compared to controls. By contrast, sensory NCS showed floor effects and were obtainable in only 13% of FRDA participants. QST thresholds for touch, vibration, and cooling were higher at the hand and foot in FRDA than controls. Reductions in ENFDs were present in more severely affected individuals with FRDA (Friedreich’s Ataxia Rating Scale (FARS) >60) compared to matched controls, although skin biopsies were not well tolerated in children. MC densities, ENFDs, and touch and vibration thresholds were associated with clinical disease severity (FARS and modified FARS) and duration since symptom onset. Interpretation MC density, ENFD, and QST thresholds provide structural and physiologic markers of sensory involvement in FRDA. Longitudinal evaluation is needed to determine whether these measures can identify changes associated with disease progression or treatment.

Objective To evaluate in-vivo reflectance confocal microscopy (RCM) of Meissner's corpuscles (MC) in diabetic distal symmetric polyneuropathy (DSP). Methods Forty-three adults with diabetes and 21 control subjects underwent RCM of MC density at the fingertip of digit V, thenar eminence (TE), and arch of the foot, ankle skin biopsy for epidermal nerve fiber density (ENFD), electrophysiological studies, monofilament threshold testing, and timed vibration at the toe. Subjects with diabetes were subdivided into groups with and without clinical DSP using the American Academy of Neurology (AAN) case definition and neuropathy outcomes were compared across groups. Results Both diabetic groups (with and without AAN clinical DSP criteria) had objective evidence of peripheral sensory involvement using conventional sensory measures, although those with clinical DSP criteria had greater abnormalities. MC densities were lower in the entire diabetic group at the TE and digit V relative to controls. MC densities at all imaging sites were associated with corresponding conventional sensory measures. MC densities were reduced in subjects without AAN clinical DSP criteria at the TE and digit V compared to controls whereas conventional upper limb sensory measures did not differ between these groups. Conclusions In-vivo RCM of MC density at digit V is a non-invasive, painless, objective marker in diabetes that offers a window into early large fiber sensory nerve terminal loss. Further studies are needed to determine whether RCM of MCs can identify quantitative changes in DSP associated with disease progression or treatment.

Introduction This study aims to assess the frequency, location, severity, duration, and fluctuation over time of muscle cramps in Charcot Marie Tooth Disease (CMT).

This study determines the impact of symptoms associated with Charcot-Marie-Tooth disease on quality-of-life. Charcot-Marie-Tooth patients in the Inherited Neuropathies Consortium Rare Diseases Clinical Research Network Contact Registry were surveyed. The survey inquired about 214 symptoms and 20 themes previously identified as important to Charcot-Marie-Tooth patients through patient interviews. Symptom population impact was calculated as the prevalence multiplied by the relative importance of each symptom identified. Prevalence and symptom impact were analyzed by age, symptom duration, gender, Charcot-Marie-Tooth type, and employment status. 407 participants returned the survey, identifying foot and ankle weakness (99.7%) and impaired balance (98.6%) as the most prevalent themes. Foot and ankle weakness and limitations with mobility were the themes with the highest impact. Both symptom prevalence and impact gradually increased with age and symptom duration. Several themes were more prevalent in women with Charcot-Marie-Tooth, including activity limitations, pain, fatigue, hip-thigh weakness, and gastrointestinal issues. All of the themes, except emotional or body image issues, were more prevalent among unemployed individuals. There were minimal differences in symptom prevalence between Charcot-Marie-Tooth types. There are multiple symptoms that impact Charcot-Marie-Tooth quality-of-life in adults. These symptoms have different levels of importance, are readily recognized by patients, and represent critical areas of Charcot-Marie-Tooth health.

Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons.

A subtelomeric region, 4q35.2, is implicated in facioscapulohumeral muscular dystrophy (FSHD), a dominant disease thought to involve local pathogenic changes in chromatin. FSHD patients have too few copies of a tandem 3.3-kb repeat (D4Z4) at 4q35.2. No phenotype is associated with having few copies of an almost identical repeat at 10q26.3. Standard expression analyses have not given definitive answers as to the genes involved. To investigate the pathogenic effects of short D4Z4 arrays on gene expression in the very gene-poor 4q35.2 and to find chromatin landmarks there for transcription control, unannotated genes and chromatin structure, we mapped DNaseI-hypersensitive (DH) sites in FSHD and control myoblasts. Using custom tiling arrays (DNase-chip), we found unexpectedly many DH sites in the two large gene deserts in this 4-Mb region. One site was seen preferentially in FSHD myoblasts. Several others were mapped >0.7 Mb from genes known to be active in the muscle lineage and were also observed in cultured fibroblasts, but not in lymphoid, myeloid or hepatic cells. Their selective occurrence in cells derived from mesoderm suggests functionality. Our findings indicate that the gene desert regions of 4q35.2 may have functional significance, possibly also to FSHD, despite their paucity of known genes.

In facioscapulohumeral muscular dystrophy (FSHD) the majority of patients carry a D4Z4 macrosatellite repeat contraction in the subtelomere of chromosome 4q. Several disease mechanisms have been proposed to explain how repeat contraction causes muscular dystrophy. All proposed mechanisms foresee a change from a closed to a more open chromatin structure followed by loss of control over expression of genes in or proximal to D4Z4. Initially, a distance and residual repeat size-dependent upregulation of the candidate genes FRG2, FRG1 and ANT1 was observed, but most successive expression studies failed to support transcriptional upregulation of 4qter genes. Moreover, chromatin studies do not provide evidence for a cis-spreading mechanism operating at 4qter in FSHD. In part, this inconsistency may be explained by differences in the techniques used, and the use of RNA samples obtained from different muscle groups. The aim of this study is to comprehensively and uniformly study the expression of the FSHD candidate genes FRG1, FRG2, CRYM, ANT1, ALP, PITX1 and LRP2BP at the RNA and protein level in identically processed primary myoblasts, myotubes and quadriceps muscle. Expression was compared between samples obtained from FSHD patients and normal controls with samples from myotonic dystrophy type 1 patients as disease controls. No consistent changes in RNA or protein expression levels were observed between the samples. The one exception was a selective increase in FRG2 mRNA expression in FSHD myotubes. This study provides further evidence that there is no demonstrable consistent, large magnitude, overexpression of any of the FSHD candidate genes.

DNA methylation and chromatin DNaseI sensitivity were analyzed in and adjacent to D4Z4 repeat arrays, which consist of 1 to approximately 100 tandem 3.3-kb units at subtelomeric 4q and 10q. D4Z4 displayed hypomethylation in some cancers and hypermethylation in others relative to normal tissues. Surprisingly, in cancers with extensive D4Z4 methylation there was a barrier to hypermethylation spreading to the beginning of this disease-associated array (facioscapulohumeral muscular dystrophy, FSHD) despite sequence conservation in repeat units throughout the array. We infer a different chromatin structure at the proximal end of the array than at interior repeats, consistent with results from chromatin DNaseI sensitivity assays indicating a boundary element near the beginning of the array. The relative chromatin DNaseI sensitivity in FSHD and control myoblasts and lymphoblasts was as follows: a non-genic D4Z4-adjacent sequence (p13E-11, array-proximal)> untranscribed gene standards > D4Z4 arrays> constitutive heterochromatin (satellite 2; P < 10(-4) for all comparisons). Cancers displaying D4Z4 hypermethylation also exhibited a hypermethylation-resistant subregion within the 3.3-kb D4Z4 repeat units. This subregion contains runs of G that form G-quadruplexes in vitro. Unusual DNA structures might contribute to topological constraints that link short 4q D4Z4 arrays to FSHD and make long ones phenotypically neutral.

Synaptotagmin 2 is a synaptic vesicle protein that functions as a calcium sensor for neurotransmission but has not been previously associated with human disease. Via whole-exome sequencing, we identified heterozygous missense mutations in the C2B calcium-binding domain of the gene encoding Synaptotagmin 2 in two multigenerational families presenting with peripheral motor neuron syndromes. An essential calcium-binding aspartate residue, Asp307Ala, was disrupted by a c.920A>C change in one family that presented with an autosomal-dominant presynaptic neuromuscular junction disorder resembling Lambert-Eaton myasthenic syndrome. A c.923C>T variant affecting an adjacent residue (p.Pro308Leu) produced a presynaptic neuromuscular junction defect and a dominant hereditary motor neuropathy in a second family. Characterization of the mutation homologous to the human c.920A>C variant in Drosophila Synaptotagmin revealed a dominant disruption of synaptic vesicle exocytosis using this transgenic model. These findings indicate that Synaptotagmin 2 regulates neurotransmitter release at human peripheral motor nerve terminals. In addition, mutations in the Synaptotagmin 2 C2B domain represent an important cause of presynaptic congenital myasthenic syndromes and link them with hereditary motor axonopathies., National Institutes of Health (U.S.) (NIH Grant NS40296), Picower Institute for Learning and Memory (Picower Neurological Disease Research Fund), JPB Foundation

Disease severity of childhood Charcot-Marie-Tooth disease (CMT) has not been extensively characterized, either within or between types of CMT to date.To assess the variability of disease severity in a large cohort of children and adolescents with CMT.A cross-sectional study was conducted among 520 children and adolescents aged 3 to 20 years at 8 universities and hospitals involved in the Inherited Neuropathies Consortium between August 6, 2009, and July 31, 2014, in Australia, Italy, the United Kingdom, and the United States. Data analysis was conducted from August 1, 2014, to December 1, 2015.Scores on the Charcot-Marie-Tooth Disease Pediatric Scale (CMTPedS), a well-validated unidimensional clinical outcome measure to assess disease severity. This instrument includes 11 items assessing fine and gross motor function, sensation, and balance to produce a total score ranging from 0 (unaffected) to 44 (severely affected).Among the 520 participants (274 males) aged 3 to 20 years, CMT type 1A (CMT1A) was the most prevalent type (252 [48.5%]), followed by CMT2A (31 [6.0%]), CMT1B (15 [2.9%]), CMT4C (13 [2.5%]), and CMTX1 (10 [1.9%]). Disease severity ranged from 1 to 44 points on the CMTPedS (mean [SD], 21.5 [8.9]), with ankle dorsiflexion strength and functional hand dexterity test being most affected. Participants with CMT1B (mean [SD] CMTPedS score, 24.0 [7.4]), CMT2A (29.7 [7.1]), and CMT4C (29.8 [8.6]) were more severely affected than those with CMT1A (18.9 [7.7]) and CMTX1 (males: 15.3 [7.7]; females: 13.0 [3.6]) (P .05). Scores on the CMTPedS tended to worsen principally during childhood (ages, 3-10 years) for participants with CMT4C and CMTX1 and predominantly during adolescence for those with CMT1B and CMT2A (ages, 11-20 years), while CMT1A worsened consistently throughout childhood and adolescence. For individual items, participants with CMT4C recorded more affected functional dexterity test scores than did those with all other types of CMT (P .05). Participants with CMT1A and CMTX1 performed significantly better on the 9-hole peg test and balance test than did those with all other types of CMT (P .05). Participants with CMT2A had the weakest grip strength (P .05), while those with CMT2A and CMT4C exhibited the weakest ankle plantarflexion and dorsiflexion strength, as well as the lowest long jump and 6-minute walk test distances (P .05). Multiple regression modeling identified increasing age (r = 0.356, β = 0.617, P .001) height (r = 0.251, β = 0.309, P = .002), self-reported foot pain (r = 0.162, β = .114, P = .009), and self-reported hand weakness (r = 0.243, β = 0.203, P .001) as independent predictors of disease severity.These results highlight the phenotypic variability within CMT genotypes and mutation-specific manifestations between types. This study has identified distinct functional limitations and self-reported impairments to target in future therapeutic trials.

Background Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, DUX4, that can encode a protein containing two homeodomains. A DUX4 transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how. Methods Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods. Results Many of the ~17,000 examined genes were differentially expressed (> 2-fold, p < 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked DUX4 RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types. Conclusions DUX4's pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated DUX4 expression at the myoblast or myotube stages. Our model could explain why DUX4's inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.