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The article explores a lengthy poem about the life of St. Thomas Aquinas written in Lima and published in Spain by the Dominican friar Adrián de Alecio (El Angélico, 1645). It starts by describing the author and his milieu. The following is an evaluation of the extent to which the text takes up and adapts the writing models and hagiographic sources from the centre of the Empire. Finally, the article aims to place the poem in the axis formed by the poetic figures of Lope de Vega and Luis de Góngora.

Background: Recently, common genetic risk factors for intracranial aneurysm (IA) and aneurysmal subarachnoid hemorrhage (ASAH) were found to explain a large amount of disease heritability and therefore have potential to be used for genetic risk prediction. We constructed a genetic risk score to (1) predict ASAH incidence and IA presence (combined set of unruptured IA and ASAH) and (2) assess its association with patient characteristics. Methods: A genetic risk score incorporating genetic association data for IA and 17 traits related to IA (so-called metaGRS) was created using 1161 IA cases and 407 392 controls from the UK Biobank population study. The metaGRS was validated in combination with risk factors blood pressure, sex, and smoking in 828 IA cases and 68 568 controls from the Nordic HUNT population study. Furthermore, we assessed association between the metaGRS and patient characteristics in a cohort of 5560 IA patients. Results: Per SD increase of metaGRS, the hazard ratio for ASAH incidence was 1.34 (95% CI, 1.20–1.51) and the odds ratio for IA presence 1.09 (95% CI, 1.01–1.18). Upon including the metaGRS on top of clinical risk factors, the concordance index to predict ASAH hazard increased from 0.63 (95% CI, 0.59–0.67) to 0.65 (95% CI, 0.62–0.69), while prediction of IA presence did not improve. The metaGRS was statistically significantly associated with age at ASAH (β=−4.82×10 −3 per year [95% CI, −6.49×10 −3 to −3.14×10 −3 ]; P =1.82×10 −8 ), and location of IA at the internal carotid artery (odds ratio=0.92 [95% CI, 0.86–0.98]; P =0.0041). Conclusions: The metaGRS was predictive of ASAH incidence, although with limited added value over clinical risk factors. The metaGRS was not predictive of IA presence. Therefore, we do not recommend using this metaGRS in daily clinical care. Genetic risk does partly explain the clinical heterogeneity of IA warranting prioritization of clinical heterogeneity in future genetic prediction studies of IA and ASAH.

Objetivo: apresentar as percepções de homens jovens sobre a vivência da paternidade. Método: estudo qualitativo, realizado através de entrevista com 12 jovens de idade entre 18 e 24 anos, captados pela técnica de bola de neve e saturação teórica. O tratamento dos dados deu-se pela análise de conteúdo. Resultados: os jovens pais encontravam-se em cenário socioeconômico desfavorecido. A paternidade foi associada a isolamento e perdas, mas também à renovação e novas perspectivas. O provimento financeiro do filho foi um aspecto relacionado a exigências do modelo de masculinidade e paternidade vigentes, o que pode dificultar a vivência de outras formas de ser pai. Considerações finais: é necessária a implementação de políticas públicas que possibilitem aos rapazes gerenciarem sua vida reprodutiva de forma saudável e responsável, estabelecendo discussões quanto a gênero e masculinidade, para favorecer a vivência de uma paternidade menos calcada nas normas de masculinidade vigentes.

This paper explores the use of nanofiltration (NF) and Reverse Osmosis (RO) to remove the pharmaceutical Fluoxetine (FLX) from water. This substance can be found in rivers and lakes and requires studies involving the application of efficient technologies for its removal or mitigation. The study evaluated NF and RO membranes as an alternative to remove Fluoxetine from water. NF removed fluoxetine within the range between 50 and 60%, and RO demonstrated higher efficiency in removing the drug (98.8%). RO is a suitable method as a complementary treatment of water to ensure a lower concentration of FLX in water. Keywords: emerging pollutants, environment, membrane technology, water treatment.

ImportanceInternational guidelines recommend avoiding intravenous thrombolysis (IVT) in patients with ischemic stroke who have a recent intake of a direct oral anticoagulant (DOAC).ObjectiveTo determine the risk of symptomatic intracranial hemorrhage (sICH) associated with use of IVT in patients with recent DOAC ingestion.Design, Setting, and ParticipantsThis international, multicenter, retrospective cohort study included 64 primary and comprehensive stroke centers across Europe, Asia, Australia, and New Zealand. Consecutive adult patients with ischemic stroke who received IVT (both with and without thrombectomy) were included. Patients whose last known DOAC ingestion was more than 48 hours before stroke onset were excluded. A total of 832 patients with recent DOAC use were compared with 32 375 controls without recent DOAC use. Data were collected from January 2008 to December 2021.ExposuresPrior DOAC therapy (confirmed last ingestion within 48 hours prior to IVT) compared with no prior oral anticoagulation.Main Outcomes and MeasuresThe main outcome was sICH within 36 hours after IVT, defined as worsening of at least 4 points on the National Institutes of Health Stroke Scale and attributed to radiologically evident intracranial hemorrhage. Outcomes were compared according to different selection strategies (DOAC-level measurements, DOAC reversal treatment, IVT with neither DOAC-level measurement nor idarucizumab). The association of sICH with DOAC plasma levels and very recent ingestions was explored in sensitivity analyses.ResultsOf 33 207 included patients, 14 458 (43.5%) were female, and the median (IQR) age was 73 (62-80) years. The median (IQR) National Institutes of Health Stroke Scale score was 9 (5-16). Of the 832 patients taking DOAC, 252 (30.3%) received DOAC reversal before IVT (all idarucizumab), 225 (27.0%) had DOAC-level measurements, and 355 (42.7%) received IVT without measuring DOAC plasma levels or reversal treatment. The unadjusted rate of sICH was 2.5% (95% CI, 1.6-3.8) in patients taking DOACs compared with 4.1% (95% CI, 3.9-4.4) in control patients using no anticoagulants. Recent DOAC ingestion was associated with lower odds of sICH after IVT compared with no anticoagulation (adjusted odds ratio, 0.57; 95% CI, 0.36-0.92). This finding was consistent among the different selection strategies and in sensitivity analyses of patients with detectable plasma levels or very recent ingestion.Conclusions and RelevanceIn this study, there was insufficient evidence of excess harm associated with off-label IVT in selected patients after ischemic stroke with recent DOAC ingestion.

Investigation of multicollinearity allows parameters in multivariate analysis to be estimated with higher precision and with biological interpretation. In order to generate reliable estimates of the degree of multicollinearity, it is necessary to use appropriate sample size. Thus, the objectives of this study were to determine the sample size (number of plants) necessary to estimate the indicators of the degree of multicollinearity - condition number (CN), correlation matrix determinant (DET), and variance inflation factor (VIF) - in morphological traits of rye and to verify the variability of the sample size between the indicators. Five and three uniformity trials were conducted with the cultivars BRS Progresso and Temprano, respectively. Eight morphological traits were evaluated in 780 plants in eight trials. For each trial, 22 cases were selected among the 28 formed by the combination of eight traits, taken six by six, totaling 176 cases. In each case, 197 sample sizes were planned (20, 25, 30, ..., 1,000 plants) and in each size 2,000 resampling procedures with replacement were performed, CN, DET, and VIF were determined and the average among 2,000 estimates was calculated. For each case and indicator (CN, DET, and VIF), the sample size was determined through three models: modified maximum curvature method and linear and quadratic segmented models with plateau response. There is variability between sample sizes between indicators, with larger sample sizes required for DET, followed by CN and VIF, in that order, with at least 180, 116 and 85 plants, respectively. A investigação da multicolinearidade permite que parâmetros em análises multivariadas sejam estimados com maior precisão e com interpretação biológica. Para ter confiabilidade nas estimativas do grau de multicolinearidade, é necessário utilizar adequado tamanho de amostra. Assim, os objetivos deste trabalho foram determinar o tamanho de amostra (número de plantas) necessário para a estimação dos indicadores do grau de multicolinearidade - número de condição (NC), determinante da matriz de correlação (DET) e fator de inflação da variância (FIV) - em caracteres morfológicos de centeio e verificar a variabilidade do tamanho de amostra entre os indicadores. Foram conduzidos cinco e três ensaios de uniformidade com as cultivares BRS Progresso e Temprano, respectivamente. Foram avaliados oito caracteres morfológicos em 780 plantas em oito ensaios. Para cada ensaio, foram selecionados 22 casos entre os 28 formados pela combinação de oito caracteres, tomados seis a seis, totalizando 176 casos. Para cada caso, foram planejados 197 tamanhos de amostra (20, 25, 30, ..., 1.000 plantas) e para cada tamanho foram realizadas 2.000 reamostragens, com reposição, determinados o NC, DET e FIV e calculada a média das 2.000 estimativas. Após, para cada caso e indicador, foi determinado o tamanho de amostra, por meio de três modelos: método da máxima curvatura modificado e modelos linear e quadrático segmentados com resposta em platô. Há variabilidade entre os tamanhos de amostra entre os indicadores, com necessidade de maiores tamanhos de amostra para DET, seguido de NC e FIV, nessa ordem, com no mínimo de 180, 116 e 85 plantas, respectivamente.

Introduction: Brazilian agriculture has developed to such an extent in the last 40 years that the country will become one of the world’s largest food suppliers in the future. Indeed, to keep up this production, agriculture makes intensive use of fertilizers and agricultural pesticides, helping Brazil become one of the main pesticide consumers worldwide. Ethylenebisdithiocarbamates are a group of fungicides that have been widely utilized around the world, and Mancozeb is one of their main representatives. Therefore, a pilot experimental model was created to evaluate the potential hepatotoxic effect of Mancozeb. Materials and Methods: An experimental study was performed with 27 male Wistar rats, divided into 3 groups of 9 rats. Control Group received saline solution, Intervention Group I received 250 mg/kg once a week and Intervention Group II received 500 mg/kg also once a week. The treatment was performed for 12 weeks. Anthropometric measurements were performed and the marker of biological exposure in urine was dosed, biochemical tests, evaluation micronucleus count and comet assay, oxidative stress markers and histological assessment of the liver were done. Results: The hepatotoxic effect of exposure to Mancozeb was confirmed by different tests: anthropometric measures, genotoxicity, and oxidative stress. Statistical significance was found when exposed groups were compared to the control. These results were supported by microscopic evaluation of the liver tissue, where histological changes were found such as inflammatory infiltrate and balloonization in the treated groups. Conclusion: Experimental model was effective to demonstrate the deleterious effect on the liver due to exposure to Mancozeb.

Electronic cigarettes (e-cigarettes) are battery-powered devices containing a liquid based on propylene glycol or vegetable glycerin, compounds which, when vaporized, act as a vehicle for nicotine, flavours, and other chemical components. These devices have been marketed without clear evidence of risks, long-term safety, and efficacy as a means of traditional smoking cessation. Recent clinical studies have shown how the use of the e-cigarette, combined with adequate psychological support, can be effective in reducing traditional smoking but not nicotine addiction. However, meta-analyses of observational studies have not confirmed this efficacy. Several studies have also highlighted an increase in sympathetic tone, vascular stiffness, and endothelial dysfunction, all factors associated with an increased cardiovascular risk. Clinicians, therefore, should carefully monitor the possible risks to public health deriving from the use of e-cigarettes and should discourage non-smokers and adolescents from using such devices. Finally, particular attention should be paid to smokers so that the combined use of electronic and traditional cigarettes can be limited as much as possible.

Background: Subclinical vascular brain lesions are highly prevalent in elderly patients with stroke. Little is known about predisposing factors and their impact on long-term outcome of patients with stroke at a young age. Methods: We quantified magnetic resonance-defined subclinical vascular brain lesions, including lacunes and white matter hyperintensities, perivascular spaces and cerebral microbleeds, and assessed total small-vessel disease (SVD) score in patients with first-ever acute ischemic stroke aged 18 to 45 years, and followed them up, as part of the multicentre Italian Project on Stroke in Young Adults. The primary end point was a composite of ischemic stroke, transient ischemic attack, myocardial infarction, or other arterial events. We assessed the predictive accuracy of magnetic resonance features and whether the addition of these markers improves outcome prediction over a validated clinical tool, such as the Italian Project on Stroke in Young Adults score. Results: Among 591 patients (males, 53.8%; mean age, 37.5±6.4 years), 117 (19.8%) had subclinical vascular brain lesions. Family history of stroke was associated with lacunes (odds ratio, 2.24 [95% CI, 1.30–3.84]) and total SVD score (odds ratio, 2.06 [95% CI, 1.20–3.53] for score≥1), hypertension with white matter hyperintensities (odds ratio, 2.29 [95% CI, 1.22–4.32]). After a median follow-up of 36.0 months (25th–75th percentile, 38.0), lacunes and total SVD score were associated with primary end point (hazard ratio, 2.13 [95% CI, 1.17–3.90] for lacunes; hazard ratio, 2.17 [95% CI, 1.20–3.90] for total SVD score ≥1), and the secondary end point brain ischemia (hazard ratio, 2.55 [95% CI, 1.36–4.75] for lacunes; hazard ratio, 2.61 [95% CI, 1.42–4.80] for total SVD score ≥1). The predictive performances of the models, including magnetic resonance features were comparable to those of the random model. Adding individual magnetic resonance features to the Italian Project on Stroke in Young Adults score did not improve model prediction. Conclusions: Subclinical vascular brain lesions affect ≈2 in 10 young adults with ischemic stroke. Although lacunes and total SVD score are associated with thrombotic recurrence, they do not improve accuracy of outcome prediction over validated clinical predictors.

Spontaneous cervical artery dissection (sCeAD) is the most common cause of ischemic stroke at a young age, but its pathogenetic mechanism and risk factors are not fully elucidated. It is reasonable to think that bleeding propensity, vascular risk factors such as hypertension and head or neck trauma, and constitutional weakness of the arterial wall together play a role in the pathogenesis of sCeAD. Hemophilia A is known to be an X-linked condition that leads to spontaneous bleeding in various tissues and organs. To date, a few cases of acute arterial dissection in patients with hemophilia have been reported, but the relationship between these two diseases has not been studied so far. In addition, there are no guidelines indicating the best antithrombotic treatment option in these patients. We report the case of a man with hemophilia A who developed sCeAD and transient oculo-pyramidal syndrome and was treated with acetylsalicylic acid. We also review previous published cases of arterial dissection in patients with hemophilia, discussing the potential pathogenetic mechanism underlying this rare association and potential antithrombotic therapeutic options.

This clinical prospective study evaluated the clinical and radiographic peri-implant conditions of two unsplinted implants immediate loaded with mandibular overdentures, as well, evaluated the oral health related quality of life (OHRQoL) in these patients. Materials and Methods: Ten totally edentulous patients received 20 implants with ball attachment, being 6 immediate loaded (test group) and 4 conventional loaded (control group). For OHRQoL assessment, the OHIP-EDENT questionnaire was applied before the implants insertion, 3 and 6 months after. After 6 months of implant insertion, the following clinical parameters were evaluated: probing depth, width of the keratinized mucosa, modified bleeding index, modified plaque index. The marginal bone loss was obtained by analyses of periapical radiographs at the day of implant insertion, 3 and 6 months after. Results: There was a significant improvement of quality of life at 3 months in immediate loaded group and at 6 months at the conventional loaded group comparing to initial time (P

A hipótese de investigação deste artigo postula a existência de isotopias dominantes e imagens-símbolos que cruzam todas as teorias de conspiração, independentemente de sua especificidade ideológica ou contextual. Assim, para detectar um núcleo duro ideal do universo semântico dos discursos conspiratórios, no qual convergem as várias vertentes das teorias de conspiração, assumimos que o seu denominador comum poderia ser encontrado no raciocínio figurativo, o que implica partilhar as mesmas imagens para explicar os acontecimentos actuais. O melhor campo para validar a nossa hipótese é constituído pela representação visual da pandemia de Covid-19 que circula nas redes sociais. O corpus a partir do qual a análise foi realizada cobre o período de 15 de Fevereiro de 2020 a 15 de Dezembro de 2021, e conta com milhares de publicações online. Dentro do corpus foram selecionados os posts sobre a pandemia de Covid-19 e aqueles sobre o lockdown de Março de 2020, feitos pelos principais jornais italianos, figuras institucionais e influenciadores digitais, relatados pela OMS como pontos cruciais de disseminação de desinformação, ao lado das facções conspiratórias italianas que costumam divulgar fake news no Facebook, Instagram, Telegram e Twitter.

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.

O bruxismo é o ato de ranger os dentes de forma subconsciente e não-funcional que costuma ocorrer durante o sono, mas também pode ocorrer durante o dia. Essa condição parafuncional afeta o indivíduo de diversas formas. O desgaste dentário é proveniente quase que inteiramente dessas atividades parafuncionais e como consequência deste há perda de dimensão vertical de oclusão (DVO). Somente com o restabelecimento de uma DVO correta o Cirurgião-Dentista pode planejar a reabilitação estética no sorriso do paciente. Dentre as alternativas restauradoras estéticas estão as resinas compostas, que oferecem uma gama de cores e de translucidez e podem ser indicadas para diversas situações. Assim, este artigo tem como objetivo relatar um caso clínico de reabilitação oral, através da confecção de facetas estéticas em resina composta em dentes anteriores superiores após restabelecimento de dimensão vertical de oclusão, em um paciente jovem que possuía perda de dimensão vertical de oclusão por bruxismo noturno e diurno.

AimFunctional traits shape the distribution of taxa across environments. However, it remains unclear whether trait and environmental niche evolution are correlated, and what happened first: trait change facilitating environment shifts (pre-adaptation) or environmental change leading to trait change (adaptation). We focus on a species-rich Neotropical legume radiation to shed light on this enigma.LocationNeotropics.Time PeriodCenozoic.Major Taxa StudiedFabaceae: Papilionoidae: Swartzia.MethodsWe assembled leaflet, fruit and petal size data from monographs and herbarium collections for 86 to 96% of the c. 180 Swartzia species, inferred a dated Swartzia phylogenetic tree from existing DNA sequences covering 38% of the species and integrated these with distribution, soil and climate data. We used phylogenetic linear regression to quantify trait–environment relationships and applied comparative methods to evaluate modes of correlated evolution between traits and environments.ResultsLeaflet and petal size were strongly linked to climate, while fruit size was not associated with climate or soil characteristics. Evolutionary transitions to relatively low rainfall and low temperature environments were conditional on the evolution of small leaflets, whereas transitions to wet and warm environments were preceded by the evolution of larger leaflets. In contrast, transitions to the warmest or coldest environments were followed, rather than preceded, by petal loss.Main ConclusionOur results show that the macroevolution of functional traits has influenced the broad-scale distribution of Swartzia across Neotropical rainforest, seasonally dry, montane and inundated habitats. We suggest that trait evolution is conditional on environmental change but both pre-adaptive and adaptive processes may occur. These processes are important to understand the distribution of diversity at both regional (e.g. Amazonia) and global biogeographical scales.

Multicollinearity must be diagnosed in multivariate analyses. Among the indicators, the condition number can be used to quantify the degree of multicollinearity. Hence, this study sought to determine the number of measurements (trials) necessary to estimate the number of condition in linear correlation matrices between rye traits. Five uniformity trials were carried out with ‘BRS Progresso’ rye, and eight morphological traits and eight productive traits were evaluated, forming two groups. In each group of traits, six cases (combinations of traits) were planned and the multicollinearity diagnosis was performed. Repeatability analyses were performed using the following methods: analysis of variance, principal component analysis, and structural analysis, and the number of measurements (trials) was determined for different levels of precision. A higher condition number of repeatability coefficients was obtained by the principal component methods (based on correlation and variance and covariance matrices) and structural analysis based on the variance and covariance matrix. A greater number of measurements (trials) is necessary to estimate the number of conditions in productive traits compared to morphological ones. One trial is enough to efficiently estimate the condition number with a minimum accuracy of 80% in morphological and productive traits of rye, whereas at least three trials are required for 95% accuracy.

BACKGROUND AND PURPOSE This study aimed to investigate the effect of endovascular treatment (EVT, with or without intravenous thrombolysis [IVT]) versus IVT alone on outcomes in patients with acute ischemic stroke (AIS) and intracranial large vessel occlusion (LVO) attributable to cervical artery dissection (CeAD). METHODS This multinational cohort study was conducted based on prospectively collected data from the EVA-TRISP (EndoVAscular treatment and ThRombolysis for Ischemic Stroke Patients) collaboration. Consecutive patients (2015-2019) with AIS-LVO attributable to CeAD treated with EVT and/or IVT were included. Primary outcome measures were (1) favorable 3-month outcome (modified Rankin Scale score 0-2) and (2) complete recanalization (thrombolysis in cerebral infarction scale 2b/3). Odds ratios with 95% confidence intervals (OR [95% CI]) from logistic regression models were calculated (unadjusted, adjusted). Secondary analyses were performed in the patients with LVO in the anterior circulation (LVOant) including propensity score matching. RESULTS Among 290 patients, 222 (76.6%) had EVT and 68 (23.4%) IVT alone. EVT-treated patients had more severe strokes (National Institutes of Health Stroke Scale score, median [interquartile range]: 14 [10-19] vs. 4 [2-7], P

Turner syndrome (TS) is associated with aortic coarctation, dissection and dilation/aneurysm. Predictors of dissection are not well delineated, making decisions regarding prophylactic root replacement challenging. In other disorders, arterial tortuosity is an imaging biomarker associated with increased risk for aortic dissection and adverse cardiovascular events. We aimed to determine if, in TS, arterial tortuosity was associated with aortic dilation or aortic events. We performed a retrospective cohort analysis of unselected women and children with TS who underwent cardiovascular magnetic resonance angiography (MRA) for a prior prospective study. We calculated tortuosity indices including vertebral artery tortuosity index, aortic arch tortuosity index, thoracic aortic tortuosity index (ATI-D), and aortic tortuosity index to the celiac artery (ATI-C). We compared tortuosity in TS patients against age and gender matched controls. We evaluated univariable and multivariable associations between the tortuosity indices and aortic root and ascending aorta size as defined by z-scores, which give a sense of how far a measurement deviates from the mean. We also studied associations between tortuosity and need for aortic root replacement or aortic dissection. Of 184 subjects, with median age 34 years, mean general aortic root z-score was 0.1 ± 1.2 and mean general ascending aortic z-score was 0.4 ± 1.5. Three patients had aortic dissection, and one had prophylactic root replacement, which all occurred prior to first MRA. Vertebral tortuosity index, ATI-D, and ATI-C all increased with age (p 0.0001) for all. ATI-C was associated with larger general ascending z-score. In multivariable analysis, ATI-C remained independently associated with larger ascending aortic z-scores. The relationship between aortic indices and surgery/dissection could not be evaluated since all were collected post-surgery/dissection. Thoracic aortic tortuosity as measured by ATI-C is independently associated with larger ascending aortic dimensions. In this population with only three aortic dissections occurring prior to imaging assessment, we could not assess for associations between aortic tortuosity and dissection. Studies including more patients with aortic dissection are needed to draw further conclusions.

Background Despite the widespread use of optical coherence tomography (OCT) for imaging of keratinocyte carcinoma, we lack an expert consensus on the characteristic OCT features of basal cell carcinoma (BCC), an internationally vetted set of OCT terms to describe various BCC subtypes, and an educational needs assessment. Objectives To identify relevant BCC features in OCT images, propose terminology based on inputs from an expert panel and identify content for a BCC-specific curriculum for OCT trainees. Methods Over three rounds, we conducted a Delphi consensus study on BCC features and terminology between March and September 2020. In the first round, experts were asked to propose BCC subtypes discriminable by OCT, provide OCT image features for each proposed BCC subtypes and suggest content for a BCC-specific OCT training curriculum. If agreement on a BCC-OCT feature exceeded 67%, the feature was accepted and included in a final review. In the second round, experts had to re-evaluate features with less than 67% agreement and rank the ten most relevant BCC OCT image features for superficial BCC, nodular BCC and infiltrative and morpheaphorm BCC subtypes. In the final round, experts received the OCT-BCC consensus list for a final review, comments and confirmation. Results The Delphi included six key opinion leaders and 22 experts. Consensus was found on terminology for three OCT BCC image features: (i) hyporeflective areas, (ii) hyperreflective areas and (iii) ovoid structures. Further, the participants ranked the ten most relevant image features for nodular, superficial, infiltrative and morpheaform BCC. The target group and the key components for a curriculum for OCT imaging of BCC have been defined. Conclusion We have established a set of OCT image features for BCC and preferred terminology. A comprehensive curriculum based on the expert suggestions will help implement OCT imaging of BCC in clinical and research settings.

Background: The “1-3-6-12-day rule” for starting direct oral anticoagulants (DOACs) in patients with nonvalvular atrial fibrillation after acute ischemic stroke or transient ischemic attack recommends timings that may be later than used in clinical practice. We investigated more practical optimal timing of DOAC initiation according to stroke severity. Methods: The combined data of prospective registries in Japan, Stroke Acute Management with Urgent Risk-factor Assessment and Improvement-nonvalvular atrial fibrillation (September 2011 to March 2014) and RELAXED (February 2014 to April 2016) were used. Patients were divided into transient ischemic attack and 3 stroke subgroups by the National Institutes of Health Stroke Scale score: mild (0–7), moderate (8–15), and severe (≥16). The early treatment group was defined as patients starting DOACs earlier than the median initiation day in each subgroup. Outcomes included a composite of recurrent stroke or systemic embolism, ischemic stroke, and severe bleeding within 90 days. Six European prospective registries were used for validation. Results: In the 1797 derivation cohort patients, DOACs were started at median 2 days after transient ischemic attack and 3, 4, and 5 days after mild, moderate, and severe strokes, respectively. Stroke or systemic embolism was less common in Early Group (n=785)—initiating DOACS within 1, 2, 3, and 4 days, respectively—than Late Group (n=1012) (1.9% versus 3.9%; adjusted hazard ratio, 0.50 [95% CI, 0.27–0.89]), as was ischemic stroke (1.7% versus 3.2%, 0.54 [0.27–0.999]). Major bleeding was similarly common in the 2 groups (0.8% versus 1.0%). On validation, both ischemic stroke (2.4% versus 2.2%) and intracranial hemorrhage (0.2% versus 0.6%) were similarly common in Early (n=547) and Late (n=1483) Groups defined using derivation data. Conclusions: In Japanese and European populations, early DOAC initiation within 1, 2, 3, or 4 days according to stroke severity seemed to be feasible to decrease the risk of recurrent stroke or systemic embolism and no increase in major bleeding. These findings support ongoing randomized trials to better establish the optimal timing of DOAC initiation.

Os cães errantes podem ser importantes fontes de infecções ao ser humano, sobretudo na contaminação das areias das praias. O estudo teve por objetivo avaliar a presença de parasitos de características zoonóticas na praia de Laguna, Santa Catarina. Amostras de areia da praia do Mar Grosso foram coletadas e analisadas em diferentes estações do ano entre 2018 a 2019. As amostras foram processadas no Laboratório do Centro de Educação Superior da Região Sul (CERES) da Universidade do Estado de Santa Catarina (UDESC), pelo método de sedimentação. As larvas presentes nas amostras foram imobilizadas com uso de lugol e analisadas ao microscópio. Das 2000 amostras de areia, 19,10% apresentaram larvas de Ancylostoma spp., 7,25% de larvas de Toxocara spp. 1,75% ambas. A estação do inverno apresentou maior ocorrência de larvas de ancilostomídeos com 30,6%, já as larvas de ascarídeos no verão com 18,8%. A média de larvas por lâmina foi de 15 e todas estavam vivas. No período de verão observou-se que o número de larvas foi maior e com comportamento mais ativo, observado antes do uso de lugol. As larvas de Ancylostoma spp. sobreviveram por 62 dias e 94 dias de Toxocara spp. Foram identificados ovos de Ancylostoma spp. e Toxocara spp. nas amostras. A presença de ovos e larvas presentes em fezes de cães na areia da praia estiveram presentes em todas as estações do ano em diferentes temperaturas, assim estes animais transitando pela areia podem ser uma ameaça as pessoas que desfrutam deste ambiente para o trabalho ou lazer.

No contexto de uma América Latina pós-moderna, representações artísticas produzidas por mulheres buscam romper com discursos sacralizados pela tradição do cânone ocidental, principalmente em relação às identidades femininas. Em Mulheres empilhadas (2019), Patrícia Melo mobiliza categorias como “mulher” e “violência” para pensar na sociedade brasileira como partícipe de um sistema moderno colonial de gênero. Nesse sentido, pretendemos explicitar e analisar as estratégias narrativas utilizadas pela autora, com destaque à diversidade de formas discursivas e ao posicionamento da protagonista, em direção à expansão das potencialidades da literatura de autoria feminina. Partimos de teorias feministas de cunho decolonial; portanto, autoras como Zilá Bernd (1998), Tânia Pellegrini (2001), Zulma Palermo (2005), María Lugones (2014) e Florencia Garramuño (2014) fundamentam as discussões propostas.

BioLaw Journal - Rivista di BioDiritto, N. 1 (2023)

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and highly heterogeneous neoplasms whose incidence has markedly increased over the last decades. A grading system based on the tumor cells’ proliferation index predicts high-risk for G3 NETs. However, low-to-intermediate grade (G1/G2) NETs have an unpredictable clinical course that varies from indolent to highly malignant. Cultures of human cancer cells enable to perform functional perturbation analyses that are instrumental to enhance our understanding of cancer biology. To date, no tractable and reliable long-term culture of G1/G2 NET has been reported to permit disease modeling and pharmacological screens. Here, we report of the first long-term culture of a G2 metastatic small intestinal NET that preserves the main genetic drivers of the tumor and retains expression patterns of the endocrine cell lineage. Replicating the tissue, this long-term culture showed a low proliferation index, and yet it could be propagated continuously without dramatic changes in the karyotype. The model was readily available for pharmacological screens using targeted agents and as expected, showed low tumorigenic capacity in vivo. Overall, this is the first long-term culture of NETs to faithfully recapitulate many aspects of the original neuroendocrine tumor.

La estimulación temprana consiste en prevenir los probables déficits en el desarrollo psicomotor del nacido prematuro hospitalizado, busca crear un ambiente estimulante adaptado a las capacidades de respuestas inmediatas del prematuro. Está indicada como forma de rehabilitación o para seguimiento evolutivo y la misma no presiona ni acelera ningún proceso de desarrollo humano. Este trabajo pretende registrar los cambios provocados por esta terapia por lo que se evaluará a 20 prematuros del HPMI. Se hará una investigación no experimental descriptiva longitudinal, teniendo un enfoque cuantitativo. Early childhood stimulation consists in preventing probable deficits in the psychomotor development of the hospitalized premature infant, it seeks to create a stimulating environment adapted to the immediate response capacities of the premature infant. It is indicated as a form of rehabilitation or for evolutionary monitoring and it does not pressure or accelerate any process of human development. This work aims to record the changes caused by this therapy, which is why 20 premature infants will be evaluated for HPMI. A longitudinal descriptive non-experimental research will be carried out, taking a quantitative approach. Fil: Chávez, Agustín. Universidad Católica de Salta. Escuela Universitaria de Ciencias de la Salud; Argentina. Fil: Poma Villar, Candelaria. Universidad Católica de Salta. Facultad de Ciencias de la Salud; Argentina. Fil: Pezzini, Aparicio Camila. Universidad Católica de Salta. Facultad de Ciencias de la Salud; Argentina.

COVID-19 has been considered a vascular disease, and inflammation, intravascular coagulation, and consequent thrombosis may be associated with endothelial dysfunction. These changes, in addition to hypoxia, may be responsible for pathological angiogenesis. This research investigated the impact of COVID-19 on vascular function by analyzing post-mortem lung samples from 24 COVID-19 patients, 10 H1N1pdm09 patients, and 11 controls. We evaluated, through the immunohistochemistry technique, the tissue immunoexpressions of biomarkers involved in endothelial dysfunction, microthrombosis, and angiogenesis (ICAM-1, ANGPT-2, and IL-6, IL-1β, vWF, PAI-1, CTNNB-1, GJA-1, VEGF, VEGFR-1, NF-kB, TNF-α and HIF-1α), along with the histopathological presence of microthrombosis, endothelial activation, and vascular layer hypertrophy. Clinical data from patients were also observed. The results showed that COVID-19 was associated with increased immunoexpression of biomarkers involved in endothelial dysfunction, microthrombosis, and angiogenesis compared to the H1N1 and CONTROL groups. Microthrombosis and vascular layer hypertrophy were found to be more prevalent in COVID-19 patients. This study concluded that immunothrombosis and angiogenesis might play a key role in COVID-19 progression and outcome, particularly in patients who die from the disease.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with few available therapeutic options. Two transcriptional cancer cell states have been consistently reported in PDAC, with the basal-like/squamous phenotype displaying a more aggressive biological behavior. Genetic and epigenetic dysregulation of the axon guidance pathway are common in PDAC, yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in sustaining the progression of PDAC. We integrated available transcriptomic datasets of human PDAC within situhybridization analyses of patients’ tissues to find that SEMA3A is expressed by stromal cells and selectively enriched in epithelial cells of the basal-like/squamous subtype. We found that both cell-intrinsic and cell extrinsic factors instructing the basal-like/squamous subtype induce expression of SEMA3A in PDAC cells.In vitro, SEMA3A promoted cell migration as well as anoikis resistance. At molecular level, these phenotypes were associated with increased FAK signaling and enrichment of gene programs related to cytoskeleton remodeling. Accordingly, SEMA3A provided mouse PDAC cells with greater metastatic competence. In mouse orthotopic allografts, SEMA3A remodeled the TME by favoring infiltration of tumor-associated macrophages and exclusion of T cells. Mechanistically, SEMA3A functioned as chemoattractant for macrophages and favored their polarization towards an M2-like phenotype. In SEMA3Ahightumors, depletion of macrophages resulted in greater intratumor infiltration by CD8+ T cells and increased sensitivity of these tumors to chemotherapy. Overall, we show that SEMA3A contributes to the malignant phenotype of basal-like PDAC.

During the storage period of dry tobacco and its derivatives, insect pests such as species of Ephestia Guenée (Lepidoptera: Pyralidae), popularly known as moths, cause damage to the product, being controlled mainly with physical practices and synthetic chemicals, although with limitations on their use and results. Some biological control agents, such as the parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae), have the potential to control these pests. This study aimed to evaluate the effect of H. hebetor release in tobacco farms and industrial warehouses, on the infestation of Ephestia spp. adults. Parasitoids were released in tobacco farmers and tobacco industry warehouses between 2016-2018, comprising two years/crop seasons. Each release consisted of 1,000 parasitoids (four times) in tobacco farmers warehouses (70 m2) and 30,000 (five times) in tobacco industry warehouses (8,000 m2). Pheromone-baited sticky traps were used to weekly monitor the average number of adults of Ephestia spp. in warehouses with (WP) and without parasitoid release (NP). The average number of adults of Ephestia spp. captured in the traps in WP environments at farmers and industry level was significantly lower than the average captured in NP from the third and fifth weeks. In the following weeks, the averages of Ephestia spp. were always significantly lower in the WP environments until the end of monitoring. Therefore, the use of H. hebetor for moth control should be considered as part of a biological control program in stored tobacco environments.

Intrathecal inflammation plays a key role in the pathogenesis of multiple sclerosis (MS). To better elucidate its relationship with peripheral inflammation, we investigated the correlation between cerebrospinal fluid (CSF) and serum levels of 61 inflammatory proteins. Paired CSF and serum samples were collected from 143 treatment-naïve MS patients at diagnosis. A customized panel of 61 inflammatory molecules was analyzed by a multiplex immunoassay. Correlations between serum and CSF expression levels for each molecule were performed by Spearman’s method. The expression of sixteen CSF proteins correlated with their serum expression (p-value < 0.001): only five molecules (CXCL9, sTNFR2, IFNα2, Pentraxin-3, and TSLP) showed a Rho value >0.40, suggesting moderate CSF/serum correlation. No correlation between inflammatory serum patterns and Qalb was observed. Correlation analysis of serum expression levels of these sixteen proteins with clinical and MRI parameters pinpointed a subset of five molecules (CXCL9, sTNFR2, IFNα2, IFNβ, and TSLP) negatively correlating with spinal cord lesion volume. However, following FDR correction, only the correlation of CXCL9 remained significant. Our data support the hypothesis that the intrathecal inflammation in MS only partially associates with the peripheral one, except for the expression of some immunomodulators that might have a key role in the initial MS immune response.

ObjectiveTo investigate the age-dependent impact of traditional stroke risk factors on the occurrence of intracerebral haemorrhage (ICH).MethodsWe performed a case–control analysis, comparing consecutive patients with ICH with age-matched and sex-matched stroke-free controls, enrolled in the setting of the Multicenter Study on Cerebral Hemorrhage in Italy (MUCH-Italy) between 2002 and 2014 by multivariable logistic regression model within subgroups stratified by age quartiles (Q1–Q4).ResultsWe analysed 3492 patients and 3492 controls. The impact of untreated hypertension on the risk of ICH was higher in the lower than in the upper age quartile (OR 11.64, 95% CI 7.68 to 17.63 in Q1 vs OR 6.05, 95% CI 3.09 to 11.85 in Q4 with intermediate ORs in Q2 and Q3), while the opposite trend was observed for untreated hypercholesterolaemia (OR 0.63, 95% CI 0.45 to 0.97 in Q1 vs OR 0.36, 95% CI 0.26 to 0.56 in Q4 with intermediate ORs in Q2 and Q3). The effect of untreated diabetes and excessive alcohol intake was detected only in the older age group (OR 3.63, 95% CI 1.22 to 10.73, and OR 1.69, 95% CI 1.13 to 2.51, respectively).ConclusionsOur findings provide evidence of age-dependent differences in the effects of susceptibility factors on the risk of ICH.

Background and purpose Whether the reported association between migraine with aura (MA) and cardioembolic stroke may be explained by a higher rate of atrial fibrillation (AF) or by other potential cardiac sources of cerebral embolism remains to be determined.Methods In the setting of a single centre cohort study of consecutive patients with acute brain ischaemia stratified by migraine status, the association between AF as well as patent foramen ovale (PFO) and migraine was explored.Results In all, 1738 patients (1017 [58.5%] men, mean age 67.9 +/- 14.9 years) qualified for the analysis. Aging was inversely associated with migraine, whilst women had a >3-fold increased disease risk (odds ratio [OR] 3.82, 95% confidence interval [CI] 2.58-5.66). No association between AF and history of migraine or its pathogenic subtypes was detected. Conversely, migraine was associated with PFO, both in the entire cohort (OR 1.84, 95% CI 1.07-3.16) and in patients aged

Background and Purpose: Clinical trials on stroke prevention in patients with atrial fibrillation have consistently shown clinical benefit from either warfarin or non–vitamin K antagonist oral anticoagulants (NOACs). NOAC-treated patients have consistently reported to be at lower risk for intracerebral hemorrhage (ICH) than warfarin-treated patients. The aims of this prospective, multicenter, multinational, unmatched, case-control study were (1) to investigate for risk factors that could predict ICH occurring in patients with atrial fibrillation during NOAC treatment and (2) to evaluate the role of CHA 2 DS 2 -VASc and HAS-BLED scores in the same setting. Methods: Cases were consecutive patients with atrial fibrillation who had ICH during NOAC treatment. Controls were consecutive patients with atrial fibrillation who did not have ICH during NOAC treatment. As within the CHA 2 DS 2 -VASc and HAS-BLED scores there are some risk factors in common, several multivariable logistic regression models were performed to identify independent prespecified predictors for ICH events. Results: Four hundred nineteen cases (mean age, 78.8±8.1 years) and 1526 controls (mean age, 76.0±10.3 years) were included in the study. From the different models performed, independent predictors of ICH were increasing age, concomitant use of antiplatelet agents, active malignancy, high risk of fall, hyperlipidemia, low clearance of creatinine, peripheral artery disease, and white matter changes. Low doses of NOACs (given according to label or not) and congestive heart failure were inversely associated with the risk of ICH. HAS-BLED and CHA 2 DS 2 -VASc scores performed poorly in predicting ICH with areas under the curves of 0.496 (95% CI, 0.468–0.525) and 0.530 (95% CI, 0.500–0.560), respectively. Conclusions: Several risk factors were associated to ICH in patients treated with NOACs for stroke prevention but not HAS-BLED and CHA 2 DS 2 -VASc scores.

Background: In patients with atrial fibrillation who suffered an ischemic stroke while on treatment with nonvitamin K antagonist oral anticoagulants, rates and determinants of recurrent ischemic events and major bleedings remain uncertain. Methods: This prospective multicenter observational study aimed to estimate the rates of ischemic and bleeding events and their determinants in the follow-up of consecutive patients with atrial fibrillation who suffered an acute cerebrovascular ischemic event while on nonvitamin K antagonist oral anticoagulant treatment. Afterwards, we compared the estimated risks of ischemic and bleeding events between the patients in whom anticoagulant therapy was changed to those who continued the original treatment. Results: After a mean follow-up time of 15.0±10.9 months, 192 out of 1240 patients (15.5%) had 207 ischemic or bleeding events corresponding to an annual rate of 13.4%. Among the events, 111 were ischemic strokes, 15 systemic embolisms, 24 intracranial bleedings, and 57 major extracranial bleedings. Predictive factors of recurrent ischemic events (strokes and systemic embolisms) included CHA 2 DS 2 -VASc score after the index event (odds ratio [OR], 1.2 [95% CI, 1.0–1.3] for each point increase; P =0.05) and hypertension (OR, 2.3 [95% CI, 1.0–5.1]; P =0.04). Predictive factors of bleeding events (intracranial and major extracranial bleedings) included age (OR, 1.1 [95% CI, 1.0–1.2] for each year increase; P =0.002), history of major bleeding (OR, 6.9 [95% CI, 3.4–14.2]; P =0.0001) and the concomitant administration of an antiplatelet agent (OR, 2.8 [95% CI, 1.4–5.5]; P =0.003). Rates of ischemic and bleeding events were no different in patients who changed or not changed the original nonvitamin K antagonist oral anticoagulants treatment (OR, 1.2 [95% CI, 0.8–1.7]). Conclusions: Patients suffering a stroke despite being on nonvitamin K antagonist oral anticoagulant therapy are at high risk of recurrent ischemic stroke and bleeding. In these patients, further research is needed to improve secondary prevention by investigating the mechanisms of recurrent ischemic stroke and bleeding.

A partir da narrativa O caso dos falcões-peregrinos (2019), do escritor pernambucano Severino Rodrigues, o artigo investiga os elementos clássicos, modernos e contemporâneos no que diz respeito ao gênero policial, bem como aspectos da literatura juvenil brasileira. A discussão parte de teóricos como Bordieu (1996) e Bakhtin (2006), em relação aos gêneros literários; Todorov (2006) e Fajardo (2018), no que concerne à ficção policial; e, para fundamentar a literatura juvenil, Lajolo e Zilberman (2007) e Luft (2010). Pode-se constatar que a obra analisada justapõe aspectos do romance policial clássico e do moderno, transformando-se em uma narrativa de suspense contemporâneo.

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.

Objective To characterize patients with acute ischemic stroke related to SARS-CoV-2 infection and assess the classification performance of clinical and laboratory parameters in predicting in-hospital outcome of these patients. Methods In the setting of the STROKOVID study including patients with acute ischemic stroke consecutively admitted to the ten hub hospitals in Lombardy, Italy, between March 8 and April 30, 2020, we compared clinical features of patients with confirmed infection and non-infected patients by logistic regression models and survival analysis. Then, we trained and tested a random forest (RF) binary classifier for the prediction of in-hospital death among patients with COVID-19. Results Among 1013 patients, 160 (15.8%) had SARS-CoV-2 infection. Male sex (OR 1.53; 95% CI 1.06–2.27) and atrial fibrillation (OR 1.60; 95% CI 1.05–2.43) were independently associated with COVID-19 status. Patients with COVID-19 had increased stroke severity at admission [median NIHSS score, 9 (25th to75th percentile, 13) vs 6 (25th to75th percentile, 9)] and increased risk of in-hospital death (38.1% deaths vs 7.2%; HR 3.30; 95% CI 2.17–5.02). The RF model based on six clinical and laboratory parameters exhibited high cross-validated classification accuracy (0.86) and precision (0.87), good recall (0.72) and F1-score (0.79) in predicting in-hospital death. Conclusions Ischemic strokes in COVID-19 patients have distinctive risk factor profile and etiology, increased clinical severity and higher in-hospital mortality rate compared to non-COVID-19 patients. A simple model based on clinical and routine laboratory parameters may be useful in identifying ischemic stroke patients with SARS-CoV-2 infection who are unlikely to survive the acute phase.

The aim of the study was to identify, synthesize and classify the software currently available that can help in the task of phonetic transcription of the spontaneous speech of pre-school children to evaluate the development of children's language. A systematic review was performed for articles published, for the 10-year period (June 2010 to June 2020), without restrictions as to location and language, using the Cochrane, Pubmed and Web of Science databases. The terms used in the search strategies were "phonological", "phonetic", "transcription", "computer" and "software". The studies were selected by two independent reviewers using pre-defined search strategies. In the initial search, after the exclusion of duplicates, 534 articles were found. By reading their titles and abstracts, 46 articles related to the theme were left, which were then read in full. After reading, 24 articles were included in the study. The results revealed a total of seven software available for the phonetic transcription of spontaneous speech from preschoolers used for different analyses: LENA and Timestamper (for babbling and pre-linguistic vocalizations), ELAN (for gestural communication, extralinguistic elements and the situational context), Phon (for phonetic and phonological analyses), CLAN and SALT (for morphosyntactic, grammatical and semantic aspects) and Praat (for acoustic measurements). Through this systematic review, it can be concluded that there are advantages to using software for phonetic transcription, sample storage, and child language analysis, especially concerning standardization and reliability for spontaneous speech samples. Phonetic transcription still relies on the ability and subjectivity of a human transcriber. The tools found in the software provide support to facilitate using phonetic symbols, audio segmentation and pairing to writing, and analysis of speech data.