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Abstract Background Facioscapulohumeral dystrophy (FSHD) is a myopathy characterized by the loss of repressive epigenetic features affecting the D4Z4 locus (4q35). The assessment of DNA methylation at two regions (DUX4-PAS and DR1) of D4Z4 locus proved to be an effective method to detect epigenetic signatures compatible with FSHD. The present study aims at validating the employment of this method into clinical practice and improving the protocol by refining the classification thresholds of 4qA/4qA patients. To this purpose, 218 subjects with clinical suspicion of FSHD collected in 2022–2023 were analyzed. Each participant underwent in parallel the traditional FSHD molecular testing (D4Z4 sizing) and the proposed methylation assay. The results provided by both analyses were compared to evaluate the concordance and calculate the performance metrics of the methylation test. Results Among the 218 subjects, the 4q variant type distribution was 54% 4qA/4qA, 43% 4qA/4qB and 3% 4qB/4qB. The methylation analysis was performed only on carriers of at least one 4qA allele. After refining the classification threshold, the test reached the following performance metrics: sensitivity = 0.90, specificity = 1.00 and accuracy = 0.93. These results confirmed the effectiveness of the methylation assay in identifying patients with genetic signature compatible with FSHD1 and FSHD2 based on their DUX4-PAS and DR1 profile, respectively. The methylation data were also evaluated with respect to the clinical information. Conclusions The study confirmed the ability of the method to accurately identify methylation profiles compatible with FSHD genetic signatures considering the 4q genotype. Moreover, the test allows the detection of hypomethylated profiles in asymptomatic patients, suggesting its potential application in identifying preclinical conditions in patients with positive family history and FSHD genetic signatures. Furthermore, the present work emphasizes the importance of interpreting methylation profiles considering the patients’ clinical data.

Abstract Background In facioscapulohumeral muscular dystrophy (FSHD), it is not known whether physical activity (PA) practiced at young age is associated with the clinical presentation of disease. To assess this issue, we performed a retrospective cohort study concerning the previous practice of sports and, among them, those with medium-high cardiovascular commitment in clinically categorized carriers of a D4Z4 reduced allele (DRA). Methods People aged between 18 and 60 were recruited as being DRA carriers. Subcategory (classical phenotype, A; incomplete phenotype, B; asymptomatic carriers, C; complex phenotype, D) and FSHD score, which measures muscle functional impairment, were assessed for all participants. Information on PAs was retrieved by using an online survey dealing with the practice of sports at a young age. Results 368 participants were included in the study, average age 36.6 years (SD = 9.4), 47.6% male. The FSHD subcategory A was observed in 157 (42.7%) participants with average (± SD) FSHD score of 5.8 ± 3.0; the incomplete phenotype (category B) in 46 (12.5%) participants (average score 2.2 ± 1.7) and the D phenotype in 61 (16.6%, average score 6.5 ± 3.8). Asymptomatic carriers were 104 (subcategory C, 28.3%, score 0.0 ± 0.2). Time from symptoms onset was higher for patients with A (15.8 ± 11.1 years) and D phenotype (13.3 ± 11.9) than for patients with B phenotype (7.3 ± 9.0). The practice of sports was associated with lower FSHD score (-17%) in participants with A phenotype (MR = 0.83, 95% CI = 0.73–0.95, p = 0.007) and by 33% in participants with D phenotype (MR = 0.67, 95% CI = 0.51–0.89, p = 0.006). Conversely, no improvement was observed in participants with incomplete phenotype with mild severity (B). Conclusions PAs at a young age are associated with a lower clinical score in the adult A and D FSHD subcategories. These results corroborate the need to consider PAs at the young age as a fundamental indicator for the correct clinical stratification of the disease and its possible evolution.

Background: Amyotrophic lateral sclerosis (ALS) is a progressive disease for which no curative treatment is currently available. Objective: This study aimed to investigate whether cortico-spinal transcranial direct current stimulation (tDCS) could mitigate symptoms in ALS patients via a randomized, double-blind, sham-controlled trial, followed by an open-label phase. Methods: Thirty-one participants were randomized into two groups for the initial controlled phase. At baseline (T0), Group 1 received placebo stimulation (sham tDCS), while Group 2 received cortico-spinal stimulation (real tDCS) for five days/week for two weeks (T1), with an 8-week (T2) follow-up (randomized, double-blind, sham-controlled phase). At the 24-week follow-up (T3), all participants (Groups 1 and 2) received a second treatment of anodal bilateral motor cortex and cathodal spinal stimulation (real tDCS) for five days/week for two weeks (T4). Follow-up evaluations were performed at 32-weeks (T5) and 48-weeks (T6) (open-label phase). At each time point, clinical assessment, blood sampling, and intracortical connectivity measures using transcranial magnetic stimulation (TMS) were evaluated. Additionally, we evaluated survival rates. Results: Compared to sham stimulation, cortico-spinal tDCS significantly improved global strength, caregiver burden, and quality of life scores, which correlated with the restoration of intracortical connectivity measures. Serum neurofilament light levels decreased among patients who underwent real tDCS but not in those receiving sham tDCS. The number of completed 2-week tDCS treatments significantly influenced patient survival. Conclusions: Cortico-spinal tDCS may represent a promising therapeutic and rehabilitative approach for patients with ALS. Further larger-scale studies are necessary to evaluate whether tDCS could potentially impact patient survival. Clinical trial registration: NCT04293484.

Abstract 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.

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype–phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is α-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype.

Amyloidoses represent a group of diseases characterized by the pathological accumulation in the extracellular area of insoluble misfolded protein material called “amyloid”. The damage to the tissue organization and the direct toxicity of the amyloidogenic substrates induce progressive dysfunctions in the organs involved. They are usually multisystem diseases involving several vital organs, such as the peripheral nerves, heart, kidneys, gastrointestinal tract, liver, skin, and eyes. Transthyretin amyloidosis (ATTR) is related to abnormalities of transthyretin (TTR), a protein that acts as a transporter of thyroxine and retinol and is produced predominantly in the liver. ATTR is classified as hereditary (ATTRv) and wild type (ATTRwt). ATTRv is a severe systemic disease of adults caused by mutations in the TTR gene and transmitted in an autosomal dominant manner with incomplete penetrance. Some pathogenic variants in TTR are preferentially associated with a neurological phenotype (progressive peripheral sensorimotor polyneuropathy); others are more frequently associated with restrictive heart failure. However, many mutations express a mixed phenotype with neurological and cardiological involvement. ATTRv is now a treatable disease. A timely and definite diagnosis is essential in view of the availability of effective therapies that have revolutionized the management of affected patients. The purpose of this review is to familiarize the clinician with the disease and with the correct diagnostic pathways in order to obtain an early diagnosis and, consequently, the possibility of an adequate treatment.

Abstract Background Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease whose spreading and progression mechanisms are still unclear. The ability to predict ALS prognosis would improve the patients’ quality of life and support clinicians in planning treatments. In this paper, we investigate ALS evolution trajectories using Process Mining (PM) techniques enriched to both easily mine processes and automatically reveal how the pathways differentiate according to patients’ characteristics. Methods We consider data collected in two distinct data sources, namely the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) dataset and a real-world clinical register (ALS–BS) including data of patients followed up in two tertiary clinical centers of Brescia (Italy). With a focus on the functional abilities progressively impaired as the disease progresses, we use two Process Discovery methods, namely the Directly-Follows Graph and the CareFlow Miner, to mine the population disease trajectories on the PRO-ACT dataset. We characterize the impairment trajectories in terms of patterns, timing, and probabilities, and investigate the effect of some patients’ characteristics at onset on the followed paths. Finally, we perform a comparative study of the impairment trajectories mined in PRO-ACT versus ALS–BS. Results We delineate the progression pathways on PRO-ACT, identifying the predominant disabilities at different stages of the disease: for instance, 85% of patients enter the trials without disabilities, and 48% of them experience the impairment of Walking/Self-care abilities first. We then test how a spinal onset increases the risk of experiencing the loss of Walking/Self-care ability as first impairment (52% vs. 27% of patients develop it as the first impairment in the spinal vs. the bulbar cohorts, respectively), as well as how an older age at onset corresponds to a more rapid progression to death. When compared, the PRO-ACT and the ALS–BS patient populations present some similarities in terms of natural progression of the disease, as well as some differences in terms of observed trajectories plausibly due to the trial scheduling and recruitment criteria. Conclusions We exploited PM to provide an overview of the evolution scenarios of an ALS trial population and to preliminary compare it to the progression observed in a clinical cohort. Future work will focus on further improving the understanding of the disease progression mechanisms, by including additional real-world subjects as well as by extending the set of events considered in the impairment trajectories.

Summary: Background: Efficacy and safety of onasemnogene abeparvovec (OA) for Spinal Muscular Atrophy infants under 7 months and

The extent of nerve involvement in leprosy is highly variable in distribution and clinical presentation. Mononeuropathies, multiple mononeuropathies, and polyneuropathies can present both in the context of a cutaneous and/or systemic picture and in the form of pure neuritic leprosy (PNL). The differential diagnosis of leprosy neuropathy remains challenging because it is a very rare condition and, especially in Western countries, is often overlooked. We report one case of the polyneuropathic form of PNL (P-PNL) and one case of multiple mononeuropathy in paucibacillary leprosy. In both cases, the diagnosis was achieved by performing a sural nerve biopsy, which showed subverted structure, severe infiltration of inflammatory cells in nerve fascicles, granulomatous abnormalities, and the presence of alcohol-acid-resistant, Ziehl–Neelsen-positive bacilli inside the nerve bundles. Leprosy remains an endemic disease in many areas of the world, and globalization has led to the spread of cases in previously disease-free countries. In this perspective, our report emphasizes that the diagnostic possibility of leprosy neuropathy should always be taken into account, even in Western countries, in the differential diagnostic process of an acquired sensory polyneuropathy or multineuropathy and confirms that nerve biopsy remains a useful procedure in working up neuropathies with unknown etiology.

Pompe disease (PD) is an autosomal recessive disorder caused by mutations in the GAA gene that lead to a deficiency in the acid alpha-glucosidase enzyme. Two clinical presentations are usually considered, named infantile-onset Pompe disease (IOPD) and late-onset Pompe disease (LOPD), which differ in age of onset, organ involvement, and severity of disease. Assessment of acid alpha-glucosidase activity on a dried blood spot is the first-line screening test, which needs to be confirmed by genetic analysis in case of suspected deficiency. LOPD is a multi-system disease, thus requiring a multidisciplinary approach for efficacious management. Enzyme replacement therapy (ERT), which was introduced over 15 years ago, changes the natural progression of the disease. However, it has limitations, including a reduction in efficacy over time and heterogeneous therapeutic responses among patients. Novel therapeutic approaches, such as gene therapy, are currently under study. We provide a comprehensive review of diagnostic advances in LOPD and a critical discussion about the advantages and limitations of current and future treatments.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a rare autosomal recessive long-chain fatty acid oxidation disorder caused by mutations in the ACADVL gene. The myopathic form presents with exercise intolerance, exercise-related rhabdomyolysis, and muscle pain, usually starting during adolescence or adulthood. We report on a 17-year-old boy who has presented with exercise-induced muscle pain and fatigue since childhood. In recent clinical history, episodes of exercise-related severe hyperCKemia and myoglobinuria were reported. Electromyography was normal, and a muscle biopsy showed only “moth-eaten” fibers, and a mild increase in lipid storage in muscle fibers. NGS analysis displayed the already known heterozygote c.1769G>A variant and the unreported heterozygote c.523G>C change in ACADVL both having disease-causing predictions. Plasma acylcarnitine profiles revealed high long-chain acylcarnitine species levels, especially C14:1. Clinical, histopathological, biochemical, and genetic tests supported the diagnosis of VLCAD deficiency. Our report of a novel pathogenic missense variant in ACADVL expands the allelic heterogeneity of the disease. Since dietary treatment is the only therapy available for treating VLCAD deficiency and it is more useful the earlier it is started, prompt diagnosis is essential in order to minimize muscle damage and slow the disease progression.

Neutral lipid storage disease type M (NLSD-M) is an ultra-rare, autosomal recessive disorder that causes severe skeletal and cardiac muscle damage and lipid accumulation in all body tissues. In this hereditary pathology, the defective action of the adipose triglyceride lipase (ATGL) enzyme induces the enlargement of cytoplasmic lipid droplets and reduction in the detachment of mono- (MG) and diglycerides (DG). Although the pathogenesis of muscle fiber necrosis is unknown, some studies have shown alterations in cellular energy production, probably because MG and DG, the substrates of Krebs cycle, are less available. No tests have been tried with medium-chain fatty acid molecules to evaluate the anaplerotic effect in NLSD cells. In this study, we evaluated the in vitro effect of triheptanoin (Dojolvi®), a highly purified chemical triglyceride with seven carbon atoms, in fibroblasts obtained from five NLSD-M patients. Glycolytic and mitochondrial functions were determined by Seahorse XF Agylent Technology, and cellular viability and triglyceride content were measured through colorimetric assays. After the addition of triheptanoin, we observed an increase in glycolysis and mitochondrial respiration in all patients compared with healthy controls. These preliminary results show that triheptanoin is able to induce an anaplerotic effect in NLSD-M fibroblasts, paving the way towards new therapeutic strategies.

Abstract Facioscapulohumeral muscular dystrophy (FSHD) is a myopathy with prevalence of 1 in 20,000. Almost all patients affected by FSHD carry deletions of an integral number of tandem 3.3 kilobase repeats, termed D4Z4, located on chromosome 4q35. Assessment of size of D4Z4 alleles is commonly used for FSHD diagnosis. However, the extended molecular testing has expanded the spectrum of clinical phenotypes. In particular, D4Z4 alleles with 9–10 repeat have been found in healthy individuals, in subjects with FSHD or affected by other myopathies. These findings weakened the strict relationship between observed phenotypes and their underlying genotypes, complicating the interpretation of molecular findings for diagnosis and genetic counseling. In light of the wide clinical variability detected in carriers of D4Z4 alleles with 9–10 repeats, we applied a standardized methodology, the Comprehensive Clinical Evaluation Form (CCEF), to describe and characterize the phenotype of 244 individuals carrying D4Z4 alleles with 9–10 repeats (134 index cases and 110 relatives). The study shows that 54.5% of index cases display a classical FSHD phenotype with typical facial and scapular muscle weakness, whereas 20.1% present incomplete phenotype with facial weakness or scapular girdle weakness, 6.7% display minor signs such as winged scapula or hyperCKemia, without functional motor impairment, and 18.7% of index cases show more complex phenotypes with atypical clinical features. Family studies revealed that 70.9% of relatives carrying 9–10 D4Z4 reduced alleles has no motor impairment, whereas a few relatives (10.0%) display a classical FSHD phenotype. Importantly all relatives of index cases with no FSHD phenotype were healthy carriers. These data establish the low penetrance of D4Z4 alleles with 9–10 repeats. We recommend the use of CCEF for the standardized clinical assessment integrated by family studies and further molecular investigation for appropriate diagnosis and genetic counseling. Especially in presence of atypical phenotypes and/or sporadic cases with all healthy relatives is not possible to perform conclusive diagnosis of FSHD, but all these cases need further studies for a proper diagnosis, to search novel causative genetic defects or investigate environmental factors or co-morbidities that may trigger the pathogenic process. These evidences are also fundamental for the stratification of patients eligible for clinical trials. Our work reinforces the value of large genotype–phenotype studies to define criteria for clinical practice and genetic counseling in rare diseases.

Objective: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. Methods: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. Results: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. Conclusions: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.

Background: Four main clinical phenotypes have been traditionally described in patients mutated in SCN4A, including sodium-channel myotonia (SCM), paramyotonia congenita (PMC), Hypokaliemic type II (HypoPP2), and Hyperkaliemic/Normokaliemic periodic paralysis (HyperPP/NormoPP); in addition, rare phenotypes associated with mutations in SCN4A are congenital myasthenic syndrome and congenital myopathy. However, only scarce data have been reported in literature on large patient cohorts including phenotypes characterized by myotonia and episodes of paralysis.Methods: We retrospectively investigated clinical and molecular features of 80 patients fulfilling the following criteria: (1) clinical and neurophysiological diagnosis of myotonia, or clinical diagnosis of PP, and (2) presence of a pathogenic SCN4A gene variant. Patients presenting at birth with episodic laryngospasm or congenital myopathy-like phenotype with later onset of myotonia were considered as neonatal SCN4A.Results: PMC was observed in 36 (45%) patients, SCM in 30 (37.5%), Hyper/NormoPP in 7 (8.7%), HypoPP2 in 3 (3.7%), and neonatal SCN4A in 4 (5%). The median age at onset was significantly earlier in PMC than in SCM (p < 0.01) and in Hyper/NormoPP than in HypoPP2 (p = 0.02). Cold-induced myotonia was more frequently observed in PMC (n = 34) than in SCM (n = 23) (p = 0.04). No significant difference was found in age at onset of episodes of paralysis among PMC and PP or in frequency of permanent weakness between PP (n = 4), SCM (n = 5), and PMC (n = 10). PP was more frequently associated with mutations in the S4 region of the NaV1.4 channel protein compared to SCM and PMC (p < 0.01); mutations causing PMC were concentrated in the C-terminal region of the protein, while SCM-associated mutations were detected in all the protein domains.Conclusions: Our data suggest that skeletal muscle channelopathies associated with mutations in SCN4A represent a continuum in the clinical spectrum.

Dystrophinopathies are inherited diseases caused by mutations in the dystrophin (DMD) gene for which testing is mandatory for genetic diagnosis, reproductive choices and eligibility for personalized trials. We genotyped the DMD gene in our Italian cohort of 1902 patients (BMD n = 740, 39%; DMD n =1162, 61%) within a nationwide study involving 11 diagnostic centers in a 10-year window (2008–2017). In DMD patients, we found deletions in 57%, duplications in 11% and small mutations in 32%. In BMD, we found deletions in 78%, duplications in 9% and small mutations in 13%. In BMD, there are a higher number of deletions, and small mutations are more frequent than duplications. Among small mutations that are generally frequent in both phenotypes, 44% of DMD and 36% of BMD are nonsense, thus, eligible for stop codon read-through therapy; 63% of all out-of-frame deletions are eligible for single exon skipping. Patients were also assigned to Italian regions and showed interesting regional differences in mutation distribution. The full genetic characterization in this large, nationwide cohort has allowed us to draw several correlations between DMD/BMD genotype landscapes and mutation frequency, mutation types, mutation locations along the gene, exon/intron architecture, and relevant protein domain, with effects on population genetic characteristics and new personalized therapies.

A cytokine storm, autoimmune features and dysfunctions of myeloid cells significantly contribute to severe coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Genetic background of the host seems to be partly responsible for severe phenotype and genes related to innate immune response seem critical host determinants. The C9orf72 gene has a role in vesicular trafficking, autophagy regulation and lysosome functions, is highly expressed in myeloid cells and is involved in immune functions, regulating the lysosomal degradation of mediators of innate immunity. A large non-coding hexanucleotide repeat expansion (HRE) in this gene is the main genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), both characterized by neuroinflammation and high systemic levels of proinflammatory cytokines, while HREs of intermediate length, although rare, are more frequent in autoimmune disorders. C9orf72 full mutation results in haploinsufficiency and intermediate HREs seem to modulate gene expression as well and impair autophagy. Herein, we sought to explore whether intermediate HREs in C9orf72 may be a risk factor for severe COVID-19. Although we found intermediate HREs in only a small portion of 240 patients with severe COVID-19 pneumonia, the magnitude of risk for requiring non-invasive or mechanical ventilation conferred by harboring intermediate repeats >10 units in at least one C9orf72 allele was more than twice respect to having shorter expansions, when adjusted for age (odds ratio (OR) 2.36; 95% confidence interval (CI) 1.04–5.37, p = 0.040). The association between intermediate repeats >10 units and more severe clinical outcome (p = 0.025) was also validated in an independent cohort of 201 SARS-CoV-2 infected patients. These data suggest that C9orf72 HREs >10 units may influence the pathogenic process driving more severe COVID-19 phenotypes.

CAPN1 encodes calpain-1, a large subunit of μ-calpain, a calcium-activated cysteine protease widely present in the central nervous system. Mutations in CAPN1 have recently been identified in a complicated form of Hereditary Spastic Paraplegia (HSP) with a combination of cerebellar ataxia and corticomotor tract disorder (SPG76). Therefore, CAPN1 is now considered one of those genes that clinically manifest with a spectrum of disorders ranging from spasticity to cerebellar ataxia and represent a link between Spinocerebellar Ataxia and HSP, two groups of diseases previously considered separate but sharing pathophysiological pathways. We here describe clinical and molecular findings of two Italian adult siblings affected with a pure form of HSP and harboring the novel homozygote c.959delA variant (p.Tyr320Leufs*73) in the CAPN1 gene. Although the reason why mutations in CAPN1 may cause heterogeneous clinical pictures remains speculative, our findings confirm that the spectrum of the CAPN1-linked phenotypes includes pure HSP with onset during the third decade of life. Further studies are warrantied in order to clarify the mechanism underlying the differences in CAPN1 mutation clinical expression.

In the last years, magnetic resonance imaging (MRI) has become fundamental for the diagnosis and monitoring of myopathies given its ability to show the severity and distribution of pathology, to identify specific patterns of damage distribution and to properly interpret a number of genetic variants. The advances in MR techniques and post-processing software solutions have greatly expanded the potential to assess pathological changes in muscle diseases, and more specifically of myopathies; a number of features can be studied and quantified, ranging from composition, architecture, mechanical properties, perfusion, and function, leading to what is known as quantitative MRI (qMRI). Such techniques can effectively provide a variety of information beyond what can be seen and assessed by conventional MR imaging; their development and application in clinical practice can play an important role in the diagnostic process and in assessing disease course and treatment response. In this review, we briefly discuss the current role of muscle MRI in diagnosing muscle diseases and describe in detail the potential and perspectives of the application of advanced qMRI techniques in this field.

Lipomas have often been associated with mtDNA mutations and were mainly observed in patients with mutation in mitochondrial tRNAlysine which is also the most frequent mutation associated with MERRF. Up to date, no systematic studies have been developed in order to assess the incidence of lipomas in large cohorts of mitochondrial patients.The aim of this study is to analyze the incidence and characteristics of lipomas among an Italian cohort of patients with mitochondrial diseases. A retrospective, database-based study (Nation-wide Italian Collaborative Network of Mitochondrial Diseases) of patients with lipomas was performed. A total of 22 (1.7%) patients with lipomas have been identified among the 1,300 mitochondrial patients, enrolled in the Italian database. In about 18% multiple systemic lipomatosis (MSL) was the only clinical manifestation; 54% of patients showed a classical MERRF syndrome. Myopathy, alone or in association with other symptoms, was found in 27% of patients. Lactate was elevated in all the 12 patients in which was measured. Muscle biopsy was available in 18/22 patients: in all of them mitochondrial abnormalities were present. Eighty six percent had mutations in mtDNA coding for tRNA lysine. In most of patients, lipomas were localized along the cervical-cranial-thoracic region. In 68% of the patients were distributed symmetrically. Only two patients had lipomas in a single anatomical site (1 in right arm and 1 in gluteus maximum). MSL is often overlooked by clinicians in patients with mitochondrial diseases where the clinical picture could be dominated by a severe multi-systemic involvement. Our data confirmed that MSL is a rare sign of mitochondrial disease with a strong association between multiple lipomas and lysine tRNA mutations. MSL could be considered, even if rare, a red flag for mitochondrial disorders, even in patients with an apparently isolated MSL.

Glycogenosis VII (GSD VII) is a rare autosomal recessive glycogen storage disorder caused by mutations in the PFKM gene encoding the phosphofructokinase (PFK) enzyme. A classical form with exercise intolerance, contractures, and myoglobinuria, a severe multisystem infantile form, an hemolytic variant and a late-onset form usually presenting with muscle pain and mild fixed proximal weakness have been reported. We describe a 65-year-old man affected by muscle PFK deficiency who, since the age of 33, presented with exercise intolerance and myoglobinuria. Muscle biopsy showed a vacuolar myopathy with glycogen storage. The biochemical assay of PFK-M showed very low residual activity (6%). Genetic analysis of PFKM gene evidenced the presence of the heterozygote c.1817A>C (p.Asp543Ala) and c.488 G>A (p.Arg100Gln) pathogenic mutations. In his fifth decade, he started cyclosporine after liver transplantation for hepatocellular carcinoma and, then, amiodarone because of atrial fibrillation. In the following years, he developed a progressive and severe muscle weakness, mainly involving lower limbs, up to a loss of independent walking. Muscle MRI showed adipose substitution of both anterior and posterior thigh muscles with selective sparing of the medial compartment. Marked signs of adipose substitution were also documented in the legs with a selective replacement of gemelli and peroneus muscles. The temporal relationship between the patient's clinical worsening and chronic treatment with cyclosporine and amiodarone suggests an additive toxic damage by these two potentially myotoxic drugs determining such an unusually severe phenotype, also confirmed by muscle MRI findings.

Background. Electrotherapy is widely used in physical therapy to increase muscle mass, improve motor function, and assist physical activity in several neurologic conditions. However, concerning Spinal Muscular Atrophy (SMA), limited evidence exists on the role of electrotherapy as an adjunct for improving muscle strength and function. Case Report. An adolescent (13 y.o.) with SMA type III underwent an 18-week strengthening program divided into two stages. During Phase I (weeks: 1-8), a home-based program for quadriceps strengthening through neuromuscular electrical stimulation (NMES) was provided. In Phase II (weeks: 9-18), at-home NMES was combined with functional electrical stimulation (FES) assisting volitional cycling for a broader, systemic conditioning. The treatment improved patient’s structural and functional motor outcomes (quadriceps circumference and strength, Tinetti scale, and Hammersmith scale) as well as independence in stair climbing. Clinical Rehabilitation Impact. The purpose of this report is to raise awareness of the potential role of electrotherapy to help improving motor performance in SMA patients and, secondly, to foster further research aimed at assessing the actual contribution this intervention may have as an add-on therapy to existing care.

In recent years, an autoantibody directed against the 5′-citosolic nucleotidase1A (cN1A) was identified in the sera of sporadic inclusion body myositis (s-IBM) patients with widely variable sensitivity (33%–76%) and specificity (87%–100%). We assessed the sensitivity/specificity of anti-cN1A antibodies in an Italian cohort of s-IBM patients, searching for a potential correlation with clinical data. We collected clinical data and sera from 62 consecutive s-IBM patients and 62 other inflammatory myopathies patients. Testing for anti-cN1A antibodies was performed using a commercial ELISA. Anti-cN1A antibodies were detected in 23 s-IBM patients, resulting in a sensitivity of 37.1% with a specificity of 96.8%. Positive and negative predictive values were 92.0% and 60.6%, respectively. We did not find significant difference regarding demographic variables, nor quadriceps or finger flexor weakness. Nevertheless, we found that anti-cN1A-positive patients presented significantly lower scores in IBMFRS item 1 (swallowing, p = 0.045) and more frequently reported more severe swallowing problems, expressed as an IBMFRS item 1 score ≤ 2 (p < 0.001). We confirmed the low sensitivity and high specificity of anti-cN1A Ab in s-IBM patients with a high positive predictive value. The presence of anti-CN1A antibodies identified patients with a greater risk of more severe dysphagia.

Chronic progressive external ophthalmoplegia is a mitochondrial disorder usually caused by single or multiple mitochondrial DNA (mtDNA) deletions and, more rarely, by maternally inherited mtDNA point mutations, most frequently in tRNA genes (MTT). We report on a patient presenting with a progressive eyelid ptosis with bilateral ophthalmoparesis, dysphagia, dysphonia and mild proximal limb weakness associate with a mild movement disorder characterized by abnormal involuntary movements involving head and limbs, imbalance and gait instability. Muscle biopsy demonstrated the presence of ragged red fibers and several cytochrome-C-oxidase negative fibers. Molecular analysis showed the novel m.5613T > C heteroplasmic mutation in the mitochondrial tRNAAla gene (MTTA) which disrupts a conserved site and fulfills the accepted criteria of pathogenicity. Moreover, a 38 CAG trinucleotide repeat expansion was found on the huntingtin gene, thus configuring a singular CPEO/“reduced penetrance” Huntington disease “double trouble”. With this novel MTTA point mutation, we extend the spectrum of provisional pathogenic changes in this gene, which is a very rare site of pathogenic mutation, and confirm that clinical expression of these mutations is hardly ever heterogeneous, including myopathy and CPEO. Mitochondrial involvement is an emerging key determinant in the pathogenesis of Huntington disease and it is well known that mutant huntingtin influences the mitochondrial respiratory complexes II and III. A synergist effect of the HTT and MTTA mutations on respiratory chain function may be hypothesized in our patient and should be regarded as a spur for further studies on the mtDNA/HTT reciprocal interactions.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder characterized by progressive gait and limb ataxia, cerebellar, pyramidal and dorsal column involvement, visual defects, scoliosis, pes cavus and cardiomyopathy. It is caused by a homozygous guanine-adenine-adenine (GAA) trinucleotide repeat expansion in intron 1 of the frataxin gene (FXN) on chromosome 9q13-q21.1. Onset is usually in the first or second decade of life; however, late-onset cases of Freidreich ataxia (LOFA), after the age of 25 years, and very late-onset cases of Freidreich ataxia (VLOFA), after the age of 40 years, have been reported. VLOFA is quite rare and usually presents a milder progression of the disease. We report the case of a 64-year-old woman affected with VLOFA whose first symptoms (balance and gait disturbances) occurred at the age of 44 years. At the age of 62 years, she started complaining of a slowly progressive dysphonia showing the clinical aspects of laryngeal dystonia. Molecular analysis showed a 210- and 230-trinucleotide GAA repeat expansion in the two alleles of the FXN gene. Laryngeal dystonia has been reported only in very few cases of ataxia syndrome and never before in FRDA patients. It may represent a rare clinical manifestation of VLOFA thus confirming the high variability of the clinical spectrum of FRDA.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a devastating autosomal recessive disorder due to mutations in TYMP, which cause loss of function of thymidine phosphorylase (TP), nucleoside accumulation in plasma and tissues and mitochondrial dysfunction. The clinical picture includes progressive gastrointestinal dysmotility, cachexia, ptosis and ophthalmoparesis, peripheral neuropathy and diffuse leukoencephalopathy, which usually lead to death in early adulthood. Therapeutic options are currently available in clinical practice (allogeneic hematopoietic stem cell transplantation and carrier erythrocyte entrapped TP therapy) and newer, promising therapies are expected in the near future. However, successful treatment is strictly related to early diagnosis. We report on an incomplete MNGIE phenotype in a young man harboring the novel heterozygote c.199 C>T (Q67X) mutation in exon 2, and the previously reported c.866 A>C (E289A) mutation in exon 7 in TYMP. The correct diagnosis was achieved many years after the onset of symptoms and unfortunately, the patient died soon after diagnosis because of multiorgan failure due to severe malnutrition and cachexia before any therapeutic option could be tried. To date, early diagnosis is essential to ensure that patients have the opportunity to be treated. MNGIE should be suspected in all patients who present with both gastrointestinal and nervous system involvement, even if the classical complete phenotype is lacking.

BackgroundDegeneration of intraepidermal nerve fibers (IENF) is a hallmark of small fiber neuropathy of different etiology, whose clinical picture is dominated by neuropathic pain. It is unknown if critical illness can affect IENF.MethodsWe enrolled 14 adult neurocritical care patients with prolonged intensive care unit (ICU) stay and artificial ventilation (≥ 3 days), and no previous history or risk factors for neuromuscular disease. All patients underwent neurological examination including evaluation of consciousness, sensory functions, muscle strength, nerve conduction study and needle electromyography, autonomic dysfunction using the finger wrinkling test, and skin biopsy for quantification of IENF and sweat gland innervation density during ICU stay and at follow-up visit. Development of infection, sepsis and multiple organ failure was recorded throughout the ICU stay.ResultsOf the 14 patients recruited, 13 (93%) had infections, sepsis or multiple organ failure. All had severe and non-length dependent loss of IENF. Sweat gland innervation was reduced in all except one patient. Of the 7 patients available for follow-up visit, three complained of diffuse sensory loss and burning pain, and another three showed clinical dysautonomia.ConclusionsSmall fiber pathology can develop in the acute phase of critical illness and may explain chronic sensory impairment and pain in neurocritical care survivors. Its impact on long term disability warrants further studies involving also non-neurologic critical care patients.

Mitochondria, the powerhouse of the cell, play a critical role in several metabolic processes and apoptotic pathways. Multiple evidences suggest that mitochondria may be crucial in ageing-related neurodegenerative diseases. Moreover, mitochondrial haplogroups have been linked to multiple area of medicine, from normal ageing to diseases, including neurodegeneration. Polymorphisms within the mitochondrial genome might lead to impaired energy generation and to increased amount of reactive oxygen species, having either susceptibility or protective role in several diseases. Here, we highlight the role of the mitochondrial haplogroups in the pathogenetic cascade leading to diseases, with special attention to Alzheimer's disease.

Glycogenosis II (GSDII) is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid alpha-glucosidase and subsequent lysosomal accumulation of glycogen in skeletal, cardiac and smooth muscles. The late-onset form is characterized by wide variability of the phenotypical spectrum. Clinical findings may include muscle weakness, respiratory insufficiency, vascular abnormalities, low bone mineral density and higher risk of developing osteoporosis. Craniovertebral junction (CVJ) malformations have never been described so far. We here report on a GSDII 43-year-old woman who harbored the mutations IVS1-13T>G and c.2237G>A in the acid alpha-glucosidase gene. She recurrently suffered from headache, neck pain and dizziness. Brain MRI and CT scan showed the presence of a very rare complex CVJ malformation composed of basilar invagination, basiocciput hypoplasia, partial C1 assimilation, C1 posterior arch aplasia and C1 lateral mass hypoplasia and offset. Although we cannot rule out their coincidental occurrence, the rarity of multiple CVJ malformations in the general population as well as the well-known GSDII multisystem involvement should suggest to study the CVJ in the diagnostic process of GSDII patients in order to assess the CVJ malformation frequency in GSDII population and verify a possible relationship between these two conditions.

Glycogen storage disease type II (GSDII) is an autosomal recessive lysosomal disorder caused by mutations in the gene encoding alpha-glucosidase (GAA). The disease can be clinically classified into three types: a severe infantile form, a juvenile and an adult-onset form. Cases with juvenile or adult onset GSDII mimic limb-girdle muscular dystrophy or polymyositis and are often characterized by respiratory involvement. GSDII patients are diagnosed by biochemical assay and by molecular characterization of the GAA gene. Ascertaining a natural history of patients with heterogeneous late-onset GSDII is useful for evaluating their progressive functional disability. A significant decline is observed over the years in skeletal and respiratory muscle function. Enzyme replacement therapy (ERT) has provided encouraging results in the infantile form. It is not yet known if ERT is effective in late-onset GSDII. We examined a series of 11 patients before and after ERT evaluating muscle strength by MRC, timed and graded functional tests, 6-minute walk test (6MWT), respiratory function by spirometric parameters and quality of life. We observed a partial improvement during a prolonged follow-up from 3 to 18 months. The use of different clinical parameters in the proposed protocol seems crucial to determine the efficacy of ERT, since not all late-onset patients respond similarly to ERT.