Different saccadic profile in bulbar versus spinal-onset amyotrophic lateral sclerosis.

Two clinical phenotypes characterize the onset of amyotrophic lateral sclerosis (ALS): the spinal variant, with symptoms beginning in the limbs, and the bulbar variant, affecting firstly speech and swallowing. The two variants show some distinct features in the histopathology, localization and prognosis, but to which extent they really differ clinically and pathologically remains to be clarified. Recent neuropathological and neuroimaging studies have suggested a broader spreading of the neurodegenerative process in ALS, extending beyond the motor areas, toward other cortical and deep grey matter regions, many of which are involved in visual processing and saccadic control. Indeed, a wide range of eye movement deficits have been reported in ALS, but they have never been used to distinguish the two ALS variants. Since quantifying eye movements is a very sensitive and specific method for the study of brain networks, we compared different saccadic and visual search behaviours across spinal ALS patients (n = 12), bulbar ALS patients (n = 6) and healthy control subjects (n = 13), along with cognitive and MRI measures, with the aim to define more accurately the two patients subgroups and possibly clarify a different underlying neural impairment. We found separate profiles of visually-guided saccades between spinal (short saccades) and bulbar (slow saccades) ALS, which could result from the pathologic involvement of different pathways. We suggest an early involvement of the parieto-collicular-cerebellar network in spinal ALS and the fronto-brainstem circuit in bulbar ALS. Overall, our data confirm the diagnostic value of the eye movements analysis in ALS and add new insight on the involved neural networks.

Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD).

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is α-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype.

The unfolded protein response in amyotrophic later sclerosis: results of a phase 2 trial.

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.

A Schematic Approach to Defining the Prevalence of COL VI Variants in Five Years of Next-Generation Sequencing.

OBJECTIVE: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. METHODS: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. RESULTS: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. CONCLUSIONS: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.

A new mutation in DNM2 gene in a large Italian family.

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.

Anti-MAG IgM: differences in antibody tests and correlation with clinical findings.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Limitations in daily activities and general perception of quality of life: Long term follow-up in patients with anti-myelin-glycoprotein antibody polyneuropathy.

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.

Novel POLG mutations and variable clinical phenotypes in 13 Italian patients.

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.

Treatment for ulnar neuropathy at the elbow.

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.

Major motor-functional determinants associated with poor self-reported health-related quality of life in myasthenia gravis patients.

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.

Frequency and time to relapse after discontinuing 6-month therapy with IVIg or pulsed methylprednisolone in CIDP.

BACKGROUND: We reported that 6-month therapy with intravenous immunoglobulin (IVIg) was more frequently effective or tolerated than intravenous methylprednisolone (IVMP) in patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). We now retrospectively compared the proportion of patients who eventually worsened after discontinuing therapy and the median time to clinical worsening. METHODS: By March 2013, data were available from 41 of the 45 patients completing the trial with a median follow-up after therapy discontinuation of 42 months (range 1-60). Three patients withdrew during the original study and one failed to respond to either of the therapies. No patient received a diagnosis alternative to CIDP during the follow-up. RESULTS: Twenty-eight of the 32 patients treated with IVIg (as primary or secondary therapy after failing to respond to IVMP) improved after therapy (87.5%) as compared with 13 of the 24 patients treated with IVMP as primary or secondary therapy (54.2%). After a median follow-up of 42 months (range 1-57), 24 out of 28 patients responsive to IVIg (85.7%) worsened after therapy discontinuation. The same occurred in 10 out of 13 patients (76.9%) responsive to IVMP (p=0.659) after a median follow-up of 43 months (range 7-60). Worsening occurred 1-24 months (median 4.5) after IVIg discontinuation and 1-31 months (median 14) after IVMP discontinuation (p=0.0126). CONCLUSIONS: A similarly high proportion of patients treated with IVIg or IVMP eventually relapse after therapy discontinuation but the median time to relapse was significantly longer after IVMP than IVIg. This difference may help to balance the more frequent response to IVIg than to IVMP in patients with CIDP.

Erythropoietin in amyotrophic lateral sclerosis: a multicentre, randomised, double blind, placebo controlled, phase III study.

OBJECTIVE: To assess the efficacy of recombinant human erythropoietin (rhEPO) in amyotrophic lateral sclerosis (ALS). METHODS: Patients with probable laboratory-supported, probable or definite ALS were enrolled by 25 Italian centres and randomly assigned (1:1) to receive intravenous rhEPO 40,000 IU or placebo fortnightly as add-on treatment to riluzole 100 mg daily for 12 months. The primary composite outcome was survival, tracheotomy or >23 h non-invasive ventilation (NIV). Secondary outcomes were ALSFRS-R, slow vital capacity (sVC) and quality of life (ALSAQ-40) decline. Tolerability was evaluated analysing adverse events (AEs) causing withdrawal. The randomisation sequence was computer-generated by blocks, stratified by centre, disease severity (ALSFRS-R cut-off score of 33) and onset (spinal or bulbar). The main outcome analysis was performed in all randomised patients and by intention-to-treat for the entire population and patients stratified by severity and onset. The study is registered, EudraCT 2009-016066-91. RESULTS: We randomly assigned 208 patients, of whom 5 (1 rhEPO and 4 placebo) withdrew consent and 3 (placebo) became ineligible (retinal thrombosis, respiratory insufficiency, SOD1 mutation) before receiving treatment; 103 receiving rhEPO and 97 placebo were eligible for analysis. At 12 months, the annualised rate of death (rhEPO 0.11, 95% CI 0.06 to 0.20; placebo: 0.08, CI 0.04 to 0.17), tracheotomy or >23 h NIV (rhEPO 0.16, CI 0.10 to 0.27; placebo 0.18, CI 0.11 to 0.30) did not differ between groups, also after stratification by onset and ALSFRS-R at baseline. Withdrawal due to AE was 16.5% in rhEPO and 8.3% in placebo. No differences were found for secondary outcomes. CONCLUSIONS: RhEPO 40,000 IU fortnightly did not change the course of ALS.

Genetic counselling in ALS: facts, uncertainties and clinical suggestions.

The clinical approach to patients with amyotrophic lateral sclerosis (ALS) has been largely modified by the identification of novel genes, the detection of gene mutations in apparently sporadic patients, and the discovery of the strict genetic and clinical relation between ALS and frontotemporal dementia (FTD). As a consequence, clinicians are increasingly facing the dilemma on how to handle genetic counselling and testing both for ALS patients and their relatives. On the basis of existing literature on genetics of ALS and of other late-onset life-threatening disorders, we propose clinical suggestions to enable neurologists to provide optimal clinical and genetic counselling to patients and families. Genetic testing should be offered to ALS patients who have a first-degree or second-degree relative with ALS, FTD or both, and should be discussed with, but not offered to, all other ALS patients, with special emphasis on its major uncertainties. Presently, genetic testing should not be proposed to asymptomatic at-risk subjects, unless they request it or are enrolled in research programmes. Genetic counselling in ALS should take into account the uncertainties about the pathogenicity and penetrance of some genetic mutations; the possible presence of mutations of different genes in the same individual; the poor genotypic/phenotypic correlation in most ALS genes; and the phenotypic pleiotropy of some genes. Though psychological, social and ethical implications of genetic testing are still relatively unexplored in ALS, we recommend multidisciplinary counselling that addresses all relevant issues, including disclosure of tests results to family members and the risk for genetic discrimination.

Early sedation and clinical outcomes of mechanically ventilated patients: a prospective multicenter cohort study.

INTRODUCTION: Sedation overuse is frequent and possibly associated with poor outcomes in the intensive care unit (ICU) patients. However, the association of early oversedation with clinical outcomes has not been thoroughly evaluated. The aim of this study was to assess the association of early sedation strategies with outcomes of critically ill adult patients under mechanical ventilation (MV). METHODS: A secondary analysis of a multicenter prospective cohort conducted in 45 Brazilian ICUs, including adult patients requiring ventilatory support and sedation in the first 48 hours of ICU admissions, was performed. Sedation depth was evaluated after 48 hours of MV. Multivariate analysis was used to identify variables associated with hospital mortality. RESULTS: A total of 322 patients were evaluated. Overall, ICU and hospital mortality rates were 30.4% and 38.8%, respectively. Deep sedation was observed in 113 patients (35.1%). Longer duration of ventilatory support was observed (7 (4 to 10) versus 5 (3 to 9) days, P = 0.041) and more tracheostomies were performed in the deep sedation group (38.9% versus 22%, P = 0.001) despite similar PaO2/FiO2 ratios and acute respiratory distress syndrome (ARDS) severity. In a multivariate analysis, age (Odds Ratio (OR) 1.02; 95% confidence interval (CI) 1.00 to 1.03), Charlson Comorbidity Index >2 (OR 2.06; 95% CI, 1.44 to 2.94), Simplified Acute Physiology Score 3 (SAPS 3) score (OR 1.02; CI 95%, 1.00 to 1.04), severe ARDS (OR 1.44; CI 95%, 1.09 to 1.91) and deep sedation (OR 2.36; CI 95%, 1.31 to 4.25) were independently associated with increased hospital mortality. CONCLUSIONS: Early deep sedation is associated with adverse outcomes and constitutes an independent predictor of hospital mortality in mechanically ventilated patients.

Clinical characteristics of patients with familial amyotrophic lateral sclerosis carrying the pathogenic GGGGCC hexanucleotide repeat expansion of C9ORF72.

A large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72, a gene located on chromosome 9p21, has been recently reported to be responsible for ~40% of familial amyotrophic lateral sclerosis cases of European ancestry. The aim of the current article was to describe the phenotype of amyotrophic lateral sclerosis cases carrying the expansion by providing a detailed clinical description of affected cases from representative multi-generational kindreds, and by analysing the age of onset, gender ratio and survival in a large cohort of patients with familial amyotrophic lateral sclerosis. We collected DNA and analysed phenotype data for 141 index Italian familial amyotrophic lateral sclerosis cases (21 of Sardinian ancestry) and 41 German index familial amyotrophic lateral sclerosis cases. Pathogenic repeat expansions were detected in 45 (37.5%) patients from mainland Italy, 12 (57.1%) patients of Sardinian ancestry and nine (22.0%) of the 41 German index familial amyotrophic lateral sclerosis cases. The disease was maternally transmitted in 27 (49.1%) pedigrees and paternally transmitted in 28 (50.9%) pedigrees (P = non-significant). On average, children developed disease 7.0 years earlier than their parents [children: 55.8 years (standard deviation 7.9), parents: 62.8 (standard deviation 10.9); P = 0.003]. Parental phenotype influenced the type of clinical symptoms manifested by the child: of the 13 cases where the affected parent had an amyotrophic lateral sclerosis-frontotemporal dementia or frontotemporal dementia, the affected child also developed amyotrophic lateral sclerosis-frontotemporal dementia in nine cases. When compared with patients carrying mutations of other amyotrophic lateral sclerosis-related genes, those with C9ORF72 expansion had commonly a bulbar onset (42.2% compared with 25.0% among non-C9ORF72 expansion cases, P = 0.03) and cognitive impairment (46.7% compared with 9.1% among non-C9ORF72 expansion cases, P = 0.0001). Median survival from symptom onset among cases carrying C9ORF72 repeat expansion was 3.2 years lower than that of patients carrying TARDBP mutations (5.0 years; 95% confidence interval: 3.6-7.2) and longer than those with FUS mutations (1.9 years; 95% confidence interval: 1.7-2.1). We conclude that C9ORF72 hexanucleotide repeat expansions were the most frequent mutation in our large cohort of patients with familial amyotrophic lateral sclerosis of Italian, Sardinian and German ancestry. Together with mutation of SOD1, TARDBP and FUS, mutations of C9ORF72 account for ~60% of familial amyotrophic lateral sclerosis in Italy. Patients with C9ORF72 hexanucleotide repeat expansions present some phenotypic differences compared with patients with mutations of other genes or with unknown mutations, namely a high incidence of bulbar-onset disease and comorbidity with frontotemporal dementia. Their pedigrees typically display a high frequency of cases with pure frontotemporal dementia, widening the concept of familial amyotrophic lateral sclerosis.

Antiangiogenic VEGF isoform in inflammatory myopathies.

OBJECTIVE: To investigate expression of vascular endothelial growth factor (VEGF) antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM) and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. SUBJECTS AND METHODS: We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis) and 6 control muscle samples. TGF- ß, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. RESULTS: VEGF-A165b was significantly upregulated in IIM, as well as TGF- ß. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF- ß. CONCLUSIONS: Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides.

Study of total duration of distal compound muscle action potential in demyelinating and axonal Guillain-Barre' syndrome.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.

Krebs von den Lungen-6 (KL-6) in cerebrospinal fluid from neurosarcoidosis patients.

BACKGROUND: Krebs von den Lungen-6 (KL-6) is a high molecular weight (MW) glycoprotein mainly secreted by type II pneumocytes because of lung damage or during regeneration. Neurosarcoidosis (NS), where sarcoid granulomas involve the nervous system, occurs in 5-20% of patients with sarcoidosis. No data is currently available on KL-6 in serum or CSF of NS patients. The present study compared KL-6 concentrations in serum and CSF of NS patients versus others with neurodegenerative (ND) or chronic inflammatory demyelinating (DM) diseases. MATERIALS AND METHODS: Nine NS patients (mean age 46.2 years, range 16-61 years, M/F 5/4), nine patients with a chronic neurodegenerative disease (mean age 53.1 years, range 37-65 years, M/F 5/4) and nine patients with a chronic demyelinating disease (mean age 46.3 years, range 18-65 years, M/F 5/4) were retrospectively enrolled. RESULTS: Measurable CSF concentrations of KL-6 were detected in 7/9 NS patients but in no ND or DM patients. No significant differences in CSF concentrations of ACE were observed between the three groups (p=0.0819). In NS patients, CSF concentrations of KL-6 were directly correlated with CSF albumin index (r=0.98; p<0.0001), albumin (r=0.979, p=0.0001), IgG (r=0.928, p=0.0009) and total protein concentrations (r=0.945, p=0.0004). DISCUSSION: KL-6 is a high MW protein, under physiological conditions it is unlikely to cross the blood-brain barrier. We found KL-6 in CSF from NS and not from ND and DM patients. The finding sustains the specificity of changes in KL-6 in this granulomatous disease, suggesting it as a candidate biomarker for recognition of NS.

Using Cluster Analysis to Overcome the Limits of Traditional Phenotype-Genotype Correlations: The Example of RYR1-Related Myopathies.

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.