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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.

Degenerative tendon pathology represents one of the most frequent and disabling musculoskeletal disorders. Diagnostic radiology plays a fundamental role in the clinical evaluation of tendon pathologies. Moreover, several minimally invasive treatments can be performed under imaging guidance to treat tendon disorders, maximizing the efficacy and reducing procedural complications. In this review article we describe the most relevant diagnostic features of conventional and advanced US and MRI imaging in tendon disorders, along with the main options for image-guided intervention. (www.actabiomedica.it)

The tomb of Giovanni and his wife Maria Salviati at Cappelle Medicee in Florence was explored to investigate the skeletal remains. Anthropological and paleopathological examination defined: age at death, physical constitution and activity, skeletal diseases. The stump of the right leg was studied macroscopically, under stereoscopic microscope, at X-ray and CT scans, to detect type of injury and level of amputation. The study of the skeleton of Giovanni revealed that he was a vigorous man, 1.78 m tall, with an athletic body, estimated skeletal age of 25-30 years, medium-sized skull, narrow nose and great skull capacity (1494 cc). His well-developed upper limbs muscular insertions (deltoid, great pectoral, great dorsal, biceps, forearm muscles) and thigh muscles confirmed his great physical strength and robusticity. Strong hypertrophy of rotator cuff, great dorsal, teres minor and anconeus insertions were all present, as well as gluteal insertions to the femur, confirming he was a highly skilled horseman. The presence of numerous Schmorl’s hernias and a wedge partial collapse, with right spondylolysis, of the fifth lumbar vertebra, revealed that Giovanni had carried heavy loads since adolescence due to horse-riding and body armor. Diffuse bilateral enthesopathies were found at the clavicular insertions of deltoid and pectoralis major, as well as at the small trochanter (psoas muscle). Skeletal markers left by habitual horseback riding were all present: exostoses and ovalization of acetabula, hypertrophy of femoral rectum muscle, strong hypertrophy of the femoral biceps, great adductor, small and great gluteus, Poirier’s facet. Paleopathological investigation showed the aftermaths of several injuries: fractures of nasal septum and proximal third of the left humerus, injury from blade affecting right ulna and radius and swelling of the posterior surface of the right tibia, with underlying osteomyelitic focus in reparative phase, as well-documented on CT. The amputation level was exactly assessed: the tibia was sawn immediately below the proximal half of diaphysis and only the lateral portion was surgically treated with an horizontal cut. Only oblique splitting was found at the medial site of the tibia. At stereoscopic microscope, surgical section revealed a marked proliferation of endosteal callus, due to a previous harquebus shot injury occurred about one year before the death. Distal extremity of fibular fragment showed an oblique splitting and a horizontal cut, with no sign of reparative process in the medullar canal. Considering the morphological aspect of the tibial and fibular injury, it was due to a cannonball from a falconet of caliber 6-7 cm, as written by Benedetto Agnello in the same day of injuring. The limb had been severely damaged by a traumatic hemi-amputation when surgeon Abramo performed the intervention, consisting in a simple completion of the amputation and regularization of proximal fragments. In conclusion, paleopathological investigations lead to exclude the hypothesis of an amputation above the knee, since the surgeon Abraham performed the procedure as best as he could in conformity with surgical knowledge of that period., Biomechanical and kinesiological reasoning allow us to investigate traumas in Paleopathology. The focus of our analysis is to reconstruct pathomechanics, treatment and gait of a subject from the Late Medieval femur, which presents an important bone callus (1). The femur was discovered in a funerary crypt of the Sanctuary of Sacro Monte (Varese, Northern Italy), an important archaeological context inserted into the UNESCO heritage since 2003. The femur was studied with computer tomography and the reconstruction of the static and dynamic fictional outcomes of the lesion was performed by the Observation Gait Analysis (OGA). The OGA is the computerized analysis of the gait. This technique permits to observe movements of each articulation in the space, the posture and the gait underlining a movement strategy. The femur presents an important callus at the middle third proximal of the shaft. The fracture is oblique and caused by a direct trauma probably associated with occupational activities. The alignment of the segments in the frontal plane leads us to assume that the fracture was treated and the femur was immobilized with splints. The use of OGA allows us to understand the subject’s kind of gait after healing. Our analysis demonstrated that there was no reduction in bone mass. The deposition of new cortical bone near the fracture determines that the individual has gradually resumed loading the leg and was walking although with significant effects on posture and movement., In 2008, archaeological excavations carried out in the inner courtyard of the former Jesuit College of Alghero brought to light the San Michele cemetery. Characteristic of the site are some burial trenches, narrow and long pits containing the remains of 10 to 30 individuals, and some multiple tombs, which can be related to the plague epidemic that ravaged the city in 1582-83. The present study is focused on the demographic analysis of the 16 trenches containing 185 individuals and of one multiple tomb (T.141) with 14 individuals. In the case of the trenches it was possible to determine the sex of 178 individuals: 37 are males (20.7%), 53 females (29.8%) and 88 of undetermined sex (49.5%). As for the first two groups, 35.6% of individuals has an age comprised between 20 and 29 years; the two age ranges 30-39 and 40-49 years present the same percentage (25.6%), 8.9% are aged between 17 and 19, and finally 4.4% are over the age of 50. The undetermined sex category is represented by 81 subadults and 7 adults. As for the subadults, the most representative age group is that between 7 and 12 years old with 39.8%, followed by the 23.9% between 13 and 19 years, 18.2% between 2 and 6 years, and finally, 10.2% between 0 and 1 years. The multiple tomb 141 includes an adult individual (20-29 years) of undetermined sex, a woman of about 17 years and with a 35-week fetus in her womb, and finally 12 subadults in an age range between 0-1 years (21.4%), 2-6 years (7.1%) and 7-12 years (50.0%). The cemetery of San Michele presents some similarities with the French cemetery of Martigues struck by the plague in 1720. Also in this cemetery this type of trench burials was found, 5 in this case, with 199 individuals. The comparison between the paleodemographic curves for both cemeteries evidences a similarity of the mortality trend. The difference between normal and catastrophic cemeteries, related to a severe epidemic event, consists in the fact that while in the former there is a greater presence of infants and elderly, in the latter there is a certain homogeneity of mortality, proof of the fact that the plague kills in a random way and therefore all individuals present the same risk of death., Ossification of spinal ligaments can persist on skeletal remains requiring a differential diagnosis between diseases that can lead to spinal column ankylosis. The most relevant diseases that can lead to this condition are represented by ankylosing spondylitis (AS) and diffuse idiopathic skeletal hyperostosis (DISH). The differential diagnosis between these two conditions is discussed in two cases of our observation. Case n. 1: Policastro Bussentino, US 112 - The burial (US 112) was found in the convent of San Francesco in Policastro Bussentino, during an emergency excavation carried out in 2015. The skeleton on a stratigraphic basis is dated between 1846 and 1892. The skeleton is affected by the fusion of 12 vertebrae of the vertebral column, from the fusion of some costovertebral joints and of the sacroiliac joints. Case n. 2: Turin, Collection Marro, skeleton n.16809 - The skeleton belongs to the Egyptian Collection of the Museum of Anthropology and Ethonography of the University of Turin. It was collected during the archaeological campaign at Gebelein (Upper Egypt) in 1920 and dates back to the First Intermediate Period (2150-1990 BC). In this case, the pathology involves only the vertebral column in which the fusion of numerous vertebrae is found. AS is a progressive inflammatory disease of unknown etiology primarily affecting the diarthrodial joints of the spine, the costovertebral joints and the sacroiliac joints and usually begins in the second or third decade of life. First it affects the lumbar spine and the sacroiliac joints and progressively ascends until the entire spine and all costovertebral joints are affected. The result of this ankylosing process is the rigid, so-called bamboo spine with loss of its physiological curvatures. DISH is an ossifying diathesis producing ankylosis of the spine due to ligament ossification without intervertebral disk disease. It is not a true arthropathy because neither cartilage nor synovium are involved. It is rarely detected before the age of 40 years and the cause is unknown. The two pictures macroscopically can be confused because both involve the spine with ossification and ankylosis of it. The differential diagnosis on skeletal remains can be based on extimated age and sex and on the elements involved. AS occurs in the second or third decade of life and mainly affects men. DISH, instead, appears not before the fourth decade of age. DISH only affects the spine, while ankylosing spondylitis also affects the cost-vertebral joints and the sacro-iliac joint. The ligaments involved in the fusion of the spine are different in the two cases. DISH does not affect the intervertebral discs unlike AS which instead involves them and thus has a total fusion of the vertebral bodies. Although DISH and AS manifest in a similar manner, they are separate diseases. Both pathologies are quite common in mild and initial forms, but are rather rare in the full-blown, severe forms, which involve the whole spine. This topic appear poorly covered both in paleopathological and clinical literature, mainly consisting in small series and case reports. As a consequence, a wide inter-individual variability is present and only rarely an accurate report of the different involved ligaments is provided. In most of the cases the description is limited to a generic attribution to spinal ligaments. Differential diagnosis may be challenging if limited to anthropological examination of the skeletal remains and further radiologic and genetic tests are necessary to confirm our findings., The object of this study is the so-called “green mummy” of Bologna, a naturally mummified body that was found in the basement of an ancient mansion in Bologna in the 1920s of last century. The hard and soft tissues of the body are green for the most. They were analyzed by FTIR spectroscopy with the aim to gain information about the biochemical degradation process, to explain both the origin and the nature of the green color and to understand how it affected the body’s preservation. The corpse was found in a copper or bronze cist that, at the time of recovery, was closed with a lid but broken at the base. It is reasonable to assume that the body had undergone the processes of putrefaction, liquefaction, skeletonization and mummification within the container. Probably, the semi-fluid mass of water and decomposing substances came out from the cist through the break at its base; therefore, the not yet putrefied tissues exposed to the atmosphere of the basement, desiccated and mummified. The acidic liquids originated by the decomposition caused the container corrosion, favouring the release of copper ions. Metal ions both inhibited the enzymes responsible for chemical reactions in the early stages of the decomposition process and acted as biocides of microorganisms involved in decomposition. FTIR analysis of soft tissues, with and without green color, showed that tissues lacking the green patina were the best preserved revealing the protein structure only minimally deteriorated, contrarily to what was observed in green areas. We have hypothesized that copper ions might have caused the decarboxylation of the RCOO group of polypeptidic backbone favoring protein degradation. Therefore, copper did not favor the mummification process, which was probably due to the environmental conditions, such as the low temperatures typical of the basement and the low availability of oxygen. Due to the known biocidal action of copper ions, we assume that copper ions might have damaged dead tissue cells in the same way they damage the cell membrane of microorganisms with which it comes into contact, causing their death. After the body decomposition liquids came out from the container, copper corrosion products precipitated as copper compounds giving the remains the green color. On the soft tissues two mineral forms of copper have been found: copper carbonate and copper phosphate called malachite and sampleite. Also, FTIR analysis of bones revealed that copper did not contribute to tissue preservation. The measured mineralization index showed the alteration of both collagen and hydroxyapatite of colored bones unlike the colorless ones. A compound known as pseudomalachite was identified in the green bones, a form of hydroxyapatite where copper replace calcium. This substitution is responsible for the unusual green coloration of the bones., The new paleoradiological investigations carried out on the Egyptian mummified human remains, a head, a left hand and foot, housed in the Civic Archaeological Museum of Erba (Como, northern Italy), allowed us to acquire comprehensive digital images and to study the anthropological and paleopathological data of the subjects. The first macroscopic investigation revealed the presence of blue-glazed Faience tubes, adherent to the dorsal wrappings of the foot. This custom was particularly attested in the 26th dynasty, even if it appeared already from the 21st dynasty and seemed to continue until the Ptolemaic period. The tomographic analysis revealed the non-compatibility of the three mummified parts to a single individual, based on the different degrees of bone development and degeneration. The radiological investigation allowed us to acquire data on the embalming techniques applied, and on the health status and pathological conditions of the head. In particular, the parietal bones of the cranium exhibited two symmetrical areas of thinning and resorption of the outer table, which suggest a case of “biparietal thinning”, also known as “biparietal osteodystrophy”. The areas involved are well-circumscribed and elliptical in shape, localized between the obelion and the superior linea temporalis. Macroscopically, these areas are noted as slight depression and flattening of the outer cortical layer. The CT imagings also revealed the mature age of the individual, which, according to several studies, is compatible with this finding. A research in the literature, showed a prevalence of this condition in Egyptian individuals, associating it with different definitions and causes. Although the etiology of this affection is still not well-known, here the embalmed head of an elderly individual revealed the presence of biparietal affection, with thinning and resorption of the outer table, adding additional evidence of this finding in an Egyptian subject., The GEDEON project will allow us to broaden the knowledge about the human adaptation against changes in nutritional resources consumption. The main purpose of the project is to investigate the putative role of selective pressure that may have acted on specific genetic markers linked to changed dietary conditions. In order to reach this topic, the project aims to connect the scientific evidence obtained through osteological studies of the ancient remains dating across the major nutritional transitions, with the evaluation of genetic markers involved in metabolic pathways that may have been affected by nutrient bio-availability. The whole genome analysis of ancient selected skeletal specimens will be sequenced to compare the data obtained with those from extant people suffering of nutritional impairments, whose information on dietary requirements is available. Known polymorphisms that are classically referable to diet-derived homeostatic alterations will be selected, such as rs2066844, rs2066845 and rs2066847, whose association with inflammatory intestinal diseases (IBD) is well known. Other variants mapping on NOD2 gene (for example rs2066843 and rs2076756) will be focused, but they will represent only the starting point for the identification of causative molecular pathways modifiers. Furthermore, the markers with a well-known association with alterations such as celiac disease (CD) and primary hypolactasia (PH) will be also considered: PTPN2 and IL18RAP loci as well as various HLA system factors and the LCT gene will be primarily evaluated. This shortlist selection will constitute the beginning for the identification of several new markers to shed light on human genetic adaptation to the changed environmental conditions including the nutritional requirements., The application of the next generation sequencing techniques to the study of ancient DNA represents an outstanding improvement for clarifying complex scenarios related to genomic-based physio-pathological conditions, whose identification in ancient remains can be tricky. Indeed, the sole presence of osteological markers could be misleading for proper diagnosis due to the non-specific nature of such lesions. A thorough molecular evaluation has been performed on a skeleton of an adult woman dating to the Roman Imperial Age. The erosive and osteolytic markers located in the tarsal bones have led to hypothesize that she probably suffered from gout. In an attempt to integrate the differential diagnosis based on osteological data, whole genome sequencing analysis was performed. The bioinformatics pipeline identified the presence of two variants in the TSC2 gene, that is known to be associated with a rare genetic disorder, the Tuberous Sclerosis Complex, featured by signs that could be shared with those due to gouty arthritis. The application of these ultimate molecular techniques surely represents a successful diagnostic tool for the identification of genetic related disorders that could be only hypothesized in ancient times., Domenico Petruccelli (Raffaele da Sant’Elia a Pianisi), servant of God, was born in Sant’Elia a Pianisi (Campobasso) in 1816 in a family of honest and wealthy peasants. He lived in different convents between Puglia and Molise. Wherever he went, he won the esteem and veneration of everyone, so much so as to be called the “Holy Monk”. He died in the convent of Sant’Elia a Pianisi in 1901. In the same convent another famous monk arrived in 1904 fr. Pius from Pietrelcina (better known as Padre Pio), here the young monk breathed the aura of holiness left by the Holy Monk who died a few years earlier, drawing strength and inspiration. A first exhumation had been performed in 1934 and the skull was placed in a metal case. In 2017, 200 years after his birth, during the second exhumation and canonical recognition of the mortal remains, the skull was in good condition of conservation. It was covered in a thin layer of yellowish-white powder and traces of deteriorated fabric which formed part of the inner lining of urn. Cleaning operations The skull was cleaned. During this operation, we proceeded to collect in a container of numerous fragments adhered to the outer and inner surface of the skull. Consolidation operations The skull was impregnated with a thin protective membrane consisting of a very thin film of an acrylic resin (Paraloid B72TM) which protects it and consolidates its most delicate and fragile parts. Paleopathological study The alterations and lesions found are: in the left orbit, two holes (of undetermined origin) in the left parietal and temporal bones, in the right parietal bone, at the base of the skull, the jaw is absent. Attribution of sex We highlight a series of morphological findings characteristic of the male sex. They are: protruding glabella and sloping forehead; Rounded, wide and thick super-margin; Wide zygomatic arch that extends beyond the external acoustic meatus; Mastoid process robust, big and verticalized; Necked crest marked, wrinkled and very evident. Diagnosis of age Coronal, sagittal and lambdoidal sutures were considered, in accordance with the criteria of Acsàdi and Nemeskèri (1970) modified by Masset (1989). The endocranial closing index can be estimated at around 4, so the subject’s age is between 50 and 80 years, according to the known age of Father Raffaele of 85 years at the time of death. Craniometry The craniometric assessments carried out highlighted: a small, rounded, long, narrow, medium-high skull with rounded sagittal contours, oval and angular orbits, mean interorbitary distance, narrow nasal opening. All these characteristics allow us to state that the skull belonged to a Caucasian subject. Histological examination The fragments examined were composed of human organic material. These are extensively necrotic and rotten tissues. An important result has been achieved with the finding in the histological findings of many fungal hyphae, better highlighted with histochemical stains PAS and Grocott., Historical information related to the life of St. Mercurialis is very scarce, everything we currently know, we owe to its legenda, is contained within the manuscript Casanatense 718 dating to the 12th century. The only certain historical information concerns the ordination of one of his successors, Grato, which took place in Ravenna during the 5th century. The relics of St. Mercurialis, preserved inside the homonymous abbey, in the Cathedral of Santa Croce and in the Santissima Trinità church, during 2018 were object of the sixth canonical recognition, necessary to verify the state of conservation of the bones. Preliminary studies were performed by direct anthropological and radiological analyses by CT scan, FTIR analysis, ancient DNA and radiocarbon dating. St. Mercurialis, was about 1.60 meters tall, the age at death is 45-50 years, and he was not particularly robust, even if marked by repeated musculoskeletal stress probably linked to habitual activities such as walking and weightlifting. He suffered from osteoporosis and perhaps had some discomfort with the shoulder girdle. He had a deviated nasal septum from birth, a condition that perhaps caused him disorders such as sinusitis. He did not suffer from osteoarthritis and he had no particular indicators about deficiencies suffered during the first and last period of life. Analyses did not reveal indicators due to traumatic events and probably did not die by strangulation, as the hyoid bone was intact. FTIR analysis was carried out on the brown substance that partly covered the lower skeleton district, shows the typical spectrum of clayey materials. The characteristic bands reveal the presence of aluminum and silicon in greater quantities, and of other elements in smaller quantities. The clays constituting the soil can therefore be considered essentially illites containing kaolinite, smectite and quartz. This aspect confirms the numerous historical information concerning the floods suffered by the abbey. A patina that covered some bones was also detected. The obtained spectrum presents the typical absorptions of the vibrations of the hydrocarbon radicals CH2 and CH3, in addition to the intense absorption typical of the C-O-C group characteristic of carbohydrates. The characteristics of the spectrum therefore seem to be typical of a methylcellulose preservative. Radiocarbon dating and accelerometry mass spectrometry (AMS) dated the relics to the I-III century AD, an interesting date that is chronologically before the only historical indication we have about the life of St. Mercurialis and which collocates his episcopate into the first stages of evangelization of the Emilia Romagna Region. The preliminary analyses of ancient DNA were targeted on the hypervariable region 1 (HVR-1) of the mitochondrial DNA (mtDNA) and on Short Tandem Repeats (STRs) of the Y-chromosome and highlighted a rather pronounced diagenesis of the DNA. The subsequent analyses will be targeted to the capture of the entire mtDNA, coupled with next generation sequencing., Andrea was a Catholic priest and a member of the Order of Saint Augustine. He was born to a modest family in the village of Mascioni (northern borders of L’Aquila province) and as a child he worked as a shepherd. Around 1417 he met Augustine from Terni, Prior of the Augustinian convent in the near Montereale, and asked to enter their ranks to be ordered as a priest at the age of 25. He earned a bachelor and master’s degree in theology, becoming professor in Siena (1443), and Provincial Prior of the Umbria region. He also served as a travelling preacher in Italy and France, reforming several Augustinian monasteries in Umbria. He died on 18 april 1479 in Montereale and, according to hagiographies, his body was exposed without balsams in the conventual church for 30 days, giving off sweet odor and performing miracles. Subsequently, it was placed in the Choir until 1568, when it was displayed beneath the main altar. His beatification was celebrated by Pope Clement XIII on 18 february 1764. In 1787 the body was translated into a newly built repository, inside the new chapel dedicated to him. Documented Canonical Recogntions took place in August 1786, July 1943, June 1961, and between June and July 1989. The last was performed by one of us (MR) and the late professor Giulio Marinozzi. External examination of the body allowed to recognize a partially skeletonized mummy belonging to an old male subject (more than 70 years of age at death) and measuring 164 cm in length. The face was almost entirely covered by mummified skin, with traces of hair in the perioral region, chin, cheeks and anterior neck, according to devotional representations of the Blessed as a bearded elder. Soft tissues of forearms, hands, legs and feet appeared in a good preservation state. A large bone defect was observed in the occipital squama. Large skin cuts were observed in the anterior neck and left hemithorax. The ventral portions of the left ribs appeared cut and displaced within the thoracic cavity. Cut marks were also found on the left margin of the sternum body and on anterior branches of the pelvis. Preserved skin was observed only in the right hemithorax. No traces of internal organs were found in thorax, abdomen and pelvis. Moderate-marked osteoarthritis of the spine was noted. Unfortunately, a radiologic investigation of the body was not performed. After external examination, conservative treatment was performed. The body of Andrea revealed indisputable evidence of artificial mummification, representing the eleventh described case of an embalmed Saint or Blessed in Catholic Religion. Nine of these artificial mummies were created in central Italy (Umbria, Toscana, Abruzzo, Lazio) between the XIII and XV century. The employed evisceration procedures appear somewhat rough, without the complexity observed in other examples. It is worth to note that Montereale is located not far from L’Aquila and on the main route towards Cascia and Spoleto. The embalming of the Blessed Andrea took place only 35 years after the death and embalming of Saint Bernardino da Siena in L’Aquila and represents the second case in Abruzzo region., Liutprand, one of the greatest Longobard sovereign, was born in the early 90s of the 7th century and died in 744 at the age of about 55 years. According to the Historia Longobardorum of Paolo Diacono, he was King of Longobards from 712 to 744. The remains of King Liutprand suffered from many translations in the centuries and this is the main problem for the validation of the authenticity of the bone remains. The first grave was in the chapel of Sant’Adriano in the Longobard cemetery of Santa Maria in Pertica (Pavia). Later, in the second half of 12th century, the body of Liutprand was translated in the Basilica of San Pietro in Ciel d’Oro and located in a monumental mausoleum. New translations took place after the Council of Trento and in 1895, when the bones were placed in a niche in the floor of the church where they were found in January 2018. The bones, contained in a wooden box, appeared extremely fragmented and in a poor state of preservation. Anthropological examination highlighted the presence of bones attributable to three individuals. Most of the remains belongs to a robust male individual with strong muscular insertions, with an age at death between 40 and 50 years. There is also a second older male with strong muscle insertions, and a third adult individual of similar size. In fact, there are a pathological left tibia and some fragments of its right controlateral, and other fragments attributable to two other left tibiae. The tibia with pathological alterations presents the upper third of the diaphysis completely altered and enlarged by bone thickening due to a severe form of osteomyelitis. Bone repair is evident and the presence of a circular depression with a diameter of about 10 mm at the point of maximum thickening could represent the trace left by a pointed object that caused the perforation of the bone and the subsequent infection with osteomyelitis. The reparative process has however led to the healing of the lesion before death, which should have occurred not far from the event (maximum 2 years). Imaging studies (CT and radiological) on the skeletal remains were performed at the “San Matteo” General Hospital in Pavia. 14C dating provide a range from 430 to 640 for the first subject, 600-770 for the second and 530-670 for the last male. Isotopic data show a rather high nutritional status for the time, with a varied diet rich of meat. In conclusion, currently it is not possible to accurately define the identity of the three individuals for lack of archaeological data and for the fragmentary nature of the bones. The age of the subjects, the robust constitution and the nutritional data suggest a belonging to a high social class perhaps devoted to war activity. Future molecular studies may perhaps reveal a possible degree of kinship between the individuals and clarify the identity of the subjects., Born to one of the leading families in Besançon, Jean Bassand was a French Christian monk. After his initial profession in the Augustinian house of Saint Paul, he joined the Celestines (a branch of the Benedictine Order) in Paris, and subsequently became prior in the city of Amiens. The Celestine monks of France were a self-governing province of an Italian Benedictine reform of the late XIII century that no longer exists. They had a great influence, representing one of the most prominent observant groups in France, and an inspiration for reform movements across multiple orders. Jean Bassand represented the most important figure in the French Celestine congregation between XIV and XV century, being elected provincial prior on five occasions. He made great efforts to establish new Houses in the French province as well as abroad. The English King Henry V invited him to found and direct a friary in Isleworth near Sheen (now Richmond, London), whereas Martin I of Aragon asked him to establish the congregation in Barcelona. In 1443, he went to L’Aquila by order of the Pope Eugenius IV, to reform the monastery of Santa Maria di Collemaggio. He had troubles in this task and retreated to Rome arguing that the Aquilans were “difficult men”, but the Pope sent him back until his mission was accomplished. He died in L’Aquila on 26 August 1445. His body, covered with lime to be displayed, was found intact 18 years after. Since his death the mummy of Jean Bassand used to be kept in the Basilica of Collemaggio. After the major earthquake that struck down the city in 2009, his remains were recovered from the church to be kept in a secret location. Recently, an inspection of the body took place as a preliminary step of a forthcoming Canonical Recognition. The mummy appeared still fully dressed, with face and hands uncovered. The skin surface was extremely well preserved, and oblique illumination disclosed multiple, round, well-circumscribed plaques on the forehead, cheeks, and upper lip. Careful examination of the digital pictures enabled us to recognize at least 19 lesions. From a modern clinical viewpoint, the facial skin eruption of Jean Bassand meets most of the diagnostic criteria for multiple seborrheic keratoses. The age at death and the lifestyle of the Blessed, with frequent, long-distance travel under severe conditions, are fully compatible with this diagnosis. It is well known that male sex, increased age and sun-exposure may predispose individuals towards the development of these lesions. This case might represent the first ancient seborrheic keratosis described in the literature, although further analyses (external examination, computed tomography, histology) are needed to confirm the diagnosis. Advanced investigation methods might also enable us to understand if an ancient case harbours the same genetic mutations detected in modern patients. In this particular case, the treatment of the corpse with lime referred to in the ancient literature may have preserved the skin lesions by dehydration., This study presents the skeletal evidence for scurvy among 22 juveniles from the Longobard necropolis of Selvicciola, Italy (VII-VIII centuries AD). The paleopathological analysis revealed a combination of bilateral porous and proliferative bone lesions affecting the orbital roof (i.e., cribra orbitalia), the cranial vault (i.e., porotic hyperostosis) and some specific areas of the entire skeleton (i.e., sphenoid, hard palate, and scapula). This pattern is typical of infantile scurvy (Geber & Murphy, 2012). The investigation also revealed a bone tissue resorption of the anterior and lateral surface of thoracic and lumbar vertebral bodies and rib lesions in 17 of the 22 juveniles that showed signs of scurvy. This kind of skeletal manifestation associated with endocranial alteration and diffuse periosteal new bone formation (PNBF) is often linked to atypical or early-stage tuberculosis (Spekker et al., 2012). Our assessment suggests that the diet in Selvicciola was mainly deficient in vitamin C, causing scurvy. Among other problems, this might indicate poor sanitary conditions and further factors related to local environment and general state of health. Consequently, scurvy may have reduced the immune resilience of the juveniles leading to the development of TB in many of them (Miladinović-Radmilović & Vulović, 2015). Additional assessment via isotopic studies of the Selvicciola burial collection (Tafuri et al., 2018) indicates that the consumption of animal proteins in their diet was quite high. Hence, TB may have originated as Mycobacterium bovis (Roberts & Buikstra, 2003), coming from dietary consumption of infected animals. The preliminary macroscopic investigation of these subadult skeletons reveals lesions that suggest a complicated relationship among several factors influencing the health of these children. Diet, infection and lack of vitamins may have contributed to the poor health and death of these juveniles. Hence, showing a complicated situation in which, these children lived and dead in. This anthropological work illustrates how paleopathology can be used to interpret the health status of individuals of past communities even when the lesion evidence suggests multiple causal factors associated with death., The Institute of Clinical Pathology currently keeps the autopsy registers from 1891 to the present; the oldest one consists of some volumes in which the protocols and the related diagnoses were written by hand; for each case, a succinct clinical history preceded the external examination. The case presented here concerns obstetric pathology related to maternal post-partum death due to uncontrollable bleeding. The autopsy was carried out in 1892 on a woman suffering from rickets with scant muscular masses; the autopsy was performed 37 hours after the patient’s death. At that time (specifically from 1886 to 1905) the Director of the Unit of Clinical Pathology was Professor Vincenzo Brigidi and the autopsy room was situated near Pammatone Hospital, which was built in the district of Portoria and almost five centuries earlier, in the fifteenth century. This was the main hospital in Genoa, and played a fundamental role in local public health; the whole institute consisted of a single autopsy room. Full-blown or paucisymptomatic rickets was common in the Italian population of the nineteenth century and the first half of the twentieth century. The high incidence of this disease was due to deficiency factors or chronic nutritional stress, which led to forms of pseudo-rickets or latent rickets; this general situation required strict supervision of pregnant patients in order to avoid fatal intra-partum accidents both in the mother and in the foetus. Moreover, it is known that the skeletal lesions typical of rickets can seriously worsen the classical pattern of common brachipelvization, resulting in more serious pathologies. Anthropologically, brachipelvization and the evolution to the erect position constitute a peculiarity of our species. Over the centuries, obstetrics has developed complex studies for the evaluation of the pelvis and in particular, for the study of planes, axes and obstetric conjugates. In the nineteenth century and in the first decades of the twentieth century, pelvimetry was carefully practiced in obstetrics to monitor the pathological conditions of the pelvis. The management of postpartum haemorrhage was less theoretically developed and, in obstetric practice, was also represented a frequent cause of maternal death. In the case presented, therefore, obstetric procedures, such as sutures of the cervix of the vagina and the use of the so-called iron perchloride as a haemostatic cauterant, were used to stop bleeding. Ferric chloride is an iron salt (hence it is wrong to call it acid, as it is wrong to call it perchloride). The haemostatic action of the latter has been known for a very long time, but owing to its caustic action, which deeply manifests itself in the tissues, it has been absolutely abandoned in modern obstetric practice. Indeed, the report reveals that ferric chloride gave the tissues inside the uterus a leathery consistency, without - however - managing to save the mother’s life., The typical modern native from Trieste is tall - they are the tallest among Italians -, over 65 years old - Trieste has the highest seniority index in Italy - and loves spending time at the seaside or eating out with friends (no statistic data are available but you can witness that with a day trip to the city!). A group of 41 individuals who lived in the Early Middle Ages in Tergeste (the modern Trieste), seems to have a lot in common with the modern inhabitant of Trieste: they were tall (average Trotter and Gleser stature for male and female are 174.2 cm and 163.2 cm, respectively) and most of them were over 55 yeas old at death (more than 50% of adult males and 50% of adult females). Moreover, some of them spent a significant amount of time in the sea, which can be seen by the auditory exostosis in males and high frequency of third distal tibiae and fibulae periostitis in adult mature females – findings suggestive of a long time spent in the sea water looking for clams. Medieval archaeological layers in Trieste show plenty of shells. These people also loved eating: the four eldest male skeletons show marks of DISH, a pathology clearly associated with metabolic disorders. One of them may have died of suffocation caused by a small-size herbivore distal humerus epiphysis showing clear signs of slicing – a morsel of stew – found on C5/C6. Talking of skeletal remains, this diagnosis can be only a suggestion; nevertheless, dysphagia leading to suffocation in the elderly is a classic. These people were buried out of the walled circle of the ancient Tergeste and not so far from the Madonna del Mare, an early Christian Basilica built in the V/VI century, on the site called Domus Mariae. In 3 out of a total of 21 tombs (the number is low because of the overlapping of different bodies), we find a skull placed in a ritual position close to the left ankle of the body. The following are the combinations found and the age at death in each of these burials: female skeleton aged 30-40/skull of child aged 4; male skeleton aged 50-60/skull of female aged more than 50; male skeleton aged 30-45/skull of male aged more than 50. In the last mentioned tomb, besides the skull to the left of the ankle there are two other skulls: one between the femora, belonging to a man aged about 20-25, and the other one to the left of his skull, belonging to a child aged about 9. The skull connected to the buried skeleton presents a trepanation probably made by scarnification: an oval hole of about 17 mm x 12 mm, on the left parietal bone. The lesion edges clearly demonstrate survival after trepanation, possibly not longer than 1 or 2 months. In fact, in the inner table, near the hole, some coral-like new bone lesions reveal a meningeal involvement. Neither the skull nor the other bones reveal traumatic lesions; we found only a sternal foramen, the lack of fusion of the transverse foramens of C2 and a osteochondritis dissecans of the right capitellum., In the Middle Ages few examples of forensic medicine had appeared, even if imperfect and sporadic, in the field of healthcare art. In the Renaissance they took on greater consistency and framed themselves better in more defined limits. The two reports presented here are part of a trial against Jews in the city of Trento in 1475. This work takes into consideration what happened, the historical period, the trial and the sentences, and the subsequent historical revision. The study of these reports also analyzes the guidelines available at that time., To understand a disease of the past, historians need to distance themselves from today’s epistemological paradigm. The disease must lie within the epistemological limits in which it is explained. In Western medical texts of the past, from Hippocrates and Galen onwards, it was the sign on the patient that led to diagnosis through a deductive process. A debatable diagnosis by our criteria. It follows that the names attributed to diseases bore several meanings, also due to translations over time from one major language to another (Greek, Arabic, Latin) and moreover in relation to the authors describing the disease – physicians, authors of literary texts, chronicles, hagiographies – representatives of an inhomogeneous medical culture. This led to an acknowledgement of the limits of retrospective diagnosis with reference to the lexicon of the textual sources. A significant example regards erysipelas, which today indicates a precise bacterial disease: this term is first found in various points of the Corpus Hippocraticum, including book III of the Epidemics, in a story subsequently commented on by Galen. From the description of the signs the term is linked to different symptoms or diseases, if interpreted with our criteria. Transliterated into Latin, in mediaeval texts the term is placed under the category of the apostemata – exceedingly complex diseases or disturbances – especially after the translation of Avicenna’s Canon. The association with ignis sacer, an expression of Latin origin, in the De Medicina of Cassius Felix (5th C.) led in some cases to a semantic change, borne out by non-medical sources. Ignis sacer in fact, independently of its oldest meaning, first came into use in chronicle sources from the 11th century, to indicate “burning” epidemics, in which ergotism and gangrene in general may be recognised. Renaissance medical sources, following direct translations of Greek medical texts without mediation of the Arabic, tended in part to recover the meaning of the term erysipelas indicated by Galen. In Hoffman’s Dissertatio of 1729, albeit with due precautions, we may recognise the symptoms of present day erysipelas, although the physician’s association with Rosa and with fuoco selvatico begs consideration of the polysemy of two nosographic expressions found in “popular” culture in many areas of Europe. The aim of this presentation is to analyse the polysemy of a nosographic term that has come down to us from the Hippocratic tradition, together with its changes in meaning over time and in accordance with contexts. A paradigmatic case for highlighting the difficulty facing historians wishing to carry out a retrospective diagnosis by means of the medical lexicon., Paolo Gorini (1813-1881) was one of the first scientists who experimentedwith the“petrification” of corpses, a particular technique used to obtain an artificial preservation of bodies, which found wide application in Italy in the 19th century. This technique allowed the exact features of the deceased to be maintained and for tissue, internal organs and hair to be preserved, mostly in a state of stone hardness. This specific mechanism was based on the replacement of biological liquids with chemical preservatives obtained through intravascular injections. Paolo Gorini performed “petrification” on hundreds of specimens, onentire cadavers as well as on parts of corpses, most of which are now housed in the Paolo Gorini Anatomical Collection of Lodi. Lodi is also home to the manuscript with the two formulas used by Gorini to petrify corpses: “a sulfuric acid solution in the proportion of ten percent or an alcohol-saturated solution of mercuric bichloride and muriate of calcium in the proportion that the volume of the first is ten times that of the second”. The aim of our work is to verify the preservation status of skin that was subjected to “petrification” by Gorini. Our study was carried out on an entirely petrified body of an unknown individual held in the aforementioned collectionat Lodi. The man had been affected by a widespread bulbous-bullous infection, possibly smallpox or pellagra. A superficial fragment of skin, free of lesions, was biopsied from the latero-plantar region of the right foot. The analysis was performed using microscopic slides following the inclusion of the samples in epoxy resin, as well as by a stain with hematoxylin-eosin and Masson’s trichrome. Other sections were stained via immunohistochemical technique with anti-cytokeratin antibodies (AE1, AE3) and vimentin. The histological investigations revealed discretely preserved epithelial tissue, with a structure that is still recognizable on the tangential sections. It is possible to distinguishan easily detachable epithelium of the stratum corneum and deeper, more cohesive, layers (stratum granulosum and spinosum) in which the shadows of nuclei are still recognizable. Histochemical investigations revealed positivity for cytokeratins and negativity for vimentin. In contrast to natural or embalmed mummified bodies, historic petrified specimens have never been histologically analyzed. This first study demonstrates that the “petrification” method performed by Gorini guaranteed good skin preservation, allowing its histological, histo-chemical, metachromatic and antigenic characteristics to be maintained., Far from representing a sample of evidence without any current interest, the anatomical and pathological assemblages stored in academic structures are still a valuable scientific and cultural resource for museum collections. In fact, these findings are able to provide, through their precise historical contextualization, important data on epidemiological aspects and medical knowledge over the time. The lack of suitable places to store them and the absence of human and financial resources together with cultural and emotional barriers regarding the death, damn the biological items to the obscurity, despite attempts to enhance them through systematic activities of cataloguing, restoration, conservation and exhibition. The promotion of a newsensitivity towards these collections as well as the developmentof a network system among academic structures may promote the recovery of this biological heritage. These actions could increase the scientific value of the items as well as the the memory of the past and could consign to museums a new role of “places for the scientific reflection and the epistemological revision”. The public exhibition of the biological findings, in accordance with human dignity as well as ethical values, could be a valuable teaching resource towards the knowledge of the human body and also to promote the health awareness. The exposure of healthy organs and pathological ones –in reflecting of unhealthy behaviours and lifestyles or catastrophic natural events – may encourage a critical reflection on the culture of life. At the same time, past human stories, albeit incomplete and fragmentary, may also be an instrument of education in the culture of death and the values of solidarity. In conclusion, we suggest Museums and collections as vectors of new social relations to be shared with the “community of the living”, in order to promote acts of the highest moral value, through awareness campaigns, on the donation of the post-mortem body for study and research purposes., Pathology Museums house ancient specimens obtained during autopsies and generally used for educational purposes. The collections usually consist of dry and wet specimens showing diseases that no longer exist or with their natural course unmodified by therapies. The preservation of the macroscopic features due to the storage fluid has a great historical and paleopathological interest. In recent years, increasing attention is being paid to the study of the wet specimens by modern techniques. Ancient DNA (aDNA) has been investigated in different specimens from natural history museums, but the experience with human material is still limited. The time elapsed between death and fixation, and the chemical composition of fixation and storage fluid may irreversibly damage the DNA, thus routine techniques may result ineffective. We propose a simple and reliable approach to aDNA collection and extraction from museum wet specimens. Ten wet specimens were selected from the Pathology Collection of Turin and submitted to histopathologic re-evaluation. As the chemical composition of the storage fluids is currently unknown, pH value was measured in each specimen. Four cases representative of different classes of pH were submitted to DNA analysis by conservative sampling. Tiny fragments of tissue were frozen at -20°C to obtain sixty 10 mm-thick sections, collected in microtubes containing 1 ml of digestion solution (75 mM NaCl, 10 mM tris, 0.5 mM EDTA, pH 8.0) and 100 ml of proteinase K solution (18 mg/ml). The samples were incubated at 56°C for 48 h and 50 ml fresh of proteinase K solution were added for 72 h. 400 ml of solution were extracted with magnetic beads using a Roche MAGNA PURE COMPACT instrument. DNA quantity and quality were evaluated using the full absorption spectrum (220/340 nm) obtained by the Nanophotometer P 300 spectrophotometer. DNA concentration in ng/ml and absorbance ratio at 260/280 nm were calculated from 4 ml samples. The quality of DNA was also observed by electrophoretic run in 1.3% of agarose gel. In order to verify DNA integrity, short tandem repeat (STR) analysis was performed using the PowerPlex 16 HS system (PROMEGA) employed for personal identification. The cases were originally diagnosed as lymphosarcoma, uterine myosarcoma, esophageal, gastric, and rectal cancers, pancreas tumor, lung cancer, and pleural sarcoma. The range of pH values was comprised between 1.46 and 4.65. The pH value of the specimens submitted to DNA analysis was 2.56, 3.15, 4.45, and 4.65 and the revised diagnoses were necrotic lung carcinoma, uterine leiomyosarcoma, lung metastases from squamous carcinoma of unknown primary, and from uterine leiomyosarcoma. The first two samples gave negative results on both spectrophotometer and electrophoretic runs. The other two showed a low quantity of DNA (6 ng/ml; 7 ng/ml) with an absorbance ratio of 1.53 and 1.50 at the spectrophotometric analysis. The electrophoretic analysis showed a light band of DNA with molecular weight around 1000 bp in both samples. STR analysis displayed DNA fragmentation, evidenced by ladderization of the electropherograms result. The amplification of amelogenin STRs of chromosome X allowed the precise identification of one patient. It is well known that DNA is better preserved in alkaline medium, but its quantity and quality may be acceptable also in specimens preserved at pH around 4.5. Museum wet specimens may represent a valid source of aDNA to investigate genetic molecular features of ancient diseases. The measurement of pH value of the storage fluid may be useful as a screening method for aDNA preservation., Investigating multiple traumas observed in an individual or among members of an entire historic community has always been an area of great interest for paleopathologists and bioarchaeologists. One task faced by paleopathologists is related to the nature of the traumatic event. Such violent events can be accidental or intentional in origin. Intentional violence might be self-inflicted or an act from another person. Once this has been determined, discriminating between multiple injury events and a single event (with multiple fractures) is challenging. While assessing the skeletal collection from the post-classical necropolis of Selvicciola (Viterbo, Latium, Italy; 4th-8th centuries AD), an adult male burial revealed a unique pattern of healed injuries. This male (T 90/5) was buried without grave goods. His tomb is located far from the church, which is the centre of the necropolis. T 90/5 is part of a specific burial group of Longobards situated in South-Eastern funerary area (dated to later period of the 7th century AD). Of these elements we located 6 fractures. This included a well healed nasal fracture, right clavicle fracture, a right scapula fractured with healing along the entire superior body (glenoid fossa to vertebral border), left scapula with an acromion process fracture healed but unfused, a healed rib right fracture and a left femoral neck fracture (unfused). This last facture appears to have happened a few months before his death. The lower edge of the fracture on the femur is well healed with a line of 2 mm of new bone formation. Moreover, the periosteal surface under the neck shows eburnation compatible with the eburnation of the inner part of the head of the femur. The inner surface of the femoral head shows polished remodelled trabeculae lesion. Its creation can likely suggest that a pseudo-articulation between the edge of the diaphysis and the head of the femur was formed as a result of movement of the joint area after the fracture occurred. Also related to the health status of the male is the considerable state of DJD of long bones, as well as the significant evidence of vertebral OA and Schmorl’s nodes. Towards the end of his life this individual was suffering from a number of chronic problems, which produced skeletal lesions specific to biomechanics and old age. Some of this might have been influenced by the trauma experienced earlier in life. Likely, at the end his life his last fracture might have been due to osteoporosis of the femoral neck. The survival of this man testifies to community care and a high value given to human life. The variety of implications in this case-study inform us the care for this individual, that for most part of his life was a disable. Not only the injuries, but also the pain suffered had to affect his daily life. In the end, the protocol of cares was realized by the community at least two times; for the first pattern of trauma (nose, shoulders, clavicle and rib) and for the femur break., Rheumatoid arthritis is a chronic, systemic, inflammatory condition that starts from a synovitis, leading to diffuse erosions in the marginal area of joints and finally conducting to articular deformity and destruction of bone ends. The aetiology of the disease is unknown but multiple genetic and environmental factors have been linked to its development. According to clinical studies, 10-30% of cases undergo healing of lesions and spontaneous remission of the disease. Today’s approach to inflammatory diseases is conditioned by the early diagnosis, thanks to the evolution of the diagnostic methods and by the mitigation of drug therapies. In the past, the remission was entrusted only to the individual’s immune resistance. A case of erosive polyarthropathy has been discovered in an elderly male individual recovered from the medieval cemetery of San Biagio in Cittiglio (northern Italy). The well preserved skeleton was unearthed in the external area in front of the church access and, according to the archaeological stratigraphy, it dates back to a period between the 12th and the 13th century. The bone elements, following macroscopic and microscopic analysis, exhibit several erosive lesions with symmetrical distribution, affecting firstly the appendicular skeleton of the little joints of hands and feet and other larger joints, such as the shoulder, elbow and hip. The bony tissue involved by the erosions is the so-called “bare area”, in the marginal region of the joints, where the synovium membrane-lined bone is found. The diagnosis of this erosive polyarthritis is complicated by the mild expression of the lesions and by the presence of a subtle sclerotic border to some erosions radiographically observed. Next, a careful differential diagnosis was necessary to clarify the aetiology of the polyarthropathy; the skeletal distribution of the lesions and their macroscopic and radiological appearance are suggestive of a case of rheumatoid arthritis-like polyarthropathy. A hypothetical remission phase of the disease, as demonstrated by the frequent presence of smoothed borders and sclerosed margins on radiographic images, is also suggested. Co-existence of diffuse marginal lipping, joint degeneration and severe areas of eburnation, is also recorded, suggesting a co-morbidity of the erosive condition with osteoarthritis, which is compatible with the advanced age of the individual. With this medieval case, we present new evidence of the existence of erosive arthritis and, specifically, of rheumatoid arthritis-like polyarthropathy in Europe before the discovery of the Americas, entering into the long debate about the antiquity of the disease that, firstly, was considered as originating in the New World and subsequently spread to the Old World. On the basis of this and other already published cases, rheumatoid arthritis seems to have been present in Europe more anciently than was previously thought., Between 2012-2014 the Superintendence of Archaeology, Fine Arts and Landscape of Emilia-Romagna, has conducted archaeological fieldworks on a large Late Medieval cemetery (14th-16th century), which archaeological and documentary sources attribute to a Jewish context. The Laboratory of Bioarchaeology and Forensic Osteology of University of Bologna conducted an anthropological study on a sample of 130 individuals. This contribution aims to present four possible cases of venereal treponematoses (TT. 91, 136, 170, 187). Human skeletal remains of graves 91, 136, 170, and 187 were examined to reconstruct the biological profiles and to conduct paleopathological and tomographic analyses, given the presence of lesions on several anatomical districts. Cranial lesions were present on individuals from TT.91 (M, 25-35 years), 170 (M, 25-35 years) and 136 (M, 15-18 years), in which simultaneous destructive and proliferative processes (caries sicca) with focal destruction and remodeling of the external surface and diploe are denoted. Long bones of these three individuals also present osseous alteration such as gummatous osteoperiostitis, with an increased bone density and non-uniform thickening. Individual of T. 187 (11-12 years) presents a hole (3 cm Ø) located on frontal bone, whose margins are remodeled with proliferative processes both on ectocranial and endocranial sides. These lesions are likely linked to treponematosis (bacterial infection by Treponema), interpreted as venereal syphilis. After differential diagnoses, we suppose the individuals of TT. 91 and 170 were likely affected by an advanced stage of the infection, while individual T. 136 seems to have been affected by a tardive congenital form of the disease. Lesions of individual of T.187 suggest an infective origin, but poor bone preservation prevents a clearer interpretation. These cases of treponematosis, possibly linked to venereal syphilis, are relevant for paleoepidemic aspects, as well as contributing to only few Italian osteological cases dating to the same period., Spinal tuberculosis (STB) is a well-known disease in paleopathology. Paleopathologists have highlighted in the last decades some morphological criteria for its diagnosis. Commonly, we are witnessing the destruction of the intervertebral disc space and the adjoining vertebral bodies, the collapse of the vertebrae and the anterior wedging which lead to a structural kyphosis classifiable in gibbus deformity. Here we present the probable STB case of a male subject, 55 years, concealed between the 18th and the first half of the 20th century in the Franciskcan monastery of Azzio, Varese, Italy. The skeleton was found both in an optimal state of preservation and representation. Anthropological analysis was performed according to Buikstra and Ubelaker standards. Paleopathological diagnosis was conducted thanks to macroscopic, microscopic and radiographic analysis, also in order to perform the differential diagnosis. Even if STB was widely present in the last centuries in northwestern Italy, only few paleopathological cases was directly studied., Cystic echinococcosis (CE) is a zoonosis caused by Echinococcus granulosus. The life cycle of the parasite develops in the canids, which house the adult tapeworm in the intestine, and in the intermediate mammal hosts. Humans are occasional dead-end hosts, infected by eggs ingestion via fecal-oral route. The larvae from the digested eggs penetrate the human gut wall and are disseminated throughout the body by the blood. The soft tissues involved at the level of the capillaries may host the larvae, and the hydatid cyst can develop in different organs. The liver is the first organ that the larvae encounter through the blood stream and consequently it is the most frequently involved; it is followed by the lungs and then other organs in frequency. The hydatid cyst is a fluid-filled formation that grows centrifugally and that can survive in the intermediate host for years. In 10 years, it can growth to a diameter of 15-20 cm. Inside hyaline outer membrane, a cellular germinating layer produces microcystic structures that develop scolices. The scolices pouring out of the cyst develop one or more cysts that can reach every tissue. The life cycle is completed when the definitive host feeds on organs of the intermediate host that contain fertile metacestodes. Death of the germinating layer within the metacestode produces calcification of the cyst wall in the intermediate host. Calcified hydatid cysts found as archaeological finds are generally associated with skeletal remains in the thoraco-abdominal site. In archaeological records, the presence of echinococcosis is underestimated, and the find is relatively rare for different reasons: 1) difficulty of recognition by archaeologists; 2) need of accuracy in excavation and recovery of osteoarcheological remains; 3) fragility of calcified formation in the soil. Furthermore, the taphonomic alterations can cause the translation from the original site of the calcified formation and undermine the recognition of the organ affected. In Italy there are only two archaeological samples of calcified formation, most probably of echinococcosis origin, described in the paleopathological literature: one from Siena (13th-14th centuries) and one from Abruzzo (early 20th century). In this report we describe another calcified formation found in the archaeological excavation of the monastic site of Badia Pozzeveri, near Lucca, Tuscany, for which we propose a diagnosis of CE. This finding comes from a privileged lithic coffin built on the northern side of the monastic Church of San Pietro. The grave was used in the 13th century as collective burial by the same laical family group. Calcification, associated with a female individual of about 35-45 years, was discovered in the thoraco-abdominal region. We propose the diagnosis of hydatid cyst from Echinococcus granulosus based on gross morphology, micro-morphology, and a multicomponent approach with cone beam computed tomography, SEM/EDS and stable isotope analysis., Biparietal osteodystrophy (BO), with symmetrical and bilateral thinning of the parietal bones, is a condition rarely discussed in the paleopathological literature. In the past, it has been described as a non-metric trait, anatomical variation and development anomaly. Even though the aetiology is still unknown, today it is described as a pathological condition. In many individuals it appears to be age-related, therefore it has been attributed to osteoporosis, postmenopausal and senile atrophy. Other causes could be congenital and hereditary transmission. Here, we presented a possible case of BO, detected on the remains of a 50-years-old female subject (Tomb 8) excavated in the cemeterial area of the medieval church of Sant’Agostino in Caravate (Varese). Anthropological analysis was performed in accordance with the standards proposed by Buikstra and Ubelaker. Paleopathological conditions were evaluated macroscopically and microscopically. Moreover, CT scan was carried out to investigate pathological evidences. Even if the cause of this uncommon condition is not yet well understood, the present case is highly significant as it enters into the debate upon the aetiology of the disease., Atherosclerosis and its complications represent an important health problem throughout the modern world, although it seems to have accompanied humanity since its beginnings. Important documents have been reported in mummified remains. Atheromas may undergo extensive deposition of calcium and bone metaplasia (Monckeberg’s arteriosclerosis) and persist to the disintegration of the soft tissues. A complete 45-55-years-old male skeleton, 165 cm tall, from bishop’s Palace in Ivrea (Turin) was discovered during archaeological excavation in 2016. Radiocarbon analysis dated the skeleton to 1400-1600 AD. During skeletal preparation in laboratory, an ectopic biological calcification tubular shaped (19 mm in length and 7 mm in diameter) were detected among pelvic bones. At the macroscopic examination, the finding appears as an irregular tubular calcification; in cross section, the mineralized deposits span the entire volume of the lumen and some bony trabeculae in the central space are well defined. Severe calcification of the blood vessel is supposed. Calcification along the expected course of an artery/vessel was considered to be probable atherosclerosis. In relation to the anatomical localization it is supposed to be an atherosclerotic calcification of iliac or femoral artery. The case study reports an uncommon finding of arterial/vessel calcification detected on skeletal remains of an adult male from the post-medieval period. Since the age plays an important role in atherosclerosis, we do not exclude that vascular calcifications affected arteries in many regions of the body. The presented paleopathological specimens suggest that our knowledge of risk factors and the etiology of atherosclerosis are incomplete. A chronic inflammatory burden may have played a greater role than previously considered in ancient cultures and population including upper classes of Italian Renaissance. While increasingly prevalent with age in ancient and modern cultures, a strong gene-environmental interplay is established in the development of atherosclerosis across the lifespan. While genes create the vulnerability, the environment determines when and if atherosclerosis becomes manifest clinically.

Report Finale progetto RadIoPoGe.

Aims and objectives Methods and materials Results Conclusion Personal information References, Aims and objectives: Breast cancer is the principal cause of death from cancer among women worldwide [1, 2]. Nevertheless, early detection of breast cancer through adequate screening programs can save many lives, reducing the overall mortality [3]. The best diagnostic...

Background/introduction Description of activity and work performed Conclusion and recommendations Personal/organisational information References, Background/introduction: The University of Pisa has a long-standingexperience in the field of radiation protection. Therefore, it is not surprising that the University of Pisa Radiology team actively participated to all editions of EuroSafe Imaging poster sessions at the...