Aberrant Adenosine Triphosphate Release and Impairment of P2Y2-Mediated Signaling in Sarcoglycanopathies.

Sarcoglycanopathies, limb-girdle muscular dystrophies (LGMD) caused by genetic loss-of-function of the membrane proteins sarcoglycans (SGs), are characterized by progressive degeneration of skeletal muscle. In these disorders, muscle necrosis is associated with immune-mediated damage, whose triggering and perpetuating molecular mechanisms are not fully elucidated yet. Extracellular adenosine triphosphate (eATP) seems to represent a crucial factor, with eATP activating purinergic receptors. Indeed, in vivo blockade of the eATP/P2X7 purinergic pathway ameliorated muscle disease progression. P2X7 inhibition improved the dystrophic process by restraining the activity of P2X7 receptors on immune cells. Whether P2X7 blockade can display a direct action on muscle cells is not known yet. In this study, we investigated eATP effects in primary cultures of myoblasts isolated from patients with LGMDR3 (α-sarcoglycanopathy) and in immortalized cells isolated from a patient with LGMDR5 (γ-sarcoglycanopathy). Our results demonstrated that, owing to a reduced ecto-ATPase activity and/or an enhanced release of ATP, patient cells are exposed to increased juxtamembrane concentrations of eATP and display a higher susceptivity to eATP signals. The purinoceptor P2Y2, which proved to be overexpressed in patient cells, was identified as a pivotal receptor responsible for the enhanced ATP-induced or UTP-induced Ca2+ increase in affected myoblasts. Moreover, P2Y2 stimulation in LDMDR3 muscle cells induced chemotaxis of immune cells and release of interleukin-8. In conclusion, a higher eATP concentration and sensitivity in primary human muscle cells carrying different α-SG or γ-SG loss-of-function mutations indicate that eATP/P2Y2 is an enhanced signaling axis in cells from patients with α-/γ-sarcoglycanopathy. Understanding the basis of the innate immune-mediated damage associated with the dystrophic process may be critical in overcoming the immunologic hurdles associated with emerging gene therapies for these disorders.

Clinical and genetic spectrum of a large cohort of patients with δ-sarcoglycan muscular dystrophy.

Sarcoglycanopathies include four subtypes of autosomal recessive limb-girdle muscular dystrophies (LGMDR3, LGMDR4, LGMDR5 and LGMDR6) that are caused, respectively, by mutations in the SGCA, SGCB, SGCG and SGCD genes. Delta-sarcoglycanopathy (LGMDR6) is the least frequent and is considered an ultra-rare disease. Our aim was to characterize the clinical and genetic spectrum of a large international cohort of LGMDR6 patients and to investigate whether or not genetic or protein expression data could predict a disease's severity. This is a retrospective study collecting demographic, genetic, clinical and histological data of patients with genetically confirmed LGMDR6 including protein expression data from muscle biopsies. We contacted 128 paediatric and adult neuromuscular units around the world that reviewed genetic data of patients with a clinical diagnosis of a neuromuscular disorder. We identified 30 patients with a confirmed diagnosis of LGMDR6 of which 23 patients were included in this study. Eighty-seven per cent of the patients had consanguineous parents. Ninety-one per cent of the patients were symptomatic at the time of the analysis. Proximal muscle weakness of the upper and lower limbs was the most common presenting symptom. Distal muscle weakness was observed early over the course of the disease in 56.5% of the patients. Cardiac involvement was reported in five patients (21.7%) and four patients (17.4%) required non-invasive ventilation. Sixty per cent of patients were wheelchair-bound since early teens (median age of 12.0 years). Patients with absent expression of the sarcoglycan complex on muscle biopsy had a significant earlier onset of symptoms and an earlier age of loss of ambulation compared to patients with residual protein expression. This study confirmed that delta-sarcoglycanopathy is an ultra-rare neuromuscular condition and described the clinical and molecular characteristics of the largest yet-reported collected cohort of patients. Our results showed that this is a very severe and quickly progressive disease characterized by generalized muscle weakness affecting predominantly proximal and distal muscles of the limbs. Similar to other forms of sarcoglycanopathies, the severity and rate of progressive weakness correlates inversely with the abundance of protein on muscle biopsy.

Early Muscle MRI Findings in a Pediatric Case of Emery-Dreifuss Muscular Dystrophy Type 1.

Emery-Dreifuss muscular dystrophy (EDMD) is a rare disease characterized by early contractures, progressive muscle weakness, and cardiac abnormalities. Different subtypes of EDMD have been described, with the two most common forms represented by the X-linked EDMD1, caused by mutations in the EMD gene encoding emerin, and the autosomal EDMD2, due to mutations in the LMNA gene encoding lamin A/C. A clear definition of the magnetic resonance imaging (MRI) pattern in the two forms, and especially in the rarer EDMD1, is still lacking, although a preferential involvement of the medial head of the gastrocnemius has been suggested in EDMD2. We report a 13-year-old boy with mild limb girdle muscle weakness, elbow and ankle contractures, with absence of emerin at muscle biopsy, carrying a hemizygous frameshift mutation on the EMD gene (c.153dupC/p.Ser52Glufs*9) of maternal inheritance. Minor cardiac rhythm abnormalities were detected at 24-hour Holter electrocardiogram and required ß-blocker therapy. MRI scan of the thighs showed a mild diffuse involvement, while tibialis anterior, extensor digitorum longus, peroneus longus, and medial gastrocnemius were the most affected muscles in the leg. We also provide a review of the muscular MRI data in EDMD patients and highlight the relative heterogeneity of the MRI patterns found in EDMDs, suggesting that muscle MRI should be studied in larger EDMD cohorts to better define disease patterns and to cover the wide disease spectrum.

The SPTLC1 p.S331 mutation bridges sensory neuropathy and motor neuron disease and has implications for treatment.

AIMS: SPTLC1-related disorder is a late onset sensory-autonomic neuropathy associated with perturbed sphingolipid homeostasis which can be improved by supplementation with the serine palmitoyl-CoA transferase (SPT) substrate, l-serine. Recently, a juvenile form of motor neuron disease has been linked to SPTLC1 variants. Variants affecting the p.S331 residue of SPTLC1 cause a distinct phenotype, whose pathogenic basis has not been established. This study aims to define the neuropathological and biochemical consequences of the SPTLC1 p.S331 variant, and test response to l-serine in this specific genotype. METHODS: We report clinical and neurophysiological characterisation of two unrelated children carrying distinct p.S331 SPTLC1 variants. The neuropathology was investigated by analysis of sural nerve and skin innervation. To clarify the biochemical consequences of the p.S331 variant, we performed sphingolipidomic profiling of serum and skin fibroblasts. We also tested the effect of l-serine supplementation in skin fibroblasts of patients with p.S331 mutations. RESULTS: In both patients, we recognised an early onset phenotype with prevalent progressive motor neuron disease. Neuropathology showed severe damage to the sensory and autonomic systems. Sphingolipidomic analysis showed the coexistence of neurotoxic deoxy-sphingolipids with an excess of canonical products of the SPT enzyme. l-serine supplementation in patient fibroblasts reduced production of toxic 1-deoxysphingolipids but further increased the overproduction of sphingolipids. CONCLUSIONS: Our findings suggest that p.S331 SPTLC1 variants lead to an overlap phenotype combining features of sensory and motor neuropathies, thus proposing a continuum in the spectrum of SPTLC1-related disorders. l-serine supplementation in these patients may be detrimental.

Body mass index in type 2 spinal muscular atrophy: a longitudinal study.

The aim of this retrospective study was to review body mass index (BMI) in a large cohort of Italian pediatric type 2 spinal muscular atrophy (SMA) patients, aged between 0 and 20 years and to establish possible differences in relation to a number of variables such as ventilation, motor function, and survival motor neuron 2 gene copies. Cross-sectional data were collected from 102 patients for a total of 344 visits. Standard growth charts for height and weight were used as reference, with age adjusted BMI calculated using the Center for Disease and Prevention Children's BMI Tool. In the 344 visits, weight ranged between 3.90 and 83 kg, and the BMI between 8.4 and 31.6 with a BMI/age z-scores < - 2SD present in 28% and BMI/age z-scores > + 2SD in 9% of the measurements. The BMI/age z-scores were relatively stable < 5 years of age with an increasing number of patients < - 2SD after the age of 5, and a wider range of BMI/age z-scores after the age of 13. A difference on the BMI/age z-scores was found among the different age subgroups (< 5, 5-12, ≥ 13 years). A multivariate analysis in 58 patients with longitudinal assessments showed that baseline BMI/age z-scores and gender were significantly contributing to the changes while other variables were not. CONCLUSION: Our results confirm that careful surveillance of weight and BMI/age z-scores is needed in type 2 SMA. Further studies, including assessments of chewing and swallowing and of lean/fat body mass, will help to better understand the possible mechanisms underlying weight issues. WHAT IS KNOWN: • Feeding difficulties have been reported in a few studies and were invariably found in patients with type 1 SMA. • Type 2 SMA patients often have low BMI with a relevant number of patients requiring tube feeding. WHAT IS NEW: • Reduction in BMI/age z-score overtime appeared to depend on baseline BMI/age z-score and gender. • Patients with a low BMI/age z-score were at higher risk of developing further reduction.

Comprehensive Phenotyping of Peripheral Blood T Lymphocytes in Healthy Mice.

T lymphocytes play a central role in antigen-specific immune responses. They modulate the function of different immune cells both through a direct contact (receptor binding) and through the secretion of cytokines. At the same time, they are deeply involved in the direct killing of aberrant target cells. T lymphocytes derive from a bone marrow precursor that migrates in the thymus where the main differentiation steps take place. Mature CD4 and CD8 single-positive cells, then, leave the thymus to reach the secondary lymphoid organs. T-cell subsets and their maturation steps can be identified mainly based on the expression of extracellular markers, intracellular transcription factors and cytokine production profiles. In this review, we report, from a cytometric point of view, an overview of the most important T-cell subpopulations and their differentiation state. © 2020 International Society for Advancement of Cytometry.

New genotype-phenotype correlations in a large European cohort of patients with sarcoglycanopathy.

Sarcoglycanopathies comprise four subtypes of autosomal recessive limb-girdle muscular dystrophies (LGMDR3, LGMDR4, LGMDR5 and LGMDR6) that are caused, respectively, by mutations in the SGCA, SGCB, SGCG and SGCD genes. In 2016, several clinicians involved in the diagnosis, management and care of patients with LGMDR3-6 created a European Sarcoglycanopathy Consortium. The aim of the present study was to determine the clinical and genetic spectrum of a large cohort of patients with sarcoglycanopathy in Europe. This was an observational retrospective study. A total of 33 neuromuscular centres from 13 different European countries collected data of the genetically confirmed patients with sarcoglycanopathy followed-up at their centres. Demographic, genetic and clinical data were collected for this study. Data from 439 patients from 13 different countries were collected. Forty-three patients were not included in the analysis because of insufficient clinical information available. A total of 159 patients had a confirmed diagnosis of LGMDR3, 73 of LGMDR4, 157 of LGMDR5 and seven of LGMDR6. Patients with LGMDR3 had a later onset and slower progression of the disease. Cardiac involvement was most frequent in LGMDR4. Sixty per cent of LGMDR3 patients carried one of the following mutations, either in a homozygous or heterozygous state: c.229C>T, c.739G>A or c.850C>T. Similarly, the most common mutations in LMGDR5 patients were c.525delT or c.848G>A. In LGMDR4 patients the most frequent mutation was c.341C>T. We identified onset of symptoms before 10 years of age and residual protein expression lower than 30% as independent risk factors for losing ambulation before 18 years of age, in LGMDR3, LGMDR4 and LGMDR5 patients. This study reports clinical, genetic and protein data of a large European cohort of patients with sarcoglycanopathy. Improving our knowledge about these extremely rare autosomal recessive forms of LGMD was helped by a collaborative effort of neuromuscular centres across Europe. Our study provides important data on the genotype-phenotype correlation that is relevant for the design of natural history studies and upcoming interventional trials in sarcoglycanopathies.

Muscle inflammatory pattern in alpha- and gamma-sarcoglycanopathies.

AIM: Since the immune system plays a role in the pathogenesis of several muscular dystrophies, we aim to characterize several muscular inflammatory features in α- (LGMD R3) and γ-sarcoglycanopathies (LGMD R5). MATERIALS AND METHODS: We explored the expression of major histocompatibility complex class I molecules (MHCI), and we analyzed the composition of the immune infiltrates in muscle biopsies from 10 patients with LGMD R3 and 8 patients with LGMD R5, comparing the results to Duchenne muscular dystrophy patients (DMD). RESULTS: A consistent involvement of the immune response was observed in sarcoglycanopathies, although it was less evident than in DMD. LGMD R3-R5 and DMD shared an abnormal expression of MHCI, and the composition of the muscular immune cell infiltrate was comparable. CONCLUSION: These findings might serve as a rationale to fine-tune a disease-specific immunomodulatory regimen, particularly relevant in view of the rapid development of gene therapy for sarcoglycanopathies.

The Danger Signal Extracellular ATP Is Involved in the Immunomediated Damage of α-Sarcoglycan-Deficient Muscular Dystrophy.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

eATP/P2X7R Axis: An Orchestrated Pathway Triggering Inflammasome Activation in Muscle Diseases.

In muscle ATP is primarily known for its function as an energy source and as a mediator of the "excitation-transcription" process, which guarantees muscle plasticity in response to environmental stimuli. When quickly released in massive concentrations in the extracellular space as in presence of muscle membrane damage, ATP acts as a damage-associated molecular pattern molecule (DAMP). In experimental murine models of muscular dystrophies characterized by membrane instability, blockade of eATP/P2X7 receptor (R) purinergic signaling delayed the progression of the dystrophic phenotype dampening the local inflammatory response and inducing Foxp3+ T Regulatory lymphocytes. These discoveries highlighted the relevance of ATP as a harbinger of immune-tissue damage in muscular genetic diseases. Given the interactions between the immune system and muscle regeneration, the comprehension of ATP/purinerigic pathway articulated organization in muscle cells has become of extreme interest. This review explores ATP release, metabolism, feedback control and cross-talk with members of muscle inflammasome in the context of muscular dystrophies.

Iron Age Italic population genetics: the Piceni from Novilara (8th-7th century BC).

BACKGROUND: Archaeological data provide evidence that Italy, during the Iron Age, witnessed the appearance of the first communities with well defined cultural identities. To date, only a few studies report genetic data about these populations and, in particular, the Piceni have never been analysed. AIMS: To provide new data about mitochondrial DNA (mtDNA) variability of an Iron Age Italic population, to understand the contribution of the Piceni in shaping the modern Italian gene pool and to ascertain the kinship between some individuals buried in the same grave within the Novilara necropolis. SUBJECTS AND METHODS: In a first set of 10 individuals from Novilara, we performed deep sequencing of the HVS-I region of the mtDNA, combined with the genotyping of 22 SNPs in the coding region and the analysis of several autosomal markers. RESULTS: The results show a low nucleotide diversity for the inhabitants of Novilara and highlight a genetic affinity of this ancient population with the current inhabitants of central Italy. No family relationship was observed between the individuals analysed here. CONCLUSIONS: This study provides a preliminary characterisation of the mtDNA variability of the Piceni of Novilara, as well as a kinship assessment of two peculiar burials.

Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-ß and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy.

DAG1 haploinsufficiency is associated with sporadic and familial isolated or pauci-symptomatic hyperCKemia.

DAG1 encodes for dystroglycan, a key component of the dystrophin-glycoprotein complex (DGC) with a pivotal role in skeletal muscle function and maintenance. Biallelic loss-of-function DAG1 variants cause severe muscular dystrophy and muscle-eye-brain disease. A possible contribution of DAG1 deficiency to milder muscular phenotypes has been suggested. We investigated the genetic background of twelve subjects with persistent mild-to-severe hyperCKemia to dissect the role of DAG1 in this condition. Genetic testing was performed through exome sequencing (ES) or custom NGS panels including various genes involved in a spectrum of muscular disorders. Histopathological and Western blot analyses were performed on muscle biopsy samples obtained from three patients. We identified seven novel heterozygous truncating variants in DAG1 segregating with isolated or pauci-symptomatic hyperCKemia in all families. The variants were rare and predicted to lead to nonsense-mediated mRNA decay or the formation of a truncated transcript. In four cases, DAG1 variants were inherited from similarly affected parents. Histopathological analysis revealed a decreased expression of dystroglycan subunits and Western blot confirmed a significantly reduced expression of beta-dystroglycan in muscle samples. This study supports the pathogenic role of DAG1 haploinsufficiency in isolated or pauci-symptomatic hyperCKemia, with implications for clinical management and genetic counseling.

Gain and loss of upper limb abilities in Duchenne muscular dystrophy patients: A 24-month study.

Duchenne muscular dystrophy (DMD) is a neuromuscular condition characterized by muscle weakness. The Performance of upper limb (PUL) test is designed to evaluate upper limb function in DMD patients across three domains. The aim of this study is to identify frequently lost or gained PUL 2.0 abilities at distinct functional stages in DMD patients. This retrospective study analyzed prospectively collected data on 24-month PUL 2.0 changes related to ambulatory function. Ambulant patients were categorized based on initial 6MWT distance, non-ambulant patients by time since ambulation loss. Each PUL 2.0 item was classified as shift up, no change, or shift down. The study's cohort incuded 274 patients, with 626 paired evaluations at the 24-month mark. Among these, 55.1 % had activity loss, while 29.1 % had gains. Ambulant patients showed the lowest loss rates, mainly in the shoulder domain. The highest loss rate was in the shoulder domain in the transitioning subgroup and in elbow and distal domains in the non-ambulant patients. Younger ambulant patients demonstrated multiple gains, whereas in the other functional subgroups there were fewer gains, mostly tied to singular activities. Our findings highlight divergent upper limb domain progression, partly linked to functional status and baseline function.

Structured Light Plethysmography for Non-Invasive Assessment of Respiratory Pattern in Spinal Muscular Atrophy Type 1.

BACKGROUND: Spinal muscular atrophy (SMA) type 1 is a severe condition leading to early respiratory failure. Treatment options have become available, yet respiratory outcome measures in SMA type 1 are limited. The aim of this study was to assess the respiratory pattern in SMA type 1 patients via structured light plethysmography (SLP). SLP measures the thoraco-abdominal movements by projecting a light grid onto the anterior thoraco-abdominal surface. METHODS: Cross-sectional study of consecutive children with SMA type 1. All children underwent motor assessment (CHOP-INTEND) and one-minute tidal breathing recording by SLP in supine position while self-ventilating in room air. The Respiratory rate, the abdominal vs. chest contribution to breath (Relative Expired Abdomen%, Relative Expired Chest%) and the severity of thoraco-abdominal paradox (Phase Angle) were acquired. RESULTS: Nineteen patients were included, median (IQR) age 2.3 years (1.4-7.9). Their respiratory pattern captured via SLP showed a raised median (IQR) respiratory rate per age of 33.5 bpm (26.6-41.7), a prevalent abdominal contribution to tidal breathing with median (IQR) Relative Expired Abdomen 77% (68-90) vs. Chest 23% (10-32). Thoracoabdominal paradox was detected (median Phase Angle 48.70°) and its severity correlated negatively with CHOP-INTEND (r -0.8, p < 0.01). CONCLUSIONS: SLP captured and quantified the respiratory features of infants and children with SMA type 1.

Nusinersen mitigates neuroinflammation in severe spinal muscular atrophy patients.

BACKGROUND: Neuroinflammation contributes to the onset and progression of neurodegenerative diseases, but has not been specifically investigated in patients affected by severe and milder forms of spinal muscular atrophy (SMA). METHODS: In this two-center retrospective study, we investigated signatures of neuroinflammation in forty-eight pediatric male and female SMA1 (n = 18), male and female SMA2 (n = 19), and female SMA3 (n = 11) patients, as well as in a limited number of male and female non-neurological control subjects (n = 4). We employed a Bio-Plex multiplex system based on xMAP technology and performed targeted quantitative analysis of a wide range of pro- and anti-inflammatory cytokines (chemokines, interferons, interleukins, lymphokines and tumor necrosis factors) and neurotrophic factors in the cerebrospinal fluid (CSF) of the study cohort before and after Nusinersen treatment at loading and maintenance stages. RESULTS: We find a significant increase in the levels of several pro-inflammatory cytokines (IL-6, IFN-γ, TNF-α, IL-2, IL-8, IL-12, IL-17, MIP-1α, MCP-1, and Eotaxin) and neurotrophic factors (PDGF-BB and VEGF) in the CSF of SMA1 patients relative to SMA2 and SMA3 individuals, who display levels in the range of controls. We also find that treatment with Nusinersen significantly reduces the CSF levels of some but not all of these neuroinflammatory molecules in SMA1 patients. Conversely, Nusinersen increases the CSF levels of proinflammatory G-CSF, IL-8, MCP-1, MIP-1α, and MIP-1ß in SMA2 patients and decreases those of anti-inflammatory IL-1ra in SMA3 patients. CONCLUSIONS: These findings highlight signatures of neuroinflammation that are specifically associated with severe SMA and the neuro-immunomodulatory effects of Nusinersen therapy.

Spinal muscular atrophy (SMA) is an inherited disorder which leads to muscle weakening. Three therapies have recently been developed, including Nusinersen. However, the effect of SMA on the immune system and how this could be affected by Nusinersen is unknown. The immune system protects the body from infection and, in some disorders, misfunctions and damages the body in the absence of infection. Here, we analyze components of the immune system in body fluids from SMA patients before and after treatment with Nusinersen. The immune system was found to be more active in patients with more severe disease. Treatment with Nusinersen reduced the levels of some, but not all of these, components of the immune system. Thus, treatments that impact the immune system might improve symptoms in patients with SMA.

Long-term clinical and MRI follow-up in two POMT2-related limb girdle muscular dystrophy (LGMDR14) patients.

INTRODUCTION: POMT2-related limb girdle muscular dystrophy (LGMDR14) is a rare muscular dystrophy caused by mutations in the POMT2 gene. Thus far only 26 LGMDR14 subjects have been reported and no longitudinal natural history data are available. CASE REPORT: We describe two LGMDR14 patients followed for 20 years since infancy. Both patients presented a childhood-onset, slowly progressive pelvic girdle muscular weakness leading to loss of ambulation in the second decade in one patient, and cognitive impairment without detectable brain structural abnormalities. Glutei, paraspinal, and adductor muscles were the primarily involved muscles at MRI. DISCUSSION: This report provides natural history data on LGMDR14 subjects, with a focus on longitudinal muscle MRI. We also reviewed the LGMDR14 literature data, providing information on the LGMDR14 disease progression. Considering the high prevalence of cognitive impairment in LGMDR14 patients, a reliable application of functional outcome measures can be challenging, therefore a muscle MRI follow-up to assess disease evolution is recommended.

Myopathologic trajectory in Duchenne muscular dystrophy (DMD) reveals lack of regeneration due to senescence in satellite cells.

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscular disease, caused by mutations in the DMD gene encoding Dystrophin and affecting 1:5000 boys worldwide. Lack of Dystrophin leads to progressive muscle wasting and degeneration resulting in cardiorespiratory failure. Despite the absence of a definitive cure, innovative therapeutic avenues are emerging. Myopathologic studies are important to further understand the biological mechanisms of the disease and to identify histopathologic benchmarks for clinical evaluations. We conducted a myopathologic analysis on twenty-four muscle biopsies from DMD patients, with particular emphasis on regeneration, fibro-adipogenic progenitors and muscle stem cells behavior. We describe an increase in content of fibro-adipogenic progenitors, central orchestrators of fibrotic progression and lipid deposition, concurrently with a decline in muscle regenerative capacity. This regenerative impairment strongly correlates with compromised activation and expansion of muscle stem cells. Furthermore, our study uncovers an early acquisition of a senescence phenotype by DMD-afflicted muscle stem cells. Here we describe the myopathologic trajectory intrinsic to DMD and establish muscle stem cell senescence as a pivotal readout for future therapeutic interventions.

Longitudinal Analysis of PUL 2.0 Domains in Ambulant and Non-Ambulant Duchenne Muscular Dystrophy Patients: How do they Change in Relation to Functional Ability?

BACKGROUND: The performance of upper limb 2.0 (PUL) is widely used to assess upper limb function in DMD patients. The aim of the study was to assess 24 month PUL changes in a large cohort of DMD patients and to establish whether domains changes occur more frequently in specific functional subgroups. METHODS: The PUL was performed in 311 patients who had at least one pair of assessments at 24 months, for a total of 808 paired assessments. Ambulant patients were subdivided according to the ability to walk: >350, 250-350, ≤250 meters. Non ambulant patients were subdivided according to the time since they lost ambulation: <1, 1-2, 2-5 or >5 years. RESULTS: At 12 months, the mean PUL 2.0 change on all the paired assessments was -1.30 (-1.51--1.05) for the total score, -0.5 (-0.66--0.39) for the shoulder domain, -0.6 (-0.74--0.5) for the elbow domain and -0.1 (-0.20--0.06) for the distal domain.At 24 months, the mean PUL 2.0 change on all the paired assessments was -2.9 (-3.29--2.60) for the total score, -1.30 (-1.47--1.09) for the shoulder domain, -1.30 (-1.45--1.11) for the elbow domain and -0.4 (-1.48--1.29) for the distal domain.Changes at 12 and 24 months were statistically significant between subgroups with different functional abilities for the total score and each domain (p < 0.001). CONCLUSION: There were different patterns of changes among the functional subgroups in the individual domains. The time of transition, including the year before and after loss of ambulation, show the peak of negative changes in PUL total scores that reflect not only loss of shoulder but also of elbow activities. These results suggest that patterns of changes should be considered at the time of designing clinical trials.

Digital health and Clinical Patient Management System (CPMS) platform utility for data sharing of neuromuscular patients: the Italian EURO-NMD experience.

BACKGROUND: The development of e-health technologies for teleconsultation and exchange of knowledge is one of the core purposes of European Reference Networks (ERNs), including the ERN EURO-NMD for rare neuromuscular diseases. Within ERNs, the Clinical Patient Management System (CPMS) is a web-based platform that seeks to boost active collaboration within and across the network, implementing data sharing. Through CPMS, it is possible to both discuss patient cases and to make patients' data available for registries and databases in a secure way. In this view, CPMS may be considered a sort of a temporary storage for patients' data and an effective tool for data sharing; it facilitates specialists' consultation since rare diseases (RDs) require multidisciplinary skills, specific, and outstanding clinical experience. Following European Union (EU) recommendation, and to promote the use of CPMS platform among EURO-NMD members, a twelve-month pilot project was set up to train the 15 Italian Health Care Providers (HCPs). In this paper, we report the structure, methods, and results of the teaching course, showing that tailored, ERN-oriented, training can significantly enhance the profitable use of the CPMS. RESULTS: Throughout the training course, 45 professionals learned how to use the many features of the CPMS, eventually opening 98 panels of discussion-amounting to 82% of the total panels included in the EURO-NMD. Since clinical, genetic, diagnostic, and therapeutic data of patients can be securely stored within the platform, we also highlight the importance of this platform as an effective tool to discuss and share clinical cases, in order to ease both case solving and data storing. CONCLUSIONS: In this paper, we discuss how similar course could help implementing the use of the platform, highlighting strengths and weaknesses of e-health for ERNs. The expected result is the creation of a "map" of neuromuscular patients across Europe that might be improved by a wider use of CPMS.