Search

Background Risdiplam has been approved for Compassionate Use (CUP) since 2019, for SMA type 1 and type 2 who could not receive the approved treatment for the disease. It’s an orally administered drug targeting SMN2 gene. More than 200 patients have had access to this program in Italy, up to June 2021. Aims To describe the clinical and demographic features of patients included in CUP amongst the iSMAc’ Italian centers. Methods This is a retrospective study including demographic, neuromotor, respiratory and nutritional data, shifts, adverse events and dropouts. Results 44 out of 576 Italian iSMAc patients (7.6%) are on risdiplam (8 SMA1, 36 SMA2). Age ranges: 0-10 yrs (n = 1, SMA2); 11-17 yrs (n = 13, 4 SMA1, 9 SMA2); 18-25 yrs (n = 15, 3 SMA1, 12 SMA2); 18-25 yrs (n = 15.3 SMA1, 12 SMA2) and older than 25 yrs (n=14 SMA2). The majority (73%) are non-sitters. Concerning respiratory status, 6 patients are in invasive-ventilation, 26 use NIV and 8 patients do not require respiratory support. Regarding the nutritional status, 11 have a PEG and 12 have dysphagia. About 20% of patients switched from nusinersen for difficulties in intrathecal administration. 3 dropped-out respectively for side-effects, subjective inefficacy, shift to gene therapy. Conclusions Considering the criteria to access risdiplam so far, all but one are older than 11 yrs, have severe motor, nutritional and respiratory conditions and are naive due to inaccessibility to nusinersen for technical difficulties. After open clinical use, further follow-up data will provide information on additional reasons and number of patients accessing risdiplam., Spinal muscular atrophy (SMA) is an autosomal recessive disease where a deficient amount of SMN protein leads to progressive degeneration of bulbar and spinal motor neurons. Therapies for the restoration of SMN production are now available. Yet, fatigue and signs of impaired neuromuscular junction (NMJ) transmission have been documented as possible contributors to SMA phenotype. Amifampridine (3,4-diaminopyridine, AP), a voltage-dependent K+ channel blocker, prolongs depolarization of the presynaptic NMJ terminal, enhancing neuromuscular transmission. Here, we evaluated the safety and efficacy of AP in ambulatory patients with SMA type 3, in a 1:1 randomized, double-blind, placebo-controlled, 2-period, 2-treatment, crossover study. Type 3 SMA, able to walk unaided for 30m, entered the run-in phase during which AP was titrated up to an optimized stable dose. Then, patients with at least 3-points improvement in Hammersmith Functional Motor Score Extended (HFMSE) were randomized to receive either AP or placebo for 2 weeks, alternatively, for a total of 28 days of double-blind treatment. Efficacy was evaluated by changes from randomization of HFMSE, quality of life, 6-minute walk test. Descriptive analyses and a mixed effects linear model were used for statistic. Six patients for each sequence of treatment were randomized. Transient paresthesias (33,3%) were the only AP-related AEs reported. AP treatment led to a statistically significant improvement in HFMSE (LS Mean Difference 0.792 (0.22 to 1.37), p = 0.0083), compared to placebo, but not in the secondary endpoints. SMA-001 study provided evidence that AP was safe and effective in treating ambulatory patients affected by SMA type 3. IND/EUDRACT number: 106263 /2017-004600-22, Objective To determine the efficacy and safety of risdiplam in infants with type 1 spinal muscular atrophy (SMA) after 24 months of treatment. Background SMA is a severe, progressive neuromuscular disease caused by reduced levels of survival of motor neuron (SMN) protein due to deletions and/or mutations of the SMN1 gene. A second SMN gene, SMN2, produces only low levels of functional SMN protein. Risdiplam (EVRYSDI™) is a centrally and peripherally distributed oral SMN2 pre-mRNA splicing modifier that increases the levels of functional SMN protein. Design/methods FIREFISH (NCT02913482) is a multicenter, open-label, two-part study of risdiplam in infants with type 1 SMA and two SMN2 gene copies, aged 1-7 months at enrollment. Part 1 (n = 21) assesses the safety, tolerability and pharmacokinetics/pharmacodynamics of different risdiplam doses. Part 2 (n = 41) assesses the efficacy and safety of the Part 1-selected dose. Results The primary endpoint of Part 2 at 12 months was met (data-cut: 14th November 2019); 29% (p < 0.0001, performance criterion = 5%) of infants were able to sit without support for ≥ 5 seconds, as measured by the Bayley Scales of Infant and Toddler Development, Third Edition. This milestone was never achieved in natural history cohorts. No treatment-related safety findings leading to withdrawal were reported in Part 2. Efficacy and safety data from infants in Part 2 who have received risdiplam treatment for 24 months will be presented. Conclusions Part 2 is ongoing and will provide important data on the long-term efficacy and safety of risdiplam in infants with Type 1 SMA., Objective To retrospectively investigate long-term safety and efficacy of nusinersen in a large cohort of adult Italian SMA patients. Methods Inclusion criteria were: 1) clinical and molecular diagnosis of SMA2 or SMA3; 2) nusinersen treatment started in adult age (> 18 years); 3) clinical data available at least at baseline (beginning of treatment) and 14 months. Results We included 112 patients (15 SMA2 and 97 SMA3) with median age at first administration of 35 years (range 18-74). Median period of treatment was 30 months (range 14-38). The Hammersmith Functional Rating Scale Expanded (HFMSE) in SMA3 patients increased significantly from baseline to the end of the follow-up (p = 0.0013), with higher improvement in walkers (median +2, p = 0.0147) than in sitters (median 0, p = 0.0384). The Revised Upper Limb Module (RULM) in SMA3 significantly improved between baseline and the end of the follow-up (p = 0.0002), with higher effect in sitters (median +2, p = 0.0024) than in walkers (median 0, p = 0.0281). Conversely, SMA2 patients had no significant changes of median HFMSE and RULM over the observational period. Furthermore, six-minute walking test distance significantly increased in SMA3 walkers during the follow-up (mean +35 m, p = 0.0003). Conclusions: Our data provide the first evidence of prolonged nusinersen safety and efficacy in a large cohort of adult SMA2 and SMA3., Type 1 spinal muscular atrophy (SMA1) is the most severe and common form of SMA. Its poor life expectancy has been drastically improved after the advent of gene therapy. In our centre, we have recently treated four patients with Onasemnogene abeparvovec (Zolgensma). Two were novel diagnoses (one female, one male), the other two were already being treated with antisense oligonucleotides (males). The median age of diagnosis was 5.5 months; the median age of treatment was 25 months. Gene therapy was administered by a neurologist and the pediatric palliative care (PPC) team specialists provided the following interventions: 1) communication of diagnosis, helping the parents to choose the treatment for their baby in line with their values and beliefs; 2) training the parents to administer respiratory, nutritional and physical therapies 3) training the family paediatrician, the home care nurses, and local hospital personnel 4) performing regular visits at home to monitor the patients and further train the parents 5) guaranteeing psychological and social support. Families’ main issues were daily managing the long-term disease burden and accepting a possible variability in the therapy outcome. From the medical perspective, communication with families, verifying their global comprehension, and mediating their expectations and attitudes (especially when switching from oligonucleotides), was additionally demanding. Neurological follow-up with periodic assessments is currently ongoing. To conclude, in the growing panorama of innovative SMA therapies, PPC supportive approaches based on continuous multidisciplinary and comprehensive clinical, rehabilitative, and social strategies maintain an essential role., Background COVID-19 is caused by SARS-CoV-2 virus and in many cases lead to a pneumonia. However a number of neumuscular manifestations have been associated to SARS-CoV-2 infection. Furthermore, multiorgan symptoms after COVID-19 are being reported by increasing numbers of patients, ranging from cough to fatigue and muscle pain. However, the long-term health consequences of COVID-19 remain largely unclear. Methods We evaluated 124 patients hospitalized between march and May 2020 for SARS-COV-2 associated pneumonia at 6 and 12 months. We retrospectively collected clinical, laboratory and radiological information available. for each patient, cognitive tests, scales for depression and anxiety and a specific Fatigue Severity Scale (FSS) were performed. Results Twenty-five patients died during hospitalization. At 12 months follow up 85 patient were evaluated. Eighty-seven (70%) patiens were male and mean age was 67.3 years. During hospitalization 43 (36.5%) of patients complain of myalgia. This patients had higher CK levels than patients who did not (534 U/L vs 93 U/l, p < 0.001). At 12 months 42% of patients complain about myalgia while 34% about fatigue. Mean FSS value were 32.93, and were significatively higher in patiets who complain about fatigue (41.52 vs 27.08 p < 0.001) and Muscle pain (40.84 vs 26.80, p < 0.001) compared to who did not. Conclusions During hospitalization for COVID-19 myalgia was associated with an higher level of CK, suggesting a possible muscle involvement. At 12 month myalgia and fatigue were present in a more than a third of patient suggesting that this manifestation could be one of the main COVID-19 sequelae., Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a rare childhood autosomal recessive motor neuron disease, due to recessively inherited IGHMBP2 gene mutations. The main hallmarks of the disease are diaphragmatic palsy and progressive distal muscular atrophy and paralysis. In few cases mutations in the same gene leaded to Charcot-Marie Tooth disease type 2S (CMT2S). There is no treatment and very little data are available on the disease progression. We present the data of a European patients’ cohort composed of 15 patients with SMARD1, aged between 5 months and 21 years and 2 patients with CMT2S respectively 16 and 63 years old. Clinical data have been collected retrospectively and prospectively. For SMARD1 patients the disease onset was before 5 months, manifesting with weak crying, feeding difficulties, hypotonia, club foot and acute respiratory distress. All patients need continuous ventilatory support (12 via tracheostomy while 3 with NIV) and G-tube was necessary in 12 cases. Head control was reached only by 60% of the patients, independent sitting only by 27%. Autonomic dysfunction and scoliosis were observed in all patients. The two CMT2S patients manifested with distal motor neuropathy respectively at 8 and 10 years, both of them are still ambulant. Diseases related to IGHMBP2 mutations are very rare and with heterogeneous clinical manifestations. Our case studies broaden the knowledge relating to the phenotype and natural course of these disorders, providing useful data for the evaluation of possible future therapeutic strategies., We present updated results from ASPIRO (NCT03199469), investigating gene replacement therapy with AT132 for XLMTM patients. XLMTM, an ultra-rare, life-threatening myopathy caused by mutations in the MTM1 gene, leads to impaired neuromuscular and respiratory function, and early death. Patients enrolled required ventilator support and had no clinically significant underlying liver disease at baseline, defined as > 5x ULN ALT or AST, or hepatic peliosis by imaging. As of July 2020, efficacy data were analysed for 16 patients (n = 6, 1 x 1014 vg/kg (age 0.8-4.1 years at dosing); n = 10, 3 x 1014 vg/kg (age 1.3-6.8 years at dosing). At baseline, all patients required ventilator support (mean [SD] 22.4 [3.34] hours/day), and missed critical motor milestones. Treated patients had significant reductions in daily hours of ventilator dependence vs control (p < 0.0001 for both doses); seven achieved ventilator independence. Treated patients showed significant improvements in motor function vs control, and acquired and maintained several major motor milestones. Muscle biopsies demonstrated durable myotubularin expression with improved pathology scores at 48 weeks. Three patients treated at 3x1014 vg/kg, experienced fatal sepsis or gastrointestinal bleeding with ongoing severe cholestatic liver dysfunction. As of September 2021, the ASPIRO program is placed on a clinical hold by the FDA following the death of a newly dosed participant at 1 x 1014 vg/kg; the cause of death is still pending. Present understanding is that these events are related to a combination of gene therapy and underlying XLMTM disease process. Safety of AT132 is being closely monitored as the four patient deaths are being thoroughly investigated., Objective Limb-girdle muscular dystrophy type 2E/R4 (LGMD2E/R4) is caused by mutations in the beta-sarcoglycan gene (SGCB), resulting in loss of SGCB protein and other components of the dystrophin-associated protein complex (DAPC). LGMD 2E/R4 manifests as progressive hip/shoulder muscle weakness. This first-in-human, phase 1/2 trial (NCT03652259) evaluated SRP-9003, a self-complementary rAAVrh74.MHCK7.hSGCB construct restoring SGCB. Methods Patients aged 4-15 years with SGCB mutation (both alleles) received 1 SRP-9003 IV infusion: Cohort 1 (n = 3), 1.85 × 1013 vg/kg; Cohort 2 (n = 3), 7.41 × 1013 vg/kg. Endpoints included safety (primary), SGCB expression (secondary), and function (North Star Assessment for Limb-girdle Type Muscular Dystrophies [NSAD], time to rise [TTR], 4-stair climb [4-sc], 100-meter timed test [100 m], 10-meter timed test [10 m]). Results We report Year 2 (Y2; Cohort 1) and Year 1 (Y1; Cohort 2) results. As of January 2021, SRP-9003 was well tolerated with no new safety signals since the previous data cut (July 2020); adverse events occurred early and were manageable. Immunofluorescence showed robust SGCB expression post treatment, leading to DAPC reconstitution, maintained to Y2 (Cohort 1). SRP-9003-treated patients showed functional improvements, maintained at Y2 in Cohort 1 (NSAD, +5.7 points; TTR, -0.6 s; 4-sc, -0.3 s; 100 m, -2.8 s; 10 m, -0.2 s) and Y1 in Cohort 2 (NSAD, +4 points; TTR, -1.1 s; 4-sc, -0.4 s; 100 m, -7.9 s; 10 m, -0.6 s). Post hoc analysis showed improved NSAD outcomes versus untreated natural history cohort (9.2-point difference, Y2; 95% CI, 3.2-15.1). Conclusions Results suggest long-term efficacy of SRP-9003, supporting advancement of the clinical development program. Study Support Sarepta Therapeutics, Inc. Disclosures LRR-K, ERP, SL, DAG, ASM, EK, SN, XL, and LP are or have been employees of Sarepta Therapeutics, Inc, and may own stock in the company. LNA and LPL received fees from Sarepta Therapeutics, Inc, for licensure of the LGMD natural history data set. JRM received financial support from Sarepta Therapeutics, Inc, for travel to meetings to present any products sponsored by Sarepta. KJL, KC, NFR, MAI, and BP have no conflicts to disclose. Product is investigational only., We are working on an integrated, multiparametric approach in diagnosis and management of neuromuscular diseases (NMDs) by using a single support software platform, potentially useful in implementing diagnosis and taking care towards trials readiness. Promising preliminary results have been obtained to date with the Health360 platform under the umbrella of the InGene 2.0 project. Health360, a platform developed under the Software-as-a-Service (SaaS) principles, merges all that, with sections dedicated to the collection of personal data (under the premises of the EU 2016/679 GDPR Regulation), as well as modules devoted to biomedical images storage and interpretation. In particular, further modules, including neurological examination and functional motor tests, muscular MRI, genetic data, muscle biopsies, are under development and optimization. The possibility to upload such images in a common, user-friendly software platform, where data and image storage, as well as the analysis of images and loops can be performed in an intelligent manner, would be of extreme aid to the clinician. If confirmed on larger cohorts and with robust statistical approach, such results could drive the present tool to be used for diagnostic aims, phenotypic characterization and clinical follow-up., Objectives To collect creatinine and CK plasma concentration in a cohort of BMD patients, in order to explore their role as biomarkers and correlation with age and muscular function. Materials and methods We collected 110 CK values in a cohort of BMD patients whose muscular function was assessed by NSAA score; for 27 patients (26.5%) we had more than one value. We also collected 61 serum creatinine values. We correlated CK (log10 transformed) and creatinine values with age and muscular function of patients. Results The mean value of CK levels in our cohort was 2090 U/L (± SD 3596 U/L), while the median was 903 U/L. We expected a decrease in CK values during years; in fact, the negative association between CK values and age was strongly significant (r = -0.63, p < 0.0001). CK decrease was loosely correlated with the reduction of NSAA score (r = 0.19, p = 0.043). The mean value of creatinine levels in our cohort was 0.59 mg/dL (± SD 0.27 mg/dL), while the median was 0.49 mg/dL. The relationship between creatinine and muscular function was statistically significant, with normal creatinine values in patients with preserved muscular function, and low creatinine values in patients with deteriorated muscular function (r = 0.61, p < 0.0001). Creatinine tended to decrease with age, but in our cohort this association was not statically significant (r = 0.2, p = 0.115). Discussion and conclusions Creatine and CK plasma concentrations seem to correlate well with muscle mass and function in BMD, as observed also in other neuromuscular diseases. Future perspectives include the evaluation of potential changes of these biomarkers with novel therapeutic interventions., Titin truncating variants (TTNtv) have been associated with a dominant cardiomyopathy showing a reduced, age dependent, penetrance but they also cause recessive skeletal muscle diseases. The mechanisms explaining the TTNtv role in dominant cardiomyopathies and in recessive skeletal muscle titinopathies are still unclear. We analysed by RNA sequencing 44 skeletal muscle samples from patients with a confirmed or a suspected titinopathy, proving variants causing a premature stop codon out of the M-band to result in a nonsense mediated decay (NMD) of titin transcripts. Vice versa, stop codons in the M-band escape NMD and still result in a ‘quasi-full-length’ protein. Although most TTNtv cause splicing defects, their effect on the transcripts is highly variable. Most of the splice variants we have characterized cause in-frame losses or gains, still resulting in a near–full length protein. Moreover, some of them affect only a reduced number of transcripts and a significant amount of normal protein is still produced. A direct analysis of RNA, cDNA and protein is crucial to characterize the effect of truncating variants. These second-tier studies are mandatory to support the variant classification of DNA changes and to clarify the pathomechanisms of rare diseases., Background In order to ease diagnosis and treatment of rare diseases afflicting 30 million people in Europe, the EU had created the European Reference Networks (ERNs), connecting cross-border healthcare providers. Among the ERN features is the Clinical Patients Management System (CPMS), a digital web-based software where clinicians can discuss about patients through virtual panels, sharing patients’ data securely. The CPMS requires carefull training for its correct use, hence a twelve-month project funded by Sarepta Therapeutics was set up to improve the use of the CPMS among Euro-NMD ERN Italian members. Methods In the first two months, two medical doctors (MD) underwent a teaching course with a CPMS expert. Afterwards, the two MDs organized and managed a training course across four months with a flexible schedule to ease the course attendance. In a further phase lasting five months, the trainers focused on panels’ progression so to ease the moving forward across the panel timeline and ease panel closure by attendees. Several new panels were opened. Results At the end of the course, participants had opened 98 panels (approximately 80% of the total in the whole Euro-NMD ERN). The participants managed to access the platform and became acquainted in using it, and the course strengthened the Italian network overall. Conclusions Cross-border virtual consulting is an outstanding tool to improve the quality of health care provision. Our results show that a training course tailored to healthcare professionals might boost the usability of the CPMS, increasing its impact to European health system., Perturbation of glycolytic enzymes results in glycogen storage disorders such as Pompe disease (PD), which is an autosomal recessive metabolic pathology due to a defect of the lysosomal enzyme acid α-glucosidase (GAA), necessary for glycogen degradation. PD was the first glycogen storage disorder linked to autophagy, a key molecular mechanism that maintains cellular homeostasis and ensures correct macromolecule turnover in the cell. Despite the significant results achieved in this disease comprehension, it remains unclear how autophagy is disrupted in PD, since it is yet unknown if an excessive acceleration or reduction of this process is present. The importance of understanding autophagy dysfunction relies in the fact that this could explain the difficulties of current therapies in restoring muscle function, in particular in late onset patients. Moreover, could explain the presence of secondary symptoms in PD patients, in particular related to neuromuscular junction (NMJ) malfunctioning. Taking advantage of the zebrafish transgenic line Tg(CMV:EGFP-map1Lc3b), characterized by fluorescent autophagosomes, we generated a new zebrafish PD model, useful for the deep study of autophagic pathway. Exploiting different drugs known to have effects on autophagy, we are investigating this pathway at immunohistochemistry, biochemistry, electron microscopy and behavioral level. We believe that our findings will trigger a reassessment of the PD pathogenic mechanisms, as well as the research of new therapeutic targets addressing both glycogen accumulation and autophagy. Furthermore, this investigation will be essential to explore if the secondary symptoms of PD could be reversible., Objective In this study we applied next generation sequencing (NGS) in undiagnosed patients with suspected neuromuscular disease to investigate the prevalence of variants in collagen VI and collected clinical and instrumental data to verify the real pathogenicity of these variants. Methods As part of InGene project, we screened 241 neuromuscular genes of which COL6A1, COL6A2, COL6A3 by NGS in 580 patients with neuromuscular disorders referred to Molecular Medicine Lab of IRCCS FSM in the last five years. Variants in collagen VI were classified according to reference literature and were collected independently by clinical picture. Clinical data available were collected in a CRF and missing data were updated with referred clinician. Results We identified “probable” or “likely pathogenetic” disease-related mutations in COLVI-related genes in 47 patients (26 male and 21 female). 11 patients harboured variants in COL6A1 gene, 18 patients had a variant in COL6A2 gene, 21 patients had a variant in COL6A3 gene. Collected clinical and instrumental data partially reflect the phenotype described in literature. Conclusions This study shows both the prevalence of collagen VI variants in patients with weak muscular symptoms and highlights the advantages of NGS used as a first level diagnostic approach especially for complex genes that are difficult to study routinely. Anyhow, it also demonstrates the difficulty of considering these variants as pathogenetic without clinical and instrumental data. It may therefore be useful to outline a flow chart to verify the real pathogenicity of found variants., We evaluated bone mass acquisition and fragility fractures determinants in 26 ambulant prepubertal DMD patients treated with deflazacort. DXA measurements of total body less head (TB-BMD Z-score), lumbar spine bone mineral density (LS-BMD Z-score) and body fat (TBF%) were obtained at baseline (T0) and every year up to 3 years (T3). A delta-TBZ-score/year [(T3_TB-BMD Z-score - T0_TB-BMD Z-score)/years] was used for a better interpretation of bone changes. On day of DXA, subjects underwent antrophometric measurements and dosage of serum bone turnover biomarkers. Median age was 7.7 years (IQR 6/9,2) at T0 and 11.4 years (IQR 9.8/13.4) at T3. At T3, the incidence of fractures was 23.1%, at a median age 12.3 years (IQR 10.7/14.3). Between T0 and T4 we observed a decline of HtSD (p = 0.001) and TB-BMD Z-score (p = 0.004), an increase in TBF% (p = 0.04), while BMI SD, LS-BMD Z-score and serum biomarkers did not change significantly. Multiple regression analysis identified TBF% as the only negative predictor of TB-BMD Z-score, and higher BMI SD at T0 was associated to greater negative delta-TBZ-score/year (r -0.63; p < 0.001). Compared to fracture-free-DMD, fractured-DMD showed 2 times greater delta-TBZ-score/year (p < 0.05) and 2,3 times increased TBF%/year (p < 0.05). Fractures were predicted independently by delta-TBZ-score/year, BMI SDS and TBF% at T3. In conclusion, we demonstrated for the first time a detrimental effect of the fat tissue on bone fragility, and we showed that TB-BMD Z-score represents a reliable tool to detect bone changes in ambulant DMD, suggesting its inclusion in trials assessing the efficacy of bone treatments in DMD., Background One of the most challenging hurdles that clinical researchers face in the Duchenne muscular dystrophy (DMD) field is the considerable inter-patient variability in age at presentation and weakness progression: that is, the relevant phenotypic variability of DMD. Increasingly, it is recognized that the genetic background, i.e genetic variation in genes different from disease gene, may modulate Mendelian disease phenotype. These trans-acting variants are called genetic modifiers. Several modifiers of DMD have been identified through candidate gene studies, while genome-wide association studies have only been initially attempted. Aim This work represents a preliminary analysis of a genome-wide association study (GWAS) looking for DMD modifier loci, that plans to collect clinical data and DNA samples from ~700 DMD patients followed by Consortium of Italian Centers, that in the last decade have collaborated to studies of DMD natural history. Materials The GWAS was carried out using the high-density Illumina Infinium Omni2.5Exome-8 genotyping chip, version 1.5, and implemented a pipeline for data interpretation based on ad-hoc scripts. The association test was a regression test of age at loss of ambulation, with the following covariates: glucocorticoid treatment (at least 1 year while ambulatory) and DMD mutation type. Results So far, we genotyped about 50% of the planned total cohort. The algorithm identified an association signal with a p value of 4,2*10-8 in an intronic region at chromosome 6q22.1, whose functional meaning needs to be further elucidated. Conclusions Identified SNPs represent putative modifiers of the phenotype of DMD. We plan to validate these findings by expanding sample size, and validating top association signals in independent cohorts., Introduction and aims Mutations amenable to skipping of specific exons have been associated with different motor progression in Duchenne muscular dystrophy (DMD). Less is known about their association with long-term respiratory function. We aimed to investigate the features of respiratory progression in four DMD genotypes relevant for ongoing exon skipping therapeutic strategies. Methods This was a retrospective longitudinal study including DMD children followed by the UK North Star network and international AFM network centres (May 2003-October 2020). We included boys amenable to skip exons 44, 45, 51 or 53, older than 5 years and ambulant at first recorded visit. Subjects who were corticosteroid-naïve or enrolled in interventional clinical trials were excluded. The progression of respiratory function (absolute forced vital capacity, FVC, and calculated as percentage of predicted, FVC%) was compared across the four subgroups (skip44, skip45, skip51, skip53). Results We included 142 boys. Mean (SD) age at first visit was 8.6 (2.5) years. Median follow-up was 3(0.3-8.3) years. In skip45 and skip51 FVC% declined linearly from first recorded visit. From the age of 9 years FVC% linearly declined in all genotypes. Skip44 had the slowest (2.7%/year) and skip51 the fastest (5.9%/year) annual FVC% decline. The absolute FVC progressively increased in skip44, skip45, skip51. In skip53 FVC started declining from 14 years of age. Discussion The progression of respiratory dysfunction follows different patterns for specific genotype categories. This information is valuable for prognosis and for the evaluation of exon skipping therapies., Objective To assess the clinical course and genotype-phenotype correlations in patients with neuromuscular disorders due to LMNA/C gene mutations. Methods We analysed clinical and genetic data in a cohort of Italian paediatric patients with a neuromuscular disorder and a pathogenetic or likely pathogenetic mutation in LMNA/C. Clinical data included motor function, scoliosis, respiratory and heart function, laboratory and imaging data. Results Nine patients aged 1-20 years were included in our retrospective study. The mean age at onset was 23 ± 25 months, ranging from birth to 6 years. The patients in the cohort ranged from those with congenital muscular dystrophy (CMD) to patients with myopathic features. All the patients with myopathic features remained independently ambulant and presented a mild phenotype. None of the patients developed conduction system defects or arrythmia but merely minor heart problems (mild tricuspid insufficiency). In our CMD patients, mutations were mainly present in the first part of the coding region (IF domain), and the two patients, whose mutations were in exon 1, presented the worst phenotype. One of them lost ambulation at the age of 2 while the other never acquired it; both needed nocturnal non-invasive ventilation. Conclusions Our findings are consistent with literature, showing an evident correlation between the severity of the muscle phenotype and the protein domain affected. Half of our paediatric patients had a CMD phenotype while the others presented myopathic features highlightening, yet again, that a mutation in LMNA/C in paediatric aged patients may be associated with mild phenotypes., Introduction Defining the natural history of a disease is an essential requisite to any therapeutic intervention. Slow progression, different pathogenic mechanisms and small number of patients have been the most relevant factors interfering with definition of markers of disease progression in LGMD2A and 2B. Patients and methods Patients with LGMDR1/LGMD2A and LGMDR2/LGMD2B attending the IRCCS “E. Medea” in the last 30 years were recruited. Demographic data, muscular strength in at least 14 muscles (Medical Research Council grading); motor function (Motor Function Measures scale, 6 min walk test and Performance of Upper Limb scale); cardiopulmonary function and swallowing capability data were collected. A previously developed regression model was used to reconstruct the evolution over time of each measurements. Results 428 visits of nineteen 2A and twenty 2B patients were retrospectively analysed through the regression model to create the curves of evolution with disease duration of muscle strength, motor and cardio-pulmonary function tests. Relevant muscular and motor function alterations occurred after the first decade of disease, while mild respiratory function alterations started after the second, with preserved cardiac function. Although type 2A showed relatively stronger distal lower limb muscles, while type 2B started with relatively stronger upper limb muscles, the corresponding motor functions were similar, becoming severely compromised after 25 years of disease. Conclusions This was the longest retrospective study in types 2A and 2B. It defined muscular, motor, cardiac and respiratory function curves of disease evolution that could be used to evaluate how the natural progression is changed by therapies., Background Congenital myasthenic syndromes (CMS) are genetic diseases characterized by impairment of neuromuscular junction structure or function. CMS natural history has been poorly investigated and has not been clarified yet. Aim of this study is to longitudinally assess the disease progression of CMS in an Italian cohort of patients. Methods We included patients with a clinical and molecular diagnosis of CMS, followed in three Italian neuromuscular centres. Patients were evaluated at baseline and then once per year, with a minimum follow-up of 1 year. Neurological examination included the Myasthenia Gravis-specific Activities of Daily Living scale (MG-ADL) and the MG-composite scale (MGC). Results A total of 33 patients, including 13 males and 20 females, were enrolled. The most common mutated gene was CHRNE (n = 13 pts), followed by DOK7 (n = 4 pts) and COLQ (n = 3 pts). Mean age at first evaluation was 41.8 ± 14.4 years (range = 10-71) and mean follow-up period was 2.1 ± 0.4 years (range = 1-3). Mean MG-ADL and MGC scores at baseline were 5.9 ± 4.0 and 14.9 ± 8.2, respectively. At the end of follow-up period, a mean reduction of 1 point for MG-ADL and 2.9 points for MGC were found, with improvement usually related to treatment modifications. MG-ADL did not change between first and last evaluation in around half of the patients (n = 19), while MGC resulted stable in 11 (33.3%) patients. Conclusions Our preliminary data suggest a disease stability according to MG-ADL and MGC in most of the CMS patients over the follow-up period., Background and aims Myasthenia Gravis (MG) is an autoimmune disorder with fluctuating weakness of the scheletric muscles causing significant disability and morbidity. The development of specialized care centers for MG patients as well as the employment of new effective treatments improved the survival in MG. The most common MG therapies may worse preexisting patients’ comorbidities or they may be controindicated in such situation. We explored the frequency of comorbidities in MG compared to healthy controls (HCs) and how they are distribuited in MG according to age at disease onset, gender, and disease severity. Materials and methods Patients with MG attending to Neuromuscular Clinic of University Hospital “Paolo Giaccone” of Palermo and “SS Annunziata” Hospital of Chieti were enrolled to study whereas HCs living in Sicily and Abruzzo were collected using a web-available questionnaire. The Chi-squared test was used to compare qualitative variables and the distribution of comorbidities between groups with level of significance set to p < 0.05. Results N = 178 patients with MG (mean age 59 years, 55% male) and 178 sex- and age matched healthy controls were enrolled. The 87% of MG patients and the 76% of HCs from comorbidities (p = 0.006). Comorbidities in MG were differently distribuited according to age at disease onset and gender. In patients with MGFA class III-IV-V respiratory disorders (p = 0.009) and thymoma (p = 0.003) were more common. Conclusions MG patients showed higher prevalence of comorbidities than HCs. Assessing of MG comorbidities may allow the clinicians to optimize the MG management., Background Refractory myasthenia gravis (MG) has been poorly investigated to date. Aim To describe the clinical features of drug-refractory patients with MG in a large Italian cohort. Methods We included 756 ocular and generalized MG patients from 2 Italian Neuromuscular Centers, with MG onset between 2000 and 2018 and at least one year of follow-up. Patients were classified as refractory when remained unchanged or worsened, according to MG Foundation of America post-Interventional Status (MGFA-PIS), with persistent symptoms or drug-related side effects, after treatment with steroids and at 2 least steroid-sparing immunosuppressive agents, administered in adequate doses for an appropriate period, as established by literature. Results Mean disease duration of the whole cohort patients was 11.2 ± 5.16 years (range 1-44). Fifty-nine (7.8%) patients were identified as refractory. The mean age at onset of refractory MG was 44 ± 17.2 years (range 16-83), slightly lower than in non-refractory subgroup, although not significant. Females were predominant in both refractory and non-refractory MG patients, but females were significantly more frequent among the former subgroup (67.8%, p < 0.0001). Similarly, thymoma was more frequently observed in the refractory MG patients (18/59; p-value: 0.002). Conversely, antibodies to MuSK (7/59) were not significantly associated to refractory MG. Recurrent need of rescue therapy as intravenous immunoglobulin or plasma exchange, were significantly more frequently observed in the refractory subgroup (p-value: 0.029). Conclusions Our study emphasises that the drug-refractory patients represent a small but considerable MG subgroup with specific features, needing an adequate management and new emerging treatment options., Although gene panels, WES or WGS analysis have changed our approach to molecular diagnosis, many genetic conditions remain unsolved. To define the large rearrangements in the DMD gene which are the most common cause of dystrophinopathies including Duchenne (DMD) and Becker (BMD) muscular dystrophy, we have applied the linked-read sequencing technology developed by 10x Genomics. The distinction between alleles along the genome through phasing is the main advantage of 10x linked-read technology. As an exemplary case, we studied a DMD carrier with an unsolved genetic status, linked to a 6-year-old boy affected by an X-linked muscular dystrophy. Despite a deletion of exons 16-29 in DMD gene was responsible for BMD phenotype in male of her family, MLPA and array-CGH analysis in the carrier showed a normal dosage of these exons and an increased dosage of flanking exons 1-15 and 30-34. The linked-read WGS was able to phase both X chromosomes, showing two different rearrangements: a deletion of exons 16-29 on one allele and a de novo duplication of exons 1-34 on the other one in the DMD gene. By data analysis, this duplication not only restores the normal dosage of exons 16-29 but involves a region of 1.52 Mb spanning the DMD gene and the 5’ upstream region. In conclusion, our results demonstrate that linked-read WGS can be a useful tool for improving our understanding of unsolved genetic conditions in a very feasible way., Sarcoglycanopathies diagnosis is genetically determined but muscle analysis, by immunohistochemistry and western blot, is still mandatory for a correct diagnostic process. In sarcoglycanopathies, clinical severity is usually correlated with the quantity of residual protein even if a deficiency in a single component of the sarcoglycan protein complex generally leads to concomitant reduction or loss of other sarcoglycans. There is no current therapy for sarcoglycanopathies, one of the most innovative therapeutic approaches is based on the use of adenoviral vectors for restoring protein expression, so the availability of a method that allow objective quantification of the membrane protein, faster than western blot, could be useful in evaluating the efficacy of pharmacological treatment. Morever immunofluorescence provides the opportunity to a simultaneous evaluation of protein quantity and sarcolemma positioning, not achievable by western blot. Muscle biopsies of 13 patients affected with genetically defined sarcoglycanopathy (8 LGMD-R3, 3 LGMD-R4 and 2 LGMD-R5), were analysed histologically and for immunofluorescence and western blot expression of each of the sarcoglycans. Laminin-α2, was used as sarcolemmal reference for membrane integrity and as internal standard of fluorescent reaction. Quantification of integrated density of fluorescent signal was performed using ImageJ software. Further analysis of immunoflourescence and western blot data obtained from these patients by Bland-Altman plot, to evaluate the level of agreement of two methods, suggested that the difference was acceptable. Preliminary results show that this semi-quantitative tool could contribute to better define patient’s prognosis and could be useful to evaluate the efficacy of potential pharmacological treatments during clinical trails., Introduction The diagnosis of FacioScapuloHumeral Dystrophy (FSHD) is complicated the high clinical variability and incomplete penetrance. Therefore, the availability of reliable (epi)genetic biomarkers is desirable for providing patients with more accurate molecular diagnoses and genotype-phenotype correlations. Aim of the study The study aimed at assessing the methylation status of the D4Z4 locus with the purpose of enhancing the molecular diagnosis of FSHD. Materials and methods The study involved 307 subjects (137 with clinical diagnosis of FSHD, 20 with LGMD and 150 controls). DNA methylation levels of the DR1 region (1kb upstream of the DUX4-ORF within the D4Z4 array), the DUX4-PAS (the distal part of the array) and its CpG6 site were assessed on genomic DNA by means of Bisulfite Sequencing and Capillary Electrophoresis. Results FSHD subjects showed significantly (p < 0.01) hypomethylation levels (0.30 ± 0.09, 0.49 ± 0.13 and 0.69 ± 0.18) compared to LGMD and controls subjects (0.38 ±0.05, 0.60 ± 0.07 and 0.88 ± 0.06) for DR1, DUX4-PAS and CpG6, respectively. Interestingly, patients carrying pathogenic mutations in FSHD-associated genes displayed lower DR1 methylation (0.15 ± 0.08 vs 0.31 ± 0.05) compared to wild-type patients. These results suggested that the methylation profile of the D4Z4 represents a useful biomarker for discriminating FSHD subjects from controls or patients with other myopathies. Conclusion The present study showed that the analysis of the methylation levels of D4Z4 region could support the molecular diagnosis of FSHD, by enhancing the genotype-phenotype correlation; orienting the specialist towards a deeper genomic analysis addressed to detect pathogenic variants in causative/modifier genes or perform differential diagnosis (LGMD, other myopathies)., AIGkit is an easy-to-use mobile application, created with the aim of allowing a telematic interaction between adult patient with Pompe disease and clinicians that can be downloaded for free on smartphone. After a first phase, whose results were presented as oral communication at AIM congress in 2018 and 2019, and published as original article in Neuromuscolar Disorders, the second operational phase of the project has been activated for its use in real life and clinical practice. The pre-established goal has been therefore to create an informatic platform for the collection of clinical data where each patient can access through the personal app on their smartphone, in order to allow the registration of their data and facilitate interaction with their clinicians. The design activity and procedures, despite the difficulties encountered in the various phases also caused by the Sars-COVID-19 emergency, were carried out to meet the requirements of the privacy guarantor, in order to activate the use of the platform and the real-time sharing of the data collected remotely on the app of each patients. We are now planning the dissemination of the initiative at national level and the implementation of this project in collaboration with other clinical centers that will be able to use the servers of the Azienda Ospedaliera Universitaria Pisana (AOUP) as coordinating center, thus improving value and utility for users, both patients and clinicians., Teleosts are commonly used in biomedical research to investigate the molecular mechanisms underlying human diseases. The medaka fish (Oryzias latipes) is a well-established vertebrate model used in developmental biology and genetic studies involving fast genetic manipulation, real-time images of developing pathologies and drug efficacy tests. We explored the possibility to use it to determine the performance of intact skeletal muscle at sarcomere level. Optically transparent tails of nine days-old medaka fish larvae were mounted in a thermoregulated trough containing physiological solution between the lever arms of a strain gauge force transducer and a loudspeaker motor at a sarcomere length (SL0) of 2.0 ± 0.1μm. Tetanic contractions were elicited by trains of stimuli (200Hz for fused tetanus) at 10°C. A striation follower (Huxley et al. J Physiol 1981;317:12-13P) was used to record sarcomere length changes during contraction in a 0.8-1.0 mm long segment selected along the central region of the sample. The force-velocity (T-V) relation was determined in afterloaded contractions and the power output at each load was calculated as the product between the imposed load and the steady shortening velocity. In fixed end conditions the plateau tetanic force (62 ± 8 kPa, mean ± SEM, n = 3) was attained with a half-sarcomere shortening against the end compliances of about 8% SL0. The unloaded shortening velocity, determined by fitting Hill equation to the T-V data, and the maximum power output are 5.9 ± 0.3 L0/s and 70 mW/g, respectively. These preliminary results make the medaka fish a promising model to investigate the genotype/phenotype correlation in skeletal muscle diseases. Supported by the EJP-RD., Alpha-sarcoglycan (SGC) deficient muscular dystrophy, also called Limb Girdle muscular disease R3, is an inherited disorder resulting from mutations in the a-sarcoglycan gene and aggravated by chronic inflammation that is finely modulated by the extracellular (e)ATP/purinoreceptors axis. Genetic ablation of P2X7 and pharmacological inhibition of the eATP-P2X7 axis by the broad-spectrum antagonist oxidized-ATP alleviated dystrophic phenotypes and dampened the local inflammatory response in mice affected by Duchenne muscular dystrophy and α-sarcoglycanopathy. Aim of this study is to evaluate the therapeutic effectiveness and to characterize the underlying mechanism of A438079, a potent and selective P2X7 antagonist, in a-sarcoglycan-deficient mice (Sgca mice). Our results show that treatment of Sgca mice with A438079 ameliorated the dystrophic phenotype without any detectable side effects. Recovery was evident in the key functional and biochemical parameters such as improved muscle performance and decreased serum creatine kinase levels. The benefits of A438079 treatment were also reflected by the muscle morphology where we observed a drastic reduction of the extent of local fibrosis and inflammation. A detailed characterization of muscle inflammatory infiltrates indicated that A438079 significantly decreased the percentage of neutrophils, activated monocytes, macrophages and dendritic cells in comparison to untreated dystrophic mice (Sgca control). In contrast, immunosuppressive regulatory T cells were significantly increased in Sgca A438079-treated mice in comparison to Sgca control animals. In conclusion, the pharmacological inhibition of P2X7 by the selective antagonist A438079 might provide a safe therapeutic approach to ameliorate the dystrophic phenotype in a-sarcoglycanopathy by decreasing local fibrosis, inflammation and muscle degeneration., Pompe disease is a lysosomal storage disorder caused by acid alpha-1,4-glucosidase deficiency due to GAA mutations. Late-onset form (LOPD) displays progressive muscle weakness with respiratory involvement often leading to premature death. ERT is the only approved therapy, but this treatment is expensive and requires lifelong biweekly infusion. Some patients are non-responders, and the appearance of autoantibodies lead to therapy suspension. Additional therapies must be investigated. Antisense oligonucleotides (ASO) have been previously used to rescue the GAA leaky splicing mutation IVS1-32-13T > G (resulting in exon 2 skipping), the most frequent molecular defect found in LOPD patients worldwide. We administered modified ASO (MOE1 and MOE2, 50 nM) targeting a GAA transcriptional repressor (MOE1) and a regulator element promoting exon 2 skipping (MOE2) into fibroblasts of 4 LOPD patients. Three days after delivery both MOE1 and MOE2 increased GAA transcript levels (from +60% to +140% versus untreated), accompanied by the restoration of physiological exon 2 splicing by MOE2 (from +49% to +180% versus untreated). The efficacy of the molecular approach was validated by biochemical and immunocytochemical methods. We did observe a significant increase of GAA stability (up to 30% of control cells) and activity as well as a reduction of intracellular glycogen content. MOE2 provided superior results compared to MOE1. The effect was maintained up to 6 days after transfection. Our findings confirm and expands previous data on the efficacy of antisense strategy aiming to increase GAA activity and support the development of ASO-based therapeutic approaches in LOPD., Solve-RD is a Horizon 2020-supported project that aims to solve a large number of unsolved rare diseases. As part of the European Reference Network for Neuromuscular Disease (ERN-EURO-NMD), we have contributed to this effort by sharing WES data from a large cohort of unsolved neuromuscular patients and their affected and unaffected relatives. The fastq files from 362 individuals (204 males and 157 females), corresponding to 223 families, were uploaded into the Genome-Phenome Analysis Platform (https://platform.rd-connect.eu/) together with a comprehensive HPO-based description of the phenotype, and processed using the RD-Connect bioinformatics pipeline. Using automated data filtering for SNV-Indels variants, the reanalysis identified the causative variant in three previously unsolved cases. A missense mutation in CASQ1 (Asp244Gly) caused asymmetrical limb myopathy in a family with autosomal dominant inheritance. A de novo mutation occurred in the other two families: the first with a missense mutation in ACTA1 (Pro334Ser) that caused a congenital myopathy, the second one with a missense mutation in SYT1 (Ile368Thr) in a pediatric patient affected by severe developmental delay and hypotonia. To date, about 1.3% of our shared cases have been solved, in line with the performance obtained from the other ERNs, which altogether account for about 3% of solved cases. Additionally, shared data from all Solve-RD participants can be searched for genes, variants, and phenotypes sub-groups, helping in further investigations, as well as the definition of novel genotype-phenotype correlations and indentifying new genes involved in neuromuscular disorders., Thyrotoxic hypokalemic periodic paralysis (THPP) is an uncommon emergency mainly affecting Asian men. The classic triad for diagnosis is: paralysis, thyrotoxicosis and hypokalemia. The typical presentation consists of symmetrical proximal muscle weakness (with legs more affected than arms, with extensor muscle more involved than flexors). We present the case of a 56-years-old Asian male who was hospitalized because of sudden onset of weakness and pain of lower limbs. He reported six months history of palpitations and hands tremors. Neurological examination showed mild symmetrical paraparesis, hyporeflexia, hands tremors, no sensory or cranial nerve deficits. He had fever (body temperature 37.4°C), profuse sweating and atrial fibrillation. Magnetic resonance imaging of spine and nerve conduction studies were normal. Laboratory data revealed potassium 2.7 mmol/l (3.4-4.5), thyroid stimulating hormone < 0.005 mIU/L (0.270-4.200), thyroxine 65.3 ng/L (9.3-17.0), and triiodothyronine 16.0 ng/L (2.0-4.4), TSH receptor antibody 11.1 IU/L (< 1.8) and anti thyroperoxidase > 600.0 kIU/L (< 34.0). He was treated with potassium supplementation, Propranolol, Methimazole and Propylthiouracil with prompt improvement of symptoms. TPP is the most common form of acquired periodic paralysis, yet diagnosis may be challenging in case of new-onset thyroid disorder. Hypokaliemia linked due to hyperactivity of NaK-ATPase stimulated by thyroid hormones is the key of pathophysiology of TPP. Diagnosis is based on clinical and biochemical elements. Management of THPP includes nonselective beta-blockers, achievement of euthyroid state and eventually potassium supplementation. In conclusion, acute paralysis with hypokalemia should prompt physicians to evaluate thyroid function., Objective To describe the first Italian patient affected by “vocal cord and pharyngeal weakness with distal myopathy” (VCPDM) due to MATR3 c.254C > G (p.S85C) mutation, and to expand VCPDM phenotypic spectrum. Methods The proband underwent neurological evaluation, personal and family history investigation, lower limbs muscular magnetic resonance imaging, neurophysiological assessment, and muscle biopsy. Genetic analysis was performed by Next Generation Sequencing analysis of a panel of genes associated with distal myopathy and hereditary neuropathy. Results We detected the heterozygous c.254C > G, p.S85C MATR3 mutation in a patient affected by progressive distal muscle weakness and hypotrophy, myalgias, dysphonia, dysphagia, respiratory impairment, and sensory abnormalities. Neurophysiological assessment revealed a severe sensorimotor polyneuropathy. Variation of fiber size, central nuclei, and non-rimmed vacuoles were evident at muscle biopsy. Conclusions This finding extends MATR3-associated VCPDM phenotypic spectrum and suggests considering MATR3 analysis in suspected congenital polyneuropathies with odd features, including dysphonia, dysphagia, and respiratory insufficiency., Background The histopathological diagnosis of sporadic inclusion body myositis (sIBM) represents a challenge because the pathology can be easily missed. The presenting case is of interest because the diagnostic alterations were detected after the re-orientation of the original muscle biopsy. Case description A 75-year-old male patient underwent a forceps muscle biopsy of gastrocnemius muscle to confirm the clinical diagnosis of sIBM based on the typical distribution of muscle weakness and muscle atrophy, which developed slowly, over the course of 5 years, were asymmetric, of proximal-distal type in the lower limbs and predominant distal in the upper limbs. At the time of the last examination, the patient was unable to flex interphalangeal joints and had severe muscle weakness in hand extensors and flexors as well. He was previously surgically treated for stenosis of the lumbal part of the spinal canal without any relief. EMG was myopathic, creatine kinase was normal. Muscle biopsy demonstrated relatively few muscle fibers (cc. 50), longitudinally oriented and occasional mononuclear inflammatory cells. Rimmed vacuoles were not found. The specimen was cut in a different plane: rimmed vacuoles, with p62 positive inclusions, CD8+ lymphocytes invading non-necrotic muscle fibers and hypertrophic and atrophic muscle fibers were found, most of them cut perpendicularly. Electron microscopy revealed filamentous inclusions in occasional nuclei and in the cytoplasm. Conclusions A diagnostic yield of a single muscle biopsy may be increased by simple maneuver- reorientation of muscle biopsy. The presented case also illustrates the importance of muscle biopsy in avoiding unnecessary surgical interventions., Apoptosis Inducing Factor Mitochondria Associated 1 (AIFM1) is a mitochondrial oxidoreductase with different role in cell pathways, ranging from respiratory chain assembly, to cell death programmes. Pathogenic variants in the AIFM1 gene have been associated with four neurological diseases such as the Cowchock syndrome (CMTX4), the X-linked deafness-5 (DFNX5), the combined oxidative phosphorylation deficiency 6 (COXPD6), and the spondyloepimetaphyseal dysplasia with hypomyelinating leukodystrophy (SEMDHL). Here we report a 17-year-old boy with a mild form of DFNX5 associated with clinical and histological myopathic features, harbouring a novel missense variant in AIFM1. He presented exercise intolerance since early childhood, and at age 7 he was diagnosed with a bilateral auditory neuropathy (deafness), treated with hearing aids. Laboratory investigations, including serum CK and lactic acid were normal. Brain MRI at age 7 and muscle MRI at age 16 did not show any abnormality. An extensive electrophysiological study at age 17 ruled out a peripheral neuropathy. Muscle biopsy performed at age 11, showed myofibrillar texture abnormalities, such as moth eaten fibres and wiped out areas. Activities of the OXPHOS enzymes were normal, while further studies on apoptosis on muscle tissue are ongoing. Whole-exome sequencing (WES) revealed a missense hemizygous mutation in AIFM1, c.1552A > G (p.Lys518Glu), not previously reported in public databases. In conclusion, this case further expands the clinical phenotype associated with AIFM1 gene mutations., Megaconial congenital muscular dystrophy (CMD) is a rare form of congenital muscular dystrophy due to recessive mutations in CHKB gene, encoding Choline Kinase Beta. Since now only few cases have been described, mainly in Asian population. We described a 6-year-old boy who came to our attention for cognitive impairment and slowly progressive muscular weakness. He was the first son of non-consanguineous healthy parents coming from Sri Lanka. Neurological examination showed proximal weakness at four limbs, weak osteotendinous reflexes, Gowers’ manoeuvre and waddling gate. Creatine kinase levels were mildly increased. EMG and brain MRI were normal. Muscle biopsy on quadriceps showed a dystrophic pattern with nuclear centralization and connective tissue increase. Histological and histochemical staining were suggestive for subsarcolemmal localization and dimensional increase of mitochondria. Ultrastructural analysis confirmed the presence of enlarged (“megaconial”) mitochondria. Direct sequencing of CHKB identified two novel defects: the c.1060G > C (p.Gly354Arg) substitution and the c.448-56_29del intronic deletion, segregating from father and mother, respectively. Interestingly, subcloning of RT-PCR amplicons from muscle RNA showed that c.448-56_29del results in the partial retention (14 nucleotides) of intron 3, altering physiological splicing and transcript stability. This report confirms the importance of considering CHKB mutations in the differential diagnosis of patients presenting with muscular dystrophy and mental retardation. Molecular analysis and muscle biopsy were fundamental for the diagnostic process., Adults with DMD are a highly vulnerable population. They are living longer thanks to the improvements of the standard of care but they also experience increasingly complex health issues. To date, there is a paucity of published natural history data and a lack of evidence for managing such complex patients, as recently highlighted by a Consensus document from the UK Adult North Star Network. We present a single centre cross-sectional study on adults with DMD aged > 30 years who are being followed at our Neuromuscular Centre in Turin. Currently, we are following 14 patients with DMD over their thirties (age range: 30 years 4 months - 47 years 10 months). Seven/14 patients are 30-35 years old, and 7/14 are older than 35 years, with 3 out of 7 aged > 45 years. All patients are living with their parent(s) but three who are living in a residential facility and one who is living independently (supported by a caregiver). Data on musculoskeletal, cardiac, respiratory, nutritional/gastrointestinal function will be presented. Data on concomitant neurological (in particular epileptic) and psychiatric aspects will be presented alongside, as well as related management issues in terms of pharmacological treatments. Discussion. Our data confirm the complexity of the multidisciplinary care of patients living with DMD in adulthood. Concomitant psychiatric conditions are frequent, and their pharmacological treatment challenging, especially considering cardiac comorbidities. Management is often based on personal clinical experience. Larger natural history studies and evidence based guidelines on this topic are strongly required., Becker muscular dystrophy (BMD) is a genetic disorder with X-linked recessive inheritance, caused by mutations of Dystrophin gene located at Xp21.2. It typically presents with gradually progressive muscle weakness. Developmental delay can be a manifestation of dystrophinopathies, and the etiology is a debated and investigated topic. 7q11.23 microduplication syndrome is a is a rare syndrome resulting from the partial duplication of the long arm of chromosome 7 characterized by a highly variable phenotype with mild to moderate intellectual delay, speech disorders, and distinctive craniofacial features. We describe a 7-year-old boy with BMD, who presented with developmental delay, bicuspid aorta and interatrial septum defect. At the presentation the serum creatine kinase level was markedly elevated (1046 U/L). Multiplex ligation-dependent probe amplification revealed an exons 48-deletion in the DMD gene. The presence of additional features prompted us to request a cgh array test that revealed a paternal 1.1 Mbp duplication in 7q11.23 region (72.856,430-73.985,812). The father has an intellectual disability too which has never been evaluated by neuropsychiatrist. To our knowledge this is the first case described with the association of BMD and 7q11.23 microduplication syndrome. The case highlights the diagnostic importance of cgh array test in individuals with developmental delay and congenital anomalies and its essential role in excluding other genetic causes of developmental delay/intellectual disability in dystrophinopathies., Introduction Cognitive and behavioral difficulties (i.e. Autism spectrum Disorder, ASD) are described in DMD patients. Different expression of dystrophin isoforms in specific brain areas may play a role in these comorbidities but few imaging data are reported in literature. The aim of this study is to identify DMD specific brain abnormalities through a machine learning approach. Materials and methods 18 boys with DMD (age = 11.0 ± 3.6 yo; IQ = 72.3 ± 16.6; ASD diagnosis n = 5) and 18 controls (age = 9.5 ± 4.1 yo; IQ = 72.1 ± 22.3; ASD n = 5) underwent a Magnetic Resonance (RM) scan session with T1 and diffusion (DTI) sequences. Thickness of 360 cortical regions, volume of 40 brain structures, fractional anisotropy (FA) and mean diffusivity (MD) of 48 regions in the middle of the main fibre bundles were extracted from images. All features were corrected for age, IQ and ASD diagnosis using a linear model. Group classification was performed using a linear SVM algorithm using a balanced leave-one-out cross validation procedure. Feature analysis was performed on the basis of the corresponding forward model weights. Results the linear classifier significantly discriminates between DMD patients and controls with accuracy = 97.2% (p < 0.0005) and AUC = 99.7% (p < 0.0002). The feature weights analysis shows that the discriminative information concentrates in the DTI derived measures (23 /25 ROI weights) and usually report a reduction (in FA/TR/thickness/volume) in DMD patients with respect to the control group (22/25 ROI weights). Moreover, the most selected ROIs refer to the cerebellum (cerebellar peduncles), the brain stem (medial lemniscus, inferior part of the corticospinal tract), the cingulum, the fornix and the superior fronto-occipital fasciculus. Discussion Machine Learning approach allows to identify brain abnormalities specifically associated with DMD, i.e. not caused by a comorbid condition., Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease due to pathogenic variants in the DMD gene. DMD often is associated with cognitive and neuro-behavioural co-morbidities, which pathogenesis and genotype-phenotype relationship are only partially understood. Multiple DMD isoforms, differentially affected based on the mutation site, play a role in these co-morbidities, based on their moderate (Dp427, mainly muscle), prominent (Dp71) or exclusive (Dp140) brain expression. We used two RNAscope® ZZ probes which recognize either the full-length Dp427 transcripts (exons 37-42) or all the DMD transcripts (exons 63-75). BaseScope® ZZ probes were also designed to specifically detect and localize the Dp427b, Dp427p2, Dp140, and Dp71 DMD isoforms. Real-time PCR was performed to validate the results. Sections from normal adult human formalin-fixed cerebellum and temporal lobe were used for the analysis. Both RNAscope probes clearly showed the expression of DMD transcripts in cerebellum (molecular, granular, Purkinje, white matter) and temporal lobe (molecular, granular, pyramidal) tissue layers, including blood vessels. BaseScope® assay allowed discrimination of DMD isoforms and visualization of Dp427b, Dp427p2, Dp140, and Dp71 transcripts in both brain areas. Here we demonstrate that the in-situ RNA hybridization approach has high sensitivity in detecting both full length and short dystrophin isoforms, and low abundant transcripts in fixed brain tissues. The human brain expression map of the multiple dystrophin isoforms will help to define the regional and cellular pattern of DMD expression and may contribute to the understanding of the DMD brain co-morbidities., Background Becker muscular dystrophy (BMD) is a dystrophinopathy caused by mutations in DMD gene which allow production of a partially functional protein, thus justifying the milder phenotype typical of the disease. The molecular diagnostic process starts with the search of large gene deletions or duplications, and, when negative, it proceeds with the sequence of the entire DMD gene. Case description We studied a 26 years-old patient diagnosed at the age of 5 years-old with BMD. At the first evaluation, proximal muscular weakness at lower limbs, mild hypotrophy of pectoralis muscles and calf muscles pseudohypertrophy were detected. Current neuromotor assessment remained stable, however patient developed hypokinetic cardiomyopathy. Diagnosis was supported by immunohistochemical analysis showing fainth dystrophin expression in muscle tissue. MLPA analysis did not show any major rearrangement on DMD. Results NGS analysis revealed a novel nucleotide intronic substitution DMD:NM_004006:exon15:c.[1705-11A>G]:p.[?] which affects a splice site. RNA analysis performed on muscle tissue showed expression of two different splice isoforms. The first one uses the splice site variant as a new acceptor site and generates an aberrant mRNA encoding a truncated protein. The second one consists in an “in-frame” splice of the exon 15. Segregation analysis confirmed the carrier condition of the mother. Conclusions Our study confirms the pathogenicity of the novel intronic variant DMD:NM_004006:exon15:c.[1705-11A>G]:p.[?] in DMD gene by demonstrating its direct effect at RNA level, and defines the genotype/phenotype correlation. We also stress that the exon boundary should always be included in molecular analysis for an exhaustive diagnosis., We report a 13 years-old patient presenting one year ago with incidental finding of high creatine-kinase levels (8413 IU/L). The patient played soccer at a competitive level and was completely asymptomatic, complaining only cramps after strenuous exercise. He did not show weakness, fatigability, myalgias, myoglobinuria. Neurological examination wan normal with exception for mild sural pseudohypertrophy. Cardiological evaluation was normal. Muscle MRI showed fibroadipose substitution of glutei, paraspinal muscles, serratum and teres major. Muscle biopsy displayed mild myopathic signs and moderate reduction of alpha-sarcoglycan levels at immunohistochemistry. A severe reduction of alpha- and beta-sarcoglycan was also confirmed by Western blot analysis. NGS panel sequencing identified two mutations in the gene encoding for beta-sarcoglycan (SGCB): the previously reported known c.377_384dup microduplication and a novel T > C transition located within a putative pseudoexon. The latter variant activated the inclusion of a cryptic sequence between Exons 2 and 3 in SGCB transcript. Interestingly we detected the same variant in a previously reported Italian LGMDR4 patient in which molecular testing had only detected the c.377_384dup insertion. Molecular studies in patient’s tissues, including autoptic heart specimen, showed global reduction of SGCB mRNA and altered splicing at transcript level. The in vitro administration of an antisense morpholino sequence targeting the pseudoexon restored physiological splicing in patient’s myoblasts. Our findings prompt the analysis of a novel variant in suspected LGMDR4 patients with monoallelic SGCB variants and provides a further example of the efficacy of morpholino antisense technology for the correction of splicing molecular defects., Ullrich congenital muscular dystrophy (UCMD) is caused by mutations in COL6A1, COL6A2 or COL6A3 gene, leading to collagen VI deficiency. Either recessive (more frequent) or dominant inheritance is reported. Clinical-pathological hallmarks of UCMD include distal hyperlaxity, proximal joint contractures, early-onset rapidly progressive scoliosis, and respiratory failure. Muscle pathology is characterized by prominent interstitial fibrosis. We describe a patient who came to our observation at the age of 11.5 years, for a picture characterized by early tendon retractions, kyphoscoliosis, respiratory insufficiency (FVC 52%). Symptoms started at the age of 7-9 months; the child could not maintain the sitting position and never walked alone so the patient underwent a muscle biopsy, which showed a severe pattern of interstitial fibrosis. CK values were normal. There was no cardiac involvement. COL6A1 and COL6A2 gene analysis was negative. Over the years, muscle condition remained stable, while the vital capacity has progressively deteriorated. He died at 25 years 9 months of acute respiratory failure. Trio whole exome sequencing identified a de novo variant c.6210+1G>A at the 5’ splice site of intron 16 of the COL6A3 gene. This LoF variant, classified as pathogenic (VCV0000949S6.5), has already been reported in literature. We identified an additional case in a 4-year boy with decreased fetal movement, neonatal respiratory distress, bilateral cryptorchidism. Muscle-skeletal symptoms are present as scoliosis, adducted thumb, kyphoscoliosis, hip dysplasia hyperextensibility at wrists, central hypotonia and no cardiac involvement. These cases expands the number of patients with autosomal dominant UCMD, due to de novo mutation., Oculo-pharyngeal muscular dystrophy (OPMD) is a late-onset, inherited muscle disorder, characterized by progressive ptosis, dysphagia and variable proximal limb weakness. The highest prevalence is reported in French Canadians (1:1000) and Bukhara Jews (1:600). Few Italian cases have also been reported. OPMD is caused by short (GCN)11-17 expansions in the polyadenylate-binding protein nuclear 1 gene (PABPN1). We summarized the neurophysiological and genetic findings of 17 OPMD patients born in Abruzzo, belonging to 11 unrelated families. In our cohort, age at onset was between 42 and 74 years (mean 57.5). The M/F ratio was 1.125:1. Family history was positive in 15 patients. As expected, ptosis was the most common initial complaint. There was a 5.8-year mean delay before the onset of a second symptom, which was dysphagia in 82.4% of cases. External ophthalmoplegia was present in 64.7% of patients. Two siblings presented cognitive impairment. Genetically, we identified four genotypes, including two GCN11 homozygous patients. Mean age at first evaluation was 67.4 and mean age at genetic screening was 69.4. Creatine kinase levels were mildly elevated in seven patients. Electromyography, performed in seven patients, showed myopathic features in four cases. Five patients had ptosis surgery and one patient reported cricopharyngeal myotomy. According to our results, OPMD has an estimated minimal prevalence of 1.32 per 100.000 persons in Abruzzo. A disease cluster can be hypothesized in Caramanico, where OPMD prevalence is about 0.42%. Despite rapid diagnosis through PABPN1 gene screening, OPMD is yet under-recognized and confirmed after several years from symptoms onset., Becker muscular dystrophy (BMD) is characterized by variable disease severity and progression, prompting the identification of biomarkers for clinical trials. We used data from a recently completed Phase II study to provide a comprehensive evaluation of a cohort of patients with BMD, and assessed correlations between histological and magnetic resonance imaging (MRI) markers with muscle function and strength. Eligible patients were ambulatory males with BMD aged 18-65 years (200-450 m on 6 minute walk test). The following were measured: function tests, strength, fat-fraction quantification using chemical shift-encoded MRI (whole thigh and quadriceps), and fibrosis and muscle fiber area (MFA) of the brachial biceps. Of 70 patients screened, 51 entered the study. There was substantial heterogeneity between patients in muscle morphology (histology and MRI), with high fat replacement. Total fibrosis correlated significantly and mostly moderately with all functional endpoints, including both upper arm strength assessments (left and right elbow flexion Rho -0.574 and -0.588, respectively [both p < 0.0001]), as did MRI fat fraction (whole thigh and quadriceps), e.g., with four stair climb velocity -0.554 and -0.550, respectively (both p < 0.0001). Total fibrosis correlated significantly and moderately with both MRI fat fraction assessments (0.500 [p = 0.0003] and 0.423 [0.0024], respectively). In this BMD cohort, micro- and macroscopic morphological muscles parameters correlated (albeit moderately) with each other and with functional parameters, potentially supporting the use of MRI fat fraction and histology as surrogate outcome measures in patients with BMD, although additional research to validate this is required., Aim of the study To describe and analyse long term functional changes in Becker muscular dystrophy (BMD) through a commonly used outcome measure in neuromuscular diseases, the 6-minute walk test (6MWT). Materials and methods We selected patients coming from “Azienda Ospedale-Università di Padova” who have a molecular confirmed diagnosis of BMD. Our patients were followed up for at least 1 year by evaluating motor function through 6MWT and also North Star Ambulatory assessment (NSAA). We used a modified version of 6MWT specifically validated for DMD. Results Our cohort was composed of 105 subjects, of whom 99 patients could perform 6MWT at baseline. We divided patients according to mutational groups. The shortest distances were registered in the “del 45-48” group, (mean distance was 330.8 m ± SD 178.8 m), and the “del 45-47” group, (mean distance was 324.8 m ± SD 142.9 m). The longitudinal study included a mean number of 5.25 (± 2.95) clinical examinations for each patient and the mean follow up was 4.66 years (± 3.0 years). Longitudinal data showed a statistically significant yearly decrease in walked distance in “del 45-48”, “del 45-47”, and “del 48-49” groups. This decrease is larger if we only consider patients with a worst baseline muscular function (NSAA < 33). We considered the comparison between the distances covered between the first and last 3 minutes of 6MWT as an indicator of fatigability. In the overall BMD cohort there was a significant decrease in distance between the two halves of the test, of -3.0 ± 18.5 m (p = 0.0005). This difference was larger in patients with baseline NSAA, Deep intronic single nucleotide variants (SNVs) are the most elusive DNA variants for NGS. Thus, defining their pathogenic role remains challenging and many genetic conditions remain unsolved. Some of deep intronic variants could lead to aberrant splicing, resulting in partial or full pseudo-exon (PEs) inclusion, mainly due to the activation of cryptic intronic acceptor and/or donor splice sites and the alteration of sequence motifs recognized by enhancer or silencer splicing factors. Here, we tested a computational approach to evaluate splicing involvement of DMD deep intronic SNVs annotated in the Leiden Open Variation Database (LOVD). To prioritize our set of variants based on their probability to be involved in PEs formation, we sequentially applied four different prediction tools (SpliceAI, NNSplice Predictor, HSF and SFMap). We divided the collected LOVD variants into two groups, TRAINING (variants with known RNA effect) and TESTING (variants with unknown RNA effect), which were used respectively to validate and test our pipeline. For TRAINING group, we confirmed involvement of 77% of variants in PEs inclusion. Interestingly, we found that 72% of TESTING variants are involved in alternative splicing leading to PEs inclusion. Overall, we believe that this pipeline could be useful to confidently predict the effect of specific SNVs on alternative splicing and PEs formation, thus providing good indication for NGS variants prioritization and transcriptomic studies in muscular biopsy., The etiology of dermatomyositis is unknown but immune dysregulation plays a key role. A 68-year-old man presented with a 1-month history of skin rash, myalgia and symmetrical proximal limb weakness. He developed these symptoms about three weeks after the second dose of Vaxzevria. On examination, he showed a diffuse facial, scalp, arm and trunk rash with periorbital edema. No Gottron papules were detected. Creatine kinase levels were markedly increased. Electromyography was normal, whereas muscle biopsy revealed a perivascular mixed cell infiltrate. Based on clinical features, elevated muscle enzymes and muscle biopsy, a diagnosis of dermatomyositis was established. A full-body CT scan, performed in order to exclude a connection with malignancies, appeared unremarkable. The patient showed a gradual improvement of symptoms after treatment with intravenous methylprednisolone 80 mg for three days, then transitioned to oral prednisolone 50 mg. To the best of our knowledge, this is the first case of new-onset dermatomyositis after COVID-19 vaccination. Dermatomyositis occurring after vaccination is a well-recognized phenomenon and may be attributable to homology between vaccine components and muscle antigens, triggering an autoimmune response. Moreover, dermatomyositis has been recognized as a manifestation of COVID-19-induced muscle disease. It has been hypothesized that SARS-CoV-2 may transfer its genetic material into the muscle fibers, thus triggering a T cell-mediated viral response leading to muscle damage. In addition, three different T cell receptor epitopes “highly specific” for SARS-CoV-2 have been detected in dermatomyositis patients, reinforcing the hypothesis of molecular mimicry., In this work, we studied a novel mutation in SCN4A gene (p. N1180I), identified in two Italian families showing a peculiar phenotype characterized by the association of myopathy and myotonia (Fusco et al., Brain Dev. 2015; Rigamonti et al., Neurol Sci. 2021). The mutation N1180I was introduced into the pRc/CMV plasmid containing the cDNA encoding wild-type (WT) human Nav1.4 channels. Whole-cell sodium currents were recorded with patch-clamp technique in HEK293 cells transfected with WT or N1180I. Cells transfected with 0.5 μg/ml cDNA of N1180I did not express any current (n = 15), in contrast to 100% of WT-transfected cells. Increasing N1180I cDNA to 1 μg/ml allowed recording of sodium currents in 15 out of 98 patched-cells. Maximum current amplitude from I-V curves was reduced for N1180I (-229 ± 38.5 pA, n = 5), compared to WT (-3959 ± 615 pA, n = 17, p < 0.005). Decay of N1180I currents was significantly slowed. The voltage-dependences of activation and fast inactivation were significantly positively shifted by about 14 mV and 9 mV, respectively. The fast inactivation impairment by N1180I suggests a gain of function of Nav1.4 channels, in accord with occurrence of myotonia in the patients. However, the reduction of expression efficiency and maximum current amplitude, together with the positive shift of activation, suggest a loss of function more compatible with the presence of myopathic traits in the patients. More studies are needed to better understand the mechanisms allowing occurrence of both myotonia and myopathy in the same patient (Supported by Grant 2017-2018 from the University of Bari)., Background Familial periodic paralyses (PPs) are inherited disorders of skeletal muscle characterized by recurrent episodes of flaccid muscle weakness. PPs are classified as hypokalemic (HypoPP), normokaliemic (NormoPP) or hyperkalemic (HyperPP) according to the potassium level during the paralytic attacks. HypoPP is an autosomal dominant disease caused by mutations in the CACNA1S gene, encoding for Cav1.1 channel, (HypoPP-1), or SCN4A gene, encoding for Nav1.4 channel, (HypoPP-2). Methods In the present study, we included 60 patients with a clinical diagnosis of HypoPP. Fifty-one (85%) patients were tested using the direct sequencing (Sanger method) of all reported HypoPP mutations in CACNA1S and SCN4A genes; the remaining 9 (15%) patients were analyzed through a next-generation sequencing (NGS) panel, including the whole CACNA1S and SCN4A genes, plus other genes rarely associated to PPs. Results Fifty patients resulted mutated: 38 (76%) cases showed p.R528H and p.R1239G/H CACNA1S mutations and 12 (24%) displayed p.R669H, p.R672C/H, p.R1132G/Q and p.R1135H SCN4A mutations. Forty-one mutated cases were identified among the 51 patients managed with Sanger sequencing, while all the 9 cases directly analyzed with the NGS panel showed mutations in the hotspot regions of SCN4A and CACNA1S. Ten out of the 51 patients unresolved through the Sanger sequencing were further analyzed with the NGS panel, without the detection of any mutation. Conclusions Hence, our data suggest that in HypoPP patients the extension of genetic analysis from the hotspot regions using the Sanger method to the NGS sequencing of the entire CACNA1S and SCN4A genes does not lead to the identification of new pathological mutations., AChR and MuSK double positive myasthenia gravis has been rarely reported. Generally, it occurs in children and adults after thymectomy or immunotherapy, two young female patients has been described as double positive since the onset of the disease. We report herein a man with a very late onset myasthenia gravis (86-year-old) and coexistence of both antibodies at the time of the diagnosis with a favourable clinical outcome. Despite the presence of MuSK antibodies, he manifested no bulbar symptoms but side effects related to low dose pyridostigmine were evident. Hence, double positivity should be considered also in elderly. We suggest to detect AChR and MuSK antibodies at the time of diagnosis. Other cases of AChR and MuSK double positive myasthenia gravis could allow a better definition of this condition., Congenital myasthenic syndromes (CMS) are genetic disorders characterized by impaired transmission at the motor endplate. Clinically, these forms present with prevalent weakness of the axial muscles (hypotonia at onset) and bulbar muscles (ptosis, ophthalmoplegia, dysphagia, facial weakness). Fluctuations of symptoms, facial dimorphisms and distal weakness are other common features. CMS may respond to treatment with drugs enhancing the neuromuscular transmission and differential diagnosis with congenital myopathies is fundamental. The PREPL gene, responsible for the autosomal recessive form of CMS22, belongs to the prolyl-oligopeptidase subfamily of serine peptidases. PREPL is localized in the cytosol where it is involved in exocytosis processes. We report an 11-year-old patient with CMS, harboring a novel homozygous variant in PREPL. At onset, the patient presented with neonatal hypotonia followed by rhinolalia, bilateral ptosis and dysphagia. Muscle biopsy revealed myopathic changes with selective hypotrophy of type II fibers, in absence of specific characteristics. We performed a custom NGS panel for congenital myopathies/myasthenic syndromes which revealed the homozygous (NM_001374276.1): c.950dup (p.Glu318Argfs*6) variant in PREPL. This insertion determines a premature stop codon, likely leading to nonsense-mediated mRNA decay. The variant is not reported in GnomAD and ClinVar, and is predicted pathogenetic by in silico tools. The repetitive stimulation confirmed an alteration of the neuromuscular transmission. The patient started a treatment with pyridostigmine, which was stopped due to allergic reaction and replaced by salbutamol. This resulted in a global improvement of the generalized hypotonia and dysphagia. However, ptosis and rhinolalia remained severe, with perturbation of speech. This case underlines the importance of genetic characterization for proper clinical management and specific therapeutic strategies., Myasthenia Gravis (MG) is characterized by muscle fatigue, determined by autoantibodies against components of neuromuscular junction, like Acetylcholine Receptor (AchR) and Muscle Specific Kinase (MuSK). Therapies differ according to clinical severity, including symptomatic treatment, immunomodulant approaches and immunosuppressants. Rituximab (RTX) is a “target” immunosuppressant agent against CD20 antigen, determining selective depletion of B lymphocytes with a long-lasting reduction of humoral immune response. It is increasingly used in clinical practice as an off-label treatment of refractory MG. High efficacy has been demonstrated in anti-MuSK MG, though there is also evidence of efficacy in anti-AchR and seronegative MG. Between 2009 and 2020, 12 patients with generalized refractory MG referring to our Unit have been treated with RTX (cycles of 1000 mg IV repeated after two weeks). Among them (9 females and 3 males) 6 had anti-MuSK MG and 6 anti-AchR MG; 3 of these patients had thymoma surgically removed. Efficacy duration of a single cycle ranged from 6 months to 9 years. About 70% of patients substantially reduced dosages of immunosuppressive treatments within one year. At most recent follow up (2021) 6 patients were in Complete Stable Remission, 1 in Pharmacological Remission and 5 in Minimal Manifestation-3 status, according to MGFA Post Interventional scale. None of our patients developed myasthenic crisis or required rescue therapies since the introduction of RTX. Only one severe drug-related, but reversible, adverse event (hypertensive crisis) was observed. Our experience confirmed RTX as a safe and effective treatment for both anti-MusK and anti-AchR MG patients., Background and aims Since COVID-19 infection became a global public health problem, finding a treatment has been an emergency and vaccines are considered the only solution. In the last months, a big amount of data has been published on COVID-19 and vaccines and are currently available for the general population, but little is still known regarding patients with myasthenia gravis. Methods We performed a cross-sectional study among a cohort of patients with Myasthenia gravis attending to the Neuromuscular Clinic of the University Hospital “Paolo Giaccone” of Palermo. Patients underwent a telephonic interview through a dedicated questionary about COVID-19 infection, vaccinations, and their effects on MG. Results In our cohort 9 patients resulted positive to SARS-COV2 infection, 4 patients died for COVID-19, a patient worsened for MG, requiring respiratory support, whereas 3 patients were asymptomatic. Fifty-three patients completed the vaccination with minor side effects in 24 cases. Seventeen patients presented a worsening of symptoms. Conclusions The reduced number of adverse events in our population suggests that vaccines for SARS-cov2 are safe in myasthenic patients that could take advantage of vaccination avoiding life-threatening complications such as myasthenic crisis and COVID-19 pneumonia. The continuation of the regular and periodic clinical follow-up will provide us data on the real effectiveness of vaccine prevention in the myasthenic population., Background Congenital myasthenic syndromes (CMS) are hereditary neuromuscular junction disorders, due to defects of proteins involved in endplate development and function. Different CMS have no clear genetic cause yet, therefore many other genes will have to be discovered. Cases description We describe herein a 52-year-old woman, whose first faticability symptoms manifested at the age of 43 years old. She complained of left fingers weakness, that progressively involved the other hand and pelvic girdle muscles, with difficulty to raising from a chair and climbing stairs. Besides, she presented sporadic episodes of double vision. Clinical examination revealed waddling gait, not possible on heels, positive Gowers sign, severe weakness of flexors and extensors finger muscles, mild weakness of bilateral iliopsoas muscle and pes cavus. Results Blood investigations, including serum CK, were normal. Electromyography evidenced a myopathic pattern. Ulnar nerve repetitive stimulation recorded no alteration, but single fiber electromyography showed increased jitter and blockings. AChR-Abs and MuSK-Abs were negative. Sequence analysis of CMS-related genes resulted surprisingly normal, while it was found a stop codon mutation of Leucine Rich Repeat And Sterile Alpha Motif Containing 1 (LRSAM1) gene (c.1279C > T; p.Arg427Ter), encoding for an axonal membrane protein. Despite that, therapy with ephedrine was started with subsequent and progressive improvement of clinical manifestations. Discussion and conclusions To date, mutations of LRSAM1 gene have been reported in Charcot-Marie-Tooth type 2P. Our patient presented clinical and neurophysiological features of CMS. This case highlights the opportunity to find new molecular basis for these disorders in order to better define their pathophysiology., Objective Neuromuscular diseases (NMD) may represent a risk factor causing a more severe course and outcome of SARS-CoV-2 infection. Interestingly, we observed several cases of SARS-CoV-2 infection in Ligurian patients affected by Myasthenia Gravis from March 2020 to April 2021. Methods We collected data from 13 patients affected by Myasthenia Gravis, followed in different Ligurian hospitals. While eight patients had a mild course of SARS-CoV-2 infection, 5 patients had an unfavorable course causing the death of 4 patients and a prolonged life threating hospitalization in one patient. We analyzed their MGFA class at the moment of the infection, the maximum MGFA class reached during their clinical history, and the previous number of myasthenic crisis. We also evaluated the age at the infection, the BMI and the number of comorbidities as independent risk factors for infection severity. Results As expected, age and elevated BMI are independent risk factors for poor outcome of Covid-19 in myasthenic patients (mean age 68,3 range 47-87 years; comorbidity rate overall 53.8%). Also having 1 or more comorbidities predicts a higher hospitalization rate (7/8 patients, 85.7%). Interestingly, the five patients with an unfavorable SARS-CoV-2 infection course had a moderate MGFA class at the moment of the infection, but almost all (80%) had previous myasthenic crisis and the average maximum MGFA class reached during the clinical history was significantly higher (MGFA = 4), compared with the group with a prompt recover (MGFA = 2); p : 0.01. Conclusions Among our neuromuscular patients, SARS-CoV-2 infection had a significant impact in particular in myasthenic patients causing the death of four of them and a long hospitalization in one. Despite Myasthenia was well compensated at the moment of the infection, patients with previous myasthenic crisis and a higher MGFA maximum tended to have an unfavorable course. This correlation, already described in a large French study (Solé G et al. Impact of Coronavirus Disease 2019 in a French Cohort of Myasthenia Gravis. Neurology 2021) supports the hypothesis that autoimmune neurological diseases may be a risk factor for a severe course of SARS-Cov-2 infection., A 29-year-old woman with Neutral Lipid Storage Myopathy (NLSDM) is presented. Until 2015, she was asymptomatic except for elevated CPK. At 18 years, hyperCkemia suggested NLSDM diagnosis and sequencing analysis revealed a retrotransposal insertion in PNPLA2 gene. PNPLA2 encodes the lipase ATGL, which mutations cause NLSDM onset. In the following years, the patient has been experiencing muscle weakness in both arms, shoulders, and hands, then extended to lower extremities. Calves MRI showed lipid accumulation. At 27 years, she was put on a diet with 30 grams per day of MCT oil and 15 grams of natural fat. After beginning MCT diet, the CPK lowered, from 2640 U/l to 1424 U/l. Nevertheless, muscle weakness did not improve, as showed by GSGC score performed in 2020: Walking 10 Meters = 10 seconds; Climbing Stairs = 13 steps up = 8.10 seconds. 13 step down = 6.40 seconds; Raising from seated floor position with no hands = 2.76 seconds; Getting out of chair = .61 seconds (< less than 1 second). Following parents’ request, we planned a recruiting study for NLSDM, as previously described. We suggested performing a new MRI and skin biopsy. While the skin biopsy showed only a slight oedema, muscle MRI highlighted advanced fat substitution in both upper and lower extremities. In this patient, no beneficial effects on myopathy progression were observed after MCT diet, probably due to complete loss of ATGL production. We reported several cases in a NLSDM registry and this patient represents an NLSDM spectrum severe example., Background Muscle biopsy is considered to be the gold standard test for diagnosis of muscle disease. It is usually indicated to evaluate abnormal clinical and/ or laboratory findings suggestive of myopathy, such as HyperCKemia, myalgia, exercise intolerance, family history of myopathy, muscle weakness, and abnormal electromyography (EMG). Methods We restropectivly reviewed 889 consequential muscle biopsies performed at Center for Neuromuscular disease between 2005 and 2020. We collected several patient information including laboratory data, neurological examination, genetic and byochemical test. Results The clinical reason for performing the muscle biopsy was myalgia in 32% of cases, muscle weakness in 50.4%, muscle atrophy in 4.1% and at least 1 episode of Rhabdomyolysis in 5.1%, while 9.1% of patients had chronic CK serum elevation and 4.4% had a past diagnosis of myopathy. Mucle biopsy led to a unique diagnosis in 42.2% of cases, which the most frequent were inflammatory disease and dystrophic forms. The Area Under the Curve (AUC) for prediction of a specific bioptic diagnosis was 0.63 (0.59-0.68) for patient with weakness, 0.57 (0.53-0.61) for patients with Hyperckemia and 0.63 (0.59-0.68) for patient with myopathic EMG. The association of these 3 element results in an increase of the AUC (0.73, 0.68-0.77). A definitive diagnosis was obtained in only 21.5% of patients without specific a priori clinical suspicion. Conclusions Muscle biopsy remains a fundamental diagnostic test for the study of muscle pathologies, especially in patients with specific clinical signs. However, its role is less clear in patients with more subtle clinical elements., Introduction Respiratory insufficiency is one of the main causes of death in myotonic dystrophy type 1 (DM1). Predictive factors, rate of decline and the effects of ventilator support need to be further explored. Objectives To analyze respiratory function over time. Methods. 175 adult patients with DM1 were subjected to arterial blood gas analysis, spirometry, cough measurements, nocturnal oximetry and respiratory muscle strength testing. Results were correlated with neuromotor function and coping strategies. Results At baseline 84 of 175 had normal respiratory function (median age 38 years, median BMI 23.9, median disease duration: 11 years); 24 were already on NIV (median age: 49 years, median BMI: 26.1, median disease duration: 21.5 years) and 67 received an indication to use NIV (median age: 49 years, median BMI: 25,8, median disease duration: 14 years). After a median time of 3.85 years, 43 patients were lost to follow-up; 9 of 84 required NIV; 16 patients initially on NIV kept using NIV; only 17 of 67 with the new NIV prescription were compliant. Conclusions Respiratory involvement affects almost 50% of patients with DM1 although typical respiratory symptoms may not be present. The majority required NIV and these were the most severely affected patients and having the longest disease durations and higher BMIs than the cohort having normal respiratory function. A minority (11%) required NIV during our observational period. Only 25% was NIV compliant and this was unrelated to the patients’ coping strategies or to specific demographic or respiratory distinctive features., Background Myotonic dystrophy type 1 (DM1) is characterized by a wide phenotypic variety. There is consensus about the need for multidisciplinary management of this disease, but there is still a need to harmonize procedures of assessment and to define the best outcome measures. END-DM1 is a multicenter, international, observational and prospective study proposed by DMCRN. The NeMO Clinical Center in Milan is the Italian representative for this network. Aims to describe the NeMO’s experience in screening, evaluating and collecting data along with difficulties and strengths in following END-DM1 procedures. Methods The protocol includes: clinical assessments (medical history, physical exam, vital signs and EKG), mobility and functional measures (10MWT, 6MWT, TUG, 4 stairs, timed supine-to-sit, step test, vHOT, pick-up coins, 9NHPT), measures of strengths (QMA, MMT, IOPI) and pulmonary function, PROMs (MDHI, EAT-10, Domain Delta questionnaire, WPAI:SHP, EQ-5D-SF, Walking Scale-12, DM1-Activ), cognitive function (Cogstate), biomarker and genetic assessments (blood and urine samples and muscle biopsy). Results We recruited 65 adult onset patients (44 females, age: 44 ± 13.57): 2 were wheel-chair bound, 7 had PM/ICD, 26 cataracts and 21 NIV. Age of first symptom: 22.66 ± 13.57; age at diagnosis 31.54 ± 12.45; grip myotonia was the most frequent symptom at onset (61/65) and hand weakness often preceded limb weakness. Only 4 patients reported cognitive symptoms. Conclusions This academic registry highlights the importance of providing appropriate evaluations by trained and dedicated staff in order to guarantee quality control of data entry in preparation for clinical trials while providing care according to patients’ needs., Heart disease is a determinant of prognosis in type 1 myotonic dystrophy (DM1). Cardiac imaging, possibly including cardiac magnetic resonance (CMR), is recommended, but there is limited information on CMR findings and their prognostic significance. All DM1 patients referred to our CMR laboratory between 2009-2020 (n = 34, 21 males, aged 45 ± 12) were retrieved. At the time of CMR, 90% had neuromuscular symptoms (duration 17 ± 13 years), 13(38%) had previous reports of atrioventricular block, 30(88%) of intraventricular conduction disturbances, 4(12%) of atrial fibrillation/flutter. At CMR, 5(15%) patients had left ventricular (LV) systolic dysfunction (LVEF < 50%) and 4(12%) a depressed right ventricular (RV) function (RVEF < 50%). Compared to age- and sex-specific reference values, 12(35%) showed a decreased LV end-diastolic volume index (LVEDVi), 7(21%) a decreased LV mass index (LVMi), and 29(85%) a decreased LVMi/LVEDVi. Nine(26%) patients had mid-wall late gadolinium enhancement (LGE), and 14(41%) some areas of fatty infiltration. Native T1 in the interventricular septum (1.041 ± 53 ms) approached the upper reference limit (1.089 ms), and the extracellular volume was increased (33 ± 2%, reference < 30%). Over a median follow-up of 2.5(1.5-4.0) years, 2(6%) patients died for infectious and respiratory complications, 5(15%) underwent PM/ICD implantation and 4(12%) presented high-risk (Lown ≥ 4) ventricular ectopic beats (VEBs). Among CMR variables, high LVMi/LVEDVi emerged as univariate predictor of all-cause death (p = 0.044). At logistic regression, anteroseptal wall thickness was associated with PM/ICD implantation (p = 0.028), LGE mass with high-risk VEBs (p = 0.026). In conclusion, DM1 patients display cardiac muscle hypotrophy, fibrosis and fatty infiltration at CMR. Such changes may anticipate the worsening of electrical disturbances., We formerly reported an Italian kindred with adult-onset autosomal dominant vacuolar myopathy with 19 affected individuals over four generations. Myopathology was characterized by rimmed autophagic vacuolation and distinctive immunohistochemical features including involvement of the ubiquitin-proteasome pathway. More recently, we identified the protein accumulating within the vacuoles and pinpoint, with the use of long-read sequencing, a large coding expansion in the PLIN4 gene, member of the perilipins’ family. The accumulation of this protein within the vacuoles and in the subsarcolemmal region of the affected fibers, coincides with the immunohistological activation of the key players of the aggrephagy pathway, notably p62/SQSTM1, NBR1 and WDFY3. We present here clinical, histological and muscle imaging data of 15 affected patients carrying the PLIN4 gene expansion. Mean age at onset was 47.3 ± 10 years (range 30-66), with upper or lower limb distal muscle weakness as presenting symptoms in most of the patients. Disease progression was slow over the years, with around half of the patients developing a predominant scapulo-humeral-peroneal pattern of weakness. Five out of 15 (33.3%) patients were wheelchair-bound after a mean disease duration of 14 ± 6.6 years. No relevant heart involvement was observed. Rimmed values were detected in all muscle biopsies taken from the patients. The extent of the histopathological changes varied between patients correlating with disease severity and PLIN4 expansion entity. In conclusion, we clinically and histologically characterized a new vacuolar distal myopathy presenting in adult age, linked to a pathological expansion of the PLIN4 gene., Glycogen Storage Disease type III (GSDIII) is a rare autosomal recessive disease caused by deficiency of the glycogen debranching enzyme (AGL). Consequently, glycogen accumulates in the affected tissues, mainly damaging liver, muscles and heart (GSD IIIa); in a minority only liver (GSD IIIb). The onset is in infancy. However, the risk of hypoglycemia usually decreases with age, with progressive reduction in liver volume, during pubertal age. In adulthood, cirrhosis of the liver and/or hepatomas may appear, as well as the involvement of skeletal muscles and myocardium. We describe two siblings, male and female, with GSDIII presenting with an early skeletal muscle involvement. Male: onset in the second decade, with difficulties in walking, climbing stairs and getting up from the floor, progressively worsening, associated to muscle weakness. Severe kyphoscoliosis. CK values up to 16 times the maximum normal values. Episodes of hypoglycaemia at 30 years; hepatosplenomegaly. Hypertrophic cardiomyopathy diagnosed at 43 years. LoA at 54 years. Over the years, worsening of general conditions, and increased functional limitation in daily activities. Female: onset of muscle symptoms at the same age; LoA at 43 years. No cardiomyopathy. CK values up to 17 times the maximum. Vacuoles at muscle biopsy, with modest glycogen accumulation; debranching enzyme deficiency causing the disease. Death at 49 years, for causes unrelated to the disease. NGS analysis showed the homozygous variant c.1283G > A p.R428K in exon 10 of the AGL gene, which modifies the last nucleotide of the exon. This variant most likely affect the splicing, as SpliceAI assigns it a score of 0.72 for donor loss. The cases here reported broaden the spectrum of GSDIII clinical presentation, confirming its clinical heterogeneity and intra-familial variability., Mutations in LMNA lead to skeletal muscle laminopathies (SMLs), a group of rare disorders characterized by skeletal and cardiac muscle involvement. To date, natural history of SMLs is not clarified yet, being mainly described by retrospective studies, reporting only major events. We aimed to investigate the natural history of SMLs through a 2-year prospective study, including several clinical outcome measures. Methods Twenty-six SML patients were enrolled in the present study and assessed with the following tests: North Star Ambulatory Assessment scale (NSAA), timed tests, manual muscle testing, joint range of motion, six-minutes walking test (6MWT); respiratory evaluation including forced vital capacity (FVC) and forced expiratory volume at 1 second (FEV1); individualized neuromuscular quality of life (INQoL) questionnaires; cardiac evaluation collecting standard 12-derivations electrocardiogram, 24-hours Holter ECG monitoring and heart echo scan. Results At the baseline, clinical assessments significantly (p value < 0.05) correlated with SMLs phenotypes, showing a worst performance in Emery-Dreifuss muscular dystrophy 2 patients. The NSAA score significantly deteriorated (p value = 0.0005, mean change: 2.9 ± 0.4) during the 2-years follow up. The respiratory function through FVC (p value: 0.0086, mean change: 6.9% ± 1.4%) and FEV1 (p value: 0.0290, mean change: 6.7% ± 1.8%) significantly declined. Conversely, 6MWT and timed tests, did not significantly change. Similarly, elbow, knee and ankle joint range of motion resulted unchanged. Conclusions Disease progression of SMLs was shown with NSAA, FVC and FEV1 in a 2-year period, suggesting a slow decline of the motor and respiratory function in these patients., Introduction Progressive external ophthalmoplegia (PEO) represents the most common feature of mitochondrial disorders. When it occurs in isolation, it is defined “chronic progressive external ophthalmoplegia”(CPEO) while the combination of PEO and other features of neuromuscular and multisystem involvement (excluding CNS) is named “CPEO plus syndrome”. Clinical variability is broad even in individuals harbouring the same genotype. Recently, some studies focused the attention on muscle weakness, exercise intolerance, and muscular pain as features of mitochondrial diseases. However, fewer studies focused the attention on severity and distribution of muscle wasting and damage in PEO patients. Our aim is to analyse systemic muscles involvement with the use of muscle MRI. Materials and methods We enrolled patients diagnosed with CPEO or CPEO plus by clinical, biological and genetic tests, at Neuromuscular Centre of Careggi University Hospital, Florence. We analyzed T1-weighted sequences of the neck, shoulder girdle, paraspinal muscles, lumbar girdle, thighs, and legs, evaluating the severity of wasting and the grade of fatty tissue substitution. Results MRI images were taken from 7 patients (3 men); their age ranged between 23 and 72 years. Three patients (2 men, age 56-72yrs) had CPEO-plus; 2 harboured pathogenetic variants in POLG. In those individuals, MRI showed similar involvement of the axial, tight, and leg muscles. Studies of the remaining 4 patients, who are still awaiting molecular definition, were unremarkable. Conclusions MRI could be a useful and informative tool in the clinical evaluation of the general muscle involvement of CPEO patients. This research project is funded by Tuscany Region., Objective To verify the change in the emotional and behavioural conditions of neuromuscular disorders patients’ before and during the COVID-19 pandemic and to evaluate if the change was predicted by coping strategies. Methods We analyzed 43 participants – age range at first assessment 4-52 - out of 112 subjects recruited in the study. The emotional and behavioural conditions were assessed through standardized questionnaires: Youth Self Report (YSR), Adult Self Report (ASR), Child Behaviour Checklist (CBCL), Adult Behaviour Checklist (ABCL). The pre-pandemic coping strategies of both parents and patients were assessed through the New Italian Version of the Coping Orientation to the Problems Experienced questionnaire. The relationship between coping strategies and psychopathological profiles was investigated through correlations, while the change in the patient’s psychopathological profile was observed with repeated measures ANOVA. The predictivity of coping strategies on adaptation during pandemic was analyzed through linear regression’s analysis. Results Patients’ coping strategies are correlated with psychopathological level reported by caregivers pre pandemic. ABCL and CBCL scores reported a significant worsening in patients’ general emotional-behavioral conditions during COVID-19, even though it’s under clinical cut-off; whilst patients didn’t perceive any variation in their well-being status. The change has no causal relationship with the patients’ coping strategies. Conclusions Pre pandemic, parents’ perception of the patient’s psychopathological level depends on the coping strategies used by the subject. Caregivers perceive a worsening of the patient’s psychopathological level during the pandemic, but this is not predicted by the patient’s pre-pandemic coping strategies., We present the case of a 52 years old male patient that came to our attention for widespread myalgia, progressive thinning of muscles and muscle cramps mainly localized at the lower limbs, without significant weakness, beginning two years ago. A positive family history for myotonic dystrophy type 2 (father) was reported. He had also previously performed a brain MRI for recurrent cephalalgia, revealing multiple, large, mainly temporo-occipitally localized subharachnoid cysts along with diffuse leukodystrophyc involvement of both supra and infratentorial white matter. Based on clinical, anamnestic and imaging data, both analysis of expansions of the CCTG repeat in the CNBP gene for DM2 and an NGS analysis panel for leukodystrophies were performed; the patient tested positive for DM2 and a rare variant of unknown significance (allelic frequence 0.00026) associated with autosomal dominant microangiopathy and leukoencephalopathy was identified in the COL4A1 gene (c.2126C > T)., Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by loss of bulbar and spinal motor neurons due to defective production of SMN protein. Nusinersen, able to restore SMN transcription levels, had recently revolutionized the approach to SMA determining motor function improvement and prolonged survival. Here, we retrospectively evaluated quality of life (QoL) in a large multicenter cohort of adult SMA patients during nusinersen treatment. We included adult patients who started nusinersen treatment in adulthood. QoL was rated by the Individualized Neuromuscular Quality of Life (INQoL) questionnaire. Concurrent motor function evaluation included the Hammersmith Functional Motor Scale Expanded (HFMSE), the Revised Upper Limb Module (RULM), the six minutes walking test (6MWT). 189 completed questionnaires were collected during a 14 months’ treatment period. 78 patients were included (7 SMA2 and 69 SMA3 and 2 SMA4) with mean disease duration at first nusinersen administration of 33.2 years (± 12.5 years). All the scores for each INQoL domain (weakness, fatigue, activities, indipendence, social relationship, emotions, body images) and the derived QoL total score, significantly improved during the observation period, except the muscle locking and pain items. Changes in emotions and social relationships were more relevant in females compared to males. Social relationships were affected also by a longer disease duration (> 30 years). In SMA3 non-walker patients, activities ameliorate better compared to walkers. In our cohort, adult SMA patients showed a global improvement at the INQoL assessment over 14 months of nusinersen treatment. QoL assessment is relevant to SMA multidisciplinary evaluation., Background Patients involved in the Expanded Access Program experience with nusinersen in spinal muscular atrophy (SMA) reported variable degrees of changes in the oro-facial district with treatment, but there are no data about facial mobility and general consensus for testing it. Aims to investigate new quantitative non-invasive measures of facial mobility in SMA. Methods We assessed facial mobility through an easy-to-interpret index based on a face tracking algorithm that exploits Facial Action Coding System (FACS) in patients with SMA at rest and while performing 7 specific tasks: frowning, eye closure without exertion, eye closure with exertion, tight-lipped smile, smile, kiss, cheeks inflation. Based on the FACS encoding system, a set of 56 facial landmarks was defined and tracked in the 2D image space per each expression, using a self-developed software (TrackOnField, BBSoF S.r.l.). These landmarks defined 40 distances that were normalized to the corresponding quantities of the neutral expression. Then, a face mobility index (FMI) was calculated per each subject task. Results 62 patients were recruited: 28 females, 34 males; 8 pts SMA1, 26 SMA2, 27 SMA3, 1 SMA4; mean age 27.16 (± 18.62) years old. Preliminary data from 34 patients showed that the task involving a greater FMI was smile (17.97), followed by eye closure with exertion (15.96) as expected. Conclusions Our preliminary data suggest that this may be a potential measure to track possible alterations on facial muscles’ mobility associated to the disease and to investigate the impact of treatments aiming at improving facial mobility., Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive cortical, bulbar and spinal, motor neuron (MN) death, leading to muscle wasting. Although it is generally accepted that ALS is primarily due to MN degeneration, upcoming evidences show an early involvement of skeletal muscles too. Different causative genes associated to ALS are involved in RNA metabolism; among non-coding RNAs, long-non coding RNAs (lncRNAs) are emerging as molecular contributors to ALS pathophysiology because of their role in regulating gene expression. Here, we analyzed the expression levels of HOTTIP, MALAT1 and NEAT1 lncRNAs, known to be involved in the development and homeostasis of the skeletal muscle, in a human induced pluripotent stem cell (hiPSC) model differentiated towards a myogenic destiny through a small molecule-based protocol, obtained from ALS patients and healthy controls. The expression of key markers of skeletal muscle development was analyzed by qPCR. Further, mRNA targets for the lncRNAs were predicted in silico, and validated by qPCR. We reported a differential lncRNA and mRNA target expression pattern in ALS-mutant cell lines compared to controls, particularly at the mesodermal progenitor, early myocyte and myotube stages. Specifically, through hierarchical clustering analysis we identified specific clusters of lncRNAs/mRNA targets characterizing ALS cell lines, suggesting that an altered expression of these molecules might contribute to the disease pathogenesis. These data highlight the role of HOTTIP, MALAT1 and NEAT1 as potential important players in muscle dysregulation in ALS. Thus, they could represent possible targets for new therapeutic strategies., Spinal muscular atrophy (SMA) is an autosomal-recessive motor neuron disease causing progressive muscular atrophy and weakness. Nusinersen, an antisense oligonucleotide able to increase functional SMN protein modifying the SMN2 pre-mRNA splicing, is proven to significantly modify the natural course SMA type 1 in infants and provides benefit even in adult SMA patients. Since fatigue represents an additional dimension of impaired motor function in SMA type 3 patients, hence we aimed to investigate the impact of nusinersen on fatigability (an objective measure of fatigue) through the 6-minute-walking-test (6MWT) in SMA type 3 patients. From a large Italian cohort of patients, we selected 58 ambulant adult SMA type 3 patients being treated with nusinersen and we analyzed data on 6MWT from baseline (T0, beginning of treatment) to 14 months of treatment (T14). Mean age at baseline was 39.6 ± 16 years. No significant reduction in the meters walked from minute 1 to minute 6 was found at T0 (60.6 ± 20.9 vs 56.38 ± 27 m) and T14 (64.0 ± 19.0 vs 54.16 ± 22.8 m). The analysis of variance for repeated measures for the distance walked at every single minute of the 6MWT during follow-up did not show statistically significant differences. Moreover, we found an average trend of improvement of the total 6MWT over a period of 14-month treatment (329.2 ± 129 vs 310.3 ± 136.3 m). In conclusion, although no relevant fatigability was found in our cohort at baseline and no effect of nusinersen on fatigability through the follow-up period, our data broaden the knowledge of nusinersen effect in adult ambulant SMA patients., Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by a progressive degeneration of motor neurons (MNs). A dysregulation of microRNA (miRNA) expression in ALS has been already described, although downstream pathological events associated with MN degeneration have not been clarified yet. miRNAs are highly expressed in central nervous system thus they may play important roles in the etiology or progression of neurodegenerative diseases. In this study, we aimed at investigating whether alteration of miRNAs expression patterns in ALS-MNs may represent a common molecular feature among the different forms of the disease. We performed differential expression profile analysis of miRNAs isolated from ALS-MN and healthy subjects and we identified a small group of downregulated miRNAs in ALS-MNs. Interestingly, a dysregulation of the same subset of miRNAs has been detected in exosomes released from the same ALS-MN cultures. Since bioinformatic analysis showed that these miRNAs regulate several pathways related to MN degeneration, we investigated their potential as disease biomarkers assessing their expression level in cerebrospinal fluid (CSF) of ALS patients. We confirmed a dysregulation of these miRNAs in CSF suggesting their potential clinical relevance. Taken together our results demonstrate that the neurodegenerative phenotype in ALS can be associated with a dysregulation of miRNAs involved in the control of disease-relevant molecular pathways. The possibility of tuning entire gene networks with a specific subset of miRNAs may provide significant insights on the development of effective new miRNA-based therapies and could be useful as disease biomarkers., As an update of our study in 30 adult SMA type 3 patients we tested as potential biomarkers Neurofilament light chain (NfL) and Profilin-1 (PFN-1) during a 26 months follow-up (timepoint M6). PFN-1 is a small actin-binding protein required in both the presynaptic and the postsynaptic compartment with a role in regulating cytoskeletal architecture and dynamics of neurons (Witke, 2004). CSF NfL was tested with an enzyme-linked immunosorbent assay (ELISA) kit (UmanDiagnostics, Sweden). PFN-1 was measured with a commercial manual ELISA kit (Cusabio, China) in CSF samples of 6 patients (3 sitters 3 walkers) at time points L1, L3 and M2 and in every available serum sample from time point L1 to time point M6; 19 serum samples of healthy donors as a control group. Mean NfL at M6 was 381.83 ± 455.71 ng/l, slightly higher than baseline but not significantly different from M3. PFN-1 concentration in all 18 CSF samples was below the lower limit of quantification (< 31.25 ng/l). Serum PFN-1 at baseline was higher in SMA group than in healthy controls (mean 1016 vs 608 ng/l, p = 0.001 Student’s t test). PFN-1 showed a complex dynamic during loading phase, with a significant reduction at L4 compared to baseline. No correlation was found with NfL and motor scores at each time point. PFN-1 as an exploratory cytoskeletal biomarker changed significantly during the first two months of treatment. To our knowledge this is the first report of PFN-1 determination in serum of SMA patients., Hereditary neuropathies represent the most common inherited neurological conditions and show wide clinical and genetic heterogeneity. To date, more than 100 genes have been described to cause Charcot Marie Tooth Disease (CMT) and the related disorders, as distal hereditary motor neuropathy (dHMN) and hereditary sensory neuropathy (HSN). Next generation sequencing (NGS) has speeded up the diagnosis of hereditary diseases and customized targeted NGS panels of disease relevant genes have been developed and used in clinical practice. We describe a 45 years old patient with distal lower limb predominant sensorimotor neuropathy clinically manifested from the age of 30 and characterized by axonal disfunction with associated demyelination. NGS custom panel analysis of 77 genes associated with CMT and related conditions was performed with an Illumina custom Nextera Rapid Capture panel and sequencing on Illumina MiSeq. The analysis revealed in the AARS1 gene (OMIM * 601065) the c.986 G > A (p.Arg329His) heterozygous missense variation, a known and recurrent mutation described worldwide. Segregation analysis through Sanger sequencing in the healthy parents showed the absence of the variation in the father and an ambiguous nucleotide annotation by Sanger in the mother. Indeed the NGS analysis demonstrated in the mother DNA a clear mosaicism for the variant. NGS analysis is very sensitive in detecting mosaicisms, which should be always analysed by family segregation in order to identify the heterozygous parent and to define the risk or recurrence in the family., Biomarkers of disease progression and outcome measures still lack for ALS. Muscle MRI can play a role to track longitudinal changes and to predict response to treatment in clinical trials. We applied quantitative muscle MRI to evaluate disease progression and explore any clinical correlation in ALS. A cohort of newly-diagnosed ALS patients, longitudinally scored using the leg subscores of ALS Functional Rating Scale Revised (ALSFSr), was enrolled. The muscle MRI protocol (6-point Dixon GRE and multi-echo TSE T2w) was implemented for quantification of fat fraction (FF) and water T2 (wT2). Twelve thigh muscles six leg muscles were manually drawn for each side. Eleven age-matched healthy controls were enrolled for comparison. Fifteen patients (M/F 8/7; average age 62.2, range 29-79) diagnosed with possible (n = 2), probable (12) or definite (1) ALS were enrolled (11 spinal onset, 4 initial bulbar envolvement). All patients performed muscle MRI at T0, nine at T1 (6 mo), and seven at T2 (12 mo). At baseline wT2 was significantly elevated in ALS subjects compared to controls for several thigh and mainly for leg muscles; FF was only elevated in a few thigh muscles. wT2 decreased over time in line with worsening in the leg subscores of ALSFRSr (mainly at the leg level and in the anterior and medial compartment of the thigh); FF increased significantly in the leg muscles over time (mainly in the triceps surae). In ALS quantitative muscle MRI represents a non-invasive tool capable of describing the trajectory of pathogenic modifications in the muscle., Background Villa Rosa RehabLab, Trento Italy, aims to design 3D printed personalized Assistive Devices (AD) to facilitate and promote participation through a user-centred co-design process.; direct involvement of the user in the design process ensures a correspondence of the AD to his/her needs, aiming to empower the person in the therapeutic strategy, ensuring the AD’s continuous use and avoiding stigmatization. The use of 3D printer is increasingly popular in the medical world, particularly in rehabilitation and occupational therapy for the manufacture of personalized adaptations and assistive devices. M.A. 58 y.o., diagnosed with radiation-induced brachial plexopathy since 2012, at the initial occupational therapy interview reported difficulty in cutting hard foods, reporting pain when he presses the knife with his left arm. Patient’s quality of performance was observed and self-perception of performance and satisfaction scored using the Canadian Occupational Performance Measure (COPM). Objective Improve quality of performance and decrease pain when performing this task. Methods After ascertaining that no commercial AD was available, an AD dedicated to this function was designed by the user on paper, and afterwards the occupational therapist created a wood prototype. Once the functionality of the prototype was ascertained, the user, guided by the therapist, drew the object with desired shape and sizes using FUSION360, which was then fabricated with the 3D printer. Result The client’s quality of performance improved using the fabricated AD, as did his COPM scores. Conclusions The RehabLab and use of 3D printer can improve independence and participation with individuals with radiation-induced brachial plexopathy., Spinal muscular atrophy (SMA) is a motor neuron disease and the first known genetic cause of infant mortality. Recently approved therapies have shown efficient results only if administrated during the pre-symptomatic phase, that seldom corresponds to the period when the disease is diagnosed in patients. Therefore, the issue of a symptomatic-suitable treatment, efficient across different clinical phenotypes, is strongly present in a clinical perspective. We have already demonstrated that the ASO variant Morpholino (MO) is able to increase the production of a functional SMN protein and rescue the murine phenotype in pre-symptomatic phase, after intracerebroventricular administration. However, this treatment resulted almost ineffective if administered during the symptomatic phase. The conjugation with Cell-Penetrating Peptides (CPPs) may represent a promising therapeutic strategy to address this issue, allowing the MO to overcome the blood-brain barrier by a systemic administration thus expanding the therapeutic window. We investigated the efficacy of CPPs in delivering MO to the central nervous system in symptomatic mice through an intraperitoneal injection, demonstrating that the conjugation with r6 peptide can improve MO biodistribution and increase SMN levels, rescuing the pathological phenotype. Histological examination on SMA treated mice showed a significant increase in the number of motor neurons and innervation of neuromuscular junctions. These data were supported by a striking increase in survival and motor functions, confirming the safety and efficacy of this approach which has never been observed with other compounds in a symptomatic phase of the disease, laying the ground for the development of future clinical trials for SMA., Background While there is general consensus on the motor function scales to be used in muscle strength and functional assessment, little is available for testing of oro-facial muscles and function, yet the pre-swallowing phase of feeding is affected not only in SMA1, but also in the other types of SMA. Aims To investigate strength and function of the oro-facial and brainstem muscles in a cohort of SMA patients treated with nusinersen and in a cohort of naive patients. Methods This is a 20-months observational, multicenter parallel study involving 120 patients with all types of SMA either on treatment or not subjected to lip and tongue strength assessments, swallowing and mastication functional tests and clinical nutritional assessments. Results 140 patients have been enrolled to date. 65%are on treatment while 35% are not (48% SMA2, 31% SMA3, 21% SMA1). 95% of SMA1, 50% of SMA2 and 27% of SMA3 are underweight. 17% of SMA2 and 33% of SMA3 are overweght. IOPI lip and tongue strength (KPa) and mouth opening (cm) was 2.6 and 2 KPa and 2.5 cm for SMA1; 12.9 and 16.2 KPa and 2.6 cm for SMA2; 19 and 42.5 Ka and 4,2 cm for SMA3. SMAHI subscores referring to swallowing and bulbar functions correlated to the nutrional and swallowing abilities of the patients including PEG tubes. Conclusions Our preliminary data suggest that IOPI and SMA-HI swallowing and bulbar subscores may be potential tools to track possible changes on orofacial and orobulbar functions related to the disease itself or to treatment., Nusinersen (Spinraza) is the first drug approved in Italy in 2017 for the treatment of SMA patients. Since its approval, the drug has been administered in several authorized Italian Centers. In the Emilia-Romagna (ER) region we started to treat SMA patients in 3 Centers authorized to prescribe and administer the drug (Bologna, Reggio-Emilia, Parma), and in 2 Centers authorized for administration (Ravenna and Rimini) in collaboration with the Bologna Centre. After the consent of the Ethics Committee we collected data regarding type of SMA, age of onset and of diagnosis in a cohort of 26 patiens treated with Spinraza, in order to study a possible correlation between these variables and the response to treatment. Moreover, procedural aspects were considered: type of hospitalization, type of anesthesia and management of the post-procedural phase. The response to therapy has been periodically measured by clinical evaluations with motor function scales and systematic collection of quality of life improvements. Instrumental investigations (muscle MRI and CMAP evaluation) were performed in collaborating patients before the start of treatment and again during the follow up. Light chain neurofilaments (NFL) levels were also studied in 17 patients. The creation of a regional network among all the Centers made it possible to share the same operative procedure for the treatment with Spinraza, to get real world data on all treated patients and to reduce the burden for the families related to the procedure and to the travel, allowing patients to be treated close to home.

Muscular dystrophy (MD) is a group of neuromuscular diseases characterized by progressive muscle weakness due to various mutations in several genes involved in muscle structure and function. The age at onset, evolution and severity of the different forms of MD can vary and there is often impairment of motor function and activities of daily living. Although there have been important scientific advances with regard to pharmacological therapies for many forms of MD, rehabilitation management remains central to ensuring the patient’s psychophysical well-being. Here we report the results of an Italian consensus conference promoted by UILDM (Unione Italiana Lotta alla Distrofia Muscolare, the Italian Muscular Dystrophy Association) in order to establish general indications and agreed protocols for motor rehabilitation of the different forms of MD.

GMPPB, encoding the guanosine-diphosphate-mannose (GDP-mannose) pyrophosphorylase B protein, has recently been associated with a wide clinical spectrum ranging from severe Walker-Warburg syndrome to pseudo-metabolic myopathy and even congenital myasthenic syndromes. In our previous research, we identified 13 additional cases from 12 families and defined seven novel mutations. In line with data from the literature, patients displayed variable phenotypes and mutations in GMPPB are more common in relatively milder forms of neuromuscular disorders. However, what our study adds to this already broad clinical spectrum is the possible presence of arthrogryposis and congenital clubfoot, particularly in patients with very severe, generalized involvement, as well as nystagmus and upgaze palsy. Ataxia could be part of the clinical picture, in line with possible evidence of cerebellar atrophy. Intellectual disability was evident in all the congenital forms, predominantly affecting the language domain. The onset of motor manifestations in the LGMD group occurred at different ages and the extent of weakness was unrelated to the timing of onset of the disease. Less severe phenotypes are also observed, such as exercise intolerance and myoglobinuria or easy fatigability or asymptomatic hyperCKemia with subtle weakness, evident only on expert clinical examination. We also demonstrated that few mutations recur in the Italian GMPPB-mutated population. Our findings, combined with literature data, show that there are at least three forms of GMPPB-related myopathy: i) CMD, ii) early onset LGMD, and iii) adult onset LGMD, often with evidence of neuromuscular junction involvement., Duchenne muscular dystrophy (DMD) is not only the most common single gene disorder leading to muscle wasting, but several studies have reported cognitive difficulties and neuropsychological alterations in these patients: in fact, a possible non-progressive cognitive impairment with worse performances in verbal than in nonverbal domain has been described both in older and younger patients. Furthermore, DMD children often show a variable language involvement and suffer from deficits in executive functions, with a possible negative impact on academic skills. The lack of specific dystrophin isoforms in the brain could be related to this impairment, taking account of the involvement of cerebellum as part of a more general involvement of the cerebellar-thalamo-cortical network. Recently, this hypothesis has been supported by the results obtained in a multicenter study on a cohort of DMD boys without intellectual disability during school age. The specific selection of the DMD sample has allowed a targeted neurocognitive detection free from potential bias, characterized by an impairment of multitasking, problem solving, inhibition and working memory and an implicit learning deficit. (Battini R et al, 2018; Vicari S. et al, 2018). In the era in which the life expectancy of DMD has increased, the DMD boys, especially those without intellectual disability, are a particularly vulnerable population and a prompt recognition of neuropsychological impairments is important in order to plan early specific treatments and to avoid an high impact on daily living with results both on academic and adaptive functioning., Limb Girdle Muscular Dystrophies (LGMDs) are a genetically and clinically heterogeneous group of rare neuromuscular conditions, comprising more than 30 causative autosomal loci and a multiplicity of complex and overlapping muscular and extra-muscular phenotypes. The “greatest common denominator” of this nosographic group is the association of some degree of proximal muscle weakness, with myopathic or overtly dystrophic muscle histology. Advances in genomics and muscle imaging have increased our abilities to diagnose these conditions; but effective therapies are still lagging behind. As the first molecular and gene therapies are being approved for more common neuromuscular diseases, such as dystrophinopathies and spinal muscular atrophy, several of these conditions emerge as potential, sometimes even ideal candidates for homologous approaches. The main hurdles, along the path to effectively bringing these treatments to approval and to the clinic, lie in scarce clinical trial readiness because of partial knowledge about natural history, lack of validated and standardized outcome measures, and difficulty in recruiting large and homogeneous groups of patients. We will review chances and challenges currently faced by clinicians, researchers, and patients. In particular, we will focus on autosomal recessive forms, which are usually determined by the lack of a specific protein which future treatments will aim at re-expressing in muscle fibers., Nusinersen is the first approved treatment for spinal muscular atrophy (SMA). Interim results from the ongoing NURTURE study (NCT02386553) examining efficacy/safety of intrathecal nusinersen, initiated prior to symptom onset, in infants with 2 or 3 SMN2 copies will be presented. The study enrolled 25 infants with age ≤ 6 weeks at first dose, clinically presymptomatic, and genetically diagnosed with SMA. Primary endpoint was time to death or respiratory intervention (≥ 6 hours/day continuously for ≥ 7 days or tracheostomy). As of 15 May 2018, infants (2 copies SMN2, n = 15; 3 copies, n = 10) were enrolled. Median age at last visit was 26.0 (range 14.0-34.3) months. All infants were alive and none required permanent ventilation. Four infants (all with 2 SMN2 copies) required respiratory intervention for ≥ 6 hours/day continuously for ≥ 7 days, with all cases initiated during acute, reversible illness. All infants achieved the WHO motor milestone sitting without support and 22/25 (88%) achieved walking with assistance; 17/22 (77%) were walking alone. Phosphorylated neurofilament heavy chain levels rapidly declined during the nusinersen loading phase and then stabilized. AEs occurred in all infants; 20/25 had AEs mild/moderate in severity; 9 had SAEs. No new safety concerns were identified. Results from 15 May 2018 interim analysis, will be presented. These findings demonstrate there was continued benefit to infants who initiated nusinersen before symptom onset, emphasizing the value of early treatment and newborn screening. Updated analyses will provide further information., Spinal muscular atrophy (SMA) is a motor neuron (MN) disorder caused by mutations in Survival Motor Neuron 1 (SMN1) gene. The role of MNs vulnerability is clearly established. The involvement of other cell types in the Central Nervous System (CNS), such as astrocytes, microglia and sensory neurons, as well as outside the CNS, such as Schwann cells, muscle cells or heart, may play a role in disease initiation and/or progression, as well as in the emergence of clinical symptoms. Heart alterations have been reported in the most severe forms of SMA; caused either by congenital anomalies manifesting during cardiogenesis, or secondary to autonomic nervous system defects, and/or to respiratory dysfunction. Metabolic defects such as fasting hyperglycemia, glucose intolerance, hypersensitivity to insulin, and hyperglucagonemia have been reported. The body of evidence derived from experimental models and patients’ experience will be analyzed to weight its relevance to the current therapeutic scenario., Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by the degeneration of motor neurons leading to progressive muscle weakness and atrophy. It is caused by mutations in the SMN1 gene. Recently, the first SMA therapy based on antisense oligonucleotides, namely nusinersen, that modulates the splicing of the paralogous gene SMN2, has been approved. Moreover, the first gene therapy clinical trial using adeno-associated virus (AAV) vectors encoding SMN showed positive results in patients’ survival and motor milestones achievement. Based on these data, phase III gene therapy trials are ongoing in USA and Europe. Nevertheless, it is likely that in SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong, in particular if the patients are treated in symptomatic stages. To identify novel SMN independent therapeutic targets we recently analyzed SMA patient specific induced pluripotent stem cells (iPSC) 2/3D disease models. RNA sequencing on human SMA iPSC derived motor neurons revealed many deregulated genes, such as the neurexin and synaptotagmin families, that are implicated in critical motor neuron functions. Motif-enrichment analyses of differentially expressed/spliced genes pointed out a common motif, motif 7, which is a target of SYNCRIP. Remarkably, SYNCRIP binds only with full-length SMN, modulating several downstream motor neuron transcripts, including SMN itself. We demonstrated that SYNCRIP overexpression rescued SMA motor neurons, due to the subsequent increase in SMN and their downstream target NRXN2 and ameliorated SMN-loss-related pathological phenotypes in animal models. Thus SMN/SYNCRIP complex may represent a novel potential therapeutic target for SMA., Expanded newborn screening (ENBS) by LC-MS/MS can detect inborn errors of metabolism life-threatening in a presymptomatic phase. In 2004, with a regional law, Tuscany was the first Italian Region to screen for 40 inborn errors of metabolism including fatty acid beta-oxidation defects (FAOD). With the law 167/2016 in Italy the ENBS is part of essential levels of assistance. In Tuscany, after a 3-years pilot project, lysosomal storage disease (MPSI, Pompe and Fabry disease) are included in the panel with a regional law. FAOD may occur with neuromuscular involvement both in early and late onset. Through ENBS we have diagnosed carnitine primary, VLCAD and LCHAD defects. In some case newborns showed iperCKemia or cardiomyopathy at the recall; in one case SIDS occurred before the ENBS results. It is important the chronic management of these patients with a low-fat diet and avoidance fasting. CPT2 deficiency usually presents with rhabdomyolysis starting in adolescents and young adults; in ENBS, the (C16 + C18:1)/C2 ratio is altered and the enzymatic assays and molecular analysis confirmed the diagnosis, but we keep in mind that ENBS can be negative. Since 2014 we screened 80.000 newborns for Pompe disease with a diagnosis of one newborn affected by cardiomyopathy in which ERT was started a one month of age. We have diagnosed 9 patients with late onset form, actually in follow up. In Tuscany and Lazio a pilot project for spinal muscular atrophy NBS will start, since the recent availability of new treatment strategies can change the disease course., In the past few years, there have been several advances in the management of myasthenia gravis (MG). In patient with generalized anti-acetylcholine receptor positive MG (AChR-MG), thymectomy not only proved superior to conservative therapy, but appears to confer prolonged benefits. Increased availability of biologics provides targeted immunotherapies. Eculizumab, that inhibits complement activation, proved effective in refractory AChR-MG in a phase III trial. B cell depletion with rituximab is thought to be the most effective therapy for MG with anti-muscle specific tyrosine kinase antibodies, although a phase II trial in AChR-MG failed to meet the primary outcome. Increase of IgG clearance through blocking the neonatal Fc receptor (FcRn), is a new therapeutic tool in MG. Efgartigimod, an IgG1-derived Fc fragment, significantly improved AChR-MG and depleted specific antibodies; the anti-FcRn rozanolixizumab is currently under study. Belimumab, which targets B-cell activating factor (BAFF), was not superior to placebo in a phase II trial. Tolicizumab (IL-6 receptor antagonist), ruxolitinib (janus kinase inhibitor) and bortezomib improved MG in single case reports. A more extensive usage of new immunotherapies will require a careful patient selection, surveillance of potential side effects and cost/benefit appraisal., Neuromuscular diseases (NMDs) have been privileged by intense genetic characterization via pioneering and fruitful collaboration between neurologists and geneticists; genetic diagnosis of NMDs is now an integral part of the diagnostic flowchart, mandatory for eligibility to treatments. Next-generation sequencing (NGS) is increasingly being applied to NMD testing, leading to a remarkable amelioration of genetic diagnosis, via new genes or new phenotypes discovery; nevertheless its clinical translation is incomplete and standard molecular genetics tests are still needed. Indeed, NGS cannot always ensure an exhaustive mutation detection, since still some mutation types (as copy number variations and dynamic mutations) escape its identification. Since NGS allows high parallelism and throughput runs, several samples can (and should) be contemporary analyzed to cover the run costs. For some ultra-rare NMDs this might not be ideal and these conditions may have consequently a delayed diagnosis. Prenatal diagnosis is still mainly based on classical molecular testing, due to the pregnancy timing. DMD (or dystrophinopathies, including Duchenne and Becker muscular dystrophies, X-linked dilated cardiomyopathy, and other milder phenotypes, as quadriceps myopathy or isolated high CK) can benefit of standard diagnostic genetic tests based on CNV detection (typically MLPA ) and exon sequencing (typically by Sanger or NGS methods). Using these two strategies a genetic diagnosis of dystrophinopathies is achieved in the vast majority of patients, with a detection rate close to 99%. A few atypical mutations or unsolved cases do exist requiring a deep characterization based on RNA or genome profiling. Ideally, it would be greatly desirable to have a single genetic method able to detect all possible occurring mutation types in DMD (and more generally NMD) gene. Finally, a reflection can be made about the powerfulness of the new non-invasive prenatal diagnostic testing (NIPT), from one side, and the new personalized therapies now available, from the other. Finding synergic strategies for these two interventions in DMD/NMDs would be greatly beneficial for patients and families., Several morphological phenotypes have been associated to RYR1-recessive myopathies. We recharacterized the RYR1-recessive morphological spectrum by a large monocentric study performed on 54 muscle biopsies from a large cohort of 48 genetically confirmed patients, using histoenzymology, immunohistochemistry, and ultrastructural studies. We also analysed the level of RyR1 expression in patients’ muscle biopsies. We defined “dusty cores” the irregular areas of myofibrillar disorganisation characterised by a reddish-purple granular material deposition with uneven oxidative stain and devoid of ATPase activity, which represent the characteristic lesion in muscle biopsy in 54% of patients. We named Dusty Core Disease (DuCD) the corresponding entity of congenital myopathy. Dusty cores had peculiar histological and ultrastructural characteristics compared to the other core diseases. DuCD muscle biopsies also showed nuclear centralization and type1 fibre predominance. Dusty cores were not observed in other core myopathies and centronuclear myopathies. The other morphological groups in our cohort of patients were: Central Core (CCD: 21%), Core-Rod (C&R:15%) and Type1 predominance “plus” (T1P+:10%). DuCD group was associated to an earlier disease onset, a more severe clinical phenotype and a lowest level of RyR1 expression in muscle, compared to the other groups. Variants located in the bridge solenoid and the pore domains were more frequent in DuCD patients. In conclusion, DuCD is the most frequent histopathological presentation of RYR1-recessive myopathies. Dusty cores represent the unifying morphological lesion among the DuCD pathology spectrum and are the morphological hallmark for the recessive form of disease., The clinical demand for appropriate and precise clinical recommendations in a rehabilitative program is a daily request in management of patients with muscular dystrophies (MD) and related disorders. Internationally validated guidelines are only available for Duchenne muscular dystrophy and few additional congenital forms but it is unclear if these can easily be applied to other forms of MD. Based on these issues we revised pertinent literature contributions related to neuromuscular rehabilitation in MDs, with the final goal to draw up a largely agreed document of practical recommendations through a consensus conference methodology. This is critical for both clinicians, offering a rapid tool to counsel patients, and patients themselves, needing assurance they are receiving the best care at the best time of their disease story. The document was committed by Unione Italiana Lotta alla Distrofia Muscolare (UILDM), an Italian association of patients suffering from neuromuscular diseases, and has the purpose to provide technical, updated and detailed directions to clinicians, patients and caregivers for rehabilitation in MDs both in childhood and in adult age. It will be based on the major literatures evidences and expert opinions, will include specific definition of rules and responsibilities of the professional figures involved in the rehabilitation program, and will offer specific information on frequency, intensity, Time and Type (F.I.T.T) of physical activity. Furthermore, it will detail practical measures to manage contractures, mobility and daily activity life. We anticipate that the Italian consensus will represent a base for official standardized guidelines and a new scenario of the patient-doctor alliance., Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders affecting the neuromuscular junction (NMJ) and clinically characterized by non-progressive fluctuating muscle weakness and fatigability. CMS diagnosis requires a high clinical suspicion, because they are rare diseases and myopathic features are often more evident than myasthenic signs. To date, more than 30 genes, encoding for pre-synaptic, synaptic and post-synaptic NMJ proteins, have been implicated in CMS, which are recessively inherited in most of the cases. CMS usually present at birth or within the first 3 years of life, although around 20% of the patients display later onset, including adult age. Mutations are identified in only about 50% of CMS patients, but many novel genes have been recently discovered, most of them codifying for proteins localized at presynaptic level, as synaptobrevin-1, Munc 13-1, synaptogamin-2, laminin α5, vesicular acetylcholine transporter, synaptosomal-associated protein 25, high affinity choline transporter solute carrier family 5 member 7, myosin 9a, and rabphilin 3A. Mutations in these genes cause CMS usually presenting at birth and often in association with involvement of central nervous system, mainly showing intellectual disability, seizures or brain malformations/atrophy. CMS treatment is based on different symptomatic therapies, which could markedly improve symptoms over the time. Interestingly, drugs with positive effects on specific CMS, may worsen other CMS forms. Hence, definite genetic diagnosis is highly recommended to optimize pharmacologic treatment., Duchenne muscular dystrophy (DMD) is a progressive X-linked degenerative muscle disease due to mutations in the DMD gene. Improvements in the standard of care for DMD have led to improved survival. Nowadays the only treatments obtainable in the clinical arena for DMD are corticosteroids and the drug Ataluren in EU and also Exondys 51 in US. Nevertheless, several other therapeutic options are currently tested in clinical trials. Novel treatments for DMD have focused on reducing the dystrophic mechanism of the muscle disease, modulating utrophin protein expression, and restoring dystrophin protein expression. Among the strategies to reduce the dystrophic mechanisms are: 1) inhibiting inflammation, 2) promoting muscle growth and regeneration, 3) reducing fibrosis, and 4) facilitating mitochondrial function. The agents under investigation include a novel steroid, myostatin inhibitors, idebenone, an anti-CTGF antibody, a histone deacetylase inhibitor, and cardiosphere-derived cells. For utrophin modulation, AAV-mediated gene therapy with GALGT2 is currently being investigated to upregulate utrophin expression. Finally, the strategies for dystrophin protein restoration include 1) nonsense readthrough, 2) synthetic antisense oligonucleotides for exon skipping, and 3) AAV-mediated micro/minidystrophin gene delivery. With newer agents, we are witnessing the use of more advanced biotechnological methods. These potential breakthroughs provide significant promise., The most common form of hereditary IBM is due to mutations of the GNE gene that is involved in sialic acid (SA) biosynthesis. More than 170 different GNE mutation have been characterized to date. The cellular pathogenic mechanism/s of GNE myopathy are mostly undefined but abnormal sialylation and processing of proteins may have a critical role. Most of the evidence point out to GNE myopathy being a disease of deranged proteostasis with increased protein transcription, abnormal post-translational modifications and inadequate disposal rather than based solely on the impairment of a specific molecular player. The identification of GNE gene defect has allowed recognition of phenotypic variants although only about 20% of variability of clinical features can be explained by GNE mutations (p.D207V mutation associated with later onset of symptoms, p.V603L with an earlier onset). Therefore other epigenetic factors may modulate the clinical phenotype accounting also for heterogeneous rate of progression. Since early restoration of a normal sialylation status within cellular environment may help to recover muscle fibers homeostasis, an extended release formulation of SA to achieve a stably elevated serum level with significant muscle uptake was developed to be used in a phase 3 clinical trial. This was the largest clinical trial in GNE myopathy patients and demonstrated that SA is not superior to placebo in improving muscle strength. The study provided novel viewpoint on conducting trials in patients with distal myopathies as understanding differences between patients with GNE myopathy will help to develop better outcome measures sensitive for early detection of therapy efficacy. Oral treatment with ManNAc, a precursor in the SA biosynthetic pathway that prevented muscle weakness in a murine model of GNE myopathy, is now being studied in an open-label phase 2 study. Gene therapy is also being explored to treat GNE myopathy., Although Pompe disease has been considered for a long time a primary muscle disorder, it is becoming evident that the pathophysiological mechanism underlying the disease involves several organs and tissues such as central nervous system. Similarly to other lysosomal storage disorders (i.e. Fabry disease), in the late onset form of Pompe disease (LOPD), vascular abnormalities have been reported with a prominent involvement of the cerebrovascular system presenting with aneurysms, vertebrobasilar dolichoectasia (VBD) or dilative arteriopathy. The recurrence of these vascular anomalies is higher than in the general population. In a previous study we demonstrated the presence of unruptured intracranial aneurysms in 9.5% of examined patients as well as an elevated frequency (47%) of VBD. In a recent study we evaluated CNS involvement performing a comprehensive investigation on morphological and functional brain areas, demonstrating characteristic signs of a small vessels disease (lacunar encephalopathy) in a substantial number of LOPD patients The impact of brain damage on cognitive functions in LOPD has been evaluate in few studies revealing a moderate involvement of cognitive ability with a prevalent impairment in visual-constructive activities and executive functions. A possible physiopathological explanation is that glycogen accumulation in the vessel walls induced a dilatative arteriopathy with dolichoectasia that could be responsible for cerebrovascular alterations leading to multiple ischemic insults., Spinal muscular atrophy (SMA), is an autosomal recessive disorder caused by mutations in the survival motor neuron (SMN1) gene. In the last few years a number of therapeutic approaches have been proposed for SMA and some of them have completed phase 2 and 3 clinical trials The clinical efficacy of Nusinersen, an antisense oligonucleotide designed to increase full-length SMN protein levels, had been initially suggested by phase 2 trials in infants with infantile-onset SMA and subsequently by a phase 3 sham-controlled studies showing increased survival and significant improvements on motor functional scales in infants receiving Nusinersen compared to sham controls. The aim of EAP was to report twelve month changes after treatment with Nusinersen in a cohort of 85 type I Spinal Muscular Atrophy patients. There was a difference between baseline and the 12 month scores on both the CHOP INTEND and the HINE for the whole group (p < 0.001) as well as for the subgroups with 2 SMN2 copies (p < 0.001), and for those with 3 SMN2 copies (p < 0.001). The difference was found not only in patients younger than 210 days at baseline (p < 0.001) but also in those younger than 5 years on the CHOP INTEND and younger than 2 years on the HINE. Our results, expanding the age range and the severity of type I patients treated with Nusinersen over one year, provide additional data on the range of efficacy of the drug that will be helpful to make an informed decision on whether to start treatment in patients of different age and severity., Magnetic resonance imaging (MRI) has progressively become an important tool for the diagnosis and monitoring of neuromuscular diseases, with the ability to display the severity and distribution of pathology, to identify involvement patterns and, sometimes, to even suggest the diagnosis in certain difficult cases where other data are unequivocal. The advances in imaging techniques have importantly expanded the potential to assess the ongoing pathology in the skeletal muscle, going beyond the knowledge provided by conventional imaging. With the so called quantitative MRI (qMRI), in fact, a number of specific characteristics can today be studied and quantified, ranging from tissue composition, architecture, mechanical properties and perfusion, only to cite a few. The application in clinical practice of such advanced quantitative techniques is already leading to a further evolution in the approach to neuromuscular diseases, even considering a number of technical difficulties. Above all, the possibility to precisely track the changes of specific features of the underlying pathology in the muscle (i.e. edema, fat replacement, alteration of metabolism, diffusivity properties etc.) appears of particular interest to define and project clinical trials in this field. The presentation will particularly focus on what qMRI can effectively provide to the clinician and how it can be implemented not only as a diagnostic tool but also as a promising tracker of disease progression and of response to therapy, when available., Duchenne muscular dystrophy (DMD) is devastating lethal neuromuscular disease due to loss-of-function mutations in the DMD gene leading to a complete dystrophin deficiency in skeletal muscle. Recent years have seen a reinassance of therapeutical approaches, including the partial correction of the underlying gene defect or the treatment of its downstream consequences. The first molecular treatment for DMD approved in Italy is a small molecule (ataluren) able to read through stop codons, allowing the production of functional dystrophin in nonsense mutation DMD. This treatment has been shown to be safe and results in a significant functional improvement in patients. Antisense oligonucleotides (AO) targeted to restore the DMD frame in single or multiple exon deletion are in advanced clinical phase. Deletions amenable to exon 51 skipping are treated with eteplirsen, a morpholino antisense oligomer, which triggers excision of exon 51 during splicing, allowing for the synthesis of an internally deleted, but partially functional dystrophin. Eteplirsen has been approved by FDA but not yet by the European Medicines Agency. Other chemistries for AOs are in advanced developmental phase with the goal to enhance delivery and efficiency. Systemic adeno-associated virus (AAV)-based gene therapy for DMD is currently into phase I-II clinical trial. A rAAVrh74.MHCK7-micro-dystrophin to achieve targeted skeletal and cardiac muscle expression of a shortened functional dystrophin protein has been administered to 4 DMD boys. Preliminary results showed lack of serious adverse events and good dystrophin re-expression. The therapeutical scenario for DMD is rapidly changing including disease-modifying therapies and personalized genetic therapies., Lipids are essential for the structural and functional maintenance of cell. Some disorders of lipid metabolism may produce prevalent damage in skeletal and cardiac muscle. The spectrum of lipid myopathies (LM) is expanding with the knowledge of new molecules involved in fatty acid metabolism. Most of the LM are caused by a gene defect, rarely by a toxic cause or a deficiency in the diet. Several classifications of the LM have been proposed based on clinical or morphological features, but a classification based on genetic defect seems more complete. Clinically, LM result in acute or indolent clinical pictures, in all ages, sometime life-threatening. The muscular symptoms consist in weakness and fatigability, they can be fluctuant or fixed, rarely with muscle atrophy. When muscular symptoms are acute, occur with massive muscle necrosis (rhabdomyolysis and myoglobinuria) and are triggered by metabolic stress, as fasting, maximal exercise, infections. Muscle biopsy can show excess of lipid, but normal findings do not rule out a lipid myopathy, because some of these diseases do not causes excessive lipid storage. Acylcarnitines profile and urinary organic acids dosage can help in the diagnostic work-up of lipid myopathies before going to the genetic confirmation. Prevention of triggers should be recommended in all LM regardless of the gene involved. Basic life-saving procedures must be adopted in case of suspected LM with rhabdomyolysis. In other condition administration of deficient substances is sufficient to resolve symptoms. New molecules are been tested for the treatment in other cases., Cardiac disease is a common clinical manifestation of several neuromuscular disorders (NMDs), most notably the muscular dystrophies (MD). In recent years, cardiac involvement has been observed in a growing number of genetic muscle diseases, and considerable progress has been made in understanding the relationships between disease skeletal muscle and cardiac muscle disease. In several forms of MD, cardiac disease may even be the predominant manifestation of the underlying genetic myopathy and precede of many years the onset of skeletal muscle involvement. Heart involvement may result from pathologic changes in the myocardium and/or the cardiac conduction system. Involvement of the myocardium manifests most frequently as a cardiomyopathy evolving to the final stage of dilation and systolic dysfunction. Cardiac conduction defects, supraventricular and ventricular arrhythmias are also common cardiac manifestations of NMDs. Arrhythmias may evolve into life-threatening ventricular tachycardias, asystole, or even sudden cardiac death. Cardiac involvement carries great prognostic significance on the outcome of dystrophinopathies, laminopathies, desminopathies, nemaline myopathy, myotonic dystrophies, metabolic myopathies, Danon disease, and Barth-syndrome. However an increasing number of genes that cause muscle diseases, are continuously reported as capable of causing even cardiac involvement. The spectrum of cardiac dysfunction in these inherited muscle disorders will be presented and practical recommendations for their monitoring and management proposed. An early detection of MD-associated cardiomyopathy is of considerable importance, as a prompt institution of cardio-protective medical or supportive therapies may slow adverse cardiac remodeling and attenuate heart failure symptoms or avoid the occurrence of sudden cardiac death in these patients., As Enzyme Replacement Therapy (ERT) enables patients with classic infantile onset Pompe disease (IOPD) to reach adulthood, white-matter abnormalities are becoming increasingly evident at neuroimaging, affecting the neuropsychological development. Previously published studies in children with IOPD showed IQ (Intelligence Quotient) scores in the lower normal range without any evidence of cognitive decay over time, but recent studies in ERT treated children showed wider cognitive development range from stable and normal to declines that lead to intellectual disabilities. In our cohort, we tested 6 patients, all of them in the normal range (IQ between 85 and 121 at baseline). In two of them, a slight decline of IQ was observed after two years, but still in the normal range. These important observations need programs to capture central nervous system (CNS) involvement in larger patient cohorts, including late-surviving IOPD patients. A longitudinal multicentric study for comprehensive CNS functions evaluation in Italian children affected by Pompe disease is proposed, to collect longitudinal data with a standardized protocol, exploring cognitive features, speech and hearing functions, behavioural data, and neuroradiological features, that will be compared to motor functions; as an innovative, relevant observation, this protocol will also investigate the children adaptive behaviour, assistance needs and quality of life. Current knowledge on CNS involvement in IOPD should be included in the counselling of parents before the start of treatment, and the brain should be considered as an additional target in the development of next-generation therapeutic strategies., At present molecular diagnostics in genetic diseases is facing several challenges. Genomic investigations in human diseases are easily accessible, but the relationship between observed phenotypes and their underlying genotypes, modes of transmission and frequencies of diseases and variants maybe of difficult interpretation. Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular diseases. The clinical and molecular complexity has complicated FSHD diagnosis. The disease first presents with weakness of the facial and shoulder girdle muscles, followed by the ankle dorsiflexors and finally the proximal leg muscles. However, there are many patients who do not fit this well-known classical FSHD phenotype. Infantile and late-onset cases are not uncommon; clinical severity and sequence of involvement in different muscle groups can vary. Presently, two genetically distinct disease subtypes, FSHD1 and FSHD2, have been described. FSHD1 is associated with contractions of a polymorphic macrosatellite repeat on chromosome 4q35.2. FSHD2 defines a smaller number of affected individuals carrying two D4Z4 arrays in the healthy range. FSHD1 and FSHD2, considered clinically undistinguishable, are characterized by DNA hypomethylation of the 4q35 D4Z4 array. The phenotypic and genetic classification of patients and families will be crucial to define the natural history of disease, to propose suitable measure of outcome and to identify new susceptibility/causative factors contributing to FSHD., Lambert-Eaton myasthenic syndrome (LEMS) is a very rare pre-synaptic disorder of the neuromuscular and autonomic transmission. It is caused by antibodies interacting with the voltage gated calcium channels (VGCCs) of P/Q-, N- and R-type. Over 50% of LEMS patients have an underlying tumour, most often small-cell lung cancer (SCLC). VGCC antibodies are more frequently detected in LEMS patients with SCLC, being present in up to 100% when compared to LEMS patients without underlying cancer. However the molecular mechanisms of LEMS remains largely unknown a careful screening for the early detection of the possible associated cancer is a crucial step. Almost all patients will benefit initially from symptomatic treatment with 3,4-diaminopyridine (3,4-DAP), a potassium channel blocking agent. Often, additional treatment is required in the form of immunosuppression. Prednisolone has been shown to be effective from observational studies; to date there are no data to suggest that intravenous immunoglobulin (IVIg) infusion is an effective long-term treatment for LEMS symptoms. Plasma exchange has been used effectively as acute treatment of severe LEMS symptoms. In patients with symptoms refractory to oral immunosuppression, novel treatment approaches may be beneficial, such as the anti-CD20 monoclonal antibody agent, Rituximab., Muscle weakness and floppiness in infancy are the most common symptoms of neuromuscular disease and may result from a large set of genetic aetiologies leading to muscle dysfunction or neuronal and neuromuscular junction abnormalities. To date, more than 780 monogenic neuromuscular diseases, linked to over 4oo different genes, have been identified in humans and therapeutic opportunities have been proposed for few. Genome-editing methods, especially the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system, hold clinical potential for curing many monogenic disorders, including NMD such as Duchenne muscular dystrophy, and myotonic dystrophy type 1. Importantly, lack of curative treatments available for most neuromuscular disorders (NMD) is in part due to the lack of in vivo models that can be utilized in high-throughput approaches for discovery of effective and personalized therapeutic options. We will review current bottlenecks in making CRISPR-Cas9-mediated gene editing a therapeutic reality, show new success obtained in Duchenne muscular dystrophy, and we will outline recent strategies that implement disease modelling and open a new path to personalized therapies., Oculopharyngeal muscular dystrophy (OPMD) is a late-onset degenerative disorder clinically characterized by ptosis, dysphagia, axial and proximal limb muscles weakness. Swallowing difficulties mainly determine bad prognosis, increasing the risk for aspiration pneumonia and poor nutrition. In the majority of cases the disease shows an autosomal dominant inheritance. OPMD is caused by mutations in the poly(A)-binding protein nuclear-1 (PABPN1) gene, resulting in a short GCG expansion in the polyalanine tract of PABPN1 protein. Expression of PABPN1 appears to be ubiquitous but symptoms in OPMD are limited to skeletal muscles. At skeletal muscle level, the pathological hallmark of OPMD is the accumulation of filamentous intranuclear inclusions detecting by electron microscopy, as well as the presence of muscle fibers with rimmed vacuoles. Symptomatic treatment is gradually introduced during the progression of disease, while no pharmacological treatment is presently available. In vitro and in vivo disease models are described and novel gene and cell therapies are currently under study., Facioscapulohumeral muscular dystrophy (FSHD) is caused by a unique, non-conventional genetic mechanism, whose downstream pathophysiology is still largely elusive. Using MRI to characterize muscle involvement and follow patients up, a picture has emerged in which sequential bursts of degeneration involve individual muscles in an asynchronous manner. This peculiar radiological progression is in line with results obtained with multidisciplinary approaches, thus configuring FSHD as a “muscle by muscle” disease. In this context, a common feature of FSHD is the presence of areas of hypersignal on STIR (short-tau inversion recovery) sequences, which represent areas of muscle edema/inflammation. Imaging and molecular evidences point toward the fact that these STIR+ lesions mark a different phase of disease at single muscle level. Consequently, the detection of these abnormalities is important to monitor disease evolution. Even in the frame of this highly peculiar disease progression, which contributes to the pronounced clinical variability and atypical presentations, still muscle imaging is able to identify muscles that are more likely to be involved, and others that are selectively spared. Reasons of this different susceptibility are still unknown and definitely represent an intriguing field of research. Relevant for clinical trials, muscle imaging can be therefore useful for choosing the patients who are in an “active” phase of the disease, as well as for correctly stratifying patients, and accurately following muscle involvement over time. Longitudinal, large cohort imaging studies using both standard and quantitative MRI are definitely needed to move forward in our understanding of FSHD natural history and pathophysiology., Muscle glycogenoses are a heterogeneous group of rare disorders where skeletal muscle is primarily compromised but even other organs are often involved. These disorders are relatively rare and may show a wide range of symptoms at infancy, childhood or adulthood. More recently, increased awareness, detailed characterization of the clinical spectrum and improved diagnostic workup have made easier to recognize these clinical entities although this is still a challenge either in the infantile or in the adult cases Innovative diagnostic techniques such as use of newborn screening, Dried Blood Spot (DBS), different biochemical approaches or molecular genetic methods as NGS (Next Generation Sequencing) or whole exome/genome sequencing, are currently considered to better evaluate either known or emerging clinical entities in the field of muscle glycogenoses Nowadays, it is important to update the evaluation of these disorders, also taking into consideration the main pathogenic mechanisms, mainly involving skeletal muscle dysfunction but also other organs or apparatuses. In fact, reaching as early as possible the diagnosis, will allow physicians to early apply a specific therapy where it is already available in an attempt to limit progressive degeneration of organs., Clinical reports show high phenotypic heterogeneity in carriers of D4Z4 reduced alleles (DRA) ranging from healthy carriers to typical FSHD to complex muscular phenotypes. 3% of healthy people carry one DRA. The Italian Clinical Network for FSHD applied the CCEF, describing nine clinical categories, on 1703 individuals, 846 index cases and 857 relatives carrying one contracted alleles with 1-10 D4Z4 repeats. Subjects’ stratification based on D4Z4 allele size shows that clinical categories are distributed in all groups. Disease penetrance is reduced with 44.3% of healthy relatives. The age at evaluation of healthy relatives is not significantly different from that of relatives who developed the full disease. The facial-sparing FSHD phenotype represents 10,2% of index cases regardless the size of the D4Z4 contracted allele. Our study show that standardized clinical evaluation can facilitate the comprehension of phenotypic diversity among carriers of D4Z4 contracted alleles and guide clinical studies and research., The first titinopathy was reported by clinical delineation long before it was known to be a titinopathy. This dominant late onset distal myopathy in Finland was reported as Tibial muscular dystrophy and after the Titin (TTN) gene mutation identification in 2002, together with the childhood onset LGMD R10 (2J) in the homozygotes, only hereditary myopathy with early respiratory failure (HMERF) was associated with Titin mutations before the era of NGS. After now 7 years of using exome sequencing and NGS gene panels the variety of the phenotypes caused by TTN mutations has exploded covering currently more than 10 different disease phenotypes. The easier part of confirming TTN variants as pathogenic are the deleterious truncating variants: nonsense, frameshifts and clarified splice site mutations. Thus congenital and severe childhood diseases with biallelic deleterious mutations have been extensively reported and covered. However, there are also congenital forms without progressive loss of muscle tissue due to mutations in exons expressed in fetal development but not after birth. The big unsolved problem with variants is still how to categorize missense variants because predictive tools for pathogenicity are misleading and the gene cannot be expressed in model systems. Thus only few missense mutations have confirmed pathogenicity, above all the missenses in exon 344 causing HMERF disease, but also missenses in the last exon 364 causing distal myopathy and some missenses with confirmed pathogenicity in compound heterozygosity with known mutations. The spectrum of titinopathies now covers almost everything from fetal lethality to almost asymptomativ very late onset distal ankle weakness after age 65., Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disorder in which the classical phenotype is characterized by a progressive muscular weakness, involving facial, shoulder and upper arms muscle; the molecular feature is a contraction in D4Z4 repeat element on chromosome 4. Currently, approved treatments are unknown and disease natural history is missing and it is crucial defining modifying outcome measure in prevision of future therapeutic clinical trials. The main aim of our study is evaluating disease progression within a 5-years time period in a cohort of 246 subjects from the Italian National Registry for FSHD. Patients, all genetically confirmed, were underwent to the same protocol including clinical evaluation using FSHD Clinical Score for functional impairment, Medical Research Council (MRC) for muscle strength examination and in the second evaluation the CCEF (Comprehensive Clinical Evaluation Form), a recent published tool in which FSHD patients are divided in different subcategories according to the affected districts. Study population was divided in index case and relatives and we extrapolated the Delta FSHD Score (ΔFSHD score) between first and second control. In our follow-up study 141 index cases and 105 relatives were admitted. 38.2% of subjects did not show progression of disability; 64.9% with mild or no disability (FSHD scores 0-2) at baseline maintained the same FSHD score; a baseline FSHD score ≥ 3 was associated with a more rapid progression, with 80% of patients increasing the FSHD score (ΔFSHD score ≥ 1). Disease progression is rapidly in index cases compared to relatives (ΔFSHD score 2.3 versus 1.2) while gender effect did not observed. According MRC examination, tibialis anterior muscle was significantly more affected muscle with difference between probands and relatives (35.5% of index cases versus 4.8% relatives with MRC ≤ 3/5). A category (which consists in the complete phenotype with affected facial and shoulder girdle districts) is associated with higher FSHD score at baseline and more deterioration at follow-up; we observed a slower progression in subjects with incomplete clinical phenotype (B category, P < 0.0001). In therapeutic era for neuromuscular diseases, our study provides clues for the understanding of the pathophysiology of FSHD disorder. Differences between familiar and index cases underline importance of genetic background., Individuals with Neuromuscular Disorders (NMDs) not rarely develop primarily ventilatory impairment, although the probability of its occurrence is different, according to baseline disease. Once respiratory muscle impairment has become pronounced, patients can use both inspiratory and expiratory muscle aids to prevent the onset of acute and/or chronic progressive respiratory failure, have excellent prognoses with long-term home Non-Invasive Mechanical Ventilation (NIV) and usually do not require tracheostomy for ventilatory reasons. By releasing effective mechanical ventilation via a nasal mask or a mouthpiece, NIV is characterized by ease of administration, preservation of upper airway function and low cost. Its long-term application is indicated when spontaneous respiratory muscle efforts are unable to sustain adequate alveolar ventilation, causing chronic stable or slowly progressive ventilatory failure. Administration of NIV to NMD patients with chronic hypoventilation may be expected to improve physiologic function and quality of life as well as decrease the frequency of episodes requiring acute care facilities (1). If NIV is not well-tolerated or unsuccessful, a decision to electively perform a tracheostomy can be taken before the patient has developed major complications of chronic ventilatory insufficiency (2). The onset of Acute Respiratory Failure (ARF) may be caused by airway encumbrance with mucous, as a result of weakened respiratory muscles and an inability to cough effectively. A non-invasive approach to the management of tracheo-bronchial secretions, based on the combination of expiratory muscle aid and NIV may result in a reduced need of nasal suctioning and conventional intubation, and/or tracheostomy (3)., Multiple treatment options are emerging for muscle diseases, but conduction of clinical trials in the field is challenged by the rarity and slow progression of the diseases. There is therefore a great unmet need to define biomarkers and outcome measures that are sensitive to change in rare, slowly progressive patient populations. MRI of muscle has attracted increasing attention in recent years as a possible outcome measure. It has the advantage that it is non-invasive, almost independent of patient-cooperation, and unlike functional tests, is independent of losing skills during a study, and can therefore be applied to the whole phenotypic spectrum of a disease. Muscle disease activity and progression is reflected by increased muscle water T2 and muscle fat replacement. If therapeutic interventions are efficacious, they should reduce muscle water T2 and replacement of muscle by fat on MRI, which suggests that skeletal muscle MRI could be a suitable biomarker for treatment efficacy. The muscle fat content correlates with muscle function in most muscle diseases. Changes in muscle fat content have been detected after a year in many muscular dystrophies, in many cases before changes in muscle function can be detected. As the body of evidence favoring MRI as a sensitive outcome for change in muscle pathology is growing, it will likely play an important role as an outcome measure in future clinical trials. From a diagnostic viewpoint, muscle MRI is also helpful as individual diseases often show particular patterns of muscle involvement. Even so, there is quite a bit of overlap in several diseases, which makes MR-guided diagnosis difficult even for experts in the field. New developments in artificial intelligence tools aimed at analyzing muscle MRI show promise in providing quick and precise diagnosis for these diseases. Current status of muscle MRI as a diagnostic and biomarker tool in longitudinal studies will be reviewed., Mitochondria are the major source of ATP that is synthesized by the respiratory chain through the process of oxidative phosphorylation (OXPHOS), a complex biochemical process carried out through the dual control of physically separated, but functionally interrelated, genomes, nuclear and mitochondrial DNAs. The genetic and biochemical intricacy of mitochondrial bioenergetics explains the extreme heterogeneity of mitochondrial disorders, a group of highly invalidating human conditions, for which no effective treatment is nowadays available. In addition to bioenergetic failure, other mechanisms are probably predominant in the pathogenesis of specific syndromes, such as alterations of cellular redox status, the production of reactive oxygen species, compromised Ca2+ homeostasis, mitochondrial protein and organelle quality control, and mitochondrial pathways of apoptosis. By investigating selected families and patients, we have identified several new disease genes, each responsible of distinct defects of the respiratory chain, mtDNA metabolism, or both, associated with paediatric or adult-onset clinical presentations. Recently published and still unpublished findings will be presented and discussed. Structural analysis and the creation of ad hoc recombinant lines in yeast, flies, and mice have allowed us to dissect out the molecular consequences of the ablation or defects of some of these proteins, and their physical status in normal and disease conditions. These models have also been exploited to implement experimental therapeutic strategies, based on gene and cell replacement, or pharmacological control of mitochondrial biogenesis., Background. Limb Girdle Muscular Dystrophy type 2A (LGMD2A) is a progressive myopathy caused by deficiency of calpain 3, a calcium-dependent cysteine protease of skeletal muscle, and it represents the most frequent type of LGMD worldwide. In the last few years, muscle magnetic resonance imaging (MRI) has been proposed as a tool for identifying patterns of muscular involvement in genetically disorders, and as a biomarker of disease progression in muscle diseases. Methods. In this study, 57 molecularly confirmed LGMD2A patients from a European cohort (age range 7-78 years) underwent muscle MRI and a global evaluation of functional status (Gardner-Medwin & Walton score and ability to raise the arms). Results. We confirmed a specific pattern of fatty substitution involving predominantly the hip adductors and hamstrings in lower limbs. Spine extensors were more severely affected than spine rotators, in agreement with higher incidence of lordosis than scoliosis in LGMD2A. Hierarchical clustering of lower limb MRI scores showed that involvement of anterior thigh muscles discriminate between classes of disease progression. Severity of muscle fatty substitution was significantly correlated with CAPN3 mutations: in particular patients with no or one “null” alleles showed a milder involvement, compared to patients with two null alleles (predicting absence of calpain-3 protein). Expectedly, fat infiltration scores strongly correlated with functional measures. The “pseudocollagen” sign (central areas of sparing) was associated with longer and more severe disease course. Conclusions. We conclude that skeletal muscle MRI represents a useful tool in the diagnosis and clinical management of LGMD2A., Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive weakening and wasting of skeletal muscles, which usually leads to loss of ambulation between 12-14 years. DMD is caused by mutations in the dystrophin gene: 70% deletions, 15-30% point mutations and 10% duplications that induce a frameshift in the protein-coding sequence. Here we report on a 18-year-old DMD patient harboring exon-2 duplication and presenting with a milder-than-expected phenotype. When he was 6-year-old, a muscle biopsy was performed because of incidental detection of elevated CK. Complete absence of dystrophin was observed and the diagnosis of DMD was genetically confirmed by exon-2 duplication. At present, the patient is able to walk > 400 meters at the 6MWT and still run. Muscle biopsy was repeated when he was 15 and faint dystrophin was detected at immunostaining and western blotting. The finding of minor motor impairment after age 16 is exceptional in DMD. To investigate the influence of exon-2 duplication on the disease phenotype, we performed an explorative survey to assess the outcome of these patients. We collected data from 27 patient, whose 16 above age 14. At age 14 and 16, the percentage of ambulant patients was 50%, and still the 30% of patients could walk at age 20. In conclusion, it is important to consider such variability as a confounding factor when analyzing outcomes of newly available treatments. Moreover, a better understanding of the mechanisms that protect these patients may provide new avenues for treatment., We tested the effects of glucocorticoids (GCs) and genetic factors on respiratory and cardiac function in a large Italian DMD cohort followed by the Italian DMD Network, with validation in the CINRG Duchenne Natural History Study (DNHS). The Italian cohort comprised 327 patients with spirometric data and 374 with echocardiographic data. We used generalized estimating equations to evaluate effects of DMD mutation types and SNP modifiers: rs28357094 (SPP1), rs10880 (LTBP4), rs1883832 (CD40), and rs1815739 (ACTN3). In the Italian cohort, we estimated annual decreases of forced vital capacity (FVC, -4.2%), peak expiratory flow (PEF, -2.9%), and left ventricular ejection fraction (EF, -0.7%). GCs significantly improved respiratory (+14.5% FVC, p < 0.001) but not cardiac function. DMD mutations involving Dp140 showed a negative effect on FVC (-6.1%, p = 0.008) but not EF. Patients amenable to skipping of exon 8 had dramatically higher PEF (+23.0%). LTBP4 rs10880 was associated with preserved EF (+4.5%, p < 0.01). In the CINRG-DNHS (n = 277 with genetic data) GC treatment significantly improved not only FVC, but also EF (+2.1%, p = 0.028). The effect of DMD mutations involving Dp140 on FVC (-5.9%, p = 0.015) and that of rs10880 on EF (+2.6%, p = 0.027) were independently validated. A meta-analysis of the two cohorts showed that the minor alleles at SPP1 and CD40 SNPs were associated with reduced FVC (-4.5%, p = 0.02, and -4.8%, p = 0.006 respectively) and PEF (-6.3%, p = 0.009 and -4.1%, p = 0.024 respectively). In conclusion, GCs preserve respiratory, and probably also cardiac function; distal DMD and SPP1/CD40 SNPs affect respiratory function. LTBP4 haplotype is protective against dilated cardiomyopathy., Limb-girdle Muscular Dystrophy 1F/D2 (LGMD1F/D2) is a rare neuromuscular disorder with autosomal dominant inheritance firstly identified in an Italo-Spanish family. Clinical investigation revealed high variability in clinical symptoms and disease progression with generalized muscle atrophy as common feature. Histopathology revealed atrophy of muscle fibers, alteration in nuclear localization, aggregates of material positive for p62, desmin and myotilin. Genetic investigation identified a causative mutation in the TNPO3 gene which normally encodes for Transportin 3, a shuttle protein that transports, from cytoplasm to the nucleus, proteins involved in alternative splicing and RNA metabolism among which the splicing factor SRSF1. Even if the genetic cause of LGMD1F/D2 has been clarified, the role of TNPO3 in skeletal muscle and its participation in the pathological mechanism are still unknown. We analysed, in muscle biopsies of two LGMD1F/D2 patients, the expression of TNPO3 itself and of a selection of nuclear (SRSF1, Lamin A/C, Emerin) and sarcomeric (alpha-actinin) proteins that are related to or that could be influenced by mutated TNPO3. We observed by confocal microscopy that some of the selected proteins showed an altered expression or localization in patients’ biopsies in comparison to control muscle. Moreover, we performed an in silico study based on the analyses of databases that collect data on biological processes, protein-protein interactions and transcriptome maps; in this way we aim to identify or make predictions on the possible relationship between some selected markers of atrophy and autophagy, increased in LGMD1F/D2 patients (p62 and MuRF-1), and TNPO3 and its preferential cargo SRSF1., The recent advent of new therapies, such as the antisense oligonucleotide nusinersen, has significantly improved the natural course of spinal muscular atrophy (SMA) type 1. Tau proteins and neurofilaments are well known markers of axonal degeneration. We measured the cerebrospinal fluid (CSF) concentration of total tau (ttau), phosphorylated tau (ptau), neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) proteins in 14 patients (age range: 2-156 months) with a diagnosis of SMA type 1, at baseline and after six months of treatment with nusinersen. Patients were assessed using the functional scale “Children’s Hospital of Philadelphia Infant Test of Neuromuscular Disorders” (CHOP-INTEND). Eight out of 14 patients had a functional improvement of more than 3 points at the CHOP-INTEND. At baseline CSF ttau (p = 0.0002) and ptau (p = 0.0054) concentration showed a significant negative correlation with the age of patients and a positive correlation with the CHOP INTEND score (p = 0.0075 and p = 0.0342, respectively). After treatment the level of the tau biomarkers did not show any change, whereas NfL and pNfH concentration significantly decreased (p = 0.0001), being NfL related to age at baseline (p < 0.05). We also found a significant correlation between the decrease of NfL and the amelioration of the CHOP INTEND score in the subgroup of patients with a functional improvement above 3 points (p < 0.05). Further studies on longer treatment time-frame and larger cohorts of patients are needed to confirm our promising results., Rationale and objective. Spinal muscular atrophy type 1 (SMA1) is a rapidly progressing neurodegenerative disease caused by biallelic survival motor neuron 1 gene (SMN1) deletion/mutation. Onasemnogene abeparvovec (AVXS-101), a one-time gene-replacement therapy, treats the genetic root cause of SMA, and is designed for immediate, sustained expression of SMN protein, allowing rapid onset and durable effect. In the phase 1/2a trial (NCT02122952), 15 SMA1 patients received a one-time intravenous dose of AVXS-101 (lower dose [cohort 1]: n = 3; proposed therapeutic dose [cohort 2]: n = 12). There was dramatic event-free survival and developmental motor milestones.1 Here we report long-term follow-up study design and data. Methods. Patients in the phase 1/2a study could rollover into a long-term follow-up study (NCT03421977). The primary objective is long-term safety (incidence of serious adverse events, hospitalizations, adverse events of special interest). Patients will have annual visits for 5 years followed by annual phone contact for 10 years. Additionally, patient record transfers from their local physician and/or neurologist will be requested. Safety assessments include medical history and record review, physical examination, clinical laboratory evaluation, and pulmonary assessments. Efficacy assessments include developmental milestones (physical examination). Results. As of September 27, 2018, the oldest patients in cohort 1 and 2 were 59.2 and 52.1 months old, respectively, and free of permanent ventilation. Preliminary data (survival, developmental milestones) will be presented. Discussion and conclusions. Patients treated with a one-time dose of AVXS-101 continue to gain strength, develop, and achieve new milestones, demonstrating a long-term, durable response., Rationale and objective. Spinal muscular atrophy type 1 (SMA1) is a rapidly progressing neurodegenerative disease caused by biallelic survival motor neuron 1 gene (SMN1) deletion/mutation. Onasemnogene abeparvovec (AVXS-101) is a gene-replacement therapy treating the genetic root cause of SMA1. In the phase 1/2a study, AVXS-101 dramatically improved outcomes in SMA1 patients. We report study design and preliminary data of STR1VE, a pivotal phase 3 study (NCT03306277) of AVXS-101. Methods. STR1VE is a phase 3, open-label, one-time-infusion study in SMA1 patients < 6 months old (biallelic SMN1 mutation/deletion, 2 x SMN2). Primary outcomes are independent sitting for ≥ 30 seconds at 18 months old, and survival (avoidance of death/permanent ventilation) at 14 months. Secondary outcomes include ability to thrive and ventilatory support at 18 months. Exploratory outcomes include CHOP INTEND and Bayley score. Results. Enrollment is complete (22 patients). Mean age at symptom onset, genetic diagnosis, and enrollment was 1.9 (0-4.0), 2.1 (0.5-4.0), and 3.7 (0.5-5.9) months. At baseline, no patient required ventilatory/nutritional support; all exclusively fed by mouth. Mean baseline CHOP INTEND score was 32.6 (17.0-52.0). As of June 29, 2018, mean CHOP INTEND increase from baseline was 6.9 (-4.0-16.0, n = 20), 10.4 (2.0-18.0, n = 12), and 11.6 (-3.0-23.0, n = 9) points at 1, 2, and 3 months. Discussion and conclusions. Data from STR1VE show rapid motor function improvements (CHOP INTEND) in patients with SMA1 that parallel phase 1/2a study findings and may correlate with survival benefit, motor milestone achievement, and bulbar function improvements. Updated data will be presented., Background. Type 1 SMA is a severe neuromuscular disease caused by reduced levels of the survival of motor neuron (SMN) protein due to deletions and/or mutations of the SMN1 gene. A second SMN gene, SMN2, produces low levels of functional SMN protein. Risdiplam (RG7916/RO7034067) is an orally administered, centrally and peripherally distributed small molecule that modulates SMN2 pre-mRNA splicing to increase the levels of functional SMN protein. Methods. FIREFISH (NCT02913482) is an ongoing, multicenter, open-label, study of risdiplam in infants aged 1-7 months at enrollment with Type 1 SMA and two SMN2 gene copies. Dose-finding Part 1 (n = 21) assesses the safety, tolerability, and PK/PD of different risdiplam dose levels. Pivotal Part 2 (n = 41) is assessing the safety and efficacy of risdiplam. Results. In a Part 1 interim analysis (data-cut, 07 September 2018), 13/14 (93%) of infants had ≥ 4-point improvement in CHOP-INTEND total score from baseline at 8 months (median change: 16 points). Infants met the following HINE-2 motor milestones: full head control 6/14 (43%), horizontal or upward kicking 7/14 (50%), rolling to side or from prone to supine 4/14 (29%) and sitting with or without support 6/14 (43%). To date (data-cut, 07 September 2018), no drug-related safety findings have led to withdrawal of any infant from the study. One-year motor milestone data will be presented from FIREFISH Part 1. Conclusions. Despite not being designed to detect efficacy, risdiplam improved motor function in infants with Type 1 SMA in FIREFISH Part 1. FIREFISH Part 2 is ongoing worldwide., Duchenne Muscular Dystrophy (DMD) is a progressive, lethal X-linked neuromuscular disorder with a point prevalence of 1:5000 male newborns. The clinical diagnosis must be confirmed by dystrophin gene testing and it is achieved at the average age of 5 years, with remarkable differences among countries. Early diagnosis gives broader advantages to the patient and his family, considering the recent availability of new therapies acting at the protein restoration. Based on preliminary data obtained within the BIO-NMD EU project, we tested 82 plasma samples (3 BMD, 16 DMD, 16 female carriers, 14 controls, and 33 other neuromuscular non-DMD diseases) in 5 replicates of a multiplexed antibody suspension bead array. The array consists of magnetic beads coupled to 2 different anti-dystrophin antibodies, which recognize the N- and the C-terminus respectively. Preliminary studies show that fragments of dystrophin protein can be detected in plasma. Moreover the dystrophin abundance varies across the subjects analyzed, whit a decreased level in DMD patients compared to controls. This immunoassay test is not expensive and can be easily applied for a huge number of samples. For these reasons, this approach, if validated, might represent a very appealing protein testing for disease screening to allow an early diagnosis and monitoring drug efficiency., Targeted resequencing of genes of interest is an effective strategy for the routine diagnostics of skeletal muscle diseases. However, the current diagnostic rate is, at best, 40-50%, resulting in a large number of unsolved cases. At the same time, the massive use of large resequencing panels has increased the number of potentially causative variants and expanded the clinical phenotypes associated with the already known disease genes. Consequently, we have a limited understanding of the genotype-phenotype correlation although this is crucial for patients in order to receive optimal care and a correct prognosis. We have successfully introduced RNA sequencing in our molecular diagnostic pipeline. By analyzing RNA extracted from a skeletal muscle biopsy of myopathy patients, we identify single nucleotide variants in the coding regions; we detect and characterize splicing defects; we can observe an altered expression due to a monoallelic expression or due to variants in regulatory elements. Simultaneously, we are using transcriptome analysis to study the differential gene expression in different stages of muscle development. An interesting hypothesis, supported by our preliminary data, is that a differential exon usage in different muscles and in different developmental stages explains some of the clinical heterogeneity observed in patients. A form of titin-related arthrogryposis multiplex congenita, for example, is specifically caused by variants in exons that have a higher expression during the embryonic development of skeletal muscles. The same mechanism of alternative splicing can explain the selective muscle involvement and the specific age of onset in other genetic Mendelian and complex myopathies., Objective. HyperCKemia and proximal “Limb-Girdle” weakness represent a frequent and unspecific presentation of muscle diseases without straightforward guidelines for the clinical workout. Method: the outpatient clinic for neuromuscular diseases takes care of 800 adult patients with different neuromuscular disorders. We retrospectively analysed the clinical features and the diagnostic algorithm in 34 patients that presented with hyperCKemia and/or mild proximal “Limb-Girdle” weakness. Patients with facial involvement, distal or congenital myopathies have been excluded. The first step of analysis was the DBS for Pompe disease and nerve conduction and electromyography studies. In all patients we analysed DM2-related gene and further excluded copy number variations in DMD gene. Undiagnosed patients were investigated by target gene panels including genes associated with benign hyperCKemia or genes causing LGMDs, based on clinical presentation. For the remaining unsolved patients we proposed a muscle biopsy. Results. After the first step we identified 1 neuropathy. A confirmed genetic diagnosis was achieved in 10/33 (30.3%) patients. The diagnoses included: 4 LGMD (3 LGMD2L, 1 LGMD1C), 1 RYR1 mutation, 2 type DM2, 1 female carriers of Duchenne muscular dystrophy, 1 type V glycogenosis and 1 metabolic myopathy due to CPT2 mutation. 14 of undiagnosed patients underwent muscle biopsy, 6 of them required further investigation, while 8 evidenced only minimal changes. Conclusion. based on our experience, we propose a diagnostic approach with target gene panels as first-line tools for patients with mild and unspecific clinical presentation of muscle diseases., Mutations in the RYR1 gene are associated with a clinically heterogeneous group of neuromuscular disorders that can be challenging to diagnose because of overlapping clinical features and nonspecific muscle pathology. Moreover, recently, the RYR1-associated phenotypic spectrum has further expanded. Next-generation sequencing (NGS) is rapidly being implemented into routine clinical practice, improving the molecular diagnosis of genetically heterogeneous disorders such as inherited neuromuscular disease. By using a customized, targeted sequencing panel able to investigate the coding exons and flanking intronic regions of 241 genes we screened RYR1 and the other muscle disease genes in 400 patients with skeletal muscle disorders. We identified 71 patients carrying “probable” or “likely pathogenetic” disease-related mutations in RYR1. Fourteen patients underwent NGS analysis because of a clinical and histological suspicion of central-multimicore myopathy, and 14 because a clinical diagnosis of congenital myopathy. Also, 18 patients presented hyperckemia whereas 42 subjects presented with unclassified myopathy. Whilst 39 cases were sporadic, positive family history of muscle weakness was found in 32 patients, with autosomal dominant inheritance in 17 and autosomal recessive in 15. Detailed description of muscle weakness distribution in symptomatic patients used the Human Phenotype Ontology (HPO) nomenclature and coding system. Our study confirms that the interpretation of RYR1 variants is particularly challenging and often requires further analyses, including a comprehensive evaluation of the clinical phenotype (deep phenotyping)., Introduction. Duchenne muscular dystrophy (DMD) is the most common severe childhood form of muscular dystrophy. More than 2000 mutations of the DMD gene are responsible for progressive loss of muscle strength, and ultimately respiratory and cardiac failure. Through head-to-head comparison, functional and histological benefit across different micro-dystrophin constructs was evaluated. Methods. We designed 4 unique constructs of AAVrh74 vector, including use of the MHCK7 promoter in comparison to a less active MCK promoter with the same micro-dystrophin transgene that contains the N-terminus and spectrin repeats R1, R2, and R3, respectively (AAVrh74.MHCK7.micro-dystrophin; AAVrh74.MCK.micro-dystrophin), a mini-dystrophin construct that contains the nNOS binding site (AAVrh74.DV.minidystrophin), and a micro-dystrophin containing the C-terminus (AAVrh74.MHCK7.microdys.Cterm). To test the efficacy of the 4 constructs of AAVrh74.micro-dystrophin, we evaluated both functional and histological benefit 4 weeks post intramuscular vector delivery in the mdx mouse model. Results. Delivery of the AAVrh74.MHCK7.micro-dystrophin construct is the most advantageous in normalizing histologic and functional outcome measures among these constructs. Specific force output significantly increased in the tibialis anterior muscle compared with the other 3 constructs and there was no difference from wild-type levels. Muscle environment was normalized, as demonstrated by reductions in centralized nucleation and normalized myofiber diameters. Transgene expression through immunofluorescent staining and Western blot was significantly increased compared with the other constructs, indicating functional and histological advantages of the AAVrh74.MHCK7.micro-dystrophin construct. Conclusions. Findings from this preclinical study provided proof-of-principle for safety and efficacy of systemic delivery of AAVrh74.MHCK7.micro-dystrophin in a dose-escalation study in the mdx mouse model for DMD., Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary myopathy with autosomal mode of inheritance. FSHD has not been associated with a classical mutation within a protein-coding gene. Instead, the majority of FSHD cases (> 95%) carry a monoallelic partial deletion of tandemly arrayed D4Z4 repeats at the 4q subtelomere. Genotype-phenotype studies reveals wide clinical variability among the affected individuals and reduced penetrance in FSDH families. Moreover, alleles with reduced number of D4Z4 elements (DRA) are found in 3% of healthy subjects and not all individuals with a DRA develop FSHD. D4Z4 is a 3.3 kb CpG-rich (73%) DNA element with multiple repeat sequences normally associated with heterochromatin. A pervasive idea is that epigenetic changes in FSHD following the D4Z4 deletion lead to disease through changes in chromatin organization and consequent inappropriate expression of nearby genes. Indeed, using a permissive cell line carrying DRA and primary cells from FSHD individuals, we found that the 4q subtelomere is subdivided into discrete domains characterized by different histone modifications. These specific epigenetic signatures lead to a different expression of 4q35 genes that inversely correlates with D4Z4 size. We observed that one of these genes, FRG2, is subjected to reversible silencing and that a general de-repression in 4q35 region occurs in FSHD cells after various drug treatments. This point at the D4Z4 array as a sensitive locus, dynamically regulated, capable of responding to external stimuli, whose de-repression could be strongly correlated with the observed differences in penetrance among patients and/or with the risk to develop the disease., Among animal models for FSHD, mice overexpressing FRG1 present a progressive myopathy with features of human disease. To investigate the molecular changes occurring during disease development, we analysed gene expression profiles of skeletal muscles of mice overexpressing increasing levels of FRG1 at 28d (dystrophy onset) and at 96d (full dystrophy). We found a profound transcriptional deregulation correlating the severity of the muscle phenotype and FRG1 expression. GSEA and GO revealed alterations in pathways related to myogenesis, energy metabolism and inflammation. Indeed, genes related to adult and normal myogenesis were downregulated with a significant enrichment of genes specifically expressed during embryogenesis. In FRG1 mice at 7d and 14d the embryonic isoforms of myosin remain high instead of following the physiological downregulation occurring in WT mice, meanwhile the expression of the mature isoforms is reduced. Starting from 14d we observed the deceleration of growth curve and a reduction of muscle cross-sectional area. Moreover, FRG1 muscles displayed the significant reduction of ATP and the phosphocreatine in association with the transcriptional downregulation of Glut4, HK2 and AldoA. Our results indicate that FRG1 overexpression induce the impairment of muscle maturation and energy metabolism that precedes dystrophy. Our study opens new perspectives on the molecular mechanisms at the basis of muscular dystrophies., Stim1 and Orai1 are the key proteins involved in store-operated Ca2+-entry, i.e. the process that allows intracellular stores to be refilled upon depletion. Gain of function mutations of these two proteins lead to ultra-rare syndromes (tubular aggregate myopathy, Stormorken, York) mainly characterized by muscle and platelet dysfunctions. We have recently developed a mouse model bearing the I118F mutation on Stim1, one of the most frequent mutations found in patients. Animals are smaller in size, have a normal life-span and breed normally. Heterozygous animals display a dystrophic muscle phenotype (although no aggregates are observed), perform poorly in the rotarod and threadmill but not in the hanging test and also show haematological defects, mainly referring to platelets and myeloid cells, thereby conferring face-validity to the model. Myotubes from knock-in animals show an increased Ca2+-entry upon store depletion that parallels what has been previously observed in myotubes from patients. Recently, two animal models bearing the R304W mutation, associated with Stormorken syndrome, have also been reported showing similar, yet non-superimposable phenotypes. More, importantly, medicinal chemistry and repurposing programs are undergoing to identify modulators of store-operated Ca2+-entry. These modulators are being developed for disorders with a higher prevalence but it is highly likely that they would mitigate the progression and symptoms of tubular aggregate myopathy patients. The time therefore appears mature to make headway in developing translational programs to provide pharmacological answers for tubular aggregate myopathy patients., Although muscle pain has been already reported in mitochondrial diseases (MD), no extensive studies have been performed so far in order to quantify and clinically characterize this symptom in MD patients. We reviewed clinical findings of 1398 MD patients which were collected from the database of the “Nation-wide Italian Collaborative Network of Mitochondrial Diseases”. Muscle pain was present in 11.7% of the patients listed in the database. It was usually observed in subjects affected with chronic progressive external ophthalmoplegia (cPEO) and primary myopathy without cPEO. Less frequently, it has been reported also in multisystem phenotypes such as Kearns Sayre syndrome, MERFF, MELAS, MNGIE, NARP and Leigh syndrome. Main complain was diffuse exercise-related muscle pain, but focal/multifocal and at rest myalgia were also described. No significant correlation between muscle biopsy/genotype findings and muscle pain was found, although patients with mtDNA mutations more frequently complained of muscle pain than nDNA mutated patients (67.8% vs 32.2%). Pharmacological control of pain was obtained in only 34% of them by using many different analgesic and modulating drugs. Interestingly, a higher prevalence of responder patients among the nDNA-mutated subjects respect to mtDNA-mutated ones was observed, suggesting a possible role of genotype in influencing the response to therapy. Our study demonstrated that muscle pain is a common symptom in MD patients. Subjects with a myopathic phenotype are more prone to develop muscle pain, but this is also observed in patients with a multi system involvement, representing an important and disabling symptom having poor response to current therapy., Introduction. Although found in different muscle diseases in association with other features, fiber type disproportion (FTD) represents the unique histological abnormality of a specific form of congenital myopathy. However, poor data on this disease have been reported in the literature. We aimed at clinically and histologically characterize a large cohort of Italian patients with FTD. Methods. We collected clinical, histological, molecular and imaging data from patients followed in 9 Italian tertiary referral centers for neuromuscular diseases. Inclusion criteria were: infantile and adult patients with at least 25% difference between type I and II fiber diameters, on average, and, as adjunct anomalies, fiber type predominance, central nuclei or cores. Other histological abnormalities were considered as exclusion criteria. Results. We included in this study 47 patients, 12 females and 35 males. Mean age at muscle biopsy was 6 years; onset at birth was observed in 23 (48.9%) patients, whereas late-onset (> 5years) was reported in 13 (27.7%) patients. Genetic characterization was achieved in 22 (46.8%) patients, with TPM3 (n = 8) the most frequent gene harboring pathogenic mutations, followed by MYH7(4), RYR1(3), ACTA1(2), TPM2, TNT, LMNA, DOK7 and DNM2 (1 each). About one fourth of the patients could not walk independently at the end of the follow-up period. Conclusions. The diagnosis of FTD is difficult due to lack of uniformity in clinical presentation. Diagnosis is made after excluding other causes of myopathy and on the basis of histological evidence of type 1 muscle fiber hypotrophy. Genetic analysis shows also a great heterogeneity. Our data shows that presentation at birth accounts only for half of the cases. An integrated multidisciplinary approach of neuromuscular experts, geneticists, neuropathologists, will improve and optimize the diagnosis in this group of congenital myopathies., Background. In the last few years an autoantibody directed against the 5’-citosolic nucleotidase 1A (CN1A) was identified in sera of sporadic inclusion body myopathy (s-IBM) patients. Sensitivity of anti-CN1A antibodies in s-IBM significantly varies in different studies ranging from 33 to 76% with a specificity between 87 to 100%. We evaluated anti-CN1A antibodies sensitivity and specificity in a large Italian cohort of s-IBM and looked for potential phenotypic differences between anti-cN1A positive and negative patients. Methods. We collected clinical data and serum from 55 consecutive s-IBM patients attending seven Italian neuromuscular center and 62 patients with other inflammatory myopathies. Testing for anti-cN1A autoantibodies was performed using a well characterized commercially available ELISA. Results. Anti-CN1A antibody was detected in 20 out of 55 s-IBM resulting in a sensitivity of 36.4% with a specificity of 96,8% (only two other myositis were positive). We did not find differences in terms of age at onset or disease duration. We found a significant difference regarding associated swallowing problems (item 1 of IBMFRS with lower scores in Ab-positive patients). Anti-cN1A Ab-positive patients were significantly more likely to have more severe swallowing problems, expressed as IBMFRS item 1 score ≤ 2 (55.6% vs 7.4%). Discussion. We confirm the low sensitivity and high specificity of anti-CN1A Ab in s-IBM patients with high positive predictive value. Anti-CN1A test could be helpful in diagnostic work-up to reduce delay to a definite s-IBM diagnosis. In our cohort the presence of anti-CN1A antibodies identified patients with greater risk of clinically significant dysphagia., The YARS2 gene encodes the mitochondrial tyrosyl tRNA synthetase, a key enzyme in mitochondrial protein synthesis. Pathogenic mutations in the YARS2 gene causes a clinical triad of Myopathy, Lactic Acidosis and Sideroblastic Anemia (MLASA) and have been described so far in less than 30 cases. Patients manifest multiple mitochondrial respiratory chain defects in skeletal muscle, often demonstrated by the severe loss of cytochrome c oxidase (COX) activity. Our patient is a 9 years old girl who presented soon after birth with hypoglicemic coma and lactic acidosis. This metabolic crisis was treated with bicarbonate and glucose and did not recur over time. At 2 years of age during respiratory infection she was diagnosed with sideroblastic anaemia requiring only a single blood transfusion. Thereafter she became transfusion independent spontaneously. At 4 years of age she was admitted to paediatric neurology clinic for exercise intolerance and mild hypotonia/joint laxity. In the suspect of a myopathy a muscle biopsy was performed showing severe reduction of COX activity. Mitochondrial activity dosage in muscle revealed multiple defects, while mtDNA sequencing was normal. Targeted NGS panel allowed the identification of homozygous c.933A > G, p.Asp311Glu variant in YARS2 gene. This variant has been previously reported in patients with transfusion-dependent sideroblastic anaemia and lactic acidosis without overt myopathy. Clinical picture at age 9 years shows scoliosis and mild limb girdle weakness with minimal increase of CK and lactate, and the patient is constantly treatment with bicarbonate and vitamins., Fibrous myopathy is a poorly known side-effect of chronic intramuscular drug administration. It is characterized by induration of subcutaneous and muscle tissue in the injected sites associated with muscle contractures, EMG myopathic findings and histological demonstration of increased connective tissue and fiber degeneration in muscle biopsy. We here describe a 48-year-old man with a history of intramuscular drug abuse who presented with slowly progressive asymmetric weakness and hypotrophy of the scapulohumeral girdle for about three years. A scleroderma-like skin pattern was evident especially in the forearms and thighs. Endocrine, renal and liver dysfunctions, inflammatory or rheumatological disorders, electrolyte imbalance, vitamine deficiencies, hepatitis B and HIV infection were ruled out by appropriate laboratory investigations. HCV positivity has been known for many years. Deltoid muscle biopsy showed the presence of diffuse endomysial fibrosis, muscle fiber degeneration, adipose infiltration and widespread anti MHC-I antibody membrane positivity. Immunohistochemical studies for Dystrophin, Alpha-sarcoglycan, Gamma-sarcoglycan, Beta-sarcoglycan, Delta-sarcoglycan, Caveolin-3, Dysferlin, Alpha-dextroglycan, Merosine, Collagen IV, Collagen VI and Telethonin were normal. Skin biopsy showed a reticular dermal thickening as per a sclerodermal process. Final diagnosis was a fibrous myopathy related to drug abuse. Prognosis of this rare disorder is currently not well established and treatment are limited to discontinuation of injections and physical therapy. Fibrous myopathy should be considered in patients using intramuscular drugs because timely recognition may prevent further muscle damage., A 55 year-old woman developed visual disorders characterized by diplopia, ophtalmoparesis and bilateral eyelid ptosis. Since the age of 53 she had been suffering of several episodes of melena which, in the following years, associated to early satiety, vomiting and bowel dysmotility. A myopathic pattern emerged from electromyography; furthermore, alterations at visual evoked potentials (VEP) and bilateral hearing loss at audiometric test were found. At the age of 63 she underwent surgical laparotomy due to abdominal pain and distension and a perforation of a small bowel diverticulum was found. Due to this finding a small bowel resection was performed. A 54 year-old woman developed progressive sensory loss in both of her feet, which evolved during the following months in a balance disorder associated to many episodes of falling. Electroneuromyography showed a pattern of sensory-motor axonal neuropathy, associated to alterations of motor, sensory, brainstem and VEP, as a sign of central nervous system impairment. At the age of 59 she developed dyspepsia and early postprandial sense of fullness, associated with several episodes of vomiting. A CT scan of the abdomen showed a huge stomach, which extended to the left iliac fossa. Explorative laparotomy was performed in the hypothesis of bowel obstruction, but no mechanical occlusion was found. Both these patients showed a progressive evolution of the gastrointestinal symptoms together with a worsening of the neurological status. A complete radiological and instrumental workup was performed. Finally, biochemical and genetic tests led to the diagnosis in both cases., A 66 years old man was referred to our attention because affected by a subacute and painless ophthalmoplegia, with bilateral eyelid ptosis.He was receiving Pembrolizumab-Lenvatinib therapy for a renal carcinoma.Routine laboratory analysis were in normal range, except for hyperCKemia, elevated up to more than 5000 UI/L. No facial, bulbar, proximal, distal or axial muscular weakness was detected.Electromyography (EMG) showed myopathic pattern, with spontaneous activity, in orbicularis oculi and proximal upper limbs muscles; repetitive nerve stimulation and single-fiber EMG documented normal neuromuscular transmission function, confirmed by negative acetylcholine and Musk antibodies. Full panel of myositis specific and myositis associated antibodies were normal. Cardiac and respiratory functions were preserved. Whole body muscle MRI revealed normal images, whereas orbital MRI disclosed bilateral hyperintesities in inferior rectus, medial rectus and superior oblique muscles in both T1 and STIR sequences, with mild muscle atrophy. Deltoid muscle biopsy documented mixed lymphocytic/macrophagic endomysial inflammatory infiltrates, with prevalent CD8 and CD68 cells, sometimes expressing PD-1 or PD-L1 antigens; sarcolemmal and cytoplasmic MHC-1 overexpression was observed in clusters of non necrotic cells. CD56 positive cells were observed in perifascicular regions.Patient discontinued Pembrolizumab and received corticosteroid treatment with progressive clinical improvement and CK normalization. Immune check point inhibitors are a novel class of anti-tumor agents, which have been linked to several neurological adverse events, including myositis and myasthenia; only few cases have been reported with isolated ocular myositis. Our findings support this clinical entity, suggesting that isolated ocular myositis represents a subgroup of generalised myositis with predominat ocular symptoms., Sarcoglycanopathies are a subgroup of LGMD, caused by mutations in sarcoglycan genes. They usually have childhood onset and rapidly progressive course with loss of ambulation over 12-16 years. We describe the clinical and neuroradiological course of two brothers with LGMD2D and on steroids therapy. The patients are on regular clinical, functional, cardiological, pulmonary and neuroradiological follow-up. Physiotherapy and the use of nocturnal ankle-foot orthoses were started at the age of 5y. Deflazacort was started at the age of 7y. Both children regularly attained motor skills. Diagnosis was made after incidental detection of high CK in the youngest boy. Muscle biopsy showed an almost complete absence of the sarcoglycans immunosignal. Genetic analysis detected the c.308 T > C in exon 3 and c.dupC L161L fs X28 in exon 5 of SGCA gene. The brothers are currently 12 (Pt1) and 10 (Pt2) years old. Major motor difficulties were observed at the age of 7y. In Pt1 the disease rapidly progressed and he lost walking ability at the age of 10y. Pt 2 is still ambulant. Muscle MRI confirmed the progression of the disease. No cardiological or pulmonary impairment have been observed. There are only few reports in the literature on the use of steroids in LGMD. In our patients, steroid therapy seems not to determine a clinical improvement or a delay in the course of the disease. Potentially trajectories appear unpredictable in salrcoglycanopathies in relation to DMD and more studies including a large number of patients are necessary to define a possible effect of steroids., Background. Duchenne Muscular Dystrophy (DMD) is a rapidly progressive, lethal neuromuscular disorder, present from birth, which occurs almost exclusively in males. However, to date, little is known of the burden of DMD, including cost of illness and impact on health-related quality of life (HRQoL). Material and method. A systematic review on evidence of burden and illness costs of DMD has been carried out from the interrogation of Electronic database (Pubmed and Cochrane library). Time period was October 2018 to January 2019. Keywords used were: Duchenne Muscular Dystrophy, burden of disease, economic evaluation, cost of illness. Results. 78 records were identified through database searching, 15 were duplicates and deleted from the search, records screened were 63. Records excluded were 53; exclusion criteria were: publication status, not in accordance with keywords and research objective. 10 studies were examined as results of the review: 7 Cost of Illness studies, 2 Systematic Review on Cost of Illness, 1 Cost Effectiveness Analysis. Main outcomes considered were: medical direct costs, not medical direct costs, indirect costs, health related quality of life. Only three studies were focusing on Italy. In all studies a correlation between disease progression and cost emerges with a delta cost between € 34,500 and € 63,500. Conclusions. This is the first systematic review of burden and cost in DMD in Italy. It has emerged that the economic cost of DMD climbs dramatically with disease progression. Future research is needed to investigate the quality of life impact linked to the condition progresses., Duchenne muscular dystrophy patients, due to more widespread prolonged survival, progressively experience multisystem complications. We retrospectively reviewed the charts of 132 Duchenne patients (112 alive/20 dead, age 3.5-32.3 years) referred to an Italian tertiary care center, with the aim to identify a scoring tool useful for benchmarking between national and international specialized centers for treatment of DMD. Four items were analyzed: cardiac function, respiratory function, nutrition and scoliosis. For each item, different functional parameters were considered and classified, according to clinical severity, and an incremental scoring was assigned. In addition, a global score incorporating all items was defined. The scoring system tested confirmed a documented knowledge that, despite the significant protective role of steroids, all functions deteriorate with age. The value of the global score became significantly higher since the age of 13 years. Comparing alive and dead patients at the same age range, the latter were characterized by significantly worse cardiac function, no steroid therapy and later use of respiratory assistive devices. In conclusion, the scoring system showed that cardiac dysfunction seems to be associated to premature death, while respiratory care to prolonged life. The proposed index allows aggregating and correlating different parameters, items and functions, and giving either an individual prognostic indicator of decline, either a global score to evaluate changes in clinical trials., Becker Muscular Dystrophy (BMD; OMIM #300376) represents an X-linked inherited neuromuscular disorder due to mutations in the dystrophin gene, which cause partial lack of Dystrophin in muscle fibers. Clinical phenotype is generally significantly less severe than his allelic Duchenne variant and much more heterogeneous. The most frequent presenting symptom at the onset is the presence of cramps or exercise-induced myalgias in childhood (Bushby, 1993; Magri, 2011), but can also be episodes of isolated myoglobinuria or motor difficulties such as frequent falls, slowness, difficulty in climbing stairs, waddling gait or toe-walking. Subjects who receive an earlier diagnosis often have a slight psychomotor delay. In literature (Angelini, 1994; Van den Bergen, 2013; Bello, 2016) a wide phenotypic variability was also identified. We describe an Italian multicenter case series of 163 patients with BMD characterized by the onset of symptoms and/or diagnosis (even in the absence of clear symptoms - due to hyperckemia or familiarity-) in developmental age. We propose a phenotypic analysis based on the first signs of disease, early neurodevelopmental milestones, the presence and the degree of muscular, cardiac and respiratory impairment and brain involvement (described by the type and the severity of cognitive impairment) or other neuropsychiatric comorbidities such as neurodevelopmental disorders or behavioural disturbances. Aim of the work is to identify the presence of different clinical phenotypes and disease trajectories in this population. We will pay particular attention to the possible involvement of the CNS and studying, where possible, the evolution over time even in adulthood., Variants in DMD gene may cause Duchenne, Becker muscular dystrophies or additional phenotypes comprising cardiomyopathy, hyperCKemia and/or limb-girdle muscular weakness. It depends on the modified dystrophin amount, function and distribution. Nonsense mutations are expected to result in premature termination of protein translation, and therefore be associated with severe DMD phenotype. However, it is well known that the effect of a stop codon can be rescued by an alternative splicing. In addition, a very early 5’stop can be rescued by re-initiation of translation as well as a 3’stop can produce a functional shortened dystrophin resulting in a milder phenotype. We collected DMD nonsense variants reported in HGMD, LOVD and ClinVar databases and included nonsense DMD variants found in a cohort of over 200 BMD/DMD patients. For each patient, we also extracted clinical data and dystrophin expression. By a careful analysis, we selected 841 unique stop codon variants. Among these, we identified 58 patients harboring nonsense variants with a milder phenotype. Clinical, genetics and protein data were accurate to provide a clinical diagnosis of Becker muscular dystrophy. As the distribution of nonsense variants along the dystrophin transcript associated with a BMD phenotype is non-random, we may conclude that the position of a stop codon in DMD gene can be predictive of the phenotype. This may have important implication for therapeutic use of nonsense suppression drugs able to read through stop signals and produce a functional protein., Duchenne Muscular Dystrophy (DMD) is a rare genetic neuromuscular disease affecting 1 in 3,500 male births worldwide, due to a variety of dystrophin gene mutations. Diagnostic settings include MLPA (MRC-Holland) and NGS dystrophin gene sequencing (DMD MASTR assay Multiplicom). Thanks to the International DMD project we have tested 182 patients from Eastern European and non-European countries: Poland (75), Hungary (19), Lithuania (6), Romania (55), Serbia (2), Croatia (8), Bosnia (2) Bulgaria (13) Cyprus (2) and 172 DNAs from Extra-European countries: Russia (1), Ukraina (92) and Algeria (79) were collected. In the European samples we identified 33 large del/dup (33.6%), 33 nonsense (33.6%), 17 small del/dup (18%), 16 splice site (16%) and 3 missense mutations (3%). In non-European patients we identified 73 large del/dup (62%), 20 nonsense (17%), 9 small del/dup (7.7%), 9 splice site (7.6%) and 4 missense mutations (3%). Sixty-two European patients and fifty-four Extra-European patients remained undiagnosed using routine methods, suggesting the presence of atypical mutations in the DMD gene or other genes involvement. The early identification of the underlying genetic mutation is critical to potentially affect the course of Duchenne Muscular Dystrophy as well as the choice of treatment, the setup of appropriate and effective care and the eligibility for clinical trials. Genetic counselling can also be offered to patients and families with important repercussions on reproductive choices and lifestyle planning (see details at www.ospfe.it/medicalgenetics). Acknowledgments The International DMD project is funded by PTC Therapeutics., In addition to muscular involvement, Duchenne Muscular Dystrophy (DMD) children may present cognitive impairment and behavioural disorders especially if the mutation is in the distal portion of dystrophin gene A 3 years-old DMD boy born from heterologous artificial insemination presents both disease-related neuromuscular symptoms and severe psychomotor delay, namely retardation in achieving motor milestones, severe language delay, repetitive behaviour and lack of social skills (normal audiometry). The parents reported normal neuropsychological development until the age of 12 months, regression being observed after a vaccine. The child carries a splice-site mutation (c.8390+1G > A) with deletion of exon 56 of the dystrophin gene. He recently underwent thorough neuropsychiatric examination which is suggestive for Autism Spectrum Disorder (ASD). Brain MRI did not show structural abnormalities. We were recently advised that another child, born from heterologous artificial insemination performed in the same foreign centre, is affected with DMD caused by the same mutation and is having a normal neuropsychological development. A higher (10-20%) ASD frequency has been reported in DMD patients compared with aged-matched population. In our case, the fact that a second child, most probably born from the same donor and, anyway, carrying the same mutation, is having a regular psycho-behavioral development, makes a strict Dys mutation-ASD association unlikely, though the contribution of regulatory genes cannot be excluded. The etiologic role of vaccine is improbable because the child had no acute post-vaccine reactions and brain MRI is normal., Nowadays, there is growing interest in the use of urine specimens as novel non-invasive approach to isolate patient-specific stem cells. We have already showed that both native urinary stem cells (USCs) and differentiated myogenic USCs are a good model for studying dystrophin gene expression and for prescreening studies of therapeutic molecules. Based on this previous evidence, we isolated the native USCs from a DMD patient missing the mutation identification by standard genomic methods (MLPA, NGS) in order to profile the dystrophin transcript and find the causative genetic variant. The DMD transcript analysis by FluiDMD card revealed the absence of amplification of the junction between exons 10-11. The DNA sequencing of intron 10 identified the mutation c.1149+250 C > T that generates a donor splice site. This activates two cryptic splice sites causing the creation of two alternative exons, both leading to premature stop codons. Therefore, USCs represent an extremely valuable alternative source to the muscle biopsy and offer a novel strategy to obtain ideal cells to study the molecular mechanisms of diseases. Considering this, we created a biological repository to preserve USCs from healthy and affected subjects to be used for different purposes such as diagnostic test, disease modeling and drug screening. So far, our Biorepository is composed of 37 samples among which 2 are Becker Muscular Dystrophy (BMD) patients, 17 are DMD patients and 9 samples are derived from individuals affected by other diseases such as mental retardation and dismorphism, Bethlem myopathy, peripheral neuropathy and hereditary spastic paraplegia., We present clinical, molecular and immunohistochemistry study of 9 Italian Becker Muscular Dystrophy (BMD) patients carrying nonsense or single nucleotide mutations in DMD gene, leading to a premature stop codon. From a clinical point of view, all patients are still ambulant, however 5 presented moderate phenotype and the oldest patient was not able to climb stairs or rise from the floor autonomously; CK was significantly elevated. The 2 oldest patients also displayed signs of heart involvement and were in therapy with ACE inhibitors. No patient was taking corticosteroids. Muscle biopsies showed variable degree of necrosis and degeneration of muscle fibres, which correlates with age at biopsy and CK level. Dystrophin expression was only partially reduced with immunohistochemistry (IIH) and western blot (WB). Study of the transcript via RT-PCR was also performed. It is known that nonsense and frameshift mutations in DMD potentially determine the interruption of protein synthesis and degradation of truncated dystrophin molecules, for this reason they are usually associated with Duchenne phenotype. The exact location of the mutation is considered an important factor. Nevertheless other factors, such as presence of splicing regulatory elements, can influence the ultimate outcome of nonsense mutation and they are largely unexplored. These cases underscore the importance of a complete analysis of DMD gene and its transcript in order to provide better prognostic information for dystrophin patients and genotype-phenotype correlation. Nonsense mutations in BMD patients also raise question on the possible therapeutic approaches such as molecules able to read through premature stop signals., Becker muscular dystrophy (BMD) is a genetic disorder of the skeletal muscle resulting from the altered expression of the protein dystrophin. Myocardial disease is common in BMD and usually manifests as a dilated cardiomyopathy phenotype, which is often recognized only when clinically overt. Understanding the pathophysiology of cardiac involvement in BMD could help identify subclinical myocardial damage and direct the appropriate therapeutic choices to counteract disease progression. Both in chronic heart failure and in mitochondrial diseases, it has been demonstrated that muscular damage increases ergoreflex sensitivity, causing a chronic sympatho-vagal imbalance that can negatively affect the heart. We aim to test this so called “muscle hypothesis” in the context of BMD. In our protocol patients with skeletal myopathy but without known cardiac disease will undergo a thorough cardiopulmonary evaluation including two-dimensional echocardiography, cardiac magnetic resonance imaging, 24-hour ECG recording, cardiopulmonary exercise testing, pulmonary function tests and biohumoral characterization comprehensive of high sensitivity troponin T/I, brain natriuretic peptides, catecholamines, renin, aldosterone, galectin-3 and soluble suppression of tumorigenesis 2. Finally, baro-, chemo- and ergoreflex assessment will provide indexes of the autonomic function. Clinical and instrumental data will be correlated with autonomic function tests in order to investigate the association between peripheral muscles impairment, autonomic imbalance and heart involvement., Introduction. Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by DMD gene mutations that prevent production of functional dystrophin protein. Eteplirsen is an antisense oligonucleotide approved in the US for the treatment of DMD in patients who have a confirmed mutation of the DMD gene that is amenable to exon 51 skipping. In studies of DMD patients, approximately 64% of total systemic clearance of eteplirsen 30 mg/kg was via renal excretion. Aim. Determine the pharmacokinetics (PK), safety, and tolerability of eteplirsen in men with mild or moderate renal impairment. Methods. This single-dose, parallel-group study enrolled male volunteers with mild (estimated glomerular filtration rate [eGFR] ≥ 60 to < 90 mL/min; n = 8) or moderate (eGFR ≥ 30 to < 60 mL/min; n = 8) renal impairment. Participants received intravenous eteplirsen 30 mg/kg. Postdose PK, safety, and tolerability results were compared with demographically matched men with normal renal function (n = 9). Results. All enrolled participants completed the study. Total plasma clearance decreased by 27.5% (mild group) and 60.6% (moderate group), with proportional reductions in renal clearance (22.7% and 56.6%, respectively), and higher overall exposure versus the normal group. The single intravenous dose was well tolerated by all groups. Three participants, one in each group, reported 6 treatment-related adverse events (AEs): dizziness (normal group); pyrexia (mild group); and pollakiuria, micturition urgency, and incontinence (moderate group). All AEs were mild to moderate, nonserious, and resolved. Conclusions. Eteplirsen was well tolerated across renally impaired and normal renal function groups. Eteplirsen exposure increased as a function of decrease in renal clearance. “Study sponsored by Sarepta Therapeutics, INc.”, Background. Limb Girdle Muscular Dystrophies (LGMD) are a clinically heterogeneous group of disorders presenting with a spectrum of disease severity ranging from severe childhood onset muscular dystrophy to adult-onset myopathy. LGMDs include both dominant and recessive forms. Methods. We reviewed detailed retrospective data of LGMD patients followed up at the Neuromuscular Centre at the University of Messina in the last 10 years. The cohort included 164 patients in whom the diagnosis of LGMD has been defined using a combination of clinical, biochemical, morphological and genetic studies. Results. 85 were females (51.8%) and 79 were males (48.2%). Mean age at last follow-up was 48.2 years ranging from 9 to 82 years. 99 patients of all (60.4%) were genetically confirmed. Among them, 15 patients (15.2%) were LGMD type 1 and 84 patients (84.8%) were LGMD type 2. Considering the LGMD classification, LGMD1B was the most frequent among the autosomal dominant forms, while the most represented among the autosomal recessive forms was LGMD2V, followed by LGMD2B and LGMD2A. Three patients LGMD2L presented a pseudometabolic phenotype. The age at onset, clinical progression and cardiac and respiratory involvements showed a wide variability in each LGMD subtypes. Conclusions: Given the broad clinical spectrum, the combination of clinical and laboratory tests remains critically important to guide the physician to a final diagnosis of LGMD. Considering that natural history data are still poor, a systematic exploration of different LGMD phenotypes, the search for genotype-phenotype correlation and the identification of phenotypically homogeneous subgroups could be relevant for prognostic purposes and for further therapeutic approaches., TPNO3 encodes a nuclear membrane protein of the importin family which transport serine/arginine-rich proteins into the nucleus. A heterozygous deletion (c.2771del) in the termination codon of TPNO3 has been recognized as the causative genetic defect in a large autosomal dominant family affected by a form of Limb Girdle Muscular Dystrophy, namely LGMD1F. An additional missense mutation (R818Q) in exon 20 has been recently described in a sporadic male patient presenting an adult form of slowly progressive limb girdle weakness. We report on a further LGMD patient in which we identified the same R818Q mutation in TPNO3 gene as possible causative variant. Patient is the only child of healthy unrelated parents and family history is negative for neuromuscular disorders. He was first admitted at age 4 years for global hypotonia and raised CK (6546 U/L). Muscle biopsy showed scattered hypotrophic and necrotic fibres. Mutation in DMD gene were excluded. At age 10 years neurological examination disclosed diffuse muscle hypotrophy and weakness. Axial muscles and neck extensor were particularly affected. Last neurological examination at age 33 showed waddling gait, scapular winging, rigid spine and scoliosis. Weakness and hypotrophy of limb girdle muscles were present with Gowers sign. Muscle MRI showed fatty substitution of most hip and thigh muscles sparing biceps and soleus. NGS analysis of known LGMD genes disclosed the R818Q change in TPNO3 as the only variant. This mutation is present in gnomAD with a population frequency of 0.00004215 and is predicted to be damaging. The mutation is inherited from the father., Limb Girdle Muscular Dystrophies (LGMD) type 2A and 2B are autosomal recessive disorders associated with mutations in CAPN3 and DYSF genes, respectively. Conventional analysis of these genes through direct sequencing is generally laborious and time-consuming due to their large size and mutational hot spots absence. Next-Generation Sequencing (NGS) has improved the diagnostic rate, but few LGMD2A/2B cases still remain unsolved. We applied Multiplex ligation-dependent probe amplification (MLPA) method in a cohort of 30 suspected LGMD patients without molecular confirmation (18 LGMD2A and 12 LGMD2B). In these patients Western blot analysis supported the diagnosis and confirmed the reduction of calpain-3 and dysferlin in muscle. However conventional or NGS resulted in the identification of only one or no candidate/causative mutations in the respective coding genes. MLPA allowed the molecular diagnosis in 1/18 LGMD2A (5%) and 4/12 LGMD2B (33%) patients. In particular MLPA detected: 1) an heterozygous CAPN3 deletion including exons 1 to 6 in a LGMD2A patient; 2) the heterozygous DYSF deletion of exons 25 to 27 in two patients; 3) the homozygous DYSF deletion of exon 55 in two subjects. No duplication was found. Among DYSF-deleted patients, protein deficiency degree correlated with the probability of finding mutations. We confirmed the usefulness of MLPA analysis in selected cases. MLPA should not be used as screening technique because it is tailored for the suspected candidate gene. It is strongly suggested in cases with only one mutation identified and/or protein absence at Western blot analysis. In the latter scenario, MLPA could precede gene sequencing., Limb Girdle Muscular Dystrophy type 2A (LGMD2A) is an autosomal recessive disease caused by CAPN3 mutations determining quantitative and/or qualitative defects of calpain-3 protein. We sought to characterize the clinical progression of a single-center cohort of LGMD2A patients followed at our Neuromuscular Clinic at the University of Padova, in a 2-year time frame. We evaluated 24 genetically defined LGMD2A patients at baseline and after 2 years with Manual Muscle Test (MMT), North Star Ambulatory Assessment (NSAA), Six Minute Walk Test (6MWT), timed function tests (TFTs), grip/pinch dynamometry, and Performance Upper Limb (PUL). We also considered quality of life with psychometric tests (Individualized Neuromuscular disease Quality of Life, INQoL, and Fatigue Severity Scale, FSS). Cardiologic and respiratory function were assessed. Significant changes were detected in hip adductor (right p = 0.022 and left p = 0.030), total (0.07 ± 0.19, p = 0.030), and lower limb composite (-0.03 ± 0.24, p = 0.005) MRC scores. NSAA and 6MWT show no significant 2-year change. Among TFTs, time to run/walk 10 m was the only one to increase significantly (-0.15 ± 0.36 = 0.045). PUL showed a significant decrease (total score -2.5 ± 3.9 p = 0.0078 and and elbow subscore -1.7 ± 3.3, p = 0.011). In conclusion, lower limb MRC scores, PUL, and 10 m walk/run seem the most promising outcome measures for LGMD2A clinical trials. However, sample size calculations suggest that adequately powered studies will need large samples for rare disease standards, in the order of 200-300 participants, even when the most sensitive measures are employed. Alternatively, subpopulations with the risk of fast functional worsening should be selected by inclusion criteria., Autophagy is a critical process for the removal of damaged and dysfunctional organelles, protein aggregates and cellular components from the cell. Autophagy defects have been observed in many infectious and autoimmune diseases, in tumors, in neurodegenerative disorders and in some forms of muscular distrophies. The aim of the research is study the expression by western blot of autophagy markers such as AMPK, phospho-AMPK (Thr 172), Beclin 1, LC3I, ULK Ser555, pACC (Ser 79) on samples of muscle biopsies from patients with muscle diseases DMD - BMD - LGMD - FSO - Bethlem Myopathy - Pompe disease stored in Naples Human Mutation Gen Biobank, to understand the role of autophagic signaling in the dystrophic muscle and determine the relationship between the degree of impairment and the progression of the disease.AMPK expression was first assessed in muscle samples from patients with different forms of muscular dystrophy comparing with healthy subjects. The expression resulted increased in dystrophic muscles, such as for Beclin 1 and LC3I, however, the expression of the phosphorylated form appears to be almost the same in the studied groups. The expression levels of pULK S 555 and pACC S79 in specimens of muscular dystrophies at different stages of disease were studied. The results obtained showed a decrease in their expression. These results, which to date are preliminary, show how the evaluation of autophagy is important to understand the degree of progression of the disease that could be the basis of pharmacological research able to modulate autophagic signals for a slower progression of disease., Limb-girdle muscular dystrophies (LGMDs) are primary myopathies of the pelvic and shoulder girdles characterized by progressive muscle degeneration, and aggravated by sterile inflammation. Among them, sarcoglycanopathies (SGCs) are caused by mutation in the genes coding for a-b-g-d sarcoglycans which are crucial components of the dystrophin-glycoprotein complex, a multi-subunit complex that connects the cytoskeleton of a muscle fiber to its extracellular matrix and guarantees the membrane integrity during muscle contraction. In LGMDs the underlying pathophysiological mechanism and the role of inflammation are poorly investigated and no treatment is available for these rare diseases. Aim of this study is to define the immunological signature of patients and mice affected by SGC. Immunophenotypical analysis by flow cytometry indicates that lymphoid and myeloid infiltrates were significantly higher in limb muscles and diaphragm of SGCa mice versus WT. In particular, we observed a pronounced increase of CD3+/CD4+, CD3+/CD8+, CD3+/CD4-/CD8- T lymphocytes sub populations. Analogously, the number of CD11b+/Ly6G+ neutrophils, CD11b+/Ly6G-/Ly6C+ monocytes, F4/80+ macrophages, F4/80-/CD11c+ dendritic cells and CD3+/CD1d+ NKT cells was significantly higher in SGCa mice versus WT. In contrast, no quantitative differences in lymphoid and myeloid cells were detected in the spleen and PB of SGCa and WT mice. Immunohistochemical analysis of LGMD2 patient muscular biopsies showed a significant increase of CD3+/CD4+, and CD3+/CD8+ cells in comparison to control subjects not affected by muscular dystrophy. In conclusion, this study defines the immunological signature of patients and mice affected by SGC suggesting a relevant role of inflammatory responses in the pathogenesis of these muscular dystrophies., Introduction. While signs of systemic and cerebral nervous system (CNS)’s abnormalities are occasionally reported in rare paediatric severe form of FSHD, a CNS involvement in the adult-onset classic form of FSHD has not been accurately investigated. Methods. Here, we present a neuropsychological and psychopathological evaluation protocol aimed to evaluate the cognitive and psychological functioning, also in relation to degree of motor impairment and disability, in a cohort of 20 adult patients clinically and genetically characterized. In particular, the protocol includes the evaluation of visuo-spatial short-term memory span and working memory, verbal learning, visual attention, executive functioning and cognitive processing; the protocol also investigates subclinical occurrence of psychopathological symptoms and psychological status. Results and conclusions. Patients showed a generally reduced performance in every cognitive functional area. These results are suggestive of a cognitive and psychopathological impairment in adult-onset forms of FSHD, thus encouraging the planning of future investigations to better characterized the cognitive profiles of these patients., Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is characterized, beyond selective muscular weakness and atrophy, by extra-muscular symptoms like sensorineural hearing loss, retinal vasculopathy, epilepsy and cognitive impairment. We assessed the cochlear function in a cohort of 26 FSHD1 patients and in healthy controls matched for age and gender. All subjects underwent a complete neurological and audiological examination, including pure-tone audiometry (PTA) and otoacoustic emissions (OAEs) which explore outer hair cell function. Transient evoked otoacoustic emissions (TEOAEs) and distortion product evoked otoacoustic emissions (DPOEAs), which extend the examination to a wider frequency spectrum, were recorded. PTA results of patients and controls were comparable. However, FSHD1 patients showed significantly reduced responses of both DPOAEs and TEOAEs at all frequencies, even when considering only subjects with a normal PTA or with a mild muscular involvement (FSHD score ≤ 2). No correlation between OAEs and EcoRi fragment length was found. In conclusion, cochlear echoes represent a sensitive tool in detecting subclinical outer hair cell damage in FSHD1, independently from hearing loss and severity of genotype., Facio-Scapulo-Humeral dystrophy is the third most common form of muscular dystrophy with a prevalence of 1:15000-20000 and represents a disease with multisystemic involvement. We describe a case of a 16-year-old boy affected by FSHD, inherited from his mother. Francesco regularly achieved the first stages of psychomotor development, but at primary schools he was diagnosed for specific learning disability. His physical examination detected facial hypomimia, chewing fatigue, winged scapulae, Beevor’s sign and “poly-hill” sign; proximal weakness of the limbs, with greater left impairment, difficulty in walking on the heels; FSHD score was 4/15. CCEF (Comprehensive Clinical Evaluation Form) could be classified as A3. At the age of 3, he presented focal to bilateral tonic-clonic seizures. The EEG showed paroxysmal multifocal epileptiform abnormalities, over the left parietal and the right frontal areas. Brain MRI was unrevealing. Treatment with carbamazepine (CBZ) was started with complete seizure remission. Six years after CBZ withdrawal, his school performances worsened, with deficits mainly in attention and memory skills. The EEG showed an increase of the epileptiform abnormalities with left anterior prevalence both during wakefulness and sleep. Thereafter, the patient presented episodes characterized by brief psychomotor arrest with palpebral myoclonias and myoclonic jerks of the upper limbs usually upon awakening without loss of awareness but with falling objects from the hands. We reported this case for the peculiar association of FSHD and epilepsy, and for the particular course of the epileptic phenotype at long-term follow-up., In recent years the discovery of effective therapies has marked the history of Spinal Muscular Atrophy (SMA). The only currently approved drug (from Agenzia Italiana del Farmaco in September 2017) is Nusinersen which is an antisense oligonucleotide that binds to the SMN2 pre-mRNA downstream of exon 7, leading to the translation of a fully functional SMN protein and which is administered intrathecally by lumbar puncture. The treatment has opened up a new scenario with the creation of a new phenotypic spectrum. To monitor the effects of therapy, serial evaluations have been planned: neurological exam, muscle strength (MRC), motor functional scales, timed tests, muscle MRI, CMAP and MUNE. We report the data of a cohort of 7 patients treated with Nusinersen. Six of them are affected by SMA3 with different degrees of motor impairment and duration of illness, two of them are in wheelchairs since the age of 18 and 29 years. The starting age of treatment is between 9 and 35 years. The only side effect noticed was headache post lumbar puncture in 4 subjects. The seventh patient is a 10 month old child affected by SMA1 who started therapy when he was three months old. All of the patients have received the loading dose, four of them received 6 treatments and two patients 5 treatments. Monitoring long-term outcome measures will allow us to characterize the new phenotypes of the disease and to define the efficacy spectrum of Nusinersen in patients with different ages and degrees of motor impairment., Background. SMA is caused by reduced levels of survival of motor neuron (SMN) protein due to deletions and/or mutations of the SMN1 gene. While SMN1 produces full-length SMN protein, a second gene, SMN2, produces low levels of functional SMN protein. Risdiplam (RG7916/RO7034067) is an orally administered, centrally and peripherally distributed small molecule that modulates SMN2 pre-mRNA splicing to increase the levels of functional SMN protein. Methods. SUNFISH (NCT02908685) is an ongoing multicenter, double-blind, placebo-controlled (randomized 2:1, risdiplam:placebo) in patients aged 2-25 years, with Type 2 or 3 SMA. Part 1 (n = 51) assesses safety, tolerability and PK/PD of different risdiplam dose levels. Pivotal Part 2 (n = 180) is assessing the safety and efficacy of the risdiplam dose level that was selected based on results from Part 1. Results. SUNFISH Part 1 included patients of broad age ranges and clinical characteristics (functional level, scoliosis and contractures). To date (data-cut, 06 July 2018), a sustained, > 2-fold increase in median SMN protein versus baseline was seen after 1 year of risdiplam. Adverse events have been mostly mild, resolved despite ongoing treatment and reflect the underlying disease. No drug-related safety findings have led to withdrawal. Despite not being designed and powered to detect efficacy, patients on risdiplam experienced improvement over 12 months in motor function measures versus natural history. Exploratory efficacy will be presented in patients treated for ≥1 year. Conclusions. To date, no drug-related safety findings have led to withdrawal. Risdiplam has led to sustained increases in SMN protein. Part 2 is ongoing worldwide., Background. The number of copies of SMN2 is the most important gene modifier of disease severity in SMA. Patients with 2 copies of SMN2 usually develop type 1 or type 2 SMA and only rarely present with type 3 SMA. Recent reports have demonstrated that single base substitution in SMN2, c.859G > C in exon 7 creates a new exonic splicing enhancer element improving the amount of full-length transcripts, thus resulting in the less severe phenotypes, regardless of the number of SMN2 copies. Case presentation. We present two siblings of 43 and 45 years-old (yo) bearing 2 copies of SMN2 but bearing a type 3b phenotype. Despite the identical genetic background, and the apparently similar onset at 10 years old, the progression of disease differed in these 2 siblings. The younger lost ambulation at 30 yo while the brother is still able to walk for a few meters with support. Neither show significant involvement of the upper limbs, complain of dysphagia, or have respiratory impairment. Both started nusinersen treatment at age 42 and 44 respectively. Results of baseline muscle MRI, functional motor tests, respiratory assessments, quality of life and disease burden perception were collected at each nusinersen infusion. Conclusion. We confirmed the good clinical prognosis of the c.859G > C variant in exon 7 of SMN2; however the different clinical progression over time and response to treatment in these genetically identical siblings suggests that other factors playing a role in modifying the phenotype., Spinal Muscular Atrophy (SMA) is a motor neuron disorder due to recessive mutations in SMN1, encoding for the Survival Motor Neuron (SMN) protein. Most of SMA patients harbor homozygous SMN1 deletions while SMN2 copy number predicts the clinical subtype (SMA-I, -II, -III). Few (3-5%) SMA patients display small mutations on the second allele. In the last 15 years, we achieved a molecular diagnosis in a cohort of 127 Italian SMA patients. Homozygous SMN1 deletion was detected in 120 probands (94.5%). In 7 patients a heterozygous SMN1 deletion was in compound with a point mutation. A null allele was observed in 3 SMA-I cases: c.469C > T (p.Q157*), c.888+1G > C, c.511G > T (p.E171*). The missense mutation c.389A > G (p.Y130C) was found in a SMA-III patient while c.815A > G (p.Y272C) substitution occurred in 3 probands (1 SMA-I, 2 SMA-II). The Y130C change has been previously associated with SMA-III presentations, irrespective of SMN2 copies. Conversely, Y272C severely impairs SMN oligomerization and function. Direct sequencing of SMN2 exon 7 detected the modifier variant c.859G > C in 3 SMA-III subjects (2.5% of our cohort of SMN1-deleted patients) harboring 2 SMN2 copies. This transversion is expected to increase full-length transcript originating from SMN2 alleles. Late onset and mild clinical course of our c.859G > C carriers support this conclusion. The behavior of point mutations in SMN1/SMN2 is heterogenous and requires proper validation. The identification of small mutations in SMA patients is important to improve diagnosis and prognosis, to clarify the response variability to available treatments and to design novel therapies addressing these peculiar mutations., Introduction. Spinal muscular atrophy (SMA) is a primary motor neuron disorder, however autonomic system dysfunction has been increasingly observed. Vascular perfusion abnormalities and skin involvement have been reported in few infants with severe SMA1 and are considered a feature of possible multiple organ involvement associated with severe phenotype. Case presentation. We describe a 9-months old child with diagnosis of SMA 1 (1 copy of SMN2) at birth. At 1 month, tracheostomy was performed followed by gastrostomy at 2 months. At 4 months she presented necrosis of toes followed by extensive necrosis of distal phalanges of her hands in the next month. She lost the distal phalanx of one toe at 5 months. Dystrophic epidermolysis bullosa was initially suspected, and she started treatment with fucidic acid cream with no change. At 7 months and half, when the child was admitted to our institution, she presented an hypotonic tetraplegia and clearly evident skin lesions in upper and lower limbs; she started intratechal nusinersen treatment. At 4th infusion, the skin lesions were largely improved and replaced by scar tissue. Follow-up confirmed distal and proximal necrosis resolution and an improvement in general health conditions as per parent impression. Conclusion. Based on previous studies antisense oligonucleotide leakage from the central nervous system does not reach a sufficient concentration in peripheral tissues to affect SMN2 splicing. The timing of improvement in our patient suggests a possible causal relationship to intrathecal nusinersen suggesting a centrally mediated mechanism on peripheral tissues which persists over time with repeated injections., Spinal muscular atrophy (SMA) is an autosomal recessive disorder, characterized by symmetrical muscular weakness and atrophy. The incidence is variable from 1 in 6000 live births but the heterozygotes frequency in caucasian population is about 1/64. SMN1 and SMN2 highly-homologous genes play a crucial role in SMA etiopathogenesis: SMN1 mainly homozygous deletions occur in more than 95%, while SMN2 number of copies may modulate the phenotype. The frequency of variation in SMN1 copy number per allele also varies and differs among populations. Here we report SMN genetic results obtained during the last eight years in our reference center (EURO-NMD). The most frequent reasons for referral are represented by consanguinity, positive family history for SMN1 deletion or SMA, and carrier preconception screening. We calculated the frequency of different SMN1 genotypes in 1546 tested subjects including 60 prenatal tests performed on chorionic villous samples. SMN1 heterozygous deletion were 16%, homozygous deletion 4.6% and heterozygous duplication 6.5% (3 copies of SMN1). Majority of subjects with duplication comes from North and West Africa or from Pakistan, and were tested since of consanguinity. Among SMA patients, the majority has 3 copies of SMN2 (45%), the remaining patients have 2 or 4 copies equally frequent. The high rate of SMN1 duplication occurrence highlights its importance when performing carrier testing and risk assessment. Considering the approved orphan drugs for SMA, genetic testing is now compulsory and determining SMN1 and SMN2 copy number cistronic association might be very valuable for therapy outcome interpretation., Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in survival motor neuron 1 gene (SMN1), resulting in a truncated SMN protein responsible for progressive degeneration of brain stem and spinal motor neurons. The paralogous SMN2 gene partially compensate the production of full length SMN protein, mitigating the phenotype. Antisense oligonucleotide (ASO) nusinersen (SpinrazaTM), designed to increase the transcription levels of SMN2 gene, is the only approved treatment for SMA in USA and Europe. SMN protein function in SMA pathogenesis is still not completely understood. Notably, SMN is involved in RNA processing, and it has been linked to microRNAs (miRNAs) biogenesis. MiRNAs are small, non-coding RNAs that regulate post-transcriptional gene expression; they contribute to different biological functions implicated in the pathogenesis of neuromuscular diseases, including SMA. Nonetheless, miRNAs are stable in body fluids, indicating their potential as biomarker. In the present study, we characterized the expression of selected neural and muscular-specific miRNAs in the serum of 19 pediatric SMA patients (15 type 2, 4 type 3) at baseline and after 6 months of nusinersen treatment. Molecular results were correlated with motor function assessed by the Hammersmith Motor Function Scale Expanded (HFMSE). We observed that circulating miRNA expression in SMA patients’ serum changed from baseline under nusinersen treatment, and that miRNA level changes predicted response to treatment (HFMSE scores ≥ 3 points from baseline). Our data support the potential of targeting miRNAs as non-invasive biomarkers to monitor disease progression and therapeutic response in SMA., Background. In December 2016 and in June 2017, respectively, the FDA and EMA approved Nusinersen as the first treatment for SMA. Bambino Gesù Hospital started to treat patients with the SMA type 1, from november 2016, as part of the Expanded Access Programm (EAP), and from november 2017, after the commercial approval, the later onset forms. Methods: we recruited patients followed in the Neuromuscular Unit. We activated a multidisciplinary team composed of neurologists, pneumologists, anaesthesiologists, radiologists, physiotherapists and pharmacists, in order to prepare the drug, assess the motor functional abilities and monitor the effectiveness, assess the respiratory function and the risk of sedation and perform the injection in severe scoliosis. The intratecal delivery was performed by pneumologists or neurologists. In ASA3 the intrathecal administration was performed with local, in ASA2 patients anaesthesia was induced in all patients with Propofol and the procedure was performed in NORA. In patients with complex spine due to severe scoliosis or spinal instrumentation the procedures were performed with fluoroscopic guide. Results. We treated 57 patients (29 SMAI, 15 SMAII and SMAIII). The age range was 2 m-7.9 ys. 20 patients were in NIV (8 type I, 4 type II); 13 type I patients had tracheostomy; 20 patients had scoliosis (10 SMA1, 6 SMAII, 2 SMAIII) of whom 6 were submitted to spine surgery; 8 patients received the intrathecal injection by fluoroscopic guide. In 8 SMA1 patients the treatment was stopped (in 5 for no effectiveness, in 3 for SAE). 24 patients completed 1 year of treatment. In 66% of patients improvement in muscle strength or in QOL together to stabilization of respiratory and swallowing function was served. Conclusions. Nusinersen is effective, feasible and safe even in patients with complex spinal anatomies and respiratory insufficiency. To guarantee the quality of the procedure, we recommend establishing an experienced interdisciplinary team., We report the first familial case of a novel heterozygous Gly270Val mutation in the exon 8 of the heterogeneous nuclear ribonucleoproteins hnRNPA1 associated with a 41 kb D4Z4 allele on chromosome 4q35 realizing an overlapping phenotype between facio-scapulo-humeral dystrophy and central-core myopathy. The patients, a young girl and his father, showed bilateral winged scapula with severe asymmetric atrophy of upper limb muscles and waddling gait. Muscle biopsy didn’t detected any dystrophic changes such as an increase in fibrous and adipose tissue or internal nuclei, but the most relevant alteration were numerous central-core and moath-eaten fibers evident with oxidative enzymes staining. We discuss the hypothesis that the phenotype observed in this family is the result of two genetic mutations that could determine a synergic effect and the possible role of a novel mutation of hnRNPA1 gene determining a myopathic pattern., Clinical features associated with the severe neonatal presentation of RYR1-associated myopathy include decreased fetal movement, hypotonia, poor feeding, respiratory involvement, arthrogryposis, ophthalmoplegia, femur fractures and hip dislocation at birth. We describe the clinical and genetic characteristics of a newborn male, fifth son of healthy consanguineous parents, who presented severe hypotonia, respiratory distress requiring intubation and mechanical ventilation, difficulty in suctioning and swallowing requiring nasogastric tube feeding, dysmorfic features, contractures and multiple congenital fractures of femur, tibia and homerus. Pregnancy was complicated by polyhydramnios. No reduction in fetal movement was reported. An older sister died at 34 days of age with the same clinical phenotype. CK levels were normal. EMG showed myopathic abnormalities. Muscle MRI showed marked fatty infiltration of both upper and lower limbs. Muscle biopsy was not specific but showed severe myopathic changes. Array CGH and genetic test for SMN1 were normal. Genetic analysis performed with TruSight panel showed the c.6661A > T/p.(Lys2221*) and c.13742A > G/p.(Tyr4581Cys) mutations in RYR1. Clinical course gradually improved. The baby was extubated and placed on NNIV. Dysphagia progressively ameliorated with complete restoring of oral feeding. Spontaneous movements of both upper and lower limbs improved as well as contractures. The last follow up visit was performed when the baby was 7 months old and showed that he had acquired a partial head control. Our report confirms and further expands the clinical and histological variability associated with severe congenital RYR1-associated myopathy., STIM1 is a reticular Ca2+ sensor composed of a luminal and a cytosolic domain. Dominant mutations in STIM1 are a cause of three allelic conditions: tubular aggregate myopathy, Stormorken syndrome (a complex phenotype including myopathy, hyposplenism, hypocalcaemia and bleeding diathesis), and a platelet dysfunction disorder, York platelet syndrome In the clinical study and molecular characterization of 332 patients with neuromuscular disorders we used a customized targeted multigene panel in NGS dedicated to muscle diseases and identified pathogenic rare variants in 305 cases. In this study we report on seven patients (age ranged 26-57 years) who harbored mutations in the STIM1 gene identified in our NGS study. The age of onset of the affected patients ranged from birth to adulthood. Two patients presented clinical characteristic compatible with Stormorken syndrome, three out of seven presented with congenital muscle weakness, one with adult onset of non-specified myopathy and one referred only myalgia. The ability to walk was quite conserved in all patients. In three patients muscle biopsy showed the presence of tubular aggregates, atrophy of type I fibers in two patients and non-specific myopathic signs in two. The mutations we identified were distributed throughout the gene, and five were novel mutations. Of interest, a specific variant (p.Asp84Glu) was associated with of Stormorken syndrome. Our work extends the series of mutations in the STIM1 gene and associated phenotypes., SH3 and cysteine-rich domain-containing protein 3, encoded by STAC3 gene, is a protein essential for skeletal muscle contraction, involved in excitation–contraction coupling (ECC) through a not completely understood mechanism. Native American myopathy (NAM), characterized by congenital hypotonia and weakness, cleft palate, short stature, ptosis, kyphoscoliosis, talipes deformities, and susceptibility to malignant hyperthermia (MH) has been originally described in 5 Native American families as a result of the single founder p.Trp284Ser variant in STAC3. Additional 21 families of non-Native American ethnicity, affected by congenital myopathy of variable severity, have been later described, mostly in association to the common p.Trp284Ser variant. Only 5 different pathogenic variants have been reported in STAC3 so far. Here we report an Italian baby (aged 7 months) with severe neonatal hypotonia and respiratory impairment. Muscle biopsy, performed at the age of 2 months, showed congenital fiber size disproportion with a marked type I predominance. Next generation sequencing in this patient revealed a novel homozygous c.685_686delGA variant in exon 8 of STAC3 converting the Asp229 to a premature stop codon. The deletion of the C-terminal 135 aminoacids of STAC3 by the p.(Asp229Ter) mutation causes a loss of the two tandem SH3 domains of the protein which are known to be involved in interaction with CaV1.1 and distruption of this interaction perturbs skeletal muscle EC coupling. We describe the first Italian patient with STAC3-related congenital myopathy expanding the genotypic spectrum of this gene., Ryanodine receptor 1 (RyR1) is a calcium release channel with a pivotal role in the muscular excitation-contraction coupling. RyR1 is encoded by a 106 exons gene whose mutations have been linked with a variety of myopathies and with malignant hyperthermia susceptibility (MHS). Both dominant and recessive mutations have been reported. Dominant mutations are typically associated to central core disease (CCD) and MHS. Recessive mutations are characterized by a wider histopathological spectrum, comprising CCD, multiminicore disease (MmD), centronuclear myopathy (CNM) and congenital fiber type disproportion (CFTD). Herein we present clinical, histopathological and genetic features of 50 unrelated patients carrying RYR1 recessive mutations from 12 Italian Neuromuscular Centres. Patients currently aged 2-63 years, all presented with symptoms at birth or in the very first years of life. 11 patients are not ambulant. 22 patients developed important scoliosis and joint retractions. Only 9 patients presented with ophthalmoplegia but myopathic face was evident in most cases. 10 patients require ventilation support. Three patients required PEG for feeding problems. Muscle biopsies were consistent with core or multiminicore disease in 29 cases. Four had central nuclei and 3 also had increased connective tissue and fibrosis. Predominance of type 1 fibres was another common feature. Genetic analysis revealed compound heterozygous mutations spread over the entire length of the gene in most patients. 11 patients carry homozygous mutation. Most variants were missense. 4 patients carry stop mutations on one allele and resulted to be more severely affected., ACTA1 gene encodes skeletal muscle alpha-actin, the principal actin isoform in adult skeletal muscle, which forms the core of the thin filament of the sarcomere where it interacts with a variety of proteins to produce the force for muscle contraction4. ACTA1 is implicated in several muscle disorders including nemaline, cores, rod-core or actin aggregate myopathies and fiber-type disproportion. We report clinical, muscle imaging and histopatological data from an italian family harboring a novel ACTA1 mutation with peculiar histopathological findings. Affected members showed a late-onset diffuse muscle weakness (facial, axial, proximal and distal) with muscle hypotrophy. Muscle MRI showed fibro-faty replacement in gluteus minimus, quadriceps, biceps, abductors and gastrocnemius. Muscle biopsy showed the presence of nemaline bodies with several fuzzy-dark areas at Gomori Trichrome in type1 muscle fibres, corresponding unstructured cores at electrom microscopy, with abundant electrodense material. The molecular analysis revealed a new mutation in exon 3 of the ACTA1 gene in heterozygous state, segregating with affected members in the family. This mutation has never been previously reported and this findings define a peculiar histopathological presentation of nemaline myopathies. Our findings enlarge the genetic and morphological spectrum of ACTA1 related myopathies., Congenital myopathy is an uncommon neonatal disorder defined by hypotonia and muscle weakness that can manifest in the neonatal period. Presentation with signs of global hypotonia and respiratory insufficiency is among the classic findings. We here report clinical and genetic characteristics of two newborns, whose phenotype was characterized by severe hypotonia and serious respiratory distress syndrome that required mechanical ventilation. The diagnostic procedure was completed by NGS analysis using a panel of 5228 genes (Constitutional Panels, Agilent) that identified rare mutations in RYR1 and TTN, respectively. The first patient, a premature infant male, showed a homozygous variant in exon 104 of RYR1 (p.Phe4976Leu). This variant was previously reported in affected males with severe neonatal myopathy, dysmorphism and motor-developmental delay, confirming its pathogenetic role. The second patient, a newborn female, presented two loss of function variants (p.Pro34879Glnfs*36 and p.Arg5308Stop) in exon 54 and 358 of TTN. Prior to NGS, the complete analysis of these genes was routinely impossible due to theirs giant size and complexity. Due to widespread use of NGS, TTN and RYR1 are emerging as responsible in different cases of human congenital myopathy. For these neonatal disorders, the early diagnosis and an accurate NGS-based genomic investigation help in defining disease prognosis and patient management and enable a proper genetic counselling of the reproductive risk., Collagen VI-related diseases (COL6-RD) are a group of inherited myopathies with varying degree of clinical severity, caused by mutations in the COL6A genes. As EU reference center for neuromuscular disorders and partner within the EURO-NMD, we aim at providing a nationwide study of COL6-RDs patients and an overview of COL6A genes variants. 245 patients were recruited via our Genetic Counselling Service as well as referred to us by other Italian centers (pediatrics, genetics and neurologists) and analyzed over a 12-year period (2006-2018). 222 patients were studied by standard diagnostic tools and 23 by NGS Illumina TruSeq Custom COL6A genes panel. 186 disease-causing variants, evenly distributed through the three COL6A genes, were identified in 150 patients with a detection rate of 61.3%. Using RNA tools (Custom FluiCol6 microfluidic card and RNA-Seq) we characterized the COL6A genes transcripts on urine stem cells (USCs) and fibroblasts from patients and healthy controls. The transcripts comparison with the skeletal muscle showed that some splicing choices and isoforms representation are different in USCs. Indeed, the COL6A3 full-length isoform represents the prevalent transcript in skeletal muscle while is expressed in USCs and fibroblast at very low levels. We also demonstrated that native USCs secrete functional collagen VI proteins, able to organize in the extracellular network. Our data provide a large COL6-RD patients cohort fully genetically characterized and propose native USCs as a non-invasive in vitro tool for functional studies, drug screening and validation in COL6-RDs., A cohort of patients from four different families (three Spanish and one Swedish), presenting with a novel form of adult-onset, asymmetric distal myopathy, was clinically and pathologically characterized. All of patients shared a characteristic pattern of muscle involvement with the atrophy of tibialis anterior and the initial involvement of ankle dorsiflexion later progressing to proximal limb muscles. In the most severe cases, histological analyses revealed a variation in the fiber size, internal nuclei and a large number of fibers containing rimmed vacuoles. A missense variant, c.1459T > C (p.C487R), in the alpha-actinin-2 gene (ACTN2) was identified in the probands of the three Spanish families. A second ACTN2 missense variant, c.392T > C (p.L131P), was identified in the affected members of the Swedish family. All the ACTN2 missense variants identified are fully penetrant and co-segregate with the disease in all the families enrolled. ACTN2 encodes for alpha actinin2, a protein highly expressed in the Z-disk of cardiac and skeletal muscles, where it interacts with other clinically relevant sarcomeric proteins, including titin and UDPN-acetylglucosamine 2-epimerase (GNE). Our study demonstrates that ACTN2 mutations cause a new type of dominant distal myopathy with a late-onset and a slow progression. At the same time as our study, two different ACTN2 variants (a missense and an in-frame deletion) have been reported in patients with a congenital myopathy. Further additional studies are needed to clarify the molecular mechanisms of the actinopathies and to improve our understanding of the genotype-phenotype correlation., LAMA2 mutations cause the most frequent congenital muscular dystrophy subtype MDC1A and a variety of milder phenotypes, characterized by total or partial laminin-α2 deficiency. Most common presenting symptoms are hypotonia at birth or in the first weeks of life or delayed motor milestones during the first year of age. Rare patients, among those with complete absence of laminin α2 protein on muscle biopsy, achieve independent ambulation; conversely, most patients, among those with partial laminin-α2 deficiency, achieve ambulation. In both severe and milder cases brain MRI invariably shows abnormal white matter signal intensity. We report clinical, histopathological, imaging and genetic data on two siblings with very subtle, and at first undetected, reduction in laminin-α2 expression, and brain MRI showing minor non-specific abnormalities. Clinical features in the female proband were characterized by muscle weakness involving neck and axial muscles, and pelvic girdle and distal lower limb muscles, reduced tendon reflexes and pes cavus. Clinical features in a younger brother were similar, and remained stable in both siblings during the follow up. Whole exome sequencing (WES) detected two heterozygous truncating LAMA2 mutations. Brain MRI in combination with laminin-α2 immunohistochemistry might not be sufficient and WES might be the only means to reach a diagnosis., Objective. To evaluate longitudinal functional changes in clinical outcome measures in a cohort of nusinersen-treated spinal muscular atrophy (SMA) type II and III patients in a single center. Methods. Thirty-three ambulant and non-ambulant SMA patients (4 type II and 29 type III), aged 15-68 years, were treated with nusinersen, an antisense oligonucleotide modulating pre-mRNA splicing of the survival motor neuron 2 gene (SMN2). Patients underwent intrathecal administration of 12 mg of nusinersen on days 1 (L1), 14 (L2), 28 (L3), 63 (L4) (loading doses) and approximatively every 4 months thereafter (maintenance doses) (M1, etc). The patients were clinically evaluated at L1, L4 and thereafter every 4 months using Hammersmith Functional Motor Scale-Expanded (HFMSE), Six-Minute Walk Test (6MWT), manual muscle strength evaluation according to Muscle Research Council (MRC) scale, Timed-Function Tests (TFTs) and revised Upper Limb Module (RULM). Results. All patients reported subjective benefit from the treatment. HFSME (L1-L4 p = 0.00002; L1-M1 p = 0.001, L1-M2 p = 0.029) and MRC (L1-L4 p = 0.013; L1-M1 p = 0.002, L1-M2 p = 0.007) improved significantly. RULM and time to climb 4 stairs were not significant but a positive trend was observed. The 6MWT was significant at L1-L4 (p = 0.036) and trended but did not reach significance thereafter. Conclusions. SMA type II and type III patients treated with nusinersen showed subjective and statistically significant improvement in some meaningful outcome measures., PITRM1 is a mitochondrial metallopeptidase, which digests oligopeptides including the mitochondrial targeting sequences, cleaved from proteins imported across the inner mitochondrial membrane. Mutations in PITRM1 have recently been associated with early onset autosomal recessive spinocerebellar ataxias. We describe a six-year-old boy, born from healthy unrelated parents, presenting delayed motor and language development. Since 25 months of age the child showed febrile tonic-clonic seizures with brain MRI showing a severe cerebellar atrophy and moderate atrophy of the pons with a diffuse signal alteration of cerebellar cortical and subcortical areas. At the age of 6 years neurological examination revealed ataxic and distal dyskinesias, severe intellectual disability and absent speech. Brain MRI demonstrated that the cerebellar atrophy was stable but a thalamic involvement was highlighted. Muscle biopsy showed slight mitochondrial proliferation and reduced enzyme activity of the respiratory chain complexes I+III. Using a targeted multigene panel we identified a new homozygous mutation in PITRM1. Segregation analysis showed that the mother was heterozygous whereas the father was wild-type. We then performed high-density SNP-CGH array analyses demonstrating a segmental uniparental disomy (UPD) in chromosome 10, by localizing interspersed regions of heterodisomy and isodisomy with maternal UPD10. In 10p15.3 region is located the mutated PITRM1, in homozigosity. In skin fibroblasts, we detected a significant reduction of PITRM1 protein expression in the patient, with low oxygen consumption and impaired basal respiration. This report expands the genotype-phenotype correlations of PITRM1 mutations and corroborates the need of a full molecular examination of apparently homozygous changes in mitochondrial disorders., After a traumatic brain injury, mitochondrial dysfunction occurs with an increase in reactive oxygen species and a decrease in ATP production; this can lead to headache and cognitive/behavioral deficits. Few reports in the literature show the consequences of head trauma in patients affected by mitochondrial disorders. We report the clinical history of a patient with Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS) who started to manifest behavioral changes immediately after a head trauma, to further investigate the assumption that traumatic brain injury can trigger the progression of mitochondrial disorders. A male patient affected with MELAS syndrome (3242A > G mutation) and followed in our Neuromuscular Unit reported a head trauma at the age of 43. Until that moment, no cognitive decline had been observed nor had he manifested overt MELAS syndrome symptoms. He became unconscious and ended up in a coma for about two weeks. When he woke up, a marked change in his mental status was noticed i.e. behavioral abnormalities (disinhibition, sexual delusions), personality change and cognitive impairment. His clinical condition remained stable over ten years, then a further, slowly progressive decline set in with episodes of hallucinations, loss of consciousness and severe loss of spontaneous speech. Mitochondrial dysfunction has a central role in determining cellular damage in head injuries. In our case, the trauma affected a tissue already suffering an oxidative damage and triggered a rapid disease progression causing severe sequelae. We therefore bring further evidence that head trauma is a precipitating factor in the progression of mitochondrial disorders., The NARS2 gene encodes the mitochondrial asparaginyl-tRNA synthetase, and enzyme critical in mitochondrial protein synthesis where it catalyzes the ligation of asparagine to tRNA molecules. Bi-allelic mutations in NARS2 have recently been linked to heterogeneous phenotypes including intellectual disability, epilepsy, hearing and visual impairment, myopathy, hepatic and renal failure, all combined with features of mitochondrial dysfunction. Here we describe two unrelated children presenting with developmental regression and infantile epileptic encephalopathy. Both patients presented episodes of failure to thrive and their latest neurological examination revealed apostural tetraparesis and severe intellectual disability. Brain MRI showed the presence of stroke–like lesions in Pt1 whereas in Pt2 there were symmetric lesions involving basal ganglia and the anterior surface of the cerebral peduncle, combined with bilateral cortical atrophy and marked T2 hyperintensity of the white matter. Muscle biopsy in Pt2 showed alterations of oxidative metabolism and abundant lipid droplets in most fibers. Using a multigene targeted resequencing panel, we identified four novel heterozygous mutations in NARS2: the c.716G > T/p.Gly239Val and c.749G > A/p.Arg250Gln in Pt1, and the c.688G > C/p.Gly230Arg and c.1339A > G/p.Met447Val in Pt2. The mutations segregated in healthy parents and were predictably damaging when examined in silico. In patients’ fibroblasts, we observed in both cases reduced expression of NARS2 protein, and a significantly impaired oxygen consumption and low basal ATP levels. Overall, our data suggest to consider NARS2-related disorder in infants presenting with early onset epileptic encephalopathy and failure to thrive., Paramyotonia congenita is a skeletal muscle sodium channelopathy caused by SCN4A gene mutations. The clinical picture is characterized by paradoxical myotonia, cold sensitivity, and episodes of paralysis, either spontaneous or exercise/cold induced. The classical first – line drug is mexiletine, followed by other sodium-blocker anti-arrhythmics, but this class of drugs requires cardiac monitoring and expose the patients to arrhythmia risks, especially when high dosages are required. To assess the electrical correlates of myotonia and paralysis, and to evaluate the effect of alternative treatments on membrane excitability, we adopted a standardized EMG protocol (long and short exercise test, with and without cold). We evaluated two male patients that had been previously treated with oral mexiletine, carbamazepine, and propaphenone, subsequently discontinued either due cardiac side effects or lack of efficacy. Searching for a non anti-arrhythmic alternatives, we evaluated the effects of three drugs, each administered off-label, and each with a distinct and peculiar mechanism of inactivation of the sodium channel: ranolazine, lacosamide, and buprenorphine. Of these, we found that only buprenorphine produced improvement in patient symptoms at a relatively low-dosage. By the exercise test we could confirm that improvement was not only symptomatic (and possibly related to the analgesic effect of opioids): indeed, we could detect an improvement of both cold- induced and exercise-induced paralysis, as well as improvement in myotonia., Background. Glycogenosis type 0 is a very rare metabolic myopathy due to glycogen synthase (GS) deficiency. So far, this condition have been described in few patients with childhood onset and severe cardiac involvement leading to sudden death in the first decade of life. Cases description. We report herein two unrelated adult subjects, who presented early fatigue, exercise intolerance and diffuse myalgia after brief physical exertion since infancy. Clinical examination revealed in both neck flexors and limb girdle muscles weakness. Results. Blood routine investigations were normal. Forearm test evidenced no rise of serum lactate in both patients. Electromyography showed a myopathic pattern only in one of them. Cardiac investigations including ECG, cardiac ultrasound and heart MRI were normal. Muscle MRI showed adipose substitution in thigh, gluteus and paraspinal muscles. Muscle biopsy revealed a marked depletion of glycogen at PAS stain in all fibers and negative stain for myophosphorylase. Muscle glycolytic enzymes assays were normal but glycogen synthase was virtually absent (0.001 and 0.002; n.v. 12.5 ± 1.2 nmol/min/mg). Sequence analysis of GYS1 revealed homozygous c.630G > C mutation in exon 3 (p.D145H) in a patient and IVS4+1 G > C homozygous mutation in the other one. The intronic mutation created two mRNA variants: 1) mRNA 1 with an insertion of 34 bp of intron 4 and mRNA 2 with skipping of exon 4. Conclusions. Hence, we described a mild form of GSD0, characterized by myopathy without cardiac involvement. These findings highlight the opportunity to considering this very rare condition in the evaluation of metabolic myopathies., Background and aims. Mitochondrial diseases (MDs) are a heterogeneous group of genetic disorders due to a primary defect in mitochondrial oxidative phosphorylation. The aim of this study was to describe prevalence and characteristics of SDB in a large cohort of patients with genetic confirmed MDs. Material and methods. A cohort of 103 consecutive patients affected by MDs, recruited in the Neurophysiopathology Unit of Gemelli Hospital in Rome in a period of five years, were enrolled. All patients were investigated by full night polysomnography (PSG). Results. SDB was demonstrated in 49 patients (47.6% ). As regard phenotypes, there were differences in distribution between groups: SDB was more frequently associated with progressive external ophthalmoplegia (PEO, 59%) and myoclonic epilepsy with ragged red fibres (MERRF, 54.5%). The prevalence of SDB was higher in patients with single or multiple mtDNA deletions and in m.8344A > G mutation. Interestingly, as far as the m.3243A > G mutation, patients with a predominant involvement of the central nervous system, as MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) had a lower prevalence of SDB than MIDD (maternally inherited diabetes and deafness), with percentages of 16.6 and 35.3% respectively. Conclusions. SDB has a higher prevalence in MDs compared to general population-based data. Moreover, SDB is more frequent in MDs characterized by predominantly skeletal muscle involvement. Overall, these data identify SDB as a common manifestation of MDs with predominant skeletal muscle involvement. The early identification of this disorder is crucial in the management of these fragile patients., Lipids represent the major building blocks for the synthesis of neo-generated membranes, besides having their well-known role as energy storage. It is possible to hypothesize that the regulation of lipid metabolism plays a pivotal role in the phospholipid composition and in building cellular membranes. In the regulation of triglycerides (TGs) in the cytoplasm (lipolysis) the adipose triglyceride lipase (ATGL) plays an important role. ATGL initiates the hydrolysis of TGs promoting the release of fatty acids (FAs) from lipid droplets. TG are crucial energy substrates, precursors for the synthesis of membrane lipids, and ligands of nuclear receptors. Patients with mutations in the PNPLA2 gene, that codifies for ATGL, suffer from a defect in TGs catabolism that reduce TGs release in cytoplasm and causes the neutral lipid storage disease type M (NLSD-M) characterized by lipid myopathy with significant disability. The pathogenesis of muscle damage in NLSD-M is not still well defined. We investigated whether cellular dysregulation of lipolysis in NLSD-M could induce a significant modification of cell membrane. Lipid composition of cell membranes of cultured fibroblast has been determined by gas chromatography electron ionisation mass spectrometry. The composition of cellular membranes is modified with respect to the control fibroblasts. In the membranes of NLSD-M patient there are significantly few short chain FAs, in particular C12, compared to the other FAs. It is possible to speculate that an alteration in the lipid composition of the membranes might leads to a membrane instability in NLSD-M cells. These observations need further investigations to confirm data and to assess their importance in causing muscle damage., Late onset Pompe disease (LOPD) is characterized by a wide spectrum of clinical presentations ranging from classical forms with manifested muscle weakness and/or respiratory impairment to isolated hyperckemia. A better awareness of the disease and the diffusion of newborn screening programs increased number of patients diagnosed at presymptomatic stage. The identification of these patients raises the consideration how to follow these patients in the view of an early detection of disease progression to start therapy. Herein we report on 8 patients with presymptomatic Pompe disease followed at our Neuromuscular Unit since the diagnosis was made. The patients had a mean age of 29 (range 4-58) years, a median follow-up duration of 10 (range 4-15) years. All patients were diagnosis because of isolated hyperckemia (CK range 400 to 1100 IU) and/or myalgia. Muscle biopsy revealed a vacuolar myopathy with glycogen storage in 4 pts whereas was unspecific in 3 pts, not performed in 1. Muscle GAA residual activity range from 3.8% to 15% of n.v. Patients were followed every 6-12 months with clinical examination including functional tests, pulmonary function tests (PFTs) and muscle MRI. Two patients, after respectively 9 yrs and 6 yrs of follow-up, start ERT because of detection of signs of muscle weakness and respiratory impairment. 6 pts were stable over the years with only persistent mild hyperckemia but no other signs of progression. Our data demonstrated that presymptomatic LOPD patients may remain clinically silent for decades but they should be monitored closely for overt signs of the disease to promptly start ERT., Background. Autophagic vacuolar myopathies (AVMs) are an emerging group of heterogeneous inherited muscle diseases linked to genes involved in autophagosomal-lysosomal processes in which glycogen deposition and autophagic vacuoles in muscle tissue are their pathological hallmarks. The best known AVMs are Danon disease, X-MEA and Pompe Disease (PD). Objectives. We aimed to dissect the mutational profiling of a neuromuscular patient sharing clinical, myopathological and biochemical findings with Late Onset Pompe Disease (LOPD) without GAA pathogenic mutations. Material and methods. 56 years old woman has been studied with a diagnostic protocol for suspected LOPD comprehensive of blood smears PAS-positive lymphocytes counting, DBS-GAA, muscle biopsy histological and immunofluorescence studies, GAA activity assay and expression studies on muscle homogenate, GAA sequencing, GAA-MLPA and WES. Results. The patient disclosed a limb girdle like muscular pattern with persistent hyperckemia and defective FVC. PAS-positive lymphocytes, glycogen accumulation and features of impaired autophagy were observed in muscle biopsy. A significant reduction of GAA activity was also measured. While GAA sequencing identified no pathogenic mutations, WES approach allowed to identify a peculiar mutational pattern in genes (MYOT, WDR24) cooperating in autophagic-lysosomal system. Discussion. Our data suggest that GAA reduction activity might occur in every condition of impaired autophagy. Therefore, GAA-DBS and PAS-positive lymphocytes counting should be considered as AVM markers in LOPD-like patients, including LOPD. Conclusions. Our study extend the number of phenotype-genotype compounds which can be listed as AVMs and WES is the tool with the best cost-benefit ratio to define the diagnosis., Pompe disease (PD) is an autosomal-recessive metabolic myopathy caused by deficiency of the lysosomal acid alpha-glucosidase, leading to an accumulation of glycogen mainly in muscles. Body Impedance Analysis (BIA) is a validated tool for the measurement of fat-free-mass (FFM), including muscle tissue, and fat mass (FM). Another value obtained by BIA is Phase Angle (PhA), proposed as an index of malnutrition and as a survival factor several diseases. We assessed the usefulness of BIA (BIOSMART, Eupraxia srl, Italy) in 15 pts with PD (7 males), all but two with normal BMI. We measured PhA, FFM, FM, extracellular fluids, and analysed their relationship with nutritional status (antropometric parameters, skinfold thickness, laboratory nutritional indices), disease parameters (motor, respiratory function, muscle MRI), and response to enzyme replacement treatment (ERT). Baseline PhA was below normal range in 7/15 patients and correlated with disease severity and worse ERT response (cut-off PhA separating good vs bad responders: 4.4°). In 2/11 patients in ERT and in 4/4 patients not in ERT, the PhA declined by an average of 0.6° on 3-year mean follow-up assessment. FM correlated with disease severity and MRI findings, and showed a similar trend. BIA may not be a valuable tool to assess nutritional status in PD, since it is influenced by disease parameters (muscle replaced by fat has a different impedance); however, FM, FM, and PhA correlate with disease severity and predict worse response to ERT, thus providing a cheap, fast, and easy-to-perform method to assess disease severity and ERT response., Background. McArdle disease, or glycogenosis type 5 (GSD5), the most common muscle glycogenosis usually presents as a dynamic disorder of muscle metabolism with exercise intolerance and risk of exercise induced myoglobinuria. In a substantial proportion of patients however the disease evolves into a fixed myopathic phenotype with preferential involvement of specific muscle groups. Muscle magnetic resonance imaging (M_MRI) is a powerful non-invasive tool providing valuable information on structural damage and tissue replacement increasingly used in support of the clinical evaluation. Methods. We performed whole-body MR examination in 9 adults with molecularly defined GSD5. Degree of myofibrillar edema and fatty substitution in the various muscles were graded semiquantitatively (Poliachick 2012, Fischer 2008). Demographics, laboratory values, clinical grading and objective exercise capacity indicators were assessed in the same patients and checked for correlations with the results of the imaging study. Results. Significant MRI signs of muscle structural modification were detected in 7 out of 9 subjects. The degree of change ranged from mild intrafibrillar edema to extensive fibroadipous substitution affecting mostly the posterior compartment. MRI detected changes showed a weak correlation with clinical symptoms severity and a moderate inverse-correlation with muscle power developed during exercise test (W max). No correlation could be found with age, CK levels, and peak VO2. Conclusions. Despite its definition as a mainly dynamic myopathy, GSD5 is very frequently associated with muscle structural changes that can be detected by MRI also in young subjects without apparent fixed myopathy. Maximum power expressed during standardized exercise testing seems the most reliable clinical indicator of muscle structural damage. M-MRI is a non-invasive and repeatable diagnostic tool assessing muscle structural changes that could provide efficient follow-up. Further investigation on larger cohorts of patients and into the relationship between MRI detected muscle alterations, clinical and functional indicators is warranted., Multiple Acyl-CoA dehydrogenases deficiency (MADD) is an inherited disorder of fatty acid oxidation. Age at onset is variable, ranging from neonatal to late-onset forms. The latter form is sometimes triggered by stressor agents as fasting, fever, drugs and often is responsive to riboflavin administration. Few reports of metabolic myopathies manifesting after statin administration are described but, to date, no cases of statins induced MADD have been reported. Among our cohort of 18 unrelated patients with late onset MADD, 4 pts (2F and 2M) started to complain of muscular symptoms after being exposed to statins. Symptoms were exercise intolerance, myalgia and proximal muscular weakness, appearing few weeks after treatment with Atorvastatin (2pts) or Pravastatin(1pt) and after increasing dosage of Simvastatin (1pt). Syndrome persisted after drugs withdrawal, halving statin dosage or changing type of statin (1pt). Serum CK was increased (400 to 3000) and LDH was 2 to 3 times nv in 3 patients. Serum acyl-carnitine profile showed an increased of all intermediates. Conventional EMG showed fibrillation potentials and PSW(3pts) and myogenic MUPs (1pt) while nerve conduction studies were normal. Muscle biopsy showed a vacuolar lipid storage myopathy. Patients were treated with riboflavin 400mg/die with a great response: after a month CK normalized and symptoms regressed. Our results suggest that a sudden motor impairment triggered by statin could be due to a MADD. An early recognition of this cases can allow a prompt treatment leading to a full clinical recovery., Human mitochondrial respiratory chain (MRC) complex I (CI) deficiency (MIM 252010) is the most common enzymatic defect in mitochondrial disease, ranging from mild muscle involvement to Leigh syndrome or fatal neonatal multiorgan disease. Here we describe a 72-year-old male who showed adult onset (65 years of age) of exclusive neuromuscular phenotype characterized by muscle weakness and muscle pain. Histological and histochemical analyses of a muscle biopsy from the quadriceps showed diffuse mitochondrial alterations. The biochemical assessment of the MRC revealed an isolated and severe deficiency of CI. The whole mtDNA sequence was normal. A genetic panel screening for nuclear genes encoding CI subunits and assembly factors led to the identification of two heterozygous variants in NDUFA11 (c.317C > T, p.Thr106Ile and c.394G > C, p.Ala132Pro. NGS revealed that the two variants were on different alleles.This case represents the second report of biallelic mutations in NDUFA11. So far, only one mutation was detected in this gene and was associated with an extremely severe, early onset phenotype characterized by fatal infantile metabolic acidosis or severe encephalocardiomyopathy. The two variants identified in our patients affect isoform 2 of NDUFA11, not isoform 1. This may explain the mild phenotype observed in the present patient. we speculate that the observed tissue specificity of the clinical presentation may be due to a specific or predominant expression of isoform 2 in skeletal muscle tissue. In our patient, idebenone therapy induced a mild improvement in clinically-measured muscle strength (MRC scale) and reported by himself., Glycogenosis VII (GSD VII) is an autosomal recessive glycogen storage disorder caused by mutations in the PFKM gene encoding the phosphofructokinase (PFK) enzyme. The classical form of GSDVII presents with exercise intolerance, contractures and myoglobinuria while the late-onset form is usually characterized by muscle pain and mild fixed proximal weakness. We describe a 65-year-old man affected by muscle PFK deficiency who, since the age of 33, presented with a classical form of disease showing exercise intolerance and myoglobinuria. Muscle biopsy showed a vacuolar myopathy with glycogen storage. Genetic analysis of PFKM gene displayed the presence of the heterozygote c.1817A > C (p.Asp543Ala) and c.488 G > A (p.Arg100Gln) pathogenic mutations. In his fifth decade, he started cyclosporine after liver transplantation and, then, amiodarone because of atrial fibrillation. In the following years, he developed a severe and progressive muscle weakness, mainly involving lower limbs, up to a loss of independent walking. Muscle MRI showed adipose substitution of both anterior and posterior thigh muscles with selective sparing of the medial ones. A selective replacement of gemelli and peroneus muscles and a relatively milder involvement of the soleus and tibialis anterior muscles were also reported. Face, abdominal and lumbar muscles were spared while minimal adipose involvement of the deltoid muscles were detected. The temporal relationship between the patient’s clinical worsening and starting chronic treatment with cyclosporine and amiodarone suggests an additive toxic damage by these two potentially myotoxic drugs in determining such an unusual phenotype, also confirmed by muscle MRI findings., The exact role of IgG antibodies against algucosidase alpha (anti-rhGAA) in modulating efficacy of ERT in patients with late-onset Pompe disease (LOPD) is still not fully understood. In order to assess if anti rh-GAA antibodies interfere with treatment efficacy, we analyzed clinical findings and performed serial measurements of IgG anti rh-GAA antibody titers from 64 LOPD patients treated with ERT. Two examinations (T0 and T1) were performed and the T0-T1 Delta of Six Minute Walking test (6MWT), MRC sum score (MRC), gait, stairs and chair performance (GSGC) and forced vital capacity (FVC) was considered and then related to the antibody titers. Seventy eight per cent of patients had an anti rhGAA positive titers (31% patients developed a low titer, 44% a medium titer and 3% a high titer) while almost 22% of the patients never developed antibodies. In a subgroup of patients treated less than 36 months, those with null antibody titers showed higher MRC sum score values than patients with positive titers. Differently, no statistical significance was found in relating the T0-T1 Delta differences and antibody titers for the other studied variables. Our results confirm that anti rh-GAA antibody generation did not significantly affect ERT efficacy. However, in the first 36 months of treatment, low-medium antibody titers might interfere with the clinical outcome. Therefore, a regular and careful evaluation of antibody titers, especially in cases with evidence of clinical decline despite ERT, should be conducted., We recently reported the first 2 MNGIE patients treated with orthotopic liver transplantation (OLT). We update their clinical and biochemical results, adding the findings of another transplanted MNGIE patient. At OLT patient-1 and patient-3 were at end-stage of disease (bed-restricted) while patient-2 had mild neurological symptoms and signs. OLT promptly and permanently normalized serum nucleosides in all patients. We didn’t observe substantial weight gain. Patient-1 recovered full ambulation and 2-years-after-OLT EMG showed nerve conduction improvement. He continued to complain of episodes of recurrent vomiting and pseudobstruction. Brain MRI remained unchanged. Unfortunately he died at 960 days after OLT for gastrointestinal hemorrhage. Patient-2 recovered from bilateral foot drop and at 900 days after OLT she has no major neuropathic and any gastrointestinal symptoms. 2-years-after-OLT EMG showed nerve conduction improvement. Brain MRI remained unchanged. Patient-3 is at 210 days after OLT. He suffered from pulmonary infection after surgery, needing of mechanical ventilation and tracheostomy. Currently he recovered ambulation, spontaneous ventilation and enteral nutrition. In MNGIE, OLT shows strong biochemical efficacy and the procedure and subsequent immunosuppressive therapy seem well tolerated. Plasma nucleoside clearance makes a long-term improvement of some aspects (in particular nerve functions and conductions, possibly related to mitochondrial DNA damage) but doesn’t influence other features (in particular gastrointestinal functions and leucoencephalopathy, possibly related to other thymidine phosphorylase actions), Cytochrome c oxidase subunit III (COIII or MTCO3) is 1 of 3 mitochondrial DNA (mtDNA) encoded subunits of respiratory Complex IV. To date MTCO3 mutations have been associated to Leber optic atrophy, myopathy with exercise intolerance or infantile encephalopathy Leigh like. Cerebral white matter involvement has been reported to mtDNA-related diseases with later onset as MELAS, MERRF and KSS. To date only few cases have been reported in childhood. Infantile mitochondrial leukoencepahlopathy (ML) are related to nDNA genes mutations and isolated complex deficiency. We report a 6 years-old boy affected by mild psychomotor delay, hypotonia, retinopathy, neurosensorial hypoacousia; MRI performed at 30 months of age disclosed sovratentorial cavited leukoencepahlopathy. Isolated complex IV defect was detected both in muscle and fibroblasts. Clinical evolution was stable. Screening of nuclear gene related to ML and defect of complex IV were negative while mtDNA sequence identified a novel mutation in COIII. In muscle tissue, blood, fibroblast and urinary sediment of patient nearly 100% of the mtDNA was mutated. The mutation was present in 69% of urinary sediment and 2% of peripheral leukocytes from the referred healthy patient’s mother. Nevertheless further investigations revealed peripheral sensory neuropathy and focal gliosis area a brain MRI while multievoked potential and plasmatic lactate level were normal. In the mutated cybrids from the patient, the amounts of fully assembled CIV are reduced and there is an accumulation of CIV assembly intermediates. Our report expands phenotype related to COIII mutations, lends further evidence to the expanding repertoire of mtDNA mutations in human diseases and suggests to look for variants in the mitochondrial genome when dealing with otherwise undetermined leukodystrophies of childhood., Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder characterized by clinical variability, affecting CNS skeletal and heart muscles. Cerebral involvement in DM1 is variable in cognitive impairment (i.e. executive, memory and visuo-constructive functions) and often associated with personality and behavioural dysfunctions. We observed during a 5 years follow up study 9 patients affected by DM1, that were periodically examined in our centre. All DM1 patients were genetically confirmed (age range was 18-75 years) and were assessed by psychological and neuropsychological tests. Brain MRI and serum myomiRNA were performed. At the baseline were memory, executive and visuo-constructive abilities the most compromised functions. Agnosognosia was present in more than 50% of patients both at baseline and follow up assessments. We observed that patients improved their performance in different tests, such as verbal abilities (verbal fluencies and verbal memory) and non verbal fluid intelligence, at the follow up when compared to the baseline assessment, although not statistically significant. We observed a worsening of performance in the The Rey–Osterrieth complex figure test (p < 0.05) and the working memory task (digit span memory test). Our study demonstrates that in the follow up in DM1 patients there is a slight improvement of cognitive abilities that could be useful to investigated in further studies. This suggests some differences and disease evolution is compared to other types of dementia and advance several possibly strategies to treat patients such as social intervention and CBT training., Aim of the study has been to identify phenotypic subgroups in patients with juvenile, adulthood and late-onset forms of DM1 with particular attention to exercise-related oxidative stress as epistatic phenomenon. 40 patients with DM1 were included. The different clinical forms were compared on the basis of the CTG-expansion class and the frequency of the main disease manifestations. The genetic features was also compared to muscular impairment (MIRS scale) and cardiac score. An incremental forearm exercise was used to assess biochemical markers of oxidative stress in the serum. There was no significant correlation between the CTG expansion class and the clinical form. However, when considered all together, an inverse relationship between CTG expansion class and clinical muscle scores. Increased serum levels of oxidized proteins and reduced serum levels of reduced glutathione were detected in patients compared to controls, however without any significant correlation with the size of the CTG expansion. Our study underlines the need to identify further and possibly multidimensional prognostic factors to better characterize the disease trajectory in DM1 in order to follow up and predict evolution as well as response to eventual therapies along the natural history of this disease., Case. A 31 year old woman came to our attention for a symptomatology, started from infancy, characterized by recurrent tetanic episodes and tremor especially in distal limbs and face and with a diagnosis of spasmophilia. Her father presented episodes of muscle spasms. Neurological examination was normal. Genetic test revealed a heterozygous mutation A763G in KCNA1 gene. Discussion. In 2009, a study conducted on a large Brazialian family with symptomatology similar to our patient, heterozygous mutation A763G in KCNA1 gene was found and associated with hypomagnesemia. Generally KCNA1 mutations are associated with different phonotypes, ranging from, classically, episodic ataxias, a group of diseases characterized by recurrent, discrete episodes of vertigo and ataxia, to new recently described phenotype (delay in motor development, choreoathetosis, cognitive dysfunction, transient postural abnormalities in infancy, shortening of the Achilles tendon in children, epileptic seizures, migraine, isolated neuromyotonia and myokimia). Diagnosis is primarily based on clinical and laboratory findings of hypomagnesemia, electromyographic myokymia (in some cases the myokymic activity only becomes apparent after application of regional ischemia) and genetic testing of KCNA1 gene. Our case confirms the broad phenotypic variability of this gene. In conclusion, we report an italian patient with hypomagnesemia and tetany caused by a KCNA1 mutation, and confirms the spread of the known A763G mutation, present also in Italy, and stresses the need to perform a KCNA1 test in patients with spasmophilia / myokimia and hypomagnesemia., Background. Metformin acts as a modifier of the aberrant alternative splicing of myotonic dystrophy type 1 (DM1) and reduces oxidative stress, which is enhanced in DM1. Preliminary animal and human studies showed its efficacy in improving motor function. This new mechanism of an old drug can bear a therapeutic potential for patients with DM1 Methods. METMYD is a phase III b randomized, double blind, multicenter trial (EudraCT n° 2018-000692-32) aimed at evaluating the superiority of a 24 month-treatment with metformin (500 mg, t.i.d; n = 97) over placebo (n = 97) in improving mobility and strength in DM1 patients with motor disability. The primary endpoint is a 40-meter difference in the 6-minute walk test. Secondary clinical endpoints address: 1) Dexterity and mobility; 2) Muscle quantitative testing; 3) Fatigue; 4) Quality of life. Moreover, circulating splicing products deficient in DM1, and markers of oxidative stress will be assessed before and after treatment. Expected results. This study will allow to explore the hypothesis that metformin may exert a protective effect through an additional mechanism of action. Circulating alternative splicing products may represent a surrogate outcome to validate the proposed principal action mechanism of this drug on motor functions. The expected positive results of this study will prompt the undertaking of confirmatory trials aimed at demonstrating the usefulness of metformin. Conclusions. Confirming the efficacy of metformin through its epigenetic mechanism on clinically meaningful outcome measures can provide an important therapeutic perspective for DM1 patients., Introduction. Metabolic alterations are an important feature of DM2; therefore, recognition of changes in body composition by BIA and its correlation with other disease features might be useful to assess disease severity. Methods. We obtained anthropometric measures, nutritional data, and blood tests in 18 DM2 patients. BIA was performed in all cases, recording phase angle (PA), body composition and body cell mass-index (BCMI). Motor function tests, including 30 second chair test (30SCT), functional index-2 (FI-2) for upper extremity, and the quick motor function test (QMFT) were administered and their results correlated with BIA data. Descriptive statistics and Pearson’s coefficient were performed. Results. Waist to hip circumference was above normal values in 67% of patients. Based on body mass index (BMI), 56% of patients were overweight and 28% of them were obese. By BIA, fat mass (FM) was increased in 56% and fat-free mass (FFM) was reduced in 50% of patients. Total body water (TBW) and PA were reduced in 61% of patients and the latter showed direct correlation with 30SCT, FI-2 flexion and abduction test, and QMFT. A direct correlation of BCMI with FI-2 flexion and of BMI with HOMA index was also found. Conclusions. This pilot study suggests that an alteration in the relative prevalence of FFM and TBW versus FM, as suggested by reduction of PA is a common feature of DM2 and parallels motor function tests impairment. Therefore, further studies on larger cohorts and with a prospective approach could validate BIA as an outcome measure for DM2., Introduction. Hand muscle weakness is a core feature of DM1 and outcome measures assessing finger pinch and grip strength should be available for natural history and trial studies. Methods. We prospectively evaluated self-assessed hand muscle strength with a hand-held dynamometer (HHD) in 57 adult DM1 patients of either gender. Three-finger pinch and handgrip strength were annually recorded in both hands with an up to four-year follow-up. Descriptive statistics, Student’s t test and correlation were performed. Results. Handgrip and pinch median values were markedly reduced at baseline in patients as compared to sex-matched normal values. In particular, P50 values for handgrip and pinch were 86% and 68% less than control values, respectively. Moreover, patient values for handgrip and pinch were significantly decreased by 23% and 29%, respectively, at 4 years, with a mean annual loss rate of 6-7%. Conclusion. Pinch and handgrip, measured by HHD, are valid instruments for detecting progression of muscle power loss in a short term and seem suitable for being used in clinical trials., Myotonic dystrophy type 1 is an autosomal dominant neuromuscular disorder resulting in unstable CTG repeats within the myotonic dystrophy protein kinase (DMPK) gene. The pediatric forms of myotonic dystrophy manifest much differently than the adult form of the disease. The aim of this study is to clinically and genetically characterize a cohort with pediatric myotonic dystrophy type 1 (DM1). Ten patients (8 males, 2 females) aged 1 to 30 years and regularly followed in our centre have been included. GTG expansion was confirmed in all the patients. Six patients had congenital onset with mild to severe neonatal symptoms including hypotonia, respiratory distress, sucking or swallowing difficulties, skeletal deformities. In these children musculoskeletal impairment varied from severe to moderate involvement and only three of them acquired independent ambulation. Cognitive deficits varying from severe to mild degree or behavioral disorders were observed in most of the patients. Three patients had respiratory impairment requiring NNIV. Gastrointestinal symptoms have been observed 4 patients. Endocrine or metabolic disorders have not been detected as well as cataracts. Cardiological alterations have been observed in 4 patient since the first decade. Two patients required PM implantation. The early detection of heart involvement suggests that a regular cardiological follow up has to be started in the first years of life., Background. Myotonic Dystrophy Type 1 (DM1) is an autosomal dominant multisystem disease caused by an unstable CTG repeat expansion in the 3’ UTR of the DMPK gene. DM1 is clinically heterogeneous. Outcome measures in DM1 should be explored to capture patients clinical status, to describe the natural history and to facilitate clinical trial design. Functional tests - including Six-minute walk test (6MWT), 10-meter run and time up and go (TUG) - could represent a reliable method to quantify disease progression. Longitudinal studies in DM1 patients using functional tests, manual muscle testing (MRC scale) and CTG expansion data are scarce. Here we present an observational 3-year longitudinal study to assess the role of functional tests as clinical progression biomarker in DM1. Materials and methods. Between 2015 and 2018 we longitudinally evaluated 57 DM1 patients according to a neuromuscular assessment protocol including MRC score from 20 muscle, MIRS scale and functional tests - 6MWT, 10-meter run, TUG. Data were analyzed including as covariate CTG expansion size. Results. Of the 57 DM1 patients included in the study protocol, longitudinal data were available for 49. 6MWT showed a statistically significant decrease at 3 years (-47.5m, SD +/- 87.8, p = 0.031), while no significant variations were detected on the 10 meter-run and TUG tests. Muscle strength showed a progressive significant decrement on the average MRC values during the 3-year follow-up (-0.33, SD +/- 0.23, p = 0.001). Conclusions. Our data confirm the well-known slowly progressive course of DM1 and support the use of 6MWT for clinical follow-up., Congenital myasthenic syndromes (CMS) are a group of heterogeneous inherited disorders caused by mutations in genes encoding proteins essential for the integrity of neuromuscular transmission. The number of genes known to cause CMS is currently 30. The main proteins involved in the pathogenesis of CMS are: choline acetyltransferase (ChAT), the endplate species of acetylcholinesterase (AChE), β2-laminin, the acetylcholine receptor subunits (CHRNE, CHRND), rapsyn, plectin, Na(v)1.4, the muscle specific protein kinase (MuSK), agrin, downstream of tyrosine kinase 7 (Dok-7), and glutamine-fructose-6-phosphate transaminase 1 (GFPT1). CMS are characterized by fatigable muscle weakness (e.g., ocular, bulbar, limb muscles) with onset at birth or in early childhood; rarely, symptoms may present later. Clinical, electrophysiological and morphological studies are essential for molecular studies, counseling and therapy. CHRND mutations are a rare cause for CMS but they should be considered in patients with a severe, early onset form, with respiratory distress. We describe two sisters, not twins, clinically and genetically diagnosed with CMS, carrying two new different mutations in the CHRND gene (c.730C > T; p.Arg244Cys; c.1304T > C; p.Leu435Pro). They both clinically showed at birth generalized hypotonia, sucking and swallowing difficulty, weak cry, severe eyelid ptosis and ophtalmoparesis and, during the first days of life, they experimented an acute respiratory distress requiring invasive ventilation. A therapy with pyridostigmine was started without benefit; in a second time, salbutamol was tried, showing an initial partial response. These two cases expand the clinical spectrum of CMS linked to CHRND mutations, suggesting their role in determining a lethal phenotype., Background. Muscle specific kinase Myasthenia Gravis (MuSK-MG) is a distinctive, frequently more severe, subtype of MG. The prevalence varies among countries and ethnic groups, 30% to 70% in European anti-AChR antibody negative patients and between 2.5% and 3% in Asian countries. MuSK-MG usually demonstrates an acute bulbar onset with rapid progression within a few weeks. We report clinical features, treatment outcomes and follow-up data of 31 Italian MuSK-MG patients. Patients and methods. We retrospectively evaluated 31 patients (17 F, 14 M) diagnosed with MuSK-MG who attended our Outpatient Department between January 1995 and January 2019. Ages at presentation ranged from 10 to 70 years old. According to Osserman criteria, the majority of patients at onset showed a type-2 MG (23/31), 4 patients type-1 MG, 3 patients type-3 and only one patient type-5. Results. Follow-up time ranged from six months to 23 years. Six patients experimented respiratory insufficiency. Five underwent thymectomy without benefit. Each patient assumed prednisolone with a partial initial response. 19/31 were treated with azathioprine, 3 in association with another immunosuppressant. Rituximab was given to 3 patients with severe phenotypes and 1 out of 3 showed a complete stable remission at six months follow up. Discussion. In our cohort MuSK-MG patients are 4.1% with a female/male ratio of approximately 1:1. A 12.9% of pure ocular form was found. Posterior neck variant was present at the onset in 19.3%. The long-term follow-up allows us to identify osteoporosis as the main long-term complication, frequently being cause of pathological fractures., Introduction. Myasthenia gravis (MG) with autoantibodies to muscle-specific tyrosine kinase (MuSK) represents a distinct type of disease, as compared MG with antibodies against acetylcholine receptor (AChR-Abs). The role of electrophysiological tests in these patients is still argued. Methods. We retrospectively evaluated repetitive nerve stimulation (RNS) and single-fibre electromyography (SF-EMG) reports of 63 MuSK patients and we compared them with 35 non-timomatous AChR-Abs (ACAB+) patients matched with MuSK subjects according to gender, onset age, age at diagnosis and disease severity. Results. Fifty-nine MuSK patients and 31 ACAB+ patients underwent RNS, whereas SF-EMG was tested in 17 MuSK patients and in 7 ACAB+ patients. RNS showed a significant decrement in a higher percentage of ACAB+ patients when compared to the MuSK group (p < 0.0008) whereas no significant SF-EMG difference was found (p = 0.5). In MuSK positive group, proximal muscles showed a more significant decrement (p < 0.02) whereas no significant differences were found in ACAB+ group. In MuSK patients, pathological RNS did not correlate either with MG severity (p = 0.9) or with the length of drug-free-period (p = 0.4). Jitter was increased in 14 (82.3%) MuSK and 5 (71.4%) ACAB+ patients, borderline in one MuSK patient and absent in 2 MuSK (11.7%) and 2 ACAB+ patients (28.5%). Conclusion. RNS-EMG positivity was significantly higher in AChR-Abs positive group, no difference being evident at SF-EMG. In MusK group, RNS decrement is more evident in proximal muscles and can be absent in distal ones. Neurophysiological evaluation is important for MG diagnosis but it plays a secondary role after anti-MuSK titration for MuSK MG diagnosis., Myasthenia Gravis-Inflammatory Myopathy (MG-IM) association has been described in less than 50 cases, as isolated reports or in few case series. Thymic pathology is present in about 50% of cases with muscle biopsy consistent of IM. Muscle specific antibodies (MSA) and muscle MRI involvement have not been systematically investigated. In this study we investigated the clinical, serological, pathological and muscle imaging findings from 13 patients with MG-IM, from a cohort of 441 MG patients (2,9%). Median age at disease onset of 51 year (range 24-73) with clinical follow-up of 88 months (range 31-237). IM was suspected by CK elevation in all patients (range 500-3000 UI/L). Three main categories of muscle involvement, sometimes overlapping, were observed: focal, generalized and mild/asymptomatic, leading to 3 main clinical groups (A,B,C) and 2 overlapping subgroups (A/B and B/C). Muscle biopsy confirmed IM in all patients. MSA were negative in all patients. Antibodies against the acetylcholine receptor were detected in al all patients associated with anti-Titin and -RyR1 in patients with thymoma. Thymus pathology was present in 10/13 patients. Antibodies against the acetylcholine receptor were detected in all patients, associated with anti-Titin and -RyR1 in patients with thymoma. In conclusion we redefined the clinical spectrum of muscle involvement in MG-IM association, manifesting as a continuum among 3 main clinical groups: focal, generalized and mild/subclinical muscle involvement. Minimal muscle involvement and focal myositis could be underestimated among myasthenic patients and increased clinical awareness is warranted to better recognize these clinical entities., Background. Myasthenia gravis is an autoimmune disorder of NMJ; in the last years an unexpected increased incidence of elderly-age MG was found. Very late onset MG can be underdiagnosed because some disturbances can be ascribed to more common chronic diseases. We evaluated the incidence and the features of MG, presenting after the age of 75 years, among our MG population Patients and methods. 36 patients (17F,19M) with a MG onset > 75 years (age-range 75-89) were identified. All the patients were evaluated at onset with clinical examination, QMG score, SFEMG, RNS, thorax CT, routine blood examinations, Ab AChR and Anti MUSK assays. Results. According to Osserman criteria, the majority of patients at onset showed a type-2 MG (23/35), 7 patients type-1 MG, 2 patients type-3 and only one patient type-4. Ab AChR were positive in (32/36); 4 patients were negative for Ab AChR and MuSK. Thymoma was found in 3 patients. The average time before the diagnosis was 11 months. The most common regimen of therapy was prednisone at low doses (less than 12,5mg/day); Azathyoprine (50 to 100mg) was used as steroid sparing agent. Discussion. Our findings show that the diagnosis and therapy of MG in the elderly can be difficult. Among our population, there were no patients with MuSK related MG. MG type, comorbidities (hypertension, diabetes, glaucoma, osteoporosis) should be carefully considered for a positive outcome., A 50 years-old man affected by hypertension, was diagnosed with psoriatic arthritis (PsA) at the age of 35 years. He was followed by the rheumatologists who started methotrexate (15 mg/weekly) and cyclosporine, that was interrupted 3 years later because of hypertension. Subsequently, etanercept (15mg/weekly) was introduced and continued for 10 years with an adequate reduction of pain symptoms related to PsA. Afterwards, etanercept was replaced with ustekinumab (45 mg/monthly, than 45 mg every 3 months) on Rehumatologist’s indication and continued for one year. Six months after the beginning of the ustekinumab treatment, the patient underwent to a chest CT scan for a follow up after pneumonia. The chest CT scan showed a neoplastic anterior mediastinal mass, revealed to be a thymoma at surgery. Three months later the patient was referred to our outpatient department with generalized weakness, fatigue, difficulty in raising arms and transient episodes of diplopia after physical effort. He came to our observation at the age of 50 years; a deep clinical history revealed that these symptoms had begun about 7 years before but they were ascribed to PsA. A diagnosis of anti-acetylcholine receptor antibody-positive (4 nmoli/l) myasthenia gravis was than made; methotrexate (20 mg/weekly) and prednisone (12.5 mg/daily) were started with regression of MG symptoms in three months. Our case increases the number of clinical reports of MG onset in patients with rheumatic disease treated with anti-TNF-alpha drugs and it could cast doubt on a possible role of ustekinumab in MG clinical worsening., Myastenia Gravis (MG) is an autoimmune disorder of neuromuscular transmission caused by antibodies targeting postsynaptic muscle end-plate at the neuromuscular junction leading to an impairment of signal transduction. The cardinal symptom is a fluctuating weakness of voluntary muscles. The disease is characterized by a wide spectrum of clinical presentations ranging from purely ocular symptoms to severe generalized forms with respiratory involvement. MG, however, may present at onset with atypical clinical pattern of weakness including dropped head, acute bilateral facial weakness, sudden bilateral lower limb weakness, foot drop, distal weakness without atrophy and limb-girdle muscle weakness. We describe an anti-AChR MG patient who manifested at onset nonfluctuating weakness and wasting involving shoulder girdle and proximal arm and leg muscles associated with dysphagia and bilateral calf hypertrophy. This unusual presentation can mimic clinically a primary disorder of muscle but neuromuscular junction studies (repetitive nerve stimulation and single-fiber electromyography) helped us to achieve the correct diagnosis. Our patient adds to the phenotypic variability of AchR positive MG and highlights that muscle atrophy may occur in the early stages of this condition, not only in patients with long-standing disease, in those who have been in corticosteroids for long periods of time or in patients seropositive for MuSK antibody., A 7-year-old child presented with a 2 months subtle onset, progressive lower limbs weakness. Creatine kinase (CK) level was 10,000. MLPA analysis of DMD gene showed no deletion or duplication. Muscle biopsy showed dystrophic pattern with numerous necrotizing fibers. Western blot analysis highlighted normal signals of dystrophin-associated glycoprotein complex proteins. Limb-girdle muscular dystrophy next generation sequencing panel revealed no definitely pathogenic mutations. Lower limbs muscle magnetic resonance imaging (MRI) showed proximal muscles relative hypotrophy with mild fibro-adipose substitution, but without signs of edema. Echocardiography was normal. In the next six months the patients complained of rapidly progressive fatigue, climbing stairs difficulty and running ability loss. Neurological examination evidenced four limbs muscles weakness, diffuse loss of muscles tone involving axial muscles and complete Gowers’ sign. Re-evaluation of muscle biopsy revealed anti-MHC-I antibodies sarcolemmal positivity in some fibers and complement deposition on capillaries. Autoimmune screening revealed anti-HMGCR antibodies positivity. Four limbs MRI showed the appearance of inflammatory findings. Echocardiography showed interventricular septal hypokinesis. Patient was treated with intravenous steroid then with slow tapering oral prednisone, with initial progressive improvement of symptoms and normalization of echocardiography. One month later therapy was implemented with administration of intravenous immunoglobulins every two months, with further functional recovery. Our case report confirms that HMGCR autoantibody testing should be part of the initial evaluation in pediatric patients with suspected muscular dystrophy and unrevealing genetic testing or negative family history. Early diagnosis and treatment could be the right approach, but there is still a substantial need for new data., Aquaporins are integral membrane water channels that regulate the flow of water in various tissues and organs. Aquaporin-4 (AQP4) is abundant in skeletal muscle fast fibers, where it is associated with glycolytic metabolism. AQP4 is also expressed by astroglial endfeet, where it is the target of a human autoimmune disease, neuromyelitis optica. To date, the involvement of AQP4 in idiopathic inflammatory myopathies (IIMs) has not been studied. We investigated the immunolocalization of AQP4 and inflammatory markers as CD68, CD56, MMP2, CXCL12, CXCR7, and fast myosin heavy chain in 28 human muscle biopsies obtained from diagnostic samples. The studied population was composed of 19 IIM patients diagnosed as 6 immune mediated necrotizing myopathy patients, 2 anti-synthetase myopathy patients, 6 dermatomyositis patients, a polymyositis patient, 2 inclusion body myositis patients, 2 B-cell inflammatory myopathy patients and 9 control subjects diagnosed as a genetically confirmed myoclonic epilepsy with ragged red fibers patient, a oculopharyngeal muscular dystrophy patient, a type 1 fiber predominance patient and 6 patients who underwent muscle biopsy for suspected myopathy, not confirmed after the clinical and diagnostic workout. AQP4 immunolocalization inversely correlated with MMP2 localization, number of CD68+ macrophages and CD56+ lymphocytes in IIMs. The immunolocalization of AQP4 in normal human myofibers appeared variable among different muscles and patients whereas in IIMs the expression of AQP4 was restricted to the preserved areas of muscle samples where inflammatory cells and molecules were absent. The MMP2 release could be responsible of agrin digestion and disruption of the correct AQP4 sarcolemmal localization., Congenital myasthenic syndromes (CMS) are heterogeneous neuromuscular disorders (NMD) due to mutations in over 25 genes coding for proteins involved in the neuromuscular junction structure and function. Although considered rare, collectively CMS are underestimated due to diagnostic difficulties. In recent years, next-generation sequencing (NGS) has increasingly been used in NMD and might prove to be important in the diagnosis of CMS. Between 2014 and 2018, among over 400 NMD patients referred to our center we evaluated 45 patients with clinical suspicion of CMS. In all we tested the coding regions of 241 genes associated with NMD using a targeted NGS technology. In each case we also collected clinical and laboratory data as well as the results of familial segregation analyses. The distribution of the muscle weakness was recorded using the Human Phenotype Ontology (HPO) codes and nomenclature. A molecular diagnosis was reached in 19/45 cases (42%). Variants of unknown significance were found in 19 patients, whereas 7 cases remained molecularly undefined. Pathogenic mutations were identified in genes already associated with CMS in 13 individuals whereas 6 cases showed mutations in other genes not previously linked to defects of neuromuscular junction. Major features of the diagnosed CMS cases were ptosis (HP:0000508; in 90%) and limb-girdle muscle weakness (HP:0003325; in 60%). This study highlights the advantages of NGS used as a first-tier diagnostic approach in genetically heterogeneous conditions and underlines the importance of a standardized clinical work-up as a component of the diagnostic pathway., Introduction. Congenital myasthenic syndromes (CMS) are a heterogeneous group of early-onset genetic neuromuscular transmission disorders due to mutations in proteins involved in the organization, maintenance, function, or modification of the motor endplate (endplate myopathies) CMS are clinically characterized by abnormal fatigability, or transient or permanent weakness. The field of CMS is steadily expanding. Cases reports. Here we describe 5 unrelated cases (3 F, 2 M, age of 13, 21, 28, 38, 52 years). Their family history was negative. All patients reported first symptoms in the first-second decade of life. The extra-ocular muscles involvement is a common feature, only in one case also associated with diplopia. Severe bulbar weakness at onset was reported in one patient. Muscle fatigability is the more disabling symptom. Fluctuating and/or persistent limb muscle weakness, with both distal and proximal distribution, is also observed, while respiratory involvement is not detected. Blood CPK is normal. Electrophysiological investigation supported the CMS diagnosis. Muscle biopsy showed mild myopathic changes, but in one case scattered fibers with negative acid phosphatase vacuoles. DNA analysis confirmed the genetic diagnosis, with detection of heterozygous variants in the following genes: CHRNA1, RAPSN, CHRNE. Therapy with AchEI appear partially effective in these cases, salbutamol is beneficial in one case., We present two families affected by LGMD D2, an autosomal dominant form of LGMD due to a mutation of TNPO3 gene. This rare disorder was firstly genetically identified in a large Italo-Spanish family (mother and daughter) and we present another family with a congenital myopathy-LGMD phenotype in a child and his mother of Hungarian origin. Clinical data, MRI imaging and quality of life were performed and compared in two families with transportinopathy. The onset of symptoms was often characterized by difficulty in walking or climbing stairs, collected by a standard LGMD questionnaire. Muscle MRI was quantified using Mercuri score. The assessment of Quality of Life(QoL) was done by an Individualized Neuromuscular Quality of Life(INQoL) test. In the Italo-Spanish family, the mutation was identified in a single nucleotide deletion (c.2771delA, p.X924C, exon22) in the TNPO3 gene while in Hungarian family the causative mutation of LGMD D2 is due to an heterozygous frameshift deletion c.2767delC. in exon 23. Mother and daughter of Italian-Spanish family presented marked atrophy of upper girdle muscles. The child of Hungarian family and his mother presented difficulty to stand from the floor. Muscle MRI of daughter and mother of Italo-spanish family showed pronounced atrophy in posterior thigh muscles. In son and mother of Hungarian family we observed generalized muscle atrophy. We monitored with age progression, connective and fat tissue increased, involving mostly posterior thigh, we propose that muscle MRI represents a valuable tool to document disease progression., Increased serum creatine kinase (CK) can be found in several genetically well-defined myopathies, but it can also be associated with no obvious clinical manifestations. Next-generation sequencing (NGS) has recently been proposed as a cost-effective strategy for the molecular diagnosis of inherited neuromuscular disorders. Over a two-year period, we evaluated 66 patients presenting with hyperCKemia and performed targeted NGS studies using a neuromuscular multigene panel. We identified a definitive molecular diagnosis in 33 patients. Among these patients, mutations in RYR1 were found in 11, four cases had mutations in ANO5 and four in genes encoding sarcoglycans, while three cases harbored mutations in CAPN3. Within the Regione Toscana Health project Ingene, genetic data, clinical information (signs/symptoms in the HPO language) and MRC values in 33 positive patients were analyzed using non-metric multidimentional scaling (nMDS), a well-known algorithm for multivariate analysis, and clustering techniques. The algorithm used the information about the specific gene variants (rare and common) found in each patient identifying if specific genes or mutations could cluster and present association with with clinical manifestations. With this method we identified three definite clusters characterized by presence/absence of muscle weakness and dystrophic features in muscle but not matching our earlier categorization based on clinical data only. We anticipate that a computer assisted integration of data will help identify more precisely genotype/phenotype correlations and also pinpoint possible genetic modifiers among hyperckemias., Neuromuscular Diseases require a careful study of the genotype-phenotype association to understand the peculiarities of each subject affected by such conditions, and to tailor a personalized treatment, maximizing its outcome. It is therefore pivotal to collect genetic, instrumental, muscular, physiotherapy, cardiovascular data to draw a comprehensive patient picture, defining treatment plans, monitoring the course of the pathology and helping the clinicians in the patient care. In the InGene project, an integrated tool to collect such data in neuromuscular diseases was developed. It includes several modules, with an integrated user interface. The PhysioTest module allows administering physiotherapy tests, with customized interfaces for the Performance of the Upper Limb (PUL), the Six-Minute Walk Test (6MWT), the Motor Function Measure (MFM) and the North Star Ambulatory Assessment (NSAA), as well as the Neurological Physical Examination. The GenoStore module archives the patient’s genotype and select patients with specific variants. The MRIndex module integrates the muscular MRI, providing an index for the muscular involvement, measuring the fat infiltration within the muscle. Finally, the “InGene” module allows selecting several parameters (also from clinical dataset), applying them exploratory statistical analysis of selected patients, through multivariate techniques, to retrieve patients’ clusters based on correlations between genotype and phenotype as a useful research aid tool. The platform, collecting data within an Electronic Health Record, named Health360, developed as a modular, Cloud-based, Software as a Service (SaaS) platform, represents a diagnostic aid tool for the clinicians in personalizing diagnosis and treatment plans addressed to patients with neuromuscular conditions., GNE myopathy is a rare hereditary inclusion body myopathy (HIBM) due to autosomal recessive mutations in the GNE gene, encoding for an enzyme involved in the sialic acid synthesis. Typical clinical presentation, with onset around the third decade of life, is characterized by weakness of the distal muscles of the leg and subsequent slowly progressive involvement of proximal and upper limbs muscles, generally sparing the quadriceps. Rimmed vacuoles are a distinctive finding at muscle biopsy. We detected the same homozygous GNE mutation c.1853T > C (p.Ile618Thr) in two families originating respectively from Kosovo and Albania. In the first proband, muscle weakness began at 27 years of age with bilateral foot-drop, subtle weakness of iliopsoas and adductors and mild hyperCKemia. Vacuoles and necrosis were reported at the muscle biopsy. Muscle MRI detected atrophy and fibro-fatty substitution of the medial and posterior compartments of the thighs. Inclusion body myopathy was reported in other three members of the family. The second proband was a 23 years old man with myalgia and proximo-distal muscle involvement in the four limbs, started about 3 years earlier. CK levels were up to 5000 U/L. Muscle MRI revealed selective fibro-fatty substitution in lower limbs. Muscle biopsy showed a severe myopathy with rimmed vacuoles. His 22-year-old brother was affected by mild lower limbs muscle weakness with hyperCKemia and rimmed vacuoles at muscle biopsy. Other three members of his family presented with different degrees of muscular involvement. Cardiomyopathy was detected only in one of them. Given the possibly common ethnic origin of the two families, allegedly Sinti, we suggested a possible founder GNE mutation c.1853T > C in this sub-population of Eastern Europe. Similar founder mutations are described in Japanese and Persian Jewish ethnicity., Introduction. Charcot-Marie-Tooth disease (CMT) is an inherited peripheral neuropathy with progressive length-dependent weakness and atrophy of the distal muscles of the limbs. In children there is significant phenotypic variability, which can complicate assessment, diagnosis and treatment. In this study we have examined the upper and lower limbs abnormalities in a group of children with CMT. Methods. Thirteen children (age range 6-17) were evaluated with CMTPedS, Movement ABC-2 Test, 6MWT and Gait Analysis. Results. At CMTPedS two cases were mildly affected (scores 0-14), eight cases moderately affected (15-29) and three severe affected (0-44). At Movement ABC-2 two cases showed normal behaviour, two were “at risk” and eight presented severe difficulty in movement. The patients performed a range between 379 to 513m at 6MWT. Using Gait Analysis we observed in some cases an increase of speed compared to standard walking. At the ankle joint level we documented a reduction of power (W/kg) generated during push-off with decreased of the dorsiflexor moment (N/Kg) during loading response. Discussion and conclusions. A general age-dependent correlation in the scores of CMTPedS emerges and the Movement ABC-2 revealed early difficulty on movement in upper limbs. 6MWT results were within normal range but the pattern of gait in these patients appeared altered indicating the important role of Gait Analysis will monitoring disease outcome. Our pilot study reveals the importance of investigating upper limbs impairment and deterioration of gait in children with CMT to structure timely therapeutic interventions., Introduction. Quantitative skeletal muscle MRI (mMRI) has been widely studied as a reliable outcome measure in patients with Neuromuscular Disorders (NMD). However the standard 3-point Dixon technique scoring system is not widely used in all centers for technical limitations. Conversely, we have developed a MRI-index, a measure extrapolated with an algorithm of image analysis able to derive automatically the fat percentage in mMRI images from NMD patients. Normative values, stratified for ages in healthy subjects, were acquired in our earlier studies. Methods. Using a standard mMRI protocol in the past two years, we acquired myoimaging results of 46 patients (age range 7-70) with variable disease severity. Of these cases, 39 were genetically characterized and included 27 BMD boys, 4 subjects presenting mutations in RYR1, 2 in MHY7, 2 in TPM3, 1 DMD carrier, and 3 patients harboring mutations in other genes. Images of the medial third of the thighs of each subject were taken into account for the analysis of MRI-index. Genotype and phenotype correlations were subsequently made. Results. Analyzing patients with the same genotype (BMD patients) we observed that disease severity correlates with the degree of fat percentage in muscle (high MRI-index). Considering patients with milder phenotype and no fat infiltration at qualitative imaging, MRI-index showed higher values when mutations occur in genes encoding structural muscular proteins. In asymptomatic hyperCKemia and mutations in RYR1, MRI-index was in the normal range. Conclusions. The MRI-index can be a valid tool to detect muscle involvement and early genotype-phenotype correlation in NMD., Myopathies are classified according to limb or cranial muscle affection, but the growing use of the imaging (MRI) showed that many myopathies have significant or selective involvement of the axial musculature. A 83 year-old lady developed weakness and atrophy of neck muscles with dropped head. Electromyography showed myopathic features confirmed by the deltoid muscle biopsy. A genetic panel ruled out several genetic disorders including glycogen storage disease. Case 2 was 74 year-old woman evaluated because of progressing over 5 year weakness of paraspinal muscles and forward flexion of the trunk.There were myopathic features on EMG in limb muscles and neurogenic changes in quadriceps biopsy .Case 3 was 65 year old female evaluated because of spine rigidity, persistent elevation of CK up to 800 UI/L (Normal < 145). Neurological examination showed neck extensor weakness, wasting and weakness of paraspinal muscles. Electromyography suggested chronic myopathy. Discussion The patients presented a late onset myopathy with indolent course, elevation of serum enzymes, predominant weakness of axial musculature. Such presentation led to extensive differential diagnosis including myasthenia gravis,amyotrophic lateral sclerosis, idiopathic inflammatory myopathies.Clinical presentation and ancillary data excluded these diseases. In accordance with profound weakness of neck extensor muscles and wasting of the paraspinals, MRI showed complete fatty transformation of what should be the paraspinal musculature. The diagnosis of axial myopathy presenting and progressing solely or predominantly either with neck extensor and paraspinal muscle weakness is challenging for clinicians and it might extend the clinical phenotype of myopathies with late onset., Camptocormia is characterized by the hyperflexion of the thoracolumbar spine during the upright position. Its etiologies are heterogenous, including parkinsonism and neuromuscular disorders. Here, we report three patients with camptocormia due to a late-onset axial myopathy and we describe a genetic heterogeneity of the disease. The patients are three unrelated woman with a late onset axial myopathy (ranging from 50 to 60 years of age) and in one is present bilateral atrophy of intrinsic hand muscles; the family history is negative a part in one case in which the son had CPK elevation. Next Generation Sequencing analysis of myopathy related genes was performed in the patients and identified variations in two genes: CAPN3 and SQSTM1. In the first patient homozygous p. Arg608Lys in CAPN3 was detected, previously identified in LGMD2A. In the second case, heterozygous p. Pro392Leu in SQSTM1 was identified in the patient and in her son. The variant was previously reported as cause of distal myopathy in double heterozygosity with a TIA1 gene mutation. In the last patient a double heterozygosity for p.Pro439Leu in SQSTM1 gene and p.Asp753Asn in CAPN3 gene was identified. The CAPN3 variant was previously reported in heterozygosity in LGMD patients. The SQSTM1 variant is described associated to different clinical phenotypes ranging from SLA to fronto-temporal dementia. Our findings highlight the genotypic heterogeneity of axial myopathy, speculate a digenic inheritance of the disease and broaden the phenotypic spectrum of recessive calpainopathy., Distal Spinal Muscular Atrophy (DSMA) is a genetically heterogeneous disorder affecting the lower motor neurons. We report clinical and genetic findings of an Italian family with autosomal dominant (AD) DSMA with five affected members in five generations. In all, weakness and atrophy started from leg and foot. Two have sensorineural hearing loss. The younger affected member (V-1), a 5-year-old boy, shows only some difficulties in heel walking. His 27 year-old mother (IV-5) is unable to walk on toe and heel, shows slight atrophy and weakness of the fingers extensor and intrinsic muscles of the hand and bilateral Achilles tendon retraction. His grandmother (III-2) presented a similar weakness and atrophy patterns with slow progression. Motor nerve conduction study showed reduced CMAP amplitude. Sensory conductions were normal and EMG was neurogenic. Biceps brachialis muscle biopsy was neurogenic. Muscle MRI confirmed leg muscles involvement. A mutation screen of 122 genes involved in DSMA revealed three rare heterozygous variants in MYH14 (p.R941L), MME (p.Y347C) and MFN2 (p.R663H) genes. Segregation analysis, performed in IV-5 and V-1, showed that variants in MYH14 and MME genes were present in heterozygosis. MFN2 variant was de novo in III-2. MYH14 is linked to AD distal weakness and deafness. Recently, the p.R941L mutation in MYH14 has been described in a Korean and a North American family with progressive DSMA associated to hearing loss. MME mutations were found only in homozygosis in a late-onset CMT2 phenotype, so we excluded p.Y347C variant as causative. To our knowledge, this is the first Italian family in which these mutations are reported., Mutations in TBC1-domain containing kinase (TBCK) cause a syndrome characterized by hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. Loss of TBCK was shown to decrease mammalian target of rapamycin (mTOR) signaling, increase autophagy, and decrease mitochondrial function. It remained unclear whether muscle weakness and hypotonia in individuals affected by this disorder were neurogenic or associated with progressive muscle disease. All previous muscle biopsy studies were performed in infants and were normal or inconclusive, but several groups had suspected muscle involvement. Here, we report two sisters, initially diagnosed with congenital myopathy and severe psychomotor delay, in whom a novel homozygous truncation in TBCK was found by exome sequencing. We show that muscle disease can be associated with TBCK, as muscle biopsies performed on both sisters show myopathic features. Our findings add to the variability of phenotypes associated with TBCK mutations which can be ascribed to the multiple roles of this protein in intracellular signalling and endolysosomal function in different tissues., Immunoglobulin light chain (AL) amyloidosis is the most frequent type of acquired amyloidosis due to a monoclonal misfolded insoluble light chain that forms amyloid fibrils with a tendency to tissue deposition leading to organ dysfunction. The most common encountered neurological manifestations are carpal tunnel syndrome, sensory-motor polyneuropathy and autonomic neuropathy. Muscle involvement in AL amyloidosis is a rare, under diagnosed condition and can be the initial manifestation of the disease. Affected patients usually present with proximal muscle weakness, macroglossia and, sometimes, muscle pseudohypertrophy. Diagnosis of amyloid myopathy is challenging and can be delayed if Congo red staining is not routinely included in the pathological work-up of muscle biopsy. The prompt recognition of this myopathy as presenting symptom of systemic amyloidosis is important in order to begin the treatment and improve the patient outcome. We report on two patients with multiple myeloma who manifested amyloid myopathy at different stages of their disease. Both patients underwent a muscle biopsy which allowed to reach the correct diagnosis. Congo red staining plays a crucial role in the pathological diagnosis of this disease and its systematic use can likely increase the frequency of diagnosis.

Data on the spectrum of second malignant neoplasms (SMNs) after primary childhood non-Hodgkin's lymphoma (NHL) are scarce. One-hundred-and-eighty-nine NHL patients diagnosed in a 30 years period of 1980-2010 developing an SMN were retrieved from 19 members of the European Intergroup for Childhood NHL and/or the international Berlin-Frankfurt-Münster Study Group. Five subgroups of SMNs were identified: (1) myeloid neoplasms (n = 43; 23%), (2) lymphoid neoplasms (n = 51; 27%), (3) carcinomas (n = 48; 25%), (4) central nervous system (CNS) tumors (n = 19; 10%), and (5) "other" SMNs (n = 28; 15%). In 37 patients (20%) preexisting disorders were reported with 90% having any kind of cancer predisposition syndrome (CPS). For the 189 primary NHL patients, 5-year overall survival (OS) after diagnosis of an SMN was 56 ± 4%, being worst for patients with preexisting disorders at 28 ± 8%. Five-year OS rates were 38 ± 8%, 59 ± 7%, 79 ± 8%, 34 ± 12%, and 62 ± 11%, respectively, for patients with myeloid and lymphoid neoplasms, carcinomas, CNS tumors, and "other" SMNs (p < 0.0001). Patients with SMNs after childhood NHL having a reported CPS, mostly mismatch repair disorders, carried a very poor prognosis. Moreover, although outcome was favorable in some subtypes of SMNs after childhood NHL (carcinomas, lymphoid neoplasms), other SMNs such as myeloid neoplasms and CNS tumors had a dismal prognosis.

Following publication of the original article [1], the typesetters have missed to add the below listed study group author names in XML in author group section. The study group authors have been added to the author group and are presented correctly in this correction article. A minor change has been made to the electronic supplementary files by removing the yellow highlights and included in this correction. Study Group Authors: Andrea Festa1 Gaia Claudia Viviana Viola1 Ilaria Zerbini1 Cristina Fatigoni2 Sara Levorato2 Silvano Monarca2 Tania Salvatori2 Samuele Vannini2 Gabriele Donzelli3 Francesco Bagordo4 Mattia De Giorgi4 Marcello Guido4 Alessandra Panico4 Francesca Serio4 Maria Rosaria Tumolo4 Silvia Bonetta5 Marta Gea5 Giorgio Gilli5 Cristina Pignata5 Tiziana Schilirò5 Valeria Romanazzi5 Camilla Furia6 Beatrice Bruni7 Beatrice Casini7 Study Group Author Affiliations: 1Department of Medical and Surgical Specialties, Radiological Science and Public Health, University of Brescia, 11 Viale Europa, 25123 Brescia, Italy 2Department of Pharmaceutical Sciences, Unit of Public Health, University of Perugia, Via del Giochetto, 06122 Perugia, Italy 3Department of Biology, University of Pisa, 35/39 Via S. Zeno, 56127 Pisa, Italy 4Department of Biological and Environmental Science and Technology, University of Salento, 165 Via Monteroni, 73100 Lecce, Italy 5Department of Public Health and Pediatrics, University of Torino, 94 Piazza Polonia, 10126 Turin, Italy 6Brescia Municipality, 1 Piazza Repubblica, 25100 Brescia, Italy 7Department of Translational Research, N.T.M.S., University of Pisa, 35/39 Via S. Zeno, 56127 Pisa, Italy Author details 1 Department of Medical and Surgical Specialties, Radiological Science and Public Health, University of Brescia, 11 Viale Europa, 25123 Brescia, Italy. 2 Department of Pharmaceutical Sciences, Unit of Public Health, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. 3 Department of Biology, University of Pisa, 35/39 Via S. Zeno, 56127 Pisa, Italy. 4 Department of Biological and Environmental Science and Technology, University of Salento, 165 Via Monteroni, 73100 Lecce, Italy. 5 Department of Public Health and Pediatrics, University of Torino, 94 Piazza Polonia, 10126 Turin, Italy. 6 Multi-sector and Technological Service Centre - CSMT Gestione S.c.a.r.l, 45 Via Branze, 25123 Brescia, Italy. 7 Brescia Municipality, 1 Piazza Repubblica, 25100 Brescia, Italy. 8 Department of Statistic, Computer Science, Applications, University of Florence, 59 Viale Morgagni, 50134 Florence, Italy.

Background Children are at high risk of suffering health consequences of air pollution and childhood exposure can increase the risk of developing chronic diseases in adulthood. The MAPEC_LIFE project, funded by EU Life+ Programme (LIFE12 ENV/IT/000614), aimed to evaluate the association between air pollution exposure and biomarkers of early effect in children and to propose guidance for implementing environmental policies. Methods The study was carried out on 6-8-year-old children. Micronucleus (MN) frequency was investigated in buccal cells of children and its association with air pollution exposure was assessed applying multiple Poisson regression mixed models, including socio-demographic and lifestyle factors as confounders. We also dichotomize air pollutants concentration according to the EU Ambient Air Quality Directives and WHO Air Quality Guidelines in all Poisson regression models to assess their risk predictive capacity. Results The project involved 1149 children providing buccal cells in winter and spring. 2139 biological samples were included in the analysis (1093 collected in winter, 1046 in spring). The analysis of the association between MN frequency and air quality parameters found positive associations for PM10, PM2.5, benzene, SO2 and ozone. Considering EU Directives, an association was found between MN frequency and PM10 exposure higher than the annual limit value, with an increase of the risk of 17.9% (95%CIs: 0.6-38.1%). Considering WHO Guidelines, the exposures to levels of PM10, benzene and BaP higher than the annual limits were associated with MN frequency, with a risk increase of 22.5%, 27.8% and 59.8% (95%CIs: 3.9-44.3%, 3.8-57.3%, 21.0-111.1%), respectively. Conclusions The analyses conducted showed an association between MN frequency in buccal cells of children and levels of some air pollutants, even at concentration below EU and WHO thresholds, which hence seemed to be insufficient for protecting children from this type of damage. Key messages Air pollution exposure induced chromosomal damage in buccal cells of children, even at concentration below the law limits. Early biological damage detected might be predictive of the occurrence of future harmful effects in humans, at a population level.

Late-onset Pompe disease (LOPD) is characterized by progressive muscle weakness, respiratory muscle dysfunction, and minor cardiac involvement. Although in LOPD, as in other neuromuscular diseases, controlled low impact sub-maximal aerobic exercise and functional ability exercise can improve general functioning and quality of life, as well as respiratory rehabilitation, the bulk of evidence on that is weak and guidelines are lacking. To date, there is no specific focus on rehabilitation issues in clinical recommendations for the care of patients with Pompe disease, and standard practice predominantly follows general recommendation guidelines for neuromuscular diseases. The Italian Association of Myology, the Italian Association of Pulmonologists, the Italian Society of Neurorehabilitation, and the Italian Society of Physical Medicine and Rehabilitation, have endorsed a project to formulate recommendations on practical, technical, and, whenever possible, disease-specific guidance on rehabilitation procedures in LOPD, with specific reference to the Italian scenario. In this first paper, we review available evidence on the role of rehabilitation in LOPD patients, particularly addressing the unmet needs in the management of motor and respiratory function for these patients. Late-onset Pompe disease (LOPD) is characterized by progressive muscle weakness, respiratory muscle dysfunction, and minor cardiac involvement. Although in LOPD, as in other neuromuscular diseases, controlled low impact sub-maximal aerobic exercise and functional ability exercise can improve general functioning and quality of life, as well as respiratory rehabilitation, the bulk of evidence on that is weak and guidelines are lacking. To date, there is no specific focus on rehabilitation issues in clinical recommendations for the care of patients with Pompe disease, and standard practice predominantly follows general recommendation guidelines for neuromuscular diseases. The Italian Association of Myology, the Italian Association of Pulmonologists, the Italian Society of Neurorehabilitation, and the Italian Society of Physical Medicine and Rehabilitation, have endorsed a project to formulate recommendations on practical, technical, and, whenever possible, disease-specific guidance on rehabilitation procedures in LOPD, with specific reference to the Italian scenario. In this first paper, we review available evidence on the role of rehabilitation in LOPD patients, particularly addressing the unmet needs in the management of motor and respiratory function for these patients.

Analysis and integration in a decision support system (DSS) of resource use efficiency, and environmental and socio-economic impact. Evaluation of the environmental impact.

Amphotericin B is a fungicidal substance that is treatment of choice for most systemic fungal infections affecting immunocompromised patients. However, severe side effects have limited the utility of this drug. The aim of this study was to evaluate the antifungal effect of the combination of amphotericin B with black tea or white tea and protective of citotoxic effect. The present study shows that white and black teas have additive effects with amphotericin B against some species Candida. In addition, the combination of white and black tea with amphotericin B may reduce the toxicity of amphotericin B to red blood cells. Our results suggest that white and black tea is a potential agent to combine with amphotericin for antifungal efficacy and to reduce the amphotericin dose to lessen side effects. Resumo A anfotericina B é o tratamento de escolha para a maioria das infecções fúngicas sistémicas que afetam os doentes imunocomprometidos. No entanto, efeitos secundários graves têm limitado a utilidade desta droga. O objetivo deste estudo foi avaliar o efeito antifúngico da combinação de anfotericina B com chá preto ou chá branco, bem como o efeito citotóxico desta combinação sobre hemáceas. O presente estudo demonstra que o chá branco e preto de Camellia sinensis têm efeitos aditivos com anfotericina B contra algumas espécies de Candida sp. Além disso, a combinação de chá branco e preto com anfotericina B pode reduzir a toxicidade da anfotericina B em hemáceas. Nossos resultados sugerem que o chá branco e preto são agentes potenciais para associação com anfotericina B contribuindo para eficácia antifúngica, bem como redução de toxicidade.

The Mediterranean diet (MD) is considered one of the healthiest dietary models, as it decreases the risk of chronic diseases and may modulate the organism's early response to environmental pollution. In recent decades, Mediterranean countries have been replacing their traditional diet with other less healthy eating habits, especially among children and teenagers.

Introduction Genotoxic biomarkers have been studied largely in adult population, but few studies so far have investigated them in children exposed to air pollution. Children are a high-risk group as regards the health effects of air pollution and some studies suggest that early exposure during childhood can play an important role in the development of chronic diseases in adulthood. The objective of the project is to evaluate the associations between the concentration of urban air pollutants and biomarkers of early biological effect in children, and to propose a model for estimating the global risk of early biological effects due to air pollutants and other factors in children. Methods and analysis Two biomarkers of early biological effects, DNA damage by the comet assay and the micronuclei (MN) test, will be investigated in oral mucosa cells of 6–8-year-old children. Concurrently, some toxic airborne pollutants (polycyclic aromatic hydrocarbon (PAH) and nitro-PAH) and in vitro air mutagenicity and toxicity in ultra-fine air particulates (PM0.5) will be evaluated. Furthermore, demographic and socioeconomic variables, other sources of exposures to air pollutants and lifestyle variables will be assessed by a structured questionnaire. The associations between sociodemographic, environmental and other exposure variables and biomarkers of early biological effect using univariate and multivariate models will be analysed. A tentative model for calculating the global absolute risk of having early biological effects caused by air pollution and other variables will be proposed. Ethics and dissemination The project has been approved by the Ethics Committees of the local Health Authorities. The results will be communicated to local Public Health Agencies, for supporting educational programmes and health policy strategies. LIFE+2012 Environment Policy and Governance. LIFE12 ENV/IT/000614.

Alzheimer's disease (AD) is a neurodegenerative disorder that accounts for two-thirds of dementia cases in the elderly and may show preliminary symptoms for decades. The dry leaf extract of Ginkgo biloba (EGb 761) has been used as a dietary supplement for the treatment of mental disorders in both humans and animals. Objective: This study examined the immunological effect of EGb 761 in the treatment of rats with experimental Alzheimer's disease. Methodology: The sample consisted of 40 Wistar male rats, weighing 200-250 grams and divided into 4 groups: 21 days control, 42 days control, 21 days treated, and 42 days treated. Stereotaxic surgery was performed with the coordinates of AP=-3.12, ML=3.6 mm, and DV=3.0; animals were undisturbed for 5 days after surgery and brought back to stereotaxic to be injected with 2 μL (1 mg) of Beta-amiloide25-35 toxin in the CA1 region. Animals in the treated group received 100 mg/kg of EGb 761 by gavage. Animals were euthanized at 21 and 42 days; 1 ml of blood was collected for the flow cytometry analysis of pro- and anti-inflammatory cytokines. Results: A significant difference was observed between the pro-inflammatory cytokines: IL-6 and IFN-γ showed low levels of concentration compared to IL-2 and TNF-α. The IL-4 and IL-10 anti-inflammatory cytokines were present at high levels of concentration in the 21 and 42 days treated groups. Conclusion: EGb 761 was effective in reducing serum levels of pro-inflammatory cytokines, thereby promoting a reduction in the inflammatory process generated by AD.

The MAPEC-Life project aims to study the biological effects of early exposure to air pollutants on the oral mucosa cells of school-age children in five Italian cities. A questionnaire was created to evaluate the association between outdoor and indoor airborne pollutants, lifestyle, diet and biomarker effects. The feasibility and reliability of the questionnaire were evaluated.