Search

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype–phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype–phenotype correlations, we found clustering to overcome the limits of the “single-dimension” paradigm traditionally used to describe genotype–phenotype relationships.

Electrophysiology plays a crucial role in Guillain-Barré syndrome (GBS) diagnosis and subtype classification. The aim of our study was to assess the potential role of distal compound muscle action potential (dCMAP) for early differentiation between acute inflammatory demyelinating polyneuropathy (AIDP) and axonal GBS.We retrospectively reviewed the medical records of 24 subjects with AIDP and 18 subjects with axonal GBS. We built up receiver operating characteristic curves for total dCMAP duration and negative phase of dCMAP duration, in order to derive cut-off values able to differentiate between AIDP and axonal GBS.The total duration of dCMAP was significantly prolonged in AIDP compared to axonal GBS. AUCs, odds ratio and positive predictive values were higher for total duration than for negative peak duration. Nerve conduction parameters in the lower limbs were more sensitive than those in the upper limbs in distinguishing AIDP from axonal GBS.Total duration of dCMAP dispersion may capture an adjunctive component of distal demyelination, not measured by the more traditional parameters and may thus represent a useful tool for early differentiation between AIDP and axonal GBS.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disorder characterized by degeneration of motor neurons in the cerebral cortex, brain stem, and spinal cord. Most cases of ALS appear sporadically, but 5–10% of patients have a family history of disease. Mutations in the superoxide dismutase 1 gene (SOD1) have been found in 12–23% of familial cases and in 1–2% of sporadic cases. Currently, more than 180 different SOD1 gene variants have been identified in ALS patients. Here, we describe two apparently sporadic ALS patients carrying the same SOD1 c.355G>A variant, leading to the p.V119M substitution, not previously described. Both the patients showed pure lower motor neuron phenotype. The former presented with the flail leg syndrome, a rare ALS variant, characterized by progressive distal onset weakness and atrophy of lower limbs, slow progression and better survival than typical ALS. The latter exhibited rapidly progressive weakness of upper and lower limbs, neither upper motor neuron nor bulbar involvement, and shorter survival than typical ALS. We provide an accurate description of the phenotype, and a bioinformatics analysis of the p.V119M variant on protein structure. This study may increase the knowledge about genotype-phenotype correlations in ALS and improve the approach to ALS patients.

In this study, we assessed the modifications over time of daily activities and quality of life (QoL) in 32 subjects with anti-myelin-glycoprotein (MAG) antibody neuropathy. A widespread panel including clinical scores and patient-reported questionnaires, in compliance of the terms by the International Classification of Functioning, Disability, and Health (ICF) of the World Health Organization (WHO), was employed at enrollment (T0) and at follow-up evaluation (T1) after a mean interval of 15.4 ± 5.7 months. The Sensory Modality Sum score (SMS) at four limbs showed a significant worsening over time (mean score 27.2 ± 3.9 at T0 vs 25.7 ± 3 at T1 at upper limbs, P = .03; 20.5 ± 4.8 at T0 vs 18.6 ± 5.9 at T1 at lower limbs, P = .04). The Visual Analogue Scale (VAS) for pain significantly worsened at upper limbs at T1 (mean values 0.84 ± 1.95 at T0 vs 1.78 ± 2.6 at T1, P = .03). All the other tests did not show significant differences between T0 and T1. In the subgroup who underwent rituximab (15/32 treated before T0, 3/32 patients treated between T0 and T1 with median interval of 1 year), no significant differences were observed between T0 and T1. Despite the quite long follow-up, statistical significance was not achieved either for the limited number of patients or for the lack of sensitive outcome measures. In our cohort, the significant worsening of the SMS and VAS after a median of 14 months can be considered as a reliable expression of the natural history of the disease, and suggest that these scales might represent possible outcome measures in anti-MAG antibody neuropathy.

Strong evidence suggests that endoplasmic reticulum stress plays a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) through altered regulation of proteostasis. Robust preclinical findings demonstrated that guanabenz selectively inhibits endoplasmic reticulum stress-induced eIF2α-phosphatase, allowing misfolded protein clearance, reduces neuronal death and prolongs survival in in vitro and in vivo models. However, its safety and efficacy in patients with ALS are unknown. To address these issues, we conducted a multicentre, randomized, double-blind trial with a futility design. Patients with ALS who had displayed an onset of symptoms within the previous 18 months were randomly assigned in a 1:1:1:1 ratio to receive 64 mg, 32 mg or 16 mg of guanabenz or placebo daily for 6 months as an add-on therapy to riluzole. The purpose of the placebo group blinding was to determine safety but not efficacy. The primary outcome was the proportion of patients progressing to higher stages of disease within 6 months as measured using the ALS Milano-Torino staging system, compared with a historical cohort of 200 patients with ALS. The secondary outcomes were the rate of decline in the total revised ALS functional rating scale score, slow vital capacity change, time to death, tracheotomy or permanent ventilation and serum light neurofilament level at 6 months. The primary assessment of efficacy was performed using intention-to-treat analysis. The treatment arms using 64 mg and 32 mg guanabenz, both alone and combined, reached the primary hypothesis of non-futility, with the proportions of patients who progressed to higher stages of disease at 6 months being significantly lower than that expected under the hypothesis of non-futility and a significantly lower difference in the median rate of change in the total revised ALS functional rating scale score. This effect was driven by patients with bulbar onset, none of whom (0/18) progressed to a higher stage of disease at 6 months compared with those on 16 mg guanabenz (4/8; 50%), the historical cohort alone (21/49; 43%; P = 0.001) or plus placebo (25/60; 42%; P = 0.001). The proportion of patients who experienced at least one adverse event was higher in any guanabenz arm than in the placebo arm, with higher dosing arms having a significantly higher proportion of drug-related side effects and the 64 mg arm a significantly higher drop-out rate. The number of serious adverse events did not significantly differ between the guanabenz arms and the placebo. Our findings indicate that a larger trial with a molecule targeting the unfolded protein response pathway without the alpha-2 adrenergic related side-effect profile of guanabenz is warranted., ER stress plays a key role in ALS pathogenesis through altered regulation of proteostasis. In a phase 2 futility trial, Dalla Bella et al. show that the alpha-2 adrenergic receptor agonist guanabenz reduces the proportion of patients progressing to higher stages of disease at 6 months compared to historical controls.

The Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy with great clinical and genetic heterogeneity. Mutations in DNM2 have been associated with CMT dominant intermediate B (CMTDIB). However, mutations in the same gene are known to induce also axonal CMT (CMT2M) or centronuclear myopathy. Moreover, the ability of effectively and simultaneously sequencing different CMT-related genes by next-generation sequencing approach makes it possible to detect even the presence of modifier genes that sometimes give reason of clinical variability in the context of complex phenotypes. Here, we describe an Italian family with very variable severity of phenotype among members harboring a novel DNM2 gene mutation which caused a prevalent CMT2M phenotype. The contemporary presence of a de novo variant in PRX gene in the most severely affected family member suggests a possible modulator effect of the PRX variant thus highlighting the possible impact of modifier genes in CMT.

Summary Patients with ALS show, in addition to the loss of motor neurons in the spinal cord, brainstem, and cerebral cortex, an abnormal depletion of energy stores alongside hypermetabolism. In this study, we show that bioenergetic defects and muscle remodeling occur in skeletal muscle of the SOD1G93A mouse model of ALS mice prior to disease onset and before the activation of muscle denervation markers, respectively. These changes in muscle physiology were followed by an increase in energy expenditure unrelated to physical activity. Finally, chronic treatment of SOD1G93A mice with Ranolazine, an FDA-approved inhibitor of fatty acid β-oxidation, led to a decrease in energy expenditure in symptomatic SOD1G93A mice, and this occurred in parallel with a robust, albeit temporary, recovery of the pathological phenotype., Graphical Abstract, Highlights • Metabolic switch use occurs early in the skeletal muscle of SOD1G93A mice • Mitochondrial impairment precedes locomotor deficits and evokes catabolic pathways • Sarcolipin upregulation in presymptomatic SOD1G93A mice precedes hypermetabolism • Pharmacological modulation of hypermetabolism improves locomotor performance, Drugs; Molecular Neuroscience; Cellular Neuroscience

ObjectiveTo assess the prevalence and isotypes of anti-nodal/paranodal antibodies to nodal/paranodal proteins in a large chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) cohort, compare clinical features in seronegative vs seropositive patients, and gather evidence of their isotype-specific pathogenic role.MethodsAntibodies to neurofascin-155 (Nfasc155), neurofascin-140/186 (Nfasc140/186), contactin-1 (CNTN1), and contactin-associated protein 1 (Caspr1) were detected with ELISA and/or cell-based assay. Antibody pathogenicity was tested by immunohistochemistry on skin biopsy, intraneural injection, and cell aggregation assay.ResultsOf 342 patients with CIDP, 19 (5.5%) had antibodies against Nfasc155 (n = 9), Nfasc140/186 and Nfasc155 (n = 1), CNTN1 (n = 3), and Caspr1 (n = 6). Antibodies were absent from healthy and disease controls, including neuropathies of different causes, and were mostly detected in patients with European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) definite CIDP (n = 18). Predominant antibody isotypes were immunoglobulin G (IgG)4 (n = 13), IgG3 (n = 2), IgG1 (n = 2), or undetectable (n = 2). IgG4 antibody-associated phenotypes included onset before 30 years, severe neuropathy, subacute onset, tremor, sensory ataxia, and poor response to intravenous immunoglobulin (IVIG). Immunosuppressive treatments, including rituximab, cyclophosphamide, and methotrexate, proved effective if started early in IVIG-resistant IgG4-seropositive cases. Five patients with an IgG1, IgG3, or undetectable isotype showed clinical features indistinguishable from seronegative patients, including good response to IVIG. IgG4 autoantibodies were associated with morphological changes at paranodes in patients' skin biopsies. We also provided preliminary evidence from a single patient about the pathogenicity of anti-Caspr1 IgG4, showing their ability to penetrate paranodal regions and disrupt the integrity of the Nfasc155/CNTN1/Caspr1 complex.ConclusionsOur findings confirm previous data on the tight clinico-serological correlation between antibodies to nodal/paranodal proteins and CIDP. Despite the low prevalence, testing for their presence and isotype could ultimately be part of the diagnostic workup in suspected inflammatory demyelinating neuropathy to improve diagnostic accuracy and guide treatment.Classification of evidenceThis study provides Class III evidence that antibodies to nodal/paranodal proteins identify patients with CIDP (sensitivity 6%, specificity 100%).

Objectives: The aim of the study is to analyze the ALS disease progression and respiratory function of Italian patients treated with edaravone (EVN), as well as the adherence to, and the effects of, the therapy. Methods: We performed an observational study of patients treated with EVN from May 2017 to May 2019, in 39 Italian ALS Centers. Taking into account ALS patients with at least 12months of EVN treatment, we compared the decline of ALSFRS-R and FVC with a group of matched historical controls from the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) database, using both descriptive and survival analysis approaches. Results: A total of 331 ALS Italian patients treated with EVN and 290 matched historical controls were recruited in this study. No significant differences on disease progression or respiratory function were found comparing the two cohorts in both descriptive and survival analyses. The EVN treatment was overall well tolerated. Conclusions: The study showed that EVN treatment was well tolerated. No significant differences were reported in ALS patients treated and not treated with EVN, in terms of both disease progression and respiratory function. These findings prove that further studies are required to better clarify whether EVN could be considered an effective treatment for ALS disease.

Chronic ataxic neuropathy with anti-disialosyl IgM antibodies (CANDA) is a rare disorder for which the pathological, neurophysiological, and therapeutic evidence remains anecdotal and controversial. This report on CANDA focuses on the neurophysiological patterns and treatment responses shared by two cases. One patient underwent nerve ultrasound follow-up. A comprehensive review of the literature highlighted the diverse experiences with different treatment options. Response to different therapies was similar in both patients: intravenous immunoglobulins achieved a favorable response albeit with significant wearing-off fluctuations; treatment with subcutaneous immunoglobulins (SCIg) was an effective alternative leading to a clinical response for at least 2 years. Rituximab, which was trialed in both patients, was not continued long enough to determine its efficacy in modifying the disease course and/or modulating responsiveness to immunoglobulins. Steroids caused clinical worsening in both patients. Immunoglobulin therapy appeared as the most effective in the treatment of these two patients. SCIg provided an effective treatment option for the long-term management of CANDA.

Between March 2017 and February 2018, 27 newly diagnoses patients (about 2 months) were examined in the ENT Clinic of our University Hospital. The bulbar-onset of ALS was diagnosed by neurologists.According to the endoscopic and clinical results, patients were classified in 4 classes of dysphagia. Laryngology and speech pathology assessment with spectroacuostic analysis of speech using Praat software are fundamental for the proper evaluation of dysphonia of these patients. This study was the first attempt to find a vocal indicator of advancement in swallowing dysfunction in the patient population of ALS. Preliminary findings indicate that the reduction in the maximum fundamental frequency is associated with advancement of dysphagia.Monitoring the vocal parameters could be useful in order to give an early rehabilitation aid to these patients improving their quality of life and reducing aspiration risks.

Anti-myelin-associated glycoprotein (MAG) antibody is associated with clinically heterogeneous polyneuropathies. Our purpose was to compare neuropathy phenotypes identified by different anti-MAG tests’ results. Cohort study: Sera from 40 neuropathy anti-MAG EIA positive patients were tested for anti-MAG by Western blot (WB), for anti-peripheral nerve myelin (PNM) on monkey nerve by immunofluorescence assay (IFA), and for anti-HNK1 on rat CNS slices by IFA. Anti-sulfatide antibodies, for comparison, were also tested by EIA. Among 40 anti-MAG EIA positive sera, 85% also had anti-PNM IFA reactivity and 67.5% bind HNK1 on rat CNS. Anti-HNK1 positive patients had the classical predominantly distal acquired demyelinating symmetric (DADS) neuropathy with a benign course, while anti-PNM positive but anti-HNK1 negative patients had predominantly axonal neuropathy with a high frequency of anti-sulfatide reactivity and the worst long-term prognosis. Anti-MAG EIA positive patients without anti-PNM or anti-HNK1 IFA reactivity had a CIDP-like polyneuropathy. Different methods to test for anti-MAG antibodies identify different clinical and electrophysiological findings, as well as long-term outcome. HNK1 reactivity is the strongest marker of DADS.

Objective: Myotonic dystrophy (DM) is a monogenic disorder. It is caused by expansion of a cytosine-thymineguanine triplet in the DMPK gene which encodes for myotonic dystrophy protein kinase (DMPK).Methods: A 24-year-old man with DM and the DMPK mutation presented with elevated adrenocorticotropic hormone (ACTH) levels twice (152 and 185 pg/mL; normal value is 10 to 52 pg/mL) with normal cortisol levels (134.6 and 113.0 ng/mL, or 371.3 and 311.7 nmol/L; normal values are 67 to 226 ng/mL or 184.8 to 623.5 nmol/L). ACTH, corticotropin-releasing hormone (CRH) and insulin tolerance test (ITT) demonstrated normal cortisol response to ACTH and partial response to CRH and ITT tests, and ACTH hyperresponse to CRH and ITT. We suspected ACTH and/or ACTH receptor (ACTHR) mutations and evaluated the genetic profile for pro-opiomelanocortin (POMC), melanocortin 2 receptor (MC2R) and follicle-stimulating hormone receptor (FSHR) genes.Results: No mutations were found in either the MC2R or FSHR genes. The patient was heterozygous for the c.614A>G mutation corresponding to a p.53D>G substitution with a glycine instead of an aspartate in position 53 in POMC gene. This mutation was outside the sequence for ACTH (which spans amino acids 138 to 176) but was included in the part originating the N-terminal peptide of pro-opiomelanocortin (also called pro-γ-melanocyte stimulating hormone) which spans amino acids 27 to 102 and is involved in the regulation of adrenal steroidogenesis.Conclusion: The pathologic expansion of the cytosine-thymine-guanine triplet repeat in the 3' noncoding region of DMPK could explain the hyperresponse of ACTH typical of DM. The mutation of pro-γ-melanocyte-stimulating hormone could be associated with the abnormal response of cortisol, compatible with a partial adrenal insufficiency. Other studies are necessary to demonstrate this hypothesis.Abbreviations: ACTH = adrenocorticotropic hormone CRH = corticotropin-releasing hormone DM = myotonic dystrophy DMPK = myotonic dystrophy protein kinase FSHR = follicle-stimulating hormone receptor ITT = insulin tolerance test MC2R = melanocortin 2 receptor MSH = melanocyte-stimulating hormone POMC = pro-opiomelanocortin

Besides loss of motor neurons in spinal cord, brainstem and cerebral cortex, patients with ALS show an abnormal depletion of energy stores and a parallel hypermetabolism in spite of simultaneous progression of skeletal muscle atrophy. Our results highlighted bioenergetic defects in skeletal muscle of ALS mice long before the disease onset that lead to deep modifications of muscle physiology. Muscle remodelling occurs in SOD1G93A mice before the activation of muscle denervation markers and this is followed by an increase of energy expenditure unrelated to physical activity. To this regard our attention has been focused on the identification of precocious biomarkers closely related to hypermetabolism, a phenomenon that possesses prognostic and diagnostic relevance. Finally, chronic Ranolazine treatment, a FDA-approved inhibitor of fatty acid b-oxidation, decreases energy expenditure in symptomatic SOD1G93A mice and this correlates with a robust, albeit temporary, recovery of pathological phenotype.

Rationale:Aromatase inhibitors (AIs) are a class of drugs widely used in the treatment of estrogen sensitive breast and ovarian cancer which convert testosterone to estradiol and androstenedione to estrogen. The AIs of third generation, including anastrazole, letrozole and exemestane, have a

Hereditary motor and sensory neuropathies (HMSN) are genetically heterogeneous disorders affecting peripheral motor and sensory functions. Many different pathogenic variants in several genes involved in the demyelinating, the axonal and the intermediate HMSN forms have been identified, for which all inheritance patterns have been described. The mutation screening currently available is based on Sanger sequencing and is time-consuming and relatively expensive due to the high number of genes involved and to the absence of mutational hot spots. To overcome these limitations, we have designed a custom panel for simultaneous sequencing of 28 HMSN-related genes. We have applied this panel to three representative patients with variable HMSN phenotype and uncertain diagnostic classifications. Using our NGS platform we rapidly identified three already described pathogenic heterozygous variants in MFN2, MPZ and DNM2 genes. Here we show that our pre-custom platform allows a fast, specific and low-cost diagnosis in sporadic HMSN cases. This prompt diagnosis is useful for providing a well-timed treatment, establishing a recurrence risk and preventing further investigations poorly tolerated by patients and expensive for the health system. Importantly, our study illustrates the utility and successful application of NGS to mutation screening of a Mendelian disorder with extreme locus heterogeneity.

© 2018 Elsevier Inc., To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

Although anti-myelin-associated glycoprotein (MAG) antibody neuropathy is reported as a slowly progressive disease, it can lead to significant disability and impairment of health-related quality of life (HR-QoL) and social participation. The aim of this cross-sectional study was to evaluate the functioning and HR-QoL determinants in 67 patients with anti-MAG neuropathy in terms of the International Classification of Functioning, Disability, and Health (ICF). Evaluations included: Medical Research Council (MRC) sum score, Sensory Modality Sum score (SMS), Berg balance scale (BBS), Fatigue Severity Scale (FSS), Visual Analogue Scale (VAS) for pain, 9-Hole Peg Test (9-HPT), 6-min Walk Distance (6MWD), Impact on Participation and Autonomy (IPA) and the physical component score (PCS) and mental component score (MCS) of the short-form-36 health status scale (SF-36) HR-QoL measure. In the regression models, 6MWD was the most reliable predictor of PCS, explaining the 52% of its variance, while the strongest determinants of 6MWD were BBS and FSS, explaining the 41% of its variance. Consistently, VAS and BBS were good predictor of PCS, explaining together 54% of its variance. FSS was the most reliable determinant of MCS, explaining 25% of its variance. SMS and MRC were not QoL determinants. The results of our study suggest that 6MWD and FSS might be considered as potential meaningful outcome measures in future clinical trials. Furthermore, neurorehabilitation interventions aimed at improving balance and walking performance, fatigue management, and specific pain relief therapy should be considered to ameliorate participation in social life and HR-QoL in anti-MAG neuropathy patients. Electronic supplementary material The online version of this article (10.1007/s00415-018-9081-7) contains supplementary material, which is available to authorized users.

POLG gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA (mtDNA) replication and repair. Mutations in POLG have been linked to a spectrum of clinical phenotypes, resulting in autosomal recessive or dominant mitochondrial diseases. These mutations have been associated with heterogeneous phenotypes, presenting with varying severity and at different ages of onset, ranging from the neonatal period to late adult life. We screened 13 patients for POLG mutations. All patients underwent a complete neurological examination, and in most of cases, muscle biopsy was performed. We detected 15 different variations in 13 unrelated Italian patients. Two mutations were novel and mapped in the pol domain (p.Thr989dup and p.Ala847Thr) of the enzyme. We also report new cases carrying controversial variations previously described as incompletely penetrant or a variant of unknown significance. Our study increases the range of clinical presentations associated with mutations in POLG gene, underlining some peculiar clinical features, such as PEO associated with corneal edema, and epilepsy, severe neuropathy with achalasia. The addition of two new substitutions, including the second report of an in-frame duplication, to the growing list of defects increases the value of POLG genetic diagnosis in a range of neurological presentations.

IntroductionRecent studies suggest that endoplasmic reticulum stress may play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) through an altered regulation of the proteostasis, the cellular pathway-balancing protein synthesis and degradation. A key mechanism is thought to be the dephosphorylation of eIF2α, a factor involved in the initiation of protein translation. Guanabenz is an alpha-2-adrenergic receptor agonist safely used in past to treat mild hypertension and is now an orphan drug. A pharmacological action recently discovered is its ability to modulate the synthesis of proteins by the activation of translational factors preventing misfolded protein accumulation and endoplasmic reticulum overload. Guanabenz proved to rescue motoneurons from misfolding protein stress both in in vitro and in vivo ALS models, making it a potential disease-modifying drug in patients. It is conceivable investigating whether its neuroprotective effects based on the inhibition of eIF2α dephosphorylation can change the progression of ALS.Methods and analysesProtocolised Management In Sepsis is a multicentre, randomised, double-blind, placebo-controlled phase II clinical trial with futility design. We will investigate clinical outcomes, safety, tolerability and biomarkers of neurodegeneration in patients with ALS treated with guanabenz or riluzole alone for 6 months. The primary aim is to test if guanabenz can reduce the proportion of patients progressed to a higher stage of disease at 6 months compared with their baseline stage as measured by the ALS Milano-Torino Staging (ALS-MITOS) system and to the placebo group. Secondary aims are safety, tolerability and change in at least one biomarker of neurodegeneration in the guanabenz arm compared with the placebo group. Findings will provide reliable data on the likelihood that guanabenz can slow the course of ALS in a phase III trial.Ethics and disseminationThe study protocol was approved by the Ethics Committee of IRCCS ‘Carlo Besta Foundation’ of Milan (Eudract no. 2014-005367-32 Pre-results) based on the Helsinki declaration.

ObjectiveNext-generation sequencing (NGS) was applied in molecularly undiagnosed asymptomatic or paucisymptomatic hyperCKemia to investigate whether this technique might allow detection of the genetic basis of the condition.MethodsSixty-six patients with undiagnosed asymptomatic or paucisymptomatic hyperCKemia, referred to tertiary neuromuscular centers over an approximately 2-year period, were analyzed using a customized, targeted sequencing panel able to investigate the coding exons and flanking intronic regions of 78 genes associated with limb-girdle muscular dystrophies, rhabdomyolysis, and metabolic and distal myopathies.ResultsA molecular diagnosis was reached in 33 cases, corresponding to a positive diagnostic yield of 50%. Variants of unknown significance were found in 17 patients (26%), whereas 16 cases (24%) remained molecularly undefined. The major features of the diagnosed cases were mild proximal muscle weakness (found in 27%) and myalgia (in 24%). Fourteen patients with a molecular diagnosis and mild myopathic features on muscle biopsy remained asymptomatic at a 24-month follow-up.ConclusionsThis study of patients with undiagnosed hyperCKemia, highlighting the advantages of NGS used as a first-tier diagnostic approach in genetically heterogeneous conditions, illustrates the ongoing evolution of molecular diagnosis in the field of clinical neurology. Isolated hyperCKemia can be the sole feature alerting to a progressive muscular disorder requiring careful surveillance.

In hereditary neuropathy with liability to pressure palsies (HNPP), the increase in distal motor latencies (DMLs) is often out of proportion to the slowing of conduction velocities, but the pathophysiological mechanism is still unclear. We used a combined electrophysiological and ultrasonographic (US) approach to provide insight into this issue. Twelve HNPP subjects underwent extensive electrophysiological studies and US measurements of the cross-sectional area (CSA) of several peripheral nerves. US nerve enlargement was only observed in the carpal tunnel, Guyon's canal, the elbow and the fibular head. We did not observe US abnormalities at sites where nerve entrapment is uncommon. An increase in DMLs was observed regardless of US nerve enlargement. The increased nerve CSA only in common sites of entrapment likely reflected the well-documented nerve vulnerability to mechanical stress in HNPP. No morphometric changes were seen in the distal nerve segments where compression/entrapment is unlikely, despite the fact that the DMLs were increased. These data suggest that factors other than mechanical stress are responsible for the distal slowing of action potential propagation. We speculate that a mixture of mechanical insults and an axon-initiated process in the distal nerves underlies the distal slowing and/or conduction failure in HNPP.

Background POLG-related disorders are a group of heterogeneous diseases characterized by an overlapping clinical presentations and associated with mutations in the POLG gene. POLG codes for the catalytic subunit of mitochondrial polymerase gamma (POLG), essential for mitochondrial DNA (mtDNA) replication and repair. Studies on mutator POLG mice showed an increase in oxidative stress and apoptosis. In this regard we analysed the involvement of POLG mutations in the apoptotic regulation, evaluating apoptosis in peripheral blood lymphocytes (PBLs) from patients with POLG-related diseases. Methods Cells were cultured under basal conditions and with 2-deoxy- d -ribose (dRib), a reducing sugar that induces apoptosis by oxidative stress. Apoptosis rate was assessed by flow cytometry. Phosphatidylserine translocation, mitochondrial membrane depolarization and caspase 3 activation were also analysed. Results Our data showed higher percentages of apoptosis after dRib treatment in patients with POLG mutations than in controls, while under basal culture conditions, apoptosis levels were similar in the two groups. Conclusions Cells with POLG mutations are more sensitive than control cells to oxidative stress-induced apoptosis, confirming that mtDNA mutations may have a role in mitochondrial apoptosis pathway. We also suggest that redox state homeostasis may play a crucial role in phenotypic expression of POLG-related diseases.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder. Since no diagnostic laboratory test exists, the identification of specific biomarkers could be fundamental in clinical practice. microRNAs (miRNAs) are considered promising biomarkers for neurodegenerative diseases. The aim of the study was to identify a CSF miRNA set that could differentiate ALS from non-ALS condition. miRNA profiling in CSF from ALS patients (n = 24; eight with C9orf72 expansion) and unaffected control subjects (n = 24) by quantitative reverse transcription PCR identified fourteen deregulated miRNAs. Validation experiments confirmed eight miRNAs as significantly deregulated in ALS. No significant differences were observed between ALS patients with or without C9orf72 expansion. The receiver operator characteristic (ROC) curve analyses revealed the highest diagnostic accuracy for the upregulated miR181a-5p and the downregulated miR21-5p and miR15b-5p. The miR181a-5p/miR21-5p and miR181a-5p/miR15b-5p ratios detected ALS with 90 and 85 % sensitivity and 87 and 91 % specificity, respectively, confirming the application potential as disease biomarkers. These deregulated miRNAs are implicated in apoptotic way and provide insight into processes responsible for motor neuron degeneration.

Background Ulnar neuropathy at the elbow (UNE) is the second most common entrapment neuropathy after carpal tunnel syndrome. Treatment may be conservative or surgical, but optimal management remains controversial. This is an update of a review first published in 2010 and previously updated in 2012. Objectives To determine the effectiveness and safety of conservative and surgical treatment in ulnar neuropathy at the elbow (UNE). We intended to test whether: - surgical treatment is effective in reducing symptoms and signs and in increasing nerve function; - conservative treatment is effective in reducing symptoms and signs and in increasing nerve function; - it is possible to identify the best treatment on the basis of clinical, neurophysiological, or nerve imaging assessment. Search methods On 31 May 2016 we searched the Cochrane Neuromuscular Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, AMED, CINAHL Plus, and LILACS. We also searched PEDro (14 October 2016), and the papers cited in relevant reviews. On 4 July 2016 we searched trials registries for ongoing or unpublished trials. Selection criteria The review included only randomised controlled clinical trials (RCTs) or quasi-RCTs evaluating people with clinical symptoms suggesting the presence of UNE. We included trials evaluating all forms of surgical and conservative treatments. We considered studies regarding therapy of UNE with or without neurophysiological evidence of entrapment. Data collection and analysis Two review authors independently reviewed titles and abstracts of references retrieved from the searches and selected all potentially relevant studies. The review authors independently extracted data from included trials and assessed trial quality. We contacted trial investigators for any missing information. Main results We identified nine RCTs (587 participants) for inclusion in the review, of which three studies were found at this update. The sequence generation was inadequate in one study and not described in three studies. We performed two meta-analyses to evaluate the clinical (3 trials, 261 participants) and neurophysiological (2 trials, 101 participants) outcomes of simple decompression versus decompression with submuscular or subcutaneous transposition; four trials in total examined this comparison. We found no difference between simple decompression and transposition of the ulnar nerve for both clinical improvement (risk ratio (RR) 0.93, 95% confidence interval (CI) 0.80 to 1.08; moderate-quality evidence) and neurophysiological improvement (mean difference (in m/s) 1.47, 95% CI -0.94 to 3.87). The number of participants to clinically improve was 91 out of 131 in the simple decompression group and 97 out of 130 in the transposition group. Transposition showed a higher number of wound infections (RR 0.32, 95% CI 0.12 to 0.85; moderate-quality evidence). In one trial (47 participants), the authors compared medial epicondylectomy with anterior transposition and found no difference in clinical and neurophysiological outcomes. In one trial (48 participants), the investigators compared subcutaneous transposition with submuscular transposition and found no difference in clinical outcomes. In one trial (54 participants for 56 nerves treated), the authors found no difference between endoscopic and open decompression in improving clinical function. One trial (51 participants) assessed conservative treatment in clinically mild or moderate UNE. Based on low-quality evidence, the trial authors found that information on avoiding prolonged movements or positions was effective in improving subjective discomfort. Night splinting and nerve gliding exercises in addition to information provision did not result in further improvement. One trial (55 participants) assessed the effectiveness of corticosteroid injection and found no difference versus placebo in improving symptoms at three months' follow-up. Authors' conclusions We found only two studies of treatment of ulnar neuropathy using conservative treatment as the comparator. The available comparative treatment evidence is not sufficient to support a multiple treatment meta-analysis to identify the best treatment for idiopathic UNE on the basis of clinical, neurophysiological, and imaging characteristics. We do not know when to treat a person with this condition conservatively or surgically. Moderate-quality evidence indicates that simple decompression and decompression with transposition are equally effective in idiopathic UNE, including when the nerve impairment is severe. Decompression with transposition is associated with more deep and superficial wound infections than simple decompression, also based on moderate-quality evidence. People undergoing endoscopic surgery were more likely to have a haematoma. Evidence from one small RCT of conservative treatment showed that in mild cases, information on movements or positions to avoid may reduce subjective discomfort.

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder in which disabling muscle weakness may affect health-related quality of life (HRQoL). The aim of this study was to investigate which common motor-functional deficits and corresponding severity are most determinant of poor HRQoL in these patients. In 41 patients, the dichotomized first item of the Italian Myasthenia Gravis Questionnaire (IMGQ), categorizing patients who report “good” and “poor” HRQoL, was chosen as dependent-outcome variable. All items composing the myasthenia gravis-specific scale (MG-ADL), i.e. talking, chewing, swallowing, breathing, impairment of ability to brush teeth or comb hair, impairment of ability to rise from chair, double vision, and eyelid droop were acquired as independent variables and dichotomized. Stepwise backward LR multivariable logistic regression analysis was performed. In addition, the main characteristics of patients were compared. MG-ADL items “chewing” ≥1, i.e. “fatigue chewing solid food”, and “breathing” ≥2, i.e. “shortness of breath at rest” proved to be significant determinants. Higher dose of corticosteroid therapy was significantly (p = 0.027; r s = −0.35), correlated with poor HRQoL. At diagnosis, a decremental response to repetitive nerve stimulation (RNS) from the abductor pollicis brevis was significantly more frequent in patients with poor HRQoL. In conclusion, impaired “chewing” and “breathing” functions indicate the need for careful planning of rehabilitation, re-education and patient management. Moreover, decremental response to RNS at diagnosis may identify patients at risk for poor HRQoL.

Wernicke encephalopathy (WE) is a neurological emergency due to thiamine deficiency. We aimed to identify clinical course and causes of diagnostic delay or failure of WE in a group of patients who underwent surgery for gastrointestinal tumors. A retrospective review of clinical, laboratory, neuroimaging, and therapeutic features of 10 patients with WE following abdominal surgery for cancer was carried out. Four patients died; in these subjects, diagnosis was delayed and supplementation of vitamin was absent or likely inadequate. Diagnostic delay or failure was also related to the coexistence of several medical complications at presentation masking typical symptoms of WE. In the surviving patients, outcome was influenced by promptness and type of therapy. Postoperative abdominal bleeding and number of subsequent operations may also had an effect. Postsurgical patients with gastrointestinal tumors may develop a subtle WE. The number of subsequent operations and the severity of postoperative complications may increase the risk of unrecognized WE. The disease should be suspected in postsurgical patients who have unexpected mental status changes, even under prophylactic treatment with vitamins. We suggest that prophylaxis with high doses of thiamine should be undertaken in patients with gastrointestinal tumors before surgery.

Mutations in the superoxide dismutase-1 (SOD1) gene have been found in 12–23% of patients with a diagnosis of ALS. Although the mechanism by which mutant SOD1 causes neural death remains elusive, several lines of evidence suggest that ALS is a protein-folding disease. Here we report a novel missense mutation in exon 1 of the SOD1 gene in a 68-year-old female with familial ALS characterized by spinal onset with upper and lower motor neuron signs and early neuroimaging evidence of corticospinal tract involvement. Molecular analysis identified a heterozygous mutation in codon 10, with substitution of a highly conserved glycine with arginine (G10R). Modelling of the mutant SOD1 showed a strong destabilization of the protein secondary structure that could influence the strength of the dimer interface. This property can result in a failure of the protein to fold and generation of toxic intracellular aggregates, suggesting a pathogenic role for the mutation.

Potentially causative missense variations in the ANG gene and a positive association with the synonymous rs11701-G substitution was detected mainly in Irish and Scottish ALS patients. We screened 262 Italian SOD1 negative ALS patients (250 sporadic) and 415 matched controls for sequence variations in the coding, 3'/5' UTR and 5' flanking (642 bp) regions of the ANG gene. We identified 53 sequence variations of which 46 new, 20 with a minor allele frequency (MAF) >or=0.01 and only three localised in the coding sequence, namely the missense I46V, identified in one patient and two controls, and the synonymous G86G and T97T corresponding to rs11701 and rs2228653. None of the detected SNPs or of their haplotypic combinations was significantly associated with ALS susceptibility or clinical features. In conclusion, we did not detect the association with rs11701-G or with any other newly detected variation in the ANG regulatory region. Furthermore we did not identify potentially causal mutations in the coding region.

Chromogranins were reported to interact specifically with mutant forms of superoxide dismutase that are linked to amyotrophic lateral sclerosis (ALS). Particularly, a variation c.1238C>T (p.Pro413Leu) in the chromogranin B gene, CHGB, has been associated with an earlier age at onset in both familial and sporadic ALS in French/French–Canadian populations studied. The aim of our study was to evaluate the P413L chromogranin variation in Italian patients with sporadic ALS. The study included 366 Italian patients with sporadic ALS and 382 control subjects. Genotyping of the polymorphism P413L in the CHGB gene was performed and the clinical characteristics of patients were analyzed in relation to their genotype. Our study on a cohort of Italian patients with SALS and controls failed to confirm an increased frequency of the 413L variant in SALS patients. Furthermore, we did not confirm the previous observation of a difference of age at onset between T-allele carriers and non-carriers (median age of onset 58.5 vs. 60.2 years of age, respectively). Our findings do not support the 413L variant as a risk factor for sporadic ALS in the Italian population.

Idiopathic hypertrophic cranial pachymeningitis (IHCP) is a rare, poorly understood, inflammatory disease, usually involving the dura mater of skull base, tentorium, and falx, and presenting with headache, progressive cranial nerve palsies, and cerebellar dysfunction. In four patients, the diagnosis of IHCP has been made on the basis of extensive clinical, and radiological investigation, and confirmed by dural biopsy in three patients. The clinical follow-up ranges from 24 to 120 months. At diagnosis, all the patients complained of severe, progressively increasing headache, two had simple or complex partial seizures, but none had cranial nerve palsies. Two patients had electrophysiological evidence of axonal peripheral neuropathy, biopsy-proved in one of them. In all the patients, MRI showed linear or focal thickening of the dura mater of the tentorium and/or of the convexity, sparing the skull base. In one patient, MRI findings resembled chronic subdural hematoma. Dural biopsy demonstrated fibrosis and prominent CD4+ T-cells inflammatory infiltrate. Pachymeningitis was highly responsive to steroid therapy, as was the peripheral neuropathy. In three patients, temporary steroids withdrawal led to dramatic clinical worsening including status epilepticus in one. In the patients here reported, absence of cranial nerve impairment, seizures, MRI findings resembling chronic subdural hematoma, and association with polineuropathy were unusual findings of IHCP. Moreover, the type of inflammatory infiltrate, lacking in previous reported cases, suggests a probable pathogenetic role for cell-mediated immunity of unknown origin.

Peripheral neuropathy (PN) has been found to be among the complications of hepatitis C virus (HCV) infection. Although the mechanisms of PN in patients with HCV infection are still unknown, cryoglobulinemia has been implicated as a possible cause of ischemic peripheral nerve impairment mediated by vasoconstriction, altered blood viscosity, and thrombosis of small vessels. However, available information is mostly based on referral patients with chronic HCV infection. In this study, an unselected sample of cases with newly diagnosed HCV infection has been examined to assess the prevalence and type of PN and to correlate the disease with the presence of cryoglobulinemia. The study population included 41 patients with HCV infection and no documented risk factors for PN (18 women and 23 men, aged 17 to 72 years) referred to the neurophysiology units of 7 Italian centers (Genova, Lodi, Messina, Monza, Napoli, Padova, and Siena) for a clinical and electrophysiological screening of peripheral nerve disease. The mean latency between diagnosis of HCV infection (interpreted as a surrogate of the duration of infection) and study entry was 6.8 months (range 1–12). PN was documented in 13 cases (31.7%). Of these, 7 had mononeuropathy and 6 had polyneuropathy. Cryoglobulinemia was present in 4 patients (9.8%) and 3 of them had PN (polyneuropathy 2, mononeuropathy 1). The odds ratio of PN in patients with cryoglobulinemia, adjusted for age, sex, and duration of infection, was 4.4 (95% confidence interval, CI: 0.3–68.2) and was fairly similar (4.1; 95% CI 0.3–56.3) when the analysis was limited to the presence of polyneuropathy. The lack of statistical significance may by explained by the small sample size. Based on these findings, cryoglobulinemia can be interpreted as an independent risk factor for PN at the presence of HCV infection. However, given the fairly high prevalence of PN in patients with newly diagnosed HCV infection and the high proportion of cases not attributable to cryoglobulinemia (9 out of 13), other factors should be investigated to clarify the mechanisms of HCV neuropathy.

8558 Background: The anti-CTLA-4 tremelimumab at two different dose-schedules of administration showed promising activity in second-line malignant mesothelioma (MM) patients (Calabrò et al., Lancet Oncol, 2013; Calabrò et al., Lancet Respir Med, 2015). These initial results and the efficacy of targeting the PD-1/PD-L1 axis in different tumor types, prompted the NIBIT-MESO-1 study aimed at investigating the efficacy and safety of tremelimumab combined with the anti-PD-L1 durvalumab in mm patients. We report the safety analysis from the fully-enrolled NIBIT-MESO-1 study. Methods: The NIBIT-MESO-1 is a phase II, open-label, single Center study. Forty mm patients received tremelimumab at 1 mg/Kg i.v. every 4 weeks (Q4W) for 4 doses, and durvalumab at 20 mg/Kg i.v. Q4W for 13 doses. Primary objective is immune-related (ir)-objective response rate; secondary are safey, ir-disease control rate, ir-progression free survival, and overall survival. Tumor assessment per ir-modified RECIST or ir-RECIST 1.1 for pleural or peritoneal MM, respectively, was performed at baseline and q12 weeks. Adverse events (AEs) were recorded according to CTC v4.0. (ClinicalTrials.gov Id: NCT02588131). Results: From October 2015 to October 2016, 40 mm patients (38 pleural and 2 peritoneal), median age 64 years (range 41-80), ECOG performance status 0 (n = 19) or 1 (n = 21) were enrolled in the study. mm histology was epithelioid (n = 32), biphasic (n = 5), sarcomatoid (n = 2) or undefined (n = 1). As of January 2017, 12 first or 28 second-line mm patients received a median of 5.5 doses of therapy (range = 1-13). Twenty-four patients (60%) experienced any grade irAEs: 5 patients (12.5%) had grade 3-4 AEs, the most frequent being hepatotoxicity (7.5%). AEs were generally manageable and reversible per protocol guidelines. Three patients (7.5%) were discontinued due to treatment-related AEs (1 trombocytopenia, 1 limbic encephalitis, 1 liver toxicity). Conclusions: The combination of tremelimumab and durvalumab is safe and manageable in mm patients. Clinical trial information: NCT02588131.

Background Miller Fisher syndrome is usually a monophasic disorder. Recurrent Miller Fisher syndrome is extremely rare, and all patients with recurrences have been adults. Although the optimal treatment for Miller Fisher syndrome has yet to be established, the typical therapy includes intravenous immunoglobulin or plasma exchange. The efficacy of steroids is still debated. Patients We describe two children with recurrent Miller Fisher syndrome. Episodes occurred at the age of 11.5 and 13 years in patient 1 and at the age of 8 and 13 years in patient 2. Results Clinical patterns of the first and recurrent episodes of Miller Fisher syndrome were overlapping. In both patients, steroids were effective in controlling clinical deterioration of Miller Fisher syndrome recurrences. Conclusions Recurrent Miller Fisher syndrome is a rare disorder that may occur in children. Our observations and a review of the literature suggest that there may be a small group of patients in whom steroids may be a therapeutic option when intravenous immunoglobulin fails to control clinical symptoms.

Rhabdomyolysis precipitated by multitherapy is most frequently described during statin treatment, due to impairment of statin clearance by drugs sharing cytochrome P450 biotransformation pathway. Modulation of membrane transporters for drug efflux, operated by substrates, can also affect drugs' tissue levels. We report rhabdomyolysis in an elderly patient, in multitreatment with different potentially myotoxic medications, taking place seven months after atorvastatin discontinuation. Affected by ischaemic heart disease, arterial hypertension and dementia-related behaviour disturbances, the patient was receiving angiotensin 2-receptor inhibitors, beta-blockers, vasodilators, diuretics, salycilates, allopurinol, proton pump inhibitors, antipsychotics and antidepressants. He had taken atorvastatin for 14 years, with constantly normal creatine-kinase plasma levels. Two months after addition of the antianginal drug ranolazine, creatine-kinase mildly increased and atorvastatin was withdrawn. Nonetheless, creatine-kinase progressively rose, with severe weakness and rhabdomyolysis developing seven months later. Muscle biopsy showed a necrotizing myopathy with no inflammation or autoimmune changes. After ranolazine withdrawal, creatine-kinase and myoglobin returned to normal levels and strength was restored. Several psychotropic and cardiovascular medications prescribed to the patient share either cytochrome P450 biotransformation and permeability-glycoprotein efflux transport. In the event of cardiovascular/neuropsychiatric polypharmacy in geriatric patients, the risk of muscle severe adverse effects from pharmacokinetic drug-drug interaction should be considered beyond the direct myotoxicity of statins.