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Background: The present study aimed at deriving equating norms to estimate scores on the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) based on those on the ALS Cognitive Behavioral Screen (ALS-CBS™) in an Italian cohort of non-demented ALS patients. Methods: ALS-CBS™ and ECAS scores of 293 ALS patients without frontotemporal dementia were retrospectively retrieved. Concurrent validity of the ALS-CBS™ towards the ECAS was tested by covarying for demographics, disease duration and severity, presence of C9orf72 hexanucleotide repeat expansion and behavioural features. A linear-smoothing equipercentile equating (LSEE) model was employed to derive ALS-CBS™-to-ECAS cross-walks. Gaps in LSEE-based estimation were managed via a linear regression-based equating approach. Equivalence between empirical and derived ECAS scores was tested via a two-one-sided test (TOST) procedure for the dependent sample. Results: The ALS-CBS™ predicted the ECAS (β = 0.75), accounting for the vast majority of its variance (60% out of an R 2 = 0.71). Consistently, a strong, one-to-one linear association between ALS-CBS™ and ECAS scores was detected (r = 0.84; R 2 = 0.73). The LSEE was able to estimate conversions for the full range of the ALS-CBS™, except for raw scores equal to 1 and 6 – for whom a linear equating-based equation was derived. Empirical ECAS scores were equivalent to those derived with both methods. Discussion: Italian practitioners and researchers have been herewith provided with valid, straightforward cross-walks to estimate the ECAS based on ALS-CBS™ scores in non-demented ALS patients. Conversions herewith provided will help avoid cross-sectional/longitudinal inconsistencies in test adoption within research, and possibly clinical, settings.

Abnormalities in cortical sources of resting-state eyes-closed electroencephalographic (rsEEG) rhythms recorded by hospital settings (10–20 electrode montage) with 19 scalp electrodes provide useful markers of neurophysiological dysfunctions in the vigilance regulation in patients with Alzheimer's disease dementia (ADD). Here we tested whether these markers may be effective from a few scalp electrodes towards the use of low-cost recording devices. Clinical and rsEEG data acquired in hospital settings (10–20 electrode montage) from 88 ADD participants and 68 age-, education-, and sex-matched normal elderly controls (Nold) were available in an international Eurasian database. Standard spectral FFT analysis of rsEEG data for individual delta, theta, and alpha frequency bands was from C3-P3, C4-P4, P3-O1, and P4-O2 bipolar channels. As compared to the Nold group, the ADD group showed increased delta, theta, low-frequency alpha power density and decreased high-frequency alpha power density at all those bipolar channels. The highest classification accuracy between the ADD and Nold individuals reached 90 % (area under the receiver operating characteristic curve) using Alpha2/Theta power density computed at the C3-P3 bipolar channel. Standard rsEEG power density computed from a few posterior bipolar channels successfully classified Nold and ADD individuals, thus encouraging a massive prescreening of neurophysiological mechanisms underpinning the vigilance dysregulation in underserved old seniors. Ministero della Salute

Abnormalities in cortical sources of resting-state eyes closed electroencephalographic (rsEEG) rhythms recorded by hospital settings (10-20 montage) with 19 scalp electrodes characterized Alzheimer's disease (AD) from preclinical to dementia stages. An intriguing rsEEG application is the monitoring and evaluation of AD progression in large populations with few electrodes in low-cost devices. Here we evaluated whether the above-mentioned abnormalities can be observed from fewer scalp electrodes in patients with mild cognitive impairment due to AD (ADMCI). Clinical and rsEEG data acquired in hospital settings (10-20 montage) from 75 ADMCI participants and 70 age-, education-, and sex-matched normal elderly controls (Nold) were available in an Italian-Turkish archive (PDWAVES Consortium; www.pdwaves.eu ). Standard spectral fast fourier transform (FFT) analysis of rsEEG data for individual delta, theta, and alpha frequency bands was computed from 6 monopolar scalp electrodes to derive bipolar C3-P3, C4-P4, P3-O1, and P4-O2 markers. The ADMCI group showed increased delta and decreased alpha power density at the C3-P3, C4-P4, P3-O1, and P4-O2 bipolar channels compared to the Nold group. Increased theta power density for ADMCI patients was observed only at the C3-P3 bipolar channel. Best classification accuracy between the ADMCI and Nold individuals reached 81% (area under the receiver operating characteristic curve) using Alpha2/Theta power density computed at the C3-P3 bipolar channel. Standard rsEEG power density computed from six posterior bipolar channels characterized ADMCI status. These results may pave the way toward diffuse clinical applications in health monitoring of dementia using low-cost EEG systems with a strict number of electrodes in lower- and middle-income countries.

Objectives: To evaluate the outcomes of bone regeneration using a customized titanium mesh scaffold to cover a bone graft for reconstruction of complex defects of the jaws. Materials and Methods: 19large defects were digitally reconstructed using CT scans according to the prosthetic requirements. A titanium mesh scaffold was designed to cover the bone (autologous/bovine bone particulate) graft. At least 6months after surgery, a new cone-beam CT was taken. The pre- and postoperative CT datasets were then converted into three-dimensional models and digitally aligned. The actual mesh position was compared to the virtual position to assess the reliability of the digital project. The reconstructed bone volumes (RBVs) were calculated according to the planned bone volumes (PBVs), outlining the areas under the mesh. These values were then correlated with the number of exposures, locations of atrophy, and virtually planned bone volume. Results: The mean matching value between the planned position of the mesh and the actual one was 82±13.4%. 52.3% (40% early and 60% late) exposures were observed, with 15.8% exhibiting infection. 26.3% resulted as failures. The amount of reconstructed bone volume (RBV) in respect to PBV was 65±40.5%, including failures, and 88.2±8.32% without considering the failures. The results of the exposure event were statistically significant (p=.006) in conditioning the bone volume regenerated. Conclusions: This study obtained up to 88% of bone regeneration in 74% of the cases. The failures encountered (26%) should underline the operator's expertise relevance in conditioning the final result.

BackgroundThe present investigation aimed at testing the psychometrics and diagnostics of the Italian version of the Caregiver Behavioral Questionnaire (CBQ) from the ALS Cognitive Behavioral Screen (ALS-CBS™), as well as its case–control discrimination, in a cohort of non-demented patients with ALS.MethodsThe caregivers of N = 265 non-demented patients with ALS and N = 99 healthy controls (HCs) were administered the CBQ and the Edinburgh Cognitive and Behavioural ALS Screen-Carer Interview (ECAS-CI). For N = 98 patients, an in-depth behavioural/psychopathological assessment via the Frontal Behavioural Inventory (FBI), the Dimensional Apathy Scale (DAS), the State and Trait Anxiety Inventory-Form Y (STAI-Y), and the Beck Depression Inventory (BDI) was also available. Factorial and construct validity, internal reliability, and diagnostics against an abnormal ECAS-CI score were tested in patients. Case–control discrimination was explored through logistic regression.ResultsThe CBQ was internally reliable (McDonald’s ω = 0.90) and underpinned by a simple, unidimensional structure; it converged with ECAS-CI, FBI, and DAS scores and diverged from STAI-Y and BDI ones. A cutoff of ≤ 33 accurately detected abnormal ECAS-CI scores (AUC = 0.85), yielding optimal error- and information-based diagnostics. The CBQ was independent of demographic and disease-related variables and discriminated patients from HCs (p < 0.001).DiscussionThe Italian version of the CBQ from the ALS-CBS™ is a valid, reliable, diagnostically sound, and feasible screener for detecting frontotemporal-like behavioural changes in non-demented patients with ALS. Its adoption is thus recommended within clinical practice and research in the view of providing preliminary information on whether the administration of more extensive behavioural instruments is needed.

Background: The present study aimed at (1) providing further validity and reliability evidence for the Italian version of the cognitive section of the ALS Cognitive Behavioral Screen (ALS-CBS™) and (2) testing its diagnostics within an Italian ALS cohort, as well as at (3) exploring its capability to discriminate patients from healthy controls (HCs). Methods: N = 293 non-demented ALS patients were administered the cognitive sections of the ALS-CBS™ and Edinburgh Cognitive and Behavioural ALS Screen (ECAS). N = 96 HCs demographically matched with N = 96 patients were also administered the cognitive section of the ALS-CBS™. In patients, factorial and construct validity, internal reliability, and diagnostics against a defective score on the cognitive section of the ECAS were tested. Case–control discrimination was assessed via a logistic regression. Results: ALS-CBS™ cognitive subscales were underpinned by a simple, unidimensional structure, internally reliable (McDonald’s ω = 0.74), and mostly related with ECAS executive and fluency scores (rs = 0.54–0.71). Both raw and age- and education-adjusted scores on the cognitive section of the ALS-CBS™ accurately detected ECAS-defined cognitive impairment (AUC = 0.80 and.88, respectively), yielding optimal error-based, information-based and unitary diagnostics. A cut-off of < 15.374 was identified on adjusted scores. The test was able to discriminate patients from HCs (p < 0.001). Discussion: The cognitive section of the Italian ALS-CBS™ is a valid, reliable, and diagnostically sound ALS-specific screener for detecting frontotemporal, executive-/attentive-based cognitive inefficiency in non-demented ALS patients, being also able to discriminate them from normotypical individuals.

Alzheimer's disease dementia (ADD) is the most diffuse neurodegenerative disorder belonging to mild cognitive impairment (MCI) and dementia in old persons. This disease is provoked by an abnormal accumulation of amyloid-beta and tauopathy proteins in the brain. Very recently, the first disease-modifying drug has been licensed with reserve (i.e., Aducanumab). Therefore, there is a need to identify and use biomarkers probing the neurophysiological underpinnings of human cognitive functions to test the clinical efficacy of that drug. In this regard, event-related electroencephalographic potentials (ERPs) and oscillations (EROs) are promising candidates. Here, an Expert Panel from the Electrophysiology Professional Interest Area of the Alzheimer's Association and Global Brain Consortium reviewed the field literature on the effects of the most used symptomatic drug against ADD (i.e., Acetylcholinesterase inhibitors) on ERPs and EROs in ADD patients with MCI and dementia at the group level. The most convincing results were found in ADD patients. In those patients, Acetylcholinesterase inhibitors partially normalized ERP P300 peak latency and amplitude in oddball paradigms using visual stimuli. In these same paradigms, those drugs partially normalize ERO phase-locking at the theta band (4–7 Hz) and spectral coherence between electrode pairs at the gamma (around 40 Hz) band. These results are of great interest and may motivate multicentric, double-blind, randomized, and placebo-controlled clinical trials in MCI and ADD patients for final cross-validation. United States Department of Health & Human Services National Institutes of Health (NIH) - USA

Becker muscular dystrophy (BMD) is an X-linked disease leading to muscle wasting and weakness. The decrease in lean body mass (LBM) in Duchenne muscular dystrophy, has shown correlation with loss of muscle function and bone density (BD). Myokines (including irisin) are hormones secreted by skeletal muscle that allow crosstalk between muscle and bone. The present study analyzed body composition and circulating myokine levels in a cohort of BMD patients; moreover, the association between dual energy X-ray absorptiometry (DXA) parameters, functional motor assessments, and myokine levels was investigated.All patients underwent DXA, blood samples for myokine assays, and functional motor assessments. A group of healthy controls (HCs) was also included.Thirty BMD patients, median age at evaluation 36.0 y [26.0-41.0], were included. Twenty-nine patients underwent whole-body DXA. Median value of total body Z-score was -0.70. The prevalence of low skeletal muscle mass defined as appendicular skeletal muscle mass index (ASMMI) 7.59 kg/mDXA is a useful tool to evaluate body composition in BMD patients; the decrease in BD and LBM is associated with a reduction of motor function in BMD.

Objectives: The aim was to evaluate the role of resorbable membranes applied over customized titanium meshes related to soft tissue healing and bone regeneration after vertical/horizontal bone augmentation. Materials and Methods: Thirty patients with partial edentulism of the maxilla/mandible, with vertical/horizontal reabsorption of the alveolar bone, and needing implant-supported restorations, were randomly divided into two groups: Group A was treated using only custom-made meshes (Mesh-) and Group B using custom-made meshes with cross-linked collagen membranes (Mesh+). Data collection included surgical/technical and healing complications, “pseudo-periosteum” thickness, bone density, planned bone volume (PBV), regenerated bone volume (RBV), regeneration rate (RR), vertical bone gain (VBG), and implant survival in regenerated areas. Statistical analysis was performed between the two study groups using a significance level of α=.05. Results: Regarding the healing complications, the noninferiority analysis proved to be inconclusive, despite the better results of group Mesh+ (13%) compared to group Mesh- (33%): estimated value −1.13 CI-95% from −0.44 to 0.17. Superiority approach confirmed the absence of significant differences (p=.39). RBV was 803.27mm3 and 843.13mm3, respectively, and higher RR was observed in group Mesh+ (82.3%) compared to Mesh- (74.3%), although this value did not reach a statistical significance (p=.44). All 30 patients completed the study, receiving 71 implants; 68 out of them were clinically stable and in function. Conclusion: The results showed that customized meshes alone do not appear to be inferior to customized meshes covered by cross-linked collagen membranes in terms of healing complication rates and regeneration rates, although superior results were observed in group Mesh+compared to group Mesh- for all variables.

The temporo-parietal junction (TPJ) is a cortical area contributing to a multiplicity of visual, language-related and cognitive functions. In line with this functional richness, also the organization of the underlying white matter is highly complex and includes several bundles. The few studies tackling to date the outcome and neurological burdens of surgical operations addressing TPJ document the presence of language disturbances and visual field damages, with the latter hardly recovered in time. This observation advocates for procedures identifying the optic radiation (OR) bundles crossing the white matter (WM) below TPJ. In the present study we adopted a multimodal approach to address the anatomo-functional correlates of the dorsal loop (DL) of the OR. In particular, we combined cadavers’ dissection with tractographic and electrophysiological data collected in drug-resistant epileptic patients explored by stereoelectroencephalography (SEEG). Cadaver dissection allowed us to appreciate the position and geometrical properties of the DL. More surprisingly, both tractographic and electrophysiological observations converged on a unitary picture highly coherent with the data obtained by neuroanatomical observation.The combination of diverse and multimodal observations allows to overcome the limitations intrinsic to single methodologies, and to define a unitary picture which makes it possible to investigate DL presurgically and at the individual patient level, aiming at limiting the postsurgical damages. Notwithstanding, such a combined approach could serve as a model of investigation for future neuroanatomical inquiries tackling WM fibers anatomy and function through SEEG-derived neurophysiological data.

Table S1. Cell samples used for CAGE profiling. Table S2. Public datasets used in this study. Table S3. Differentially expressed promoters in FANTOM5 NSCLC cell lines. Table S4. Up-regulated/hypomethylated promoters in NSCLC cell lines. Table S5. Epi-markers in NSCLC. Table S6. Extended information for Table 2. Table S7. Univariate and multivariate Cox regression models of TCGA LUAD and LUSC datasets. Table S8. Gene expression profiling of SAECs treated with 5-aza-dC and TSA. Table S9. Up-regulated/hypomethylated genes in NSCLC cell lines that show up-regulation by 5-aza-dC and TSA in SAECs. Table S10. Promoters and methylation array probes that overlap nine families of repetitive elements. Table S11. Promoters and methylation array probes that overlap REP522 repetitive elements. Table S12. Transcripts commonly down-regulated by two different MYEOV siRNAs in A549 cells. Table S13. The relationship between expression of 22 epi-markers and EGFR/KRAS mutation in TCGA LUAD dataset.

Lung cancer is the leading cause of cancer-related deaths worldwide. The majority of cancer driver mutations have been identified; however, relevant epigenetic regulation involved in tumorigenesis has only been fragmentarily analyzed. Epigenetically regulated genes have a great theranostic potential, especially in tumors with no apparent driver mutations. Here, epigenetically regulated genes were identified in lung cancer by an integrative analysis of promoter-level expression profiles from Cap Analysis of Gene Expression (CAGE) of 16 non–small cell lung cancer (NSCLC) cell lines and 16 normal lung primary cell specimens with DNA methylation data of 69 NSCLC cell lines and 6 normal lung epithelial cells. A core set of 49 coding genes and 10 long noncoding RNAs (lncRNA), which are upregulated in NSCLC cell lines due to promoter hypomethylation, was uncovered. Twenty-two epigenetically regulated genes were validated (upregulated genes with hypomethylated promoters) in the adenocarcinoma and squamous cell cancer subtypes of lung cancer using The Cancer Genome Atlas data. Furthermore, it was demonstrated that multiple copies of the REP522 DNA repeat family are prominently upregulated due to hypomethylation in NSCLC cell lines, which leads to cancer-specific expression of lncRNAs, such as RP1-90G24.10, AL022344.4, and PCAT7. Finally, Myeloma Overexpressed (MYEOV) was identified as the most promising candidate. Functional studies demonstrated that MYEOV promotes cell proliferation, survival, and invasion. Moreover, high MYEOV expression levels were associated with poor prognosis.Implications: This report identifies a robust list of 22 candidate driver genes that are epigenetically regulated in lung cancer; such genes may complement the known mutational drivers.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/10/1354/F1.large.jpg. Mol Cancer Res; 15(10); 1354–65. ©2017 AACR.

Figure S1. Methylation and expression levels of genes in lung cancer and normal lung tissues. Figure S2. Expression levels of epi-markers in TCGA LUAD dataset. Figure S3. Expression levels of epi-markers in TCGA LUSC dataset. Figure S4. The signature of 22 epigenetically regulated genes in non-small cell lung cancer. Figure S5. ZENBU genome browser view of RP1-90G24.10 and AL022344.4. Figure S6. ZENBU genome browser view of MYEOV. Figure S7. Higher MYEOV expression is associated with poor prognosis in NSCLC.

To define the presence and type of frontotemporal dysfunction in amyotrophic lateral sclerosis (ALS), different screening tools have been created. Currently, the most used screening tests are the Edinburgh cognitive and behavioural ALS screen (ECAS) and the ALS cognitive behavioural screen (ALS-CBS). The objective of this study was to compare the ability of ECAS and ALS-CBS in classifying non-demented ALS patients according to Strong criteria. One-hundred and fifty-four in- and out-patients with an age > 18 and a definite or probable ALS diagnosis were recruited between September 2019 and February 2020 at NeMO Clinical Centre and at Istituto Auxologico Italiano in Milan and underwent the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) and the ALS Cognitive Behavioural Screen (ALS-CBS). Exclusion criteria involved patients with a diagnosis of FTD, with a severe cognitive deterioration and/or an important behavioural impairment, with a significant psychiatric disorder or with the co-presence of another significant illness. The distribution of patients according to Strong criteria was different for ECAS and ALS-CBS and the degree of agreement between the two tests in terms of Cohen's Kappa coefficient resulted equal to 0.2047 with a 95% confidence limits interval between 0.1122 and 0.2973. This study for the first time compares the ability of ECAS and ALS-CBS in stratifying ALS patients. Further studies will be conducted to better understand the reasons underlying the differences between these two tests in classifying the different subtypes of fronto-temporal dysfunction in ALS.

STUDY OBJECTIVES: Excessive daytime sleepiness (EDS) in myotonic dystrophy type 1 is mostly of central origin but it may coexist with sleep-related breathing disorders. However, there is no consensus on the sleep protocols to be used, assessments vary, and only a minority of patients are regularly tested or are on treatment for EDS. Our study presents data on self-reported and objective EDS in adult-onset myotonic dystrophy type 1. METHODS: Sixty-three patients with adult-onset DM1 were subjected to EDS-sleep assessments (polysomnography, Multiple Sleep Latency Test, Epworth Sleepiness Scale). Correlation coefficients were computed to assess the relationship between sleep and sleepiness test results, fatigue, and quality of life. RESULTS: 33% and 48% of patients had EDS based, respectively, on the Epworth Sleepiness Scale and the Multiple Sleep Latency Test, with a low concordance between these tests (k = 0.19). Thirteen patients (20%) displayed 2 or more sleep-onset rapid eye movement periods on Multiple Sleep Latency Test. Patients having EDS by Multiple Sleep Latency Test had a shorter disease duration (P < .05), higher total sleep time and sleep efficiency and lower wake after sleep onset on polysomnography. Patients with self-reported EDS reported significantly higher fatigue score compared with patients without EDS (P < .05). No other difference was found in demographic, clinical, and respiratory features. CONCLUSIONS: EDS test results are contradictory, making treatment options difficult. Combining quantitative tests and self-reported scales may facilitate physicians in planning EDS care with patients and families. CITATION: Sansone VA, Proserpio P, Mauro L, et al. Assessment of self-reported and objective daytime sleepiness in adult-onset myotonic dystrophy type 1. J Clin Sleep Med. 2021;17(12):2383–2391.

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.

In this study, we aimed to systematically review and critically appraise the available literature concerning the effectiveness of marsupialisation and decompression on the reduction of cystic jaw lesions. The 'Preferred Reporting Items for Systematic Reviews and Meta-Analysis' guidelines were followed and the study protocol was registered at the 'International Prospective Register of Systematic Reviews' (CRD42019116099). Six main databases were searched: Embase, LILACS, PubMed, Scopus, The Cochrane Library, and Web of Science. Searches were complemented with three grey literature sources: Google Scholar, ProQuest, and Open Grey. Any reduction measures, compared with preoperative status or other procedures, were considered. Risk of bias was assessed using the Joanna Briggs Institute Critical Appraisal Tool. Thirty-one studies were included, of which five were judged with low, 24 with moderate, and two with high risk of bias. Considering surgical approach, 20 studies assessed the decompression and 11 the marsupialisation technique. Most studies considered these techniques as preliminary treatments, followed by enucleation. From 1088 lesions found, most were odontogenic keratocysts (33.8%), followed by unicystic ameloblastomas (21.0%), dentigerous cysts (20.6%), and radicular cysts (8.4%). Large lesions and younger individuals frequently presented more favourable responses to treatment and anatomical location was not associated with lesion reduction overall. The intervention duration generally ranged between one to two years. In conclusion, marsupialisation and decompression were mostly considered as preliminary treatments, followed by enucleation. Lesion reduction was generally considered insufficient for these techniques to be used as definitive therapies, although benefits concerning the diminished invasiveness of the secondary surgery were often proposed.

The aim of this study was to adapt the Facioscapulohumeral Muscular Dystrophy – Health Index (FSHD-HI) to an Italian population affected by FSHD by translating, validating, and testing this instrument in an Italian cohort. Italian FSHD patients were interviewed regarding the form and content of the translated instrument. Subsequently, forty FSHD patients were recruited to test the reliability (Intraclass Correlation Coefficient, ICC for test-retest; and Cronbach’s Alpha for Internal consistency), known groups (Mann-Whitney U test and Area Under the Curve, AUC) and concurrent validity (Pearson’s and Spearman’s Rank Correlation Coefficient) of the instrument by serially completing the FSHD-HI and an extensive set of tests measuring the neuromotor, psychological and cognitive functions, and perceived quality of life (QoL) aspects. The Italian translation of the FSHD-HI and its subscales were highly relevant to patients, had a high internal consistency (Cronbach’s Alpha = 0.90), optimal test-retest reliability (ICC= 0.95), and was significantly associated with motor function, respiratory function, and QoL assessments. Overall, the Italian FSHD-HI is a valid and well-suited measurement of the multi-dimensional aspects of disease burden in FSHD patients. Facioscapulohumeral muscular dystrophy (FSHD) negatively impacts the quality of life and increases the disease burden.It is important for the clinical community to have a valid instrument that can serially measure a patient’s perception of their multifactorial disease burden in FSHD.The Facioscapulohumeral Muscular Dystrophy – Health Index (FSHD-HI) is a valid instrument that allows patients to provide their perspective regarding their current health state.FSHD-HI-IT provides a valid option for measuring multifactorial disease burden in Italian patients with FSHD during clinical trials. Facioscapulohumeral muscular dystrophy (FSHD) negatively impacts the quality of life and increases the disease burden. It is important for the clinical community to have a valid instrument that can serially measure a patient’s perception of their multifactorial disease burden in FSHD. The Facioscapulohumeral Muscular Dystrophy – Health Index (FSHD-HI) is a valid instrument that allows patients to provide their perspective regarding their current health state. FSHD-HI-IT provides a valid option for measuring multifactorial disease burden in Italian patients with FSHD during clinical trials.

IntroductionGraph theory models a network by its nodes (the fundamental unit by which graphs are formed) and connections. ‘Degree’ hubs reflect node centrality (the connection rate), while ‘connector’ hubs are those linked to several clusters of nodes (mainly long-range connections).MethodsHere, we compared hubs modeled from measures of interdependencies of between-electrode resting-state eyes-closed electroencephalography (rsEEG) rhythms in normal elderly (Nold) and Alzheimer’s disease dementia (ADD) participants. At least 5 min of rsEEG was recorded and analyzed. As ADD is considered a ‘network disease’ and is typically associated with abnormal rsEEG delta (ResultsConvergent results showed that in both the Nold and ADD groups there were significant parietal ‘degree’ and ‘connector’ hubs derived from alpha rhythms. These hubs had a prominent outward ‘directionality’ in the two groups, but that ‘directionality’ was lower in ADD participants than in Nold participants.DiscussionIn conclusion, independent methodologies and hub definitions suggest that ADD patients may be characterized by low outward ‘directionality’ of partially preserved parietal ‘degree’ and ‘connector’ hubs derived from rsEEG alpha rhythms.

Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K

Objective: To verify the safety and potential effect on ALS progression of a low-intensity immunosuppressive regimen followed by autologous hematopoietic stem cell transplantation (aHSCT) in amyotrophic lateral sclerosis (ALS) patients. Methods: ALS eligible patients underwent a set of clinical and laboratory evaluations at T-4 (screening), T-1 (pre-treatment visit), and for the 12 consecutive months after treatment (T3, T6, T9, T12). We evaluated the tolerability of the procedure, its efficacy on clinical course and quality of life (QoL). Results: Eight of the 11 ALS patients enrolled received the established immunoablative protocol. The procedure was well tolerated and side effects were those expected. One patient died 4 months after the conditioning regimen and another patient underwent tracheotomy just before T3 for a sudden respiratory failure, but he is still alive 4 years after the procedure without being ventilated any more. A third patient died 10 months after conditioning. In the other cases, there was no statistical difference in all functional measures and QoL pre- and post-treatment; however, a transitory slopes’ reduction of ALSFRS-R and seated SVC% after the conditioning procedures was reported. Moreover, although not statistically significant, trends of reduction of CD4 + and increment of CD8 + were found. Conclusions: aHSCT was overall well tolerated, but it was not followed by any significant modification in disease progression. Considering the negative results of this small trial, further studies aimed to evaluate the possible efficacy of the aHSCT using a higher-intensity regimen should be carefully and with caution evaluated.

BackgroundMalnutrition and weight loss are negative prognostic factors for survival in patients with amyotrophic lateral sclerosis (ALS). However, energy expenditure at rest (REE) is still not included in clinical practice, and no data are available concerning hypometabolic state in ALS.ObjectiveTo evaluate in a referral cohort of patients with ALS the prevalence of hypometabolic state as compared with normometabolic and hypermetabolic states, and to correlate it with clinical phenotype, rate of progression and survival.DesignWe conducted a retrospective study examining REE measured by indirect calorimetry in patients with ALS referred to Milan, Limoges and Tours referral centres between January 2011 and December 2017. Hypometabolism and hypermetabolism states were defined when REE difference between measured and predictive values was ≤−10% and ≥10%, respectively. We evaluated the relationship between these metabolic alterations and measures of body composition, clinical characteristics and survival.ResultsEight hundred forty-seven patients with ALS were recruited. The median age at onset was 63.79 years (IQR 55.00–71.17). The male/female ratio was 1.26 (M/F: 472/375). Ten per cent of patients with ALS were hypometabolic whereas 40% were hypermetabolic. Hypometabolism was significantly associated with later need for gastrostomy, non-invasive ventilation and tracheostomy placement. Furthermore, hypometabolic patients with ALS significantly outlived normometabolic (HR=1.901 (95% CI 1.080 to 3.345), p=0.0259) and hypermetabolic (HR=2.138 (95% CI 1.154 to 3.958), p=0.0157) patients.ConclusionHypometabolism in ALS is not uncommon and is associated with slower disease progression and better survival than normometabolic and hypermetabolic subjects. Indirect calorimetry should be performed at least at time of diagnosis because alterations in metabolism are correlated with prognosis.

The present study aimed to evaluate the influence of manual expertise on static computer-aided implantology (s-CAI) in terms of accuracy and operative timings. After the cone-beam CT (CBCT) scanning of eleven mandibular models, a full-arch rehabilitation was planned, and two different skilled operators performed s-CAI. The distances between the virtual and actual implant positions were calculated considering the three spatial vectorial axes and the three-dimensional Euclidean value for the entry (E) and apical (A) points, along with the axis orientation differences (Ax). These values emerged from the overlapping of the pre-op CBCT to post-op CBCT data (method 1), from scanning the data from the laboratory scanner (method 2), and from the intra-oral scanner (method 3) and were correlated with the operators’ expertise and operative timings. The mean values for accuracy from the three methods were: E = 0.57 (0.8, 0.45, 0.47) mm, A = 0.6 (0.8, 0.48, 0.49) mm, and Ax 1.04 (1.05,1.03,1.05) ° for the expert operator; and E = 0.8 (0.9, 0.87, 0.77), A = 0.95 (1.02, 0.95, 0.89), and Ax =1.64 (1.78, 1.58, 1.58) for the novice. The mean value of the operative timings was statistically inferior for the expert operator (p < 0.05), with an improved accuracy over time for both operators. A significant difference (p < 0.05) emerged between method 1 and methods 2 and 3 for seven of the nine variables, without differences between the evaluations from the two scanners. The support from digital surgical guides does not eliminate the importance of manual expertise for the reliability and the shortening of the surgical procedure, and it requires a learning pathway over time.

Purpose To assess the impact of lung segmentation accuracy in an automatic pipeline for quantitative analysis of CT images. Methods Four different platforms for automatic lung segmentation based on convolutional neural network (CNN), region-growing technique and atlas-based algorithm were considered. The platforms were tested using CT images of 55 COVID-19 patients with severe lung impairment. Four radiologists assessed the segmentations using a 5-point qualitative score (QS). For each CT series, a manually revised reference segmentation (RS) was obtained. Histogram-based quantitative metrics (QM) were calculated from CT histogram using lung segmentationsfrom all platforms and RS. Dice index (DI) and differences of QMs (ΔQMs) were calculated between RS and other segmentations. Results Highest QS and lower ΔQMs values were associated to the CNN algorithm. However, only 45% CNN segmentations were judged to need no or only minimal corrections, and in only 17 cases (31%), automatic segmentations provided RS without manual corrections. Median values of the DI for the four algorithms ranged from 0.993 to 0.904. Significant differences for all QMs calculated between automatic segmentations and RS were found both when data were pooled together and stratified according to QS, indicating a relationship between qualitative and quantitative measurements. The most unstable QM was the histogram 90th percentile, with median ΔQMs values ranging from 10HU and 158HU between different algorithms. Conclusions None of tested algorithms provided fully reliable segmentation. Segmentation accuracy impacts differently on different quantitative metrics, and each of them should be individually evaluated according to the purpose of subsequent analyses.

Objective To evaluate the applicability of corticocortical evoked potentials (CCEP) for intraoperative monitoring of the language network in epilepsy surgery under general anesthesia. To investigate the clinical relevance on language functions of intraoperative changes of CCEP recorded under these conditions. Methods CCEP monitoring was performed in 14 epileptic patients (6 females, 4 children) during resections in the left perisylvian region under general anesthesia. Electrode strips were placed on the anterior language area (AL) and posterior language area (PL), identified by structural and functional magnetic resonance imaging. Single-pulse electric stimulations were delivered to pairs of adjacent contacts in a bipolar fashion. During resection, we monitored the integrity of the dorsal language pathway by stimulating either AL by recording CCEP from PL or vice versa, depending on stability and reproducibility of CCEP. We evaluated the first negative (N1) component of CCEP before, during, and after resection. Results All procedures were successfully completed without adverse events. The best response was obtained from AL during stimulation of PL in 8 patients and from PL during stimulation of AL in 6 patients. None of 12 patients with a postresection N1 amplitude decrease of 0%–15% from baseline presented postoperative language impairment. Decreases of 28% and 24%, respectively, of the N1 amplitude were observed in 2 patients who developed transient postoperative speech disturbances. Conclusions The application of CCEP monitoring is possible and safe in epilepsy surgery under general anesthesia. Putative AL and PL can be identified using noninvasive presurgical neuroimaging. Decrease of N1 amplitude >15% from baseline may predict postoperative language deficits.

This study examined neurophysiological (NI), split-hand (SI) and split-leg (SLI) index in patients with amyotrophic lateral sclerosis (ALS), and their correlation with functional status, disease duration, staging and survival.Eighty-two patients underwent nerve conduction study to analyze NI, SI and SLI. Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R), disease progression rate (ΔFS), Milano-Torino (MiToS) and King's staging systems, Forced Vital Capacity (FVC), and survival data were collected.Both NI and SI indices were significantly associated with ALSFRS-R, MiToS, King's and FVC. Slow progressor patients (ΔFS 0.5) reported a significantly higher NI and SI values compared to both normal (0.5 ≤ ΔFS 1.00) and fast progressors (ΔFS ≥ 1.0). After dichotomizing patients in slow progressors (ΔFS 0.5) and not-slow progressors (ΔFS ≥ 0.5), a combination of SI index and disease duration revealed to be the best prediction model to discriminate patients in accordance with their disease progression (c-index: 0.92), leading to a new prognostic index: the 'Split-Hand prognostic index' (SHpi).SI and NI are correlated with functional status and FVC. SHpi index could represent an useful tool to discriminate patients in accordance with their disease progression.These data provide novel evidence of neurophysiological indices as promising biomarkers in ALS.

The main aim of this report is to identify and describe the idea of digital leadership. For the study, the effects of digital leaders' traits and experience on the digitalization process will be examined. Different multinational and global businesses have been examined to apply the concept of digital leadership to their businesses. In this paper, with the goal of an organizational literature review, we analyze the concept of digital leadership. The paper seeks to identify the digital leadership concept; to analyze these abilities and their impacts deeply in the new digital age. As a result of detailed literature research, digitalization and technological developments lead businesses to transform organizational structures, processes, business models, and strategies. Digital leadership is crucial for organizations to survive in the new digital era by adapting and transforming business strategies. To accomplish business objectives and successfully drive the digital business transformation, digital leaders use and enhance the company's digital assets. Digital leaders have different abilities and perspectives compared to traditional leaders. However, many organizations do not understand digital leaders' value, leading to poor performance and unrecoverable failures. This research paper demonstrates a strong link between digital leadership and corporate success and productivity, following the analysis and clarification of the digital leadership concept generated in the modern age and the review of variable business strategies and positioning of the leadership concept in different sectors.

Among the new contaminants relevant for environment, one of the most significant roles is played by pharmaceuticals like antibiotic products for either human or veterinary use. Their presence could cause serious damage to bacteria and microfauna, like nematodes. Within the widely investigated nematodes, very little is known about the interaction between antibiotics and entomopathogenic nematodes (EPN). EPNs have been used for biological control of crops, due to their ability to penetrate arthropod pests and kill their hosts thanks to a complex symbiotic mechanism with specific gram-negative bacteria. Tetracycline is an antibiotic used in human and veterinary medicine, both for therapeutic purposes and for the growth of livestock. Since its action against gram-negative bacteria is documented, we verified in this study the survival, growth and pathogenicity of two species of EPNs, Steinernema vulcanicum and S. feltiae. All tests were performed with tetracycline in 1% ethanol solution and up to 300 mg/L. Apparently, this incubation did not harm the vitality of EPNs. Both S. vulcanicum as S. feltiae recovered their vitality and entomopathogenic ability after 48 h. Moreover, the latter EPN species did not grow nor reproduce in the hemolymph of the Greater Wax Moth, Galleria mellonella, and their endosymbionts did not grow on MacConkey Agar. Our results suggest that the first EPN species has always retained all its abilities and that endosymbionts have acquired resistance to tetracycline, while experiments with the second EPN species provided some contrasting results in time that will require further investigations.

Objective: To outline a practical method of performing prostate cancer radiotherapy in patients with bilateral metal hip prostheses with the standard resources available in a modern general hospital. The proposed workflow is based exclusively on magnetic resonance imaging (MRI) to avoid computed tomography (CT) artifacts. Case description: This study concerns a 73-year-old man with bilateral hip prostheses with an elevated risk prostate cancer. Magnetic resonance images with assigned electron densities were used for planning purposes, generating a synthetic CT (sCT). Imaging acquisition was performed with an optimized Dixon sequence on a 1.5T MRI scanner. The images were contoured by autosegmentation software, based on an MRI database of 20 patients. The sCT was generated assigning averaged electron densities to each contour. Two volumetric modulated arc therapy plans, a complete arc and a partial one, where the beam entrances through the prostheses were avoided for about 50° on both sides, were compared. The feasibility of matching daily cone beam CT (CBCT) with MRI reference images was also tested by visual evaluations of different radiation oncologists. Conclusions: The use of magnetic resonance images improved accuracy in targets and organs at risk (OARs) contouring. The complete arc plan was chosen because of 10% lower mean and maximum doses to prostheses with the same planning target volume coverage and OAR sparing. The image quality of the match between performed CBCTs and MRI was considered acceptable. The proposed method seems promising to improve radiotherapy treatments for this complex category of patients.

The BDNF Val66Met gene polymorphism is a relevant factor explaining inter-individual differences to TMS responses in studies of the motor system. However, whether this variant also contributes to TMS-induced memory effects, as well as their underlying brain mechanisms, remains unexplored. In this investigation, we applied rTMS during encoding of a visual memory task either over the left frontal cortex (LFC; experimental condition) or the cranial vertex (control condition). Subsequently, individuals underwent a recognition memory phase during a functional MRI acquisition. We included 43 young volunteers and classified them as 19 Met allele carriers and 24 as Val/Val individuals. The results revealed that rTMS delivered over LFC compared to vertex stimulation resulted in reduced memory performance only amongst Val/Val allele carriers. This genetic group also exhibited greater fMRI brain activity during memory recognition, mainly over frontal regions, which was positively associated with cognitive performance. We concluded that BDNF Val66Met gene polymorphism, known to exert a significant effect on neuroplasticity, modulates the impact of rTMS both at the cognitive as well as at the associated brain networks expression levels. This data provides new insights on the brain mechanisms explaining cognitive inter-individual differences to TMS, and may inform future, more individually-tailored rTMS interventions.

Oxidative stress and inflammation play a pivotal role in the development of cardiovascular diseases, an ever-growing worldwide problem. As a non-pharmacological approach, diet, especially a flavonoid-rich diet, showed promising results in the reduction of cardiovascular diseases and alleviation of their symptoms. In this study, in vitro systems based on human microvascular endothelial cells (hmvEC) and human umbilical cord endothelial cells (HUVEC) were established to determine the effect of Healthberry 865

Background: Patients with amnesic mild cognitive impairment due to Alzheimer’s disease (ADMCI) typically show a “slowing” of cortical resting-state eyes-closed electroencephalographic (rsEEG) rhythms. Some of them also show subclinical, non-convulsive, and epileptiform EEG activity (EEA) with an unclear relationship with that “slowing.” Objective: Here we tested the hypothesis that the “slowing” of rsEEG rhythms is related to EEA in ADMCI patients. Methods: Clinical and instrumental datasets in 62 ADMCI patients and 38 normal elderly (Nold) subjects were available in a national archive. No participant had received a clinical diagnosis of epilepsy. The eLORETA freeware estimated rsEEG cortical sources. The area under the receiver operating characteristic curve (AUROCC) indexed the accuracy of eLORETA solutions in the classification between ADMCI-EEA and ADMCI-noEEA individuals. Results: EEA was observed in 15% (N = 8) of the ADMCI patients. The ADMCI-EEA group showed: 1) more abnormal Aβ42 levels in the cerebrospinal fluid as compared to the ADMCI-noEEA group and 2) higher temporal and occipital delta (

Respiratory insufficiency is one of the main causes of death in myotonic dystrophy type 1 (DM1). Although there is general consensus these patients have a restrictive ventilatory pattern, hypoventilation, chronic hypercapnia and sleep disturbances, the prevalence of respiratory disease and indication for and effects of non-invasive ventilation (NIV) need to be further explored.We aim to describe the respiratory function and the need for NIV at baseline and over time in a cohort of adult patients with DM1.One hundred and fifty-one adult patients with DM1 were subjected to arterial blood gas analysis, sitting and supine forced vital capacity (FVC), peak cough expiratory flow (PCEF), nocturnal oximetry, maximal inspiratory and expiratory pressures (MIP/PEP).On first assessment 84 of 151 had normal respiratory function (median age 38 years, median BMI 23.9, median disease duration: 11 years); 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; only 17 of 67 with the new NIV prescription were adherent.We provide additional data on the natural history of respiratory function decline and treatment adherence in a relatively large cohort of well-characterized patients with DM1. A high proportion (28%) were lost to follow-up. A minority (11%) required NIV, and only 25% were treatment adherent, irrespective of specific demographics and respiratory features. Our results also confirm previous findings showing that age, disease duration and higher BMIs are predisposing factors for respiratory impairment.

Cerebrospinal and structural-molecular neuroimaging in-vivo biomarkers are recommended for diagnostic purposes in Alzheimer’s disease (AD) and other dementias; however, they do not explain the effects of AD neuropathology on neurophysiological mechanisms underpinning cognitive processes. Here, an Expert Panel from the Electrophysiology Professional Interest Area of the Alzheimer’s Association reviewed the field literature and reached consensus on the event-related electroencephalographic oscillations (EROs) that show consistent abnormalities in patients with significant cognitive deficits due to Alzheimer’s, Parkinson’s (PD), Lewy body (LBD), and cerebrovascular diseases. Converging evidence from oddball paradigms showed that, as compared to cognitively unimpaired (CU) older adults, AD patients had lower amplitude in widespread delta (>4 Hz) and theta (4–7 Hz) phase-locked EROs as a function of disease severity. Similar effects were also observed in PD, LBD, and/or cerebrovascular cognitive impairment patients. Non-phase-locked alpha (8–12 Hz) and beta (13–30 Hz) oscillations were abnormally reduced (event-related desynchronization, ERD) in AD patients relative to CU. However, studies on patients with other dementias remain lacking. Delta and theta phase-locked EROs during oddball tasks may be useful neurophysiological biomarkers of cognitive systems at work in heuristic and intervention clinical trials performed in AD patients, but more research is needed regarding their potential role for other dementias. European Committee (H2020-EU.1.3.1.H2020-MSCA-ITN-ETN-2016 project)

BACKGROUND Participation in sports is known to have positive effects on people's health and psycho-social well-being. Recently, physical activity implications for people with disabilities have been explored, showing promising results on quality of life and self-concept. However, few studies have specifically investigated the effects of participation in adaptive sports on neuromuscular patients' quality of life. OBJECTIVE To evaluate differences in psycho-social well-being between people affected by a neuromuscular disease who play wheelchair hockey and those who do not. Individuals playing an adaptive sport would report better quality of life, higher physical self-efficacy scores and more effective coping strategies, as assessed by self-reported measures. DESIGN Cross-sectional study. SETTING Data were collected during clinical follow-ups at the NEMO Clinical Center in Milan (Italy). PARTICIPANTS A total of 25 patients affected by neuromuscular diseases, aged 18 to 40 years, participated in the study. MAIN OUTCOME MEASURES The primary outcome was to compare quality of life between groups. Secondary outcomes were the comparisons of physical self-efficacy and coping strategies through self-reported measures. RESULTS Wheelchair hockey players scored significantly higher on the Quality of Life Index (specifically on the health/functioning and psychological/spiritual sub-scales) and reported better physical self-efficacy and perceived physical ability compared to the control group (i.e., patients who do not participate in any adaptive sport), controlling for age and pathology. On the contrary, no difference was found for coping strategies between the two groups. CONCLUSIONS This study identified a significant association between participation in wheelchair hockey and improved physical and psychological well-being of people affected by neuromuscular diseases, compared to those who are not involved in adaptive sports. This article is protected by copyright. All rights reserved.

Objective: To comprehensively assess whether neopterin in urine could be a candidate biomarker for determining the neuroinflammatory status in ALS. Methods: We performed an observational, cross-sectional study in 81 pALS, 68 age- and sex-comparable healthy controls (HC), 14 patients affected by MS and 24 OND patients. ALS patients underwent a neurological evaluation to assess the global functional status evaluated by Amyotrophic Lateral Sclerosis Functional Rating Scale—Revised (ALSFRS-R) and the disease progression rate. Urinary neopterin concentrations were determined by high-performance liquid chromatography method and were recorded at the time of first examination to assess their effect on disease severity and survival. Results: Urinary neopterin was significantly higher in pALS (263.90 [198.71–474.90]) compared to MS (155.28 [131.74–190.38], p =

The changes introduced with Council Directive 2013/59/Euratom will require European Member States adapt their regulations, procedures and equipment to the new high standards of radiation safety. These new requirements will have an impact, in particular, on the radiology community (including medical physics experts) and on industry. Relevant changes include new definitions, a new dose limit for the eye lens, non-medical imaging exposures, procedures in asymptomatic individuals, the use and regular review of diagnostic reference levels (including interventional procedures), dosimetric information in imaging systems and its transfer to the examination report, new requirements on responsibilities, the registry and analysis of accidental or unintended exposure and population dose evaluation (based on age and gender distribution). Furthermore, the Directive emphasises the need for justification of medical exposure (including asymptomatic individuals), introduces requirements concerning patient information and strengthens those for recording and reporting doses from radiological procedures, the use of diagnostic reference levels, the availability of dose-indicating devices and the improved role and support of the medical physics experts in imaging.

One of the main challenges in cancer surgery is to ensure the complete excision of the tumour while sparing as much healthy tissue as possible. Histopathology, the gold-standard technique used to assess the surgical margins on the excised tissue, is often impractical for intra-operative use because of the time-consuming tissue cryo-sectioning and staining, and availability of histopathologists to assess stained tissue sections. Raman micro-spectroscopy is a powerful technique that can detect microscopic residual tumours on ex vivo tissue samples with accuracy, based entirely on intrinsic chemical differences. However, raster-scanning Raman micro-spectroscopy is a slow imaging technique that typically requires long data acquisition times wich are impractical for intra-operative use. Selective-sampling Raman imaging overcomes these limitations by using information regarding the spatial properties of the tissue to reduce the number of Raman spectra. This paper reviews the latest advances in selective-sampling Raman techniques and applications, mainly based on multimodal optical imaging. We also highlight the latest results of clinical integration of a prototype device for non-melanoma skin cancer. These promising results indicate the potential impact of Raman spectroscopy for providing fast and objective assessment of surgical margins, helping surgeons ensure the complete removal of tumour cells while sparing as much healthy tissue as possible.

Objective This retrospective and exploratory study tested the accuracy of artificial neural networks (ANNs) at detecting Alzheimer’s disease patients with dementia (ADD) based on input variables extracted from resting-state electroencephalogram (rsEEG), structural magnetic resonance imaging (sMRI) or both. Methods For the classification exercise, the ANNs had two architectures that included stacked (autoencoding) hidden layers recreating input data in the output. The classification was based on LORETA source estimates from rsEEG activity recorded with 10–20 montage system (19 electrodes) and standard sMRI variables in 89 ADD and 45 healthy control participants taken from a national database. Results The ANN with stacked autoencoders and a deep leaning model representing both ADD and control participants showed classification accuracies in discriminating them of 80%, 85%, and 89% using rsEEG, sMRI, and rsEEG + sMRI features, respectively. The two ANNs with stacked autoencoders and a deep leaning model specialized for either ADD or control participants showed classification accuracies of 77%, 83%, and 86% using the same input features. Conclusions The two architectures of ANNs using stacked (autoencoding) hidden layers consistently reached moderate to high accuracy in the discrimination between ADD and healthy control participants as a function of the rsEEG and sMRI features employed. Significance The present results encourage future multi-centric, prospective and longitudinal cross-validation studies using high resolution EEG techniques and harmonized clinical procedures towards clinical applications of the present ANNs.

Featured Application: The proposed protocol, starting from a careful prediction of the final result with the ideal relationship between the prosthesis and soft tissues, aims to improve the GBR approach, particularly for critical atrophies. Background: Treating extended alveolar defects is challenging for their irregular shape and lack of hard and soft tissues. Virtual planned guided bone regeneration (GBR) with customized meshes aims to optimize the treatment by reducing the risk of dehiscence. The mucosa characteristics are crucial in preserving the bone graft covering and vitality. Methods: Two three-dimensional and extended defects, a mandibular posterior and anterior maxillary atrophy were reconstructed with a particulate graft and a digitally customized scaffold. The workflow entailed merging the pre-operatory clinical related data from intra-oral scanning with the radiologic ones from cone beam-CT. A final ideal prediction of the soft tissue relationship with the implant-borne prosthesis was virtually elaborated, conditioning the design of the titanium membrane fitting the bone defects. Results: A good matching between the scaffold and the bone surface was intra-operatory noted; no complications were registered in the first months of healing with complete integrity of the soft tissues above the graft. Conclusions: A careful evaluation of the soft tissues and a forecast of their final relationship with the implant and prosthesis can improve digital mesh/membrane manufacturing with a suitable healing process up to implant placement and loading, favoring peri-implant tissue stability over time.

The Functional ANnoTation Of the Mammalian genome (FANTOM) Consortium has continued to provide extensive resources in the pursuit of understanding the transcriptome, and transcriptional regulation, of mammalian genomes for the last 20 years. To share these resources with the research community, the FANTOM web-interfaces and databases are being regularly updated, enhanced and expanded with new data types. In recent years, the FANTOM Consortium's efforts have been mainly focused on creating new non-coding RNA datasets and resources. The existing FANTOM5 human and mouse miRNA atlas was supplemented with rat, dog, and chicken datasets. The sixth (latest) edition of the FANTOM project was launched to assess the function of human long non-coding RNAs (lncRNAs). From its creation until 2020, FANTOM6 has contributed to the research community a large dataset generated from the knock-down of 285 lncRNAs in human dermal fibroblasts; this is followed with extensive expression profiling and cellular phenotyping. Other updates to the FANTOM resource includes the reprocessing of the miRNA and promoter atlases of human, mouse and chicken with the latest reference genome assemblies. To facilitate the use and accessibility of all above resources we further enhanced FANTOM data viewers and web interfaces. The updated FANTOM web resource is publicly available at https://fantom.gsc.riken.jp/.

Amyotrophic lateral sclerosis (ALS) is a multisystemic disease compromising both the neuromuscular system and the cognitive status. Non-invasive ventilation (NIV) has been shown to improve survival and quality of life in ALS patients with respiratory failure, but scanty literature investigated which are the predictors of NIV tolerance. The aim of this study was to evaluate the impact of functional, cognitive, neurobehavioral, and respiratory status on NIV compliance and tolerance in patients with ALS. We retrospectively evaluated clinical data of ALS patients who consecutively underwent a NIV trial during hospitalization. Cognitive and neurobehavioral assessments have been performed using the Edinburgh Cognitive and Behavioral ALS Screen (ECAS), the Hospital Anxiety and Depression Scale (HADS), the Frontal Assessment Battery (FAB), the Raven's 47 Colored Progressive Matrices (PM47), and the Neurobehavioral Rating Scale Revised (NRSR). Seventy-two patients (mean age ± SD; 63.9 ± 10.6 years) were included. Patients adapted were 63/72 (87.5%). The average time of adaptation was 7.82 ± 5.27 days. The time required to reach a satisfying NIV adaptation was significantly related to the presence of sialorrhea (p = 0.02), respiratory status (Borg Dyspnoea Scale, p = 0.006, and ALS-FRS-R respiratory subscore, p = 0.03) and behavioral and cognitive impairment (NRSR-F1, p = 0.04, NRSR- F5, p = 0.04). Presence of sialorrhea and neurobehavioral impairment, and absence of respiratory symptoms are negative predictors of NIV adaptation. This study highlights the need of a multidisciplinary patient-tailored approach including cognitive-behavioral assessment and a psychological support program to optimize patient's training and compliance to NIV.

Gene expression profiles in homologous tissues have been observed to be different between species, which may be due to differences between species in the gene expression program in each cell type, but may also reflect differences in cell type composition of each tissue in different species. Here, we compare expression profiles in matching primary cells in human, mouse, rat, dog, and chicken using Cap Analysis Gene Expression (CAGE) and short RNA (sRNA) sequencing data from FANTOM5. While we find that expression profiles of orthologous genes in different species are highly correlated across cell types, in each cell type many genes were differentially expressed between species. Expression of genes with products involved in transcription, RNA processing, and transcriptional regulation was more likely to be conserved, while expression of genes encoding proteins involved in intercellular communication was more likely to have diverged during evolution. Conservation of expression correlated positively with the evolutionary age of genes, suggesting that divergence in expression levels of genes critical for cell function was restricted during evolution. Motif activity analysis showed that both promoters and enhancers are activated by the same transcription factors in different species. An analysis of expression levels of mature miRNAs and of primary miRNAs identified by CAGE revealed that evolutionary old miRNAs are more likely to have conserved expression patterns than young miRNAs. We conclude that key aspects of the regulatory network are conserved, while differential expression of genes involved in cell-to-cell communication may contribute greatly to phenotypic differences between species.