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Portable and wearable devices can monitor a number of physical performances and lately have been applied to patients with neuromuscular disorders (NMDs).We performed a systematic search of literature databases following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) principles, including all studies reporting the use of technological devices for motor function assessment in NMDs from 2000 to 2021. We also summarized the evidence on measurement properties (validity, reliability, responsiveness) of the analyzed technological outcome measures.One hundred studies fulfilled the selection criteria, most of them published in the past 10 years. We defined four categories that gathered similar technologies: gait analysis tools, for clinical assessment of pace and posture; continuous monitoring of physical activity with inertial sensors, which allow "unsupervised" activity assessment; upper limb evaluation tools, including Kinect-based outcome measures to assess the reachable workspace; and new muscle strength assessment tools, such as Myotools. Inertial sensors have the evident advantage of being applied in the "in-home" setting, which has become especially appealing during the COVID-19 pandemic, although poor evidence from psychometric property assessment and results of the analyzed studies may limit their research application. Both Kinect-based outcome measures and Myotools have already been validated in multicenter studies and different NMDs, showing excellent characteristics for application in clinical trials.This overview is intended to raise awareness on the potential of the different technology outcome measures in the neuromuscular field and to be an informative source for the design of future clinical trials, particularly in the era of telemedicine.

Background Only a few studies have reported muscle imaging data on small cohorts of patients with myotonic dystrophy type 1 (DM1). We aimed to investigate the muscle involvement in a large cohort of patients in order to refine the pattern of muscle involvement, to better understand the pathophysiological mechanisms of muscle weakness, and to identify potential imaging biomarkers for disease activity and severity. Methods One hundred and thirty-four DM1 patients underwent a cross-sectional muscle magnetic resonance imaging (MRI) study. Short tau inversion recovery (STIR) and T1 sequences in the lower and upper body were analyzed. Fat replacement, muscle atrophy and STIR positivity were evaluated using three different scales. Correlations between MRI scores, clinical features and genetic background were investigated. Results The most frequent pattern of muscle involvement in T1 consisted of fat replacement of the tongue, sternocleidomastoideus, paraspinalis, gluteus minimus, distal quadriceps and gastrocnemius medialis. Degree of fat replacement at MRI correlated with clinical severity and disease duration, but not with CTG expansion. Fat replacement was also detected in milder/asymptomatic patients. More than 80% of patients had STIR-positive signals in muscles. Most DM1 patients also showed a variable degree of muscle atrophy regardless of MRI signs of fat replacement. A subset of patients (20%) showed a 'marbled' muscle appearance. Conclusions Muscle MRI is a sensitive biomarker of disease severity alsofor the milder spectrum of disease. STIR hyperintensity seems to precede fat replacement in T1. Beyond fat replacement, STIR positivity, muscle atrophy and a 'marbled' appearance suggest further mechanisms of muscle wasting and weakness in DM1, representing additional outcome measures and therapeutic targets for forthcoming clinical trials.

Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy (BMD). In the last decade several antisense oligonucleotides drugs have been developed to induce a partially functional internally deleted dystrophin, similar to that produced in BMD, and expected to ameliorate the disease course. The pattern of dystrophin expression and functionality in dystrophinopathy patients is variable due to multiple factors, such as molecular functionality of the dystrophin and its distribution. To benchmark the success of therapeutic intervention, a clear understanding of dystrophin expression patterns in dystrophinopathy patients is vital. Recently, several groups have used innovative techniques to quantify dystrophin in muscle biopsies of children but not in patients with milder BMD. This study reports on dystrophin expression using both Western blotting and an automated, high-throughput, image analysis platform in DMD, BMD, and intermediate DMD/BMD skeletal muscle biopsies. Our results found a significant correlation between Western blot and immunofluorescent quantification indicating consistency between the different methodologies. However, we identified significant inter- and intradisease heterogeneity of patterns of dystrophin expression in patients irrespective of the amount detected on blot, due to variability in both fluorescence intensity and dystrophin sarcolemmal circumference coverage. Our data highlight the heterogeneity of the pattern of dystrophin expression in BMD, which will assist the assessment of dystrophin restoration therapies.

Objective In this study we address the automatic segmentation of selected muscles of the thigh and leg through a supervised deep learning approach. Material and methods The application of quantitative imaging in neuromuscular diseases requires the availability of regions of interest (ROI) drawn on muscles to extract quantitative parameters. Up to now, manual drawing of ROIs has been considered the gold standard in clinical studies, with no clear and universally accepted standardized procedure for segmentation. Several automatic methods, based mainly on machine learning and deep learning algorithms, have recently been proposed to discriminate between skeletal muscle, bone, subcutaneous and intermuscular adipose tissue. We develop a supervised deep learning approach based on a unified framework for ROI segmentation. Results The proposed network generates segmentation maps with high accuracy, consisting in Dice Scores ranging from 0.89 to 0.95, with respect to “ground truth” manually segmented labelled images, also showing high average performance in both mild and severe cases of disease involvement (i.e. entity of fatty replacement). Discussion The presented results are promising and potentially translatable to different skeletal muscle groups and other MRI sequences with different contrast and resolution.

The diagnosis of facioscapulohumeral muscular dystrophy (FSHD) can be challenging in patients not displaying the classical phenotype or with atypical clinical features. Despite the identification by magnetic resonance imaging (MRI) of selective patterns of muscle involvement, their specificity and added diagnostic value are unknown. We aimed to identify the radiological features more useful to distinguish FSHD from other myopathies and test the diagnostic accuracy of MRI. A retrospective cohort of 295 patients (187 FSHD, 108 non-FSHD) studied by upper and lower-limb muscle MRI was analyzed. Scans were evaluated for the presence of 15 radiological features. A random forest machine learning algorithm was used to identify the most relevant for FSHD diagnosis. Different patterns were created by their combination and diagnostic accuracy of each of them was tested. The combination of trapezius involvement and bilateral subscapularis muscle sparing achieved the best diagnostic accuracy (0.89, 95% Confidence Interval [0.85–0.92]) with 0.90 [0.85–0.94] sensitivity and 0.88 [0.80–0.93] specificity. This pattern correctly identified 91% atypical FSHD patients of our cohort. The combination of trapezius involvement, bilateral subscapularis and iliopsoas sparing and asymmetric involvement of upper and lower-limb muscles was pathognomonic for FSHD, yielding a specificity of 0.99 [0.95–1.00]. We identified MRI patterns that showed a high diagnostic power in promptly discriminating FSHD from other muscle disorders, with comparable performance irrespective of typical or atypical clinical features. Upper girdle in addition to lower-limb muscle imaging should be extensively implemented in the diagnostic workup to support or exclude a diagnosis of FSHD.

In recent years, growing evidence has suggested a prominent role of oxidative stress in the pathophysiology of several early- and adult-onset muscle disorders, although effective antioxidant treatments are still lacking. Oxidative stress causes cell damage by affecting protein function, membrane structure, lipid metabolism, and DNA integrity, thus interfering with skeletal muscle homeostasis and functionality. Some features related to oxidative stress, such as chronic inflammation, defective regeneration, and mitochondrial damage are shared among most muscular dystrophies, and Nrf2 has been shown to be a central player in antagonizing redox imbalance in several of these disorders. However, the exact mechanisms leading to overproduction of reactive oxygen species and deregulation in the cellular antioxidants system seem to be, to a large extent, disease-specific, and the clarification of these mechanisms in vivo in humans is the cornerstone for the development of targeted antioxidant therapies, which will require testing in appropriately designed clinical trials.

ObjectiveTo report on a cohort of patients diagnosed with brachio-cervical inflammatory myopathy (BCIM), with specific focus on muscle MRI and follow-up data.MethodsClinical, histopathologic, serologic, and pre- and post-treatment MRI findings of patients diagnosed with BCIM were retrospectively evaluated.ResultsSix patients, all females with a mean age at onset of 53 years (range 37–62 years), were identified. Mean diagnostic delay was 17 months, and mean follow-up was 35 months. Most common clinical features encompassed predominant involvement of neck and proximal upper limb muscles, followed by distal upper limb, facial, and bulbar muscle weakness with different severity. Lower limb involvement was rare, although present in severe cases. Muscle biopsies showed a heterogeneous degree of perivascular and endomysial inflammatory changes. Myositis-specific antibodies were absent in all patients, whereas all resulted positive for antinuclear antibodies; half of the patients had anti–acetylcholine receptor antibodies without evidence of muscle fatigability. MRI showed disproportionate involvement of upper girdle and neck muscles compared with lower limbs, with frequent hyperintensities on short-tau inversion recovery sequences. Partial clinical and radiologic improvement with steroid and immunosuppressant therapy was obtained in most patients, especially in proximal upper limb muscles, whereas neck weakness persisted.ConclusionBCIM is an inflammatory myopathy with a peculiar clinical and radiologic presentation and a relatively broad spectrum of severity. Long-term follow-up data suggest that appropriate and early treatment can prevent chronic muscle function impairment. MRI characterization can be helpful in reducing diagnostic and treatment delay with positive consequence on clinical outcome.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by a complex epigenetic mechanism finally leading to the misexpression of DUX4 in skeletal muscle. Detecting DUX4 and quantifying disease progression in FSHD is extremely challenging, thus increasing the need for surrogate biomarkers. We applied a shotgun proteomic approach with two different setups to analyze the protein repertoire of interstitial fluids obtained from 20 muscles in different disease stages classified by magnetic resonance imaging (MRI) and serum samples from 10 FSHD patients. A total of 1156 proteins were identified in the microdialysates by data independent acquisition, 130 of which only found in muscles in active disease stage. Proteomic profiles were able to distinguish FSHD patients from controls. Two innate immunity mediators (S100-A8 and A9) and Dermcidin were upregulated in muscles with active disease and selectively present in the sera of FSHD patients. Structural muscle and plasminogen pathway proteins were downregulated. Together with the upstream inhibition of myogenic factors, this suggests defective muscle regeneration and increased fibrosis in early/active FSHD. Our MRI targeted exploratory approach confirmed that inflammatory response has a prominent role, together with impaired muscle regeneration, before clear muscle wasting occurs. We also identified three proteins as tissue and possibly circulating biomarkers in FSHD.

Purpose Quantitative MRI (qMRI) plays a crucial role for assessing disease progression and treatment response in neuromuscular disorders, but the required MRI sequences are not routinely available in every center. The aim of this study was to predict qMRI values of water T2 (wT2) and fat fraction (FF) from conventional MRI, using texture analysis and machine learning. Method Fourteen patients affected by Facioscapulohumeral muscular dystrophy were imaged at both thighs using conventional and quantitative MR sequences. Muscle FF and wT2 were calculated for each muscle of the thighs. Forty-seven texture features were extracted for each muscle on the images obtained with conventional MRI. Multiple machine learning regressors were trained to predict qMRI values from the texture analysis dataset. Results Eight machine learning methods (linear, ridge and lasso regression, tree, random forest (RF), generalized additive model (GAM), k-nearest-neighbor (kNN) and support vector machine (SVM) provided mean absolute errors ranging from 0.110 to 0.133 for FF and 0.068 to 0.115 for wT2. The most accurate methods were RF, SVM and kNN to predict FF, and tree, RF and kNN to predict wT2. Conclusion This study demonstrates that it is possible to estimate with good accuracy qMRI parameters starting from texture analysis of conventional MRI.

ObjectivesTo characterise the pattern and spectrum of involvement on muscle MRI in a large cohort of patients with sarcoglycanopathies, which are limb-girdle muscular dystrophies (LGMD2C–2F) caused by mutations in one of the four genes coding for muscle sarcoglycans.MethodsLower limb MRI scans of patients with LGMD2C–2F, ranging from severe childhood variants to milder adult-onset forms, were collected in 17 neuromuscular referral centres in Europe and USA. Muscle involvement was evaluated semiquantitatively on T1-weighted images according to a visual score, and the global pattern was assessed as well.ResultsScans from 69 patients were examined (38 LGMD2D, 18 LGMD2C, 12 LGMD2E and 1 LGMD2F). A common pattern of involvement was found in all the analysed scans irrespective of the mutated gene. The most and earliest affected muscles were the thigh adductors, glutei and posterior thigh groups, while lower leg muscles were relatively spared even in advanced disease. A proximodistal gradient of involvement of vasti muscles was a consistent finding in these patients, including the most severe ones.ConclusionsMuscle involvement on MRI is consistent in patients with LGMD2C–F and can be helpful in distinguishing sarcoglycanopathies from other LGMDs or dystrophinopathies, which represent the most common differential diagnoses. Our data provide evidence about selective susceptibility or resistance to degeneration of specific muscles when one of the sarcoglycans is deficient, as well as preliminary information about progressive involvement of the different muscles over time.

Contains fulltext : 229735.pdf (Publisher’s version ) (Closed access) BACKGROUND AND PURPOSE: The aim was to define the radiological picture of facioscapulohumeral muscular dystrophy 2 (FSHD2) in comparison with FSHD1 and to explore correlations between imaging and clinical/molecular data. METHODS: Upper girdle and/or lower limb muscle magnetic resonance imaging scans of 34 molecularly confirmed FSHD2 patients from nine European neuromuscular centres were analysed. T1-weighted and short-tau inversion recovery (STIR) sequences were used to evaluate the global pattern and to assess the extent of fatty replacement and muscle oedema. RESULTS: The most frequently affected muscles were obliquus and transversus abdominis, semimembranosus, soleus and gluteus minimus in the lower limbs; trapezius, serratus anterior, latissimus dorsi and pectoralis major in the upper girdle. Iliopsoas, popliteus, obturator internus and tibialis posterior in the lower limbs and subscapularis, spinati, sternocleidomastoid and levator scapulae in the upper girdle were the most spared. Asymmetry and STIR hyperintensities were consistent features. The pattern of muscle involvement was similar to that of FSHD1, and the combined involvement of trapezius, abdominal and hamstring muscles, together with complete sparing of iliopsoas and subscapularis, was detected in 91% of patients. Peculiar differences were identified in a rostro-caudal gradient, a predominant involvement of lower limb muscles compared to the upper girdle, and in the higher percentage of STIR hyperintensities in FSHD2. CONCLUSION: This multicentre study defines the pattern of muscle involvement in FSHD2, providing useful information for diagnostics and clinical trial design. Both similarities and differences between FSHD1 and FSHD2 were detected, which is also relevant to better understand the pathogenic mechanisms underlying the FSHD-related disease spectrum.

Background Limb girdle muscular dystrophy type R1/2A (LGMDR1/ LGMD2A) is a progressive myopathy caused by deficiency of calpain 3, a calcium-dependent cysteine protease of skeletal muscle, and it represents the most frequent type of LGMD worldwide. In the last few years, muscle magnetic resonance imaging (MRI) has been proposed as a tool for identifying patterns of muscular involvement in genetic disorders and as a biomarker of disease progression in muscle diseases. In this study, 57 molecularly confirmed LGMDR1 patients from a European cohort (age range 7-78 years) underwent muscle MRI and a global evaluation of functional status (Gardner-Medwin and Walton score and ability to raise the arms). Results We confirmed a specific pattern of fatty substitution involving predominantly the hip adductors and hamstrings in lower limbs. Spine extensors were more severely affected than spine rotators, in agreement with higher incidence of lordosis than scoliosis in LGMDR1. Hierarchical clustering of lower limb MRI scores showed that involvement of anterior thigh muscles discriminates between classes of disease progression. Severity of muscle fatty substitution was significantly correlated with CAPN3 mutations: in particular, patients with no or one "null" alleles showed a milder involvement, compared to patients with two null alleles (i.e., predicting absence of calpain- 3 protein). Expectedly, fat infiltration scores strongly correlated with functional measures. The "pseudocollagen" sign (central areas of sparing in some muscle) was associated with longer and more severe disease course. Conclusions We conclude that skeletal muscle MRI represents a useful tool in the diagnostic workup and clinical management of LGMDR1.

Background: Congenital muscular dystrophies (CMD) with reduced glycosylation of alpha-dystroglycan (α-DG) are a heterogeneous group of conditions associated with mutations in six genes encoding proven or putative glycosyltransferases. Objectives: The aim of the study was to establish the prevalence of mutations in the six genes in the Italian population and the spectrum of clinical and brain MRI findings. Methods: As part of a multicentric study involving all the tertiary neuromuscular centers in Italy, FKRP , POMT1 , POMT2 , POMGnT1 , fukutin , and LARGE were screened in 81 patients with CMD and α-DG reduction on muscle biopsy (n = 76) or with a phenotype suggestive of α-dystroglycanopathy but in whom a muscle biopsy was not available for α-DG immunostaining (n = 5). Results: Homozygous and compound heterozygous mutations were detected in a total of 43/81 patients (53%), and included seven novel variants. Mutations in POMT1 were the most prevalent in our cohort (21%), followed by POMT2 (11%), POMGnT1 (10%), and FKRP (9%). One patient carried two heterozygous mutations in fukutin and one case harbored a new homozygous variant in LARGE. No clear-cut genotype-phenotype correlation could be observed with each gene, resulting in a wide spectrum of clinical phenotypes. The more severe phenotypes, however, appeared to be consistently associated with mutations predicted to result in a severe disruption of the respective genes. Conclusions: Our data broaden the clinical spectrum associated with mutations in glycosyltransferases and provide data on their prevalence in the Italian population.

Objective Therapeutic perspectives have brought attention to the development of instruments to accurately evaluate the degree of pathology in patients with facioscapulohumeral muscular dystrophy. We aimed to analyze the type and extent of muscle involvement on magnetic resonance imaging (MRI) in a large cohort of patients representative of the broad clinical spectrum of this disease. Methods Pelvic and lower limb muscle MRI scans of 269 symptomatic individuals and 19 nonpenetrant gene carriers were assessed. Comparative analysis of the upper girdle scan in 181 of these subjects was also performed. Results We found a peculiar susceptibility and resistance of particular muscles. Combined involvement of abdominal and hamstring muscles with iliopsoas sparing is common in facioscapulohumeral muscular dystrophy (67% of the patients). Adductor longus and/or rectus femoris, whose involvement can go clinically undetected, are often typically affected in early disease (69% of patients younger than 45 years). The extent of lesions on lower limb MRI showed a high correlation with overall clinical severity. One-fourth of the nonpenetrant gene carriers showed abnormalities on MRI. Hyperintensities on short-tau inversion recovery sequences, markers of active disease, were found in muscles without signs of fatty replacement in 35% of patients, representing early lesions. Interpretation Our large-scale cross-sectional data provide preliminary evidence for the usefulness of MRI in clinical trials, and set the baseline for longitudinal studies. Muscle MRI can also be used for distinguishing facioscapulohumeral muscular dystrophy from other myopathies in selected cases. Finally, our results are consistent with a model that configures facioscapulohumeral muscular dystrophy as a “muscle-by-muscle” disease. Ann Neurol 2016;79:854–864

Clinical trial preparedness in facioscapulohumeral muscular dystrophy: Clinical, tissue, and imaging outcome measures 29–30 May 2015, Rochester, New York Rabi Tawil *, George W. Padberg , Dennis W. Shaw , Silvere M. van der Maarel , Stephen J. Tapscott , The FSHD Workshop Participants 1 a Department of Neurology, University of Rochester Medical Center, Rochester, NY, USA b Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands c Department of Radiology, Seattle Children’s Hospital, Seattle, WA, USA d Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands e Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

In this study, we investigated the sequence of (Structural Maintenance of Chromosomes flexible Hinge Domain containing 1) SMCHD1 gene in a cohort of clinically defined FSHD (facioscapulohumeral muscular dystrophy) patients in order to assess the distribution of SMCHD1 variants, considering the D4Z4 fragment size in terms of repeated units (RUs; short fragment: 1–7 RU, borderline: 8-10RU and normal fragment: >11RU). The analysis of SMCHD1 revealed the presence of 82 variants scattered throughout the introns, exons and 3’untranslated region (3′UTR) of the gene. Among them, 64 were classified as benign polymorphisms and 6 as VUS (variants of uncertain significance). Interestingly, seven pathogenic/likely pathogenic variants were identified in patients carrying a borderline or normal D4Z4 fragment size, namely c.182_183dupGT (p.Q62Vfs*48), c.2129dupC (p.A711Cfs*11), c.3469G>T (p.G1157*), c.5150_5151delAA (p.K1717Rfs*16) and c.1131+2_1131+5delTAAG, c.3010A>T (p.K1004*), c.853G>C (p.G285R). All of them were predicted to disrupt the structure and conformation of SMCHD1, resulting in the loss of GHKL-ATPase and SMC hinge essential domains. These results are consistent with the FSHD symptomatology and the Clinical Severity Score (CSS) of patients. In addition, five variants (c.*1376A>C, rs7238459; c.*1579G>A, rs559994; c.*1397A>G, rs150573037; c.*1631C>T, rs193227855; c.*1889G>C, rs149259359) were identified in the 3′UTR region of SMCHD1, suggesting a possible miRNA-dependent regulatory effect on FSHD-related pathways. The present study highlights the clinical utility of next-generation sequencing (NGS) platforms for the molecular diagnosis of FSHD and the importance of integrating molecular findings and clinical data in order to improve the accuracy of genotype–phenotype correlations.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a well-recognized clinical mitochondrial disease which causes typical symptoms of short stature, failure to thrive, recurrent headaches and vomiting, stroke-like episodes causing hemiplegia or vision loss, epilepsy, hearing loss, and diabetes. Additional manifestations include other endocrinopathies and cardiac involvement. MELAS is typically caused by a mitochondrial DNA point mutation (m.3243A>G) and is therefore maternally inherited. It is not at all unusual to find additional family members who are also affected or may be oligosymptomatic with other clinical phenotypes caused by this pathogenic variant such as maternally inherited diabetes and deafness (MIDD). Recognition of MELAS is critically important to ensure proper long-term surveillance and potential therapies. MELAS leads to overall morbidity and mortality due to recurrent stroke-like episodes, cerebral atrophy, and physical and mental deterioration over time.

Background and objectiveOculopharyngeal muscular dystrophy (OPMD) is a genetic disorder caused by an abnormal expansion of GCN triplets within thePABPN1gene. Previous descriptions have focused on lower limb muscles in small cohorts of patients with OPMD, but larger imaging studies have not been performed. Previous imaging studies have been too small to be able to correlate imaging findings to genetic and clinical data.MethodsWe present cross-sectional, T1-weighted muscle MRI and CT-scan data from 168 patients with genetically confirmed OPMD. We have analysed the pattern of muscle involvement in the disease using hierarchical analysis and presented it as heatmaps. Results of the scans were correlated with genetic and clinical data.ResultsFatty replacement was identified in 96.7% of all symptomatic patients. The tongue, theadductor magnusand thesoleuswere the most commonly affected muscles. Muscle pathology on MRI correlated positively with disease duration and functional impairment.ConclusionsWe have described a pattern that can be considered characteristic of OPMD. An early combination of fat replacement in the tongue,adductor magnusandsoleuscan be helpful for differential diagnosis. The findings suggest the natural history of the disease from a radiological point of view. The information generated by this study is of high diagnostic value and important for clinical trial development.

Introduction: In sporadic inclusion-body myositis (IBM), additional tools are needed to confirm the diagnosis, particularly in clinically atypical or pathologically unproven patients. The aims of this study were to define the pattern of muscle MRI in IBM and to assess its accuracy in differentiating IBM from other myopathies that overlap with it clinically or pathologically. Methods: Blind assessment was done on the scans of 17 definite IBM, 2 possible IBM, and 118 patients with other myopathies. Results: The diagnostic accuracy to detect definite IBM was 95% for the typical pattern (with 100% specificity) and 97% for both typical and consistent patterns (with 97% specificity). Conclusions: Muscle MRI is an accurate tool for diagnostic work-up of suspected IBM patients and may be particularly helpful in patients with early disease or who lack the classical IBM pathology. Muscle Nerve, 2015

Background and purpose Juvenile- or adult-onset forms of severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency manifesting as complicated hereditary spastic paraplegia have rarely been described. Methods Two siblings with mental retardation developed a progressive spastic paraparesis in their late teens. Their diagnostic assessment included extensive neurophysiologic, neuroimaging and metabolic studies. Results Brain magnetic resonance imaging showed occipital white matter alterations, and electromyography documented a mixed polyneuropathy. Severe hyperhomocisteinemia (>150 μmol/L) associated with the characteristic amino acid profile suggested a diagnosis of severe MTHFR deficiency, confirmed by MTHFR direct sequencing. Treatment with betaine and vitamins benefitted patients' symptoms and diagnostic features. Conclusions Severe MTHFR deficiency can be a rare, treatable cause of autosomal recessive complicated hereditary spastic paraplegia. Its screening should be part of the diagnostic flowchart for these disorders.

FSHD forms are associated to D4Z4 DNA hypomethylation on 4qA allelic chromosomes. These epigenetic changes provide a transcriptionally permissive chromatin environment leading to the production of a pathogenic protein DUX4. We have shown that the epigenetic changes in FSHD, can be investigated using methylation assays specific for permissive chromosomes 4qA (PAS+). Here, we report the analysis of a large cohort of individuals consisting of 287 FSHD1, 53 FSHD2 and 165 control subjects. This analysis showed highly significant difference of methylation levels between affected and control subjects further supported by strong correlation with the number of D4Z4 repeats both in FSHD1 and FSHD2. The different haplotype distribution between affected and control groups as well as particular methylation levels of some haplotypes suggested that diagnosis by methylation analysis can be improved by developing haplotype specific assays. To this aim, we developed haplotype specific assays and additional assays that allow precise genotyping. Furthermore, we set up bisulfite methylation assays to discriminate FSHD1 from FSHD2. Finally, assessment of methylation of intra-D4Z4 region and another SMCHD1 target sequence allowed evaluation of the effect of SMCHD1 mutations. Our results can allow the development of an integrate platform of methylation and sequencing assays to accelerate and improve the diagnostic procedure of FSHD.

Recent progresses in the understanding of facioscapulohumeral muscular dystrophy (FSHD) genetics opened the way to the development of targeted therapies. However, knowledge about pathophysiology of muscle damage is still limited and there is increasing need to identify biomarkers of disease activity in the perspective of clinical trial readiness. We analyzed inflammatory mediators in the interstitial fluid of muscles with different MRI signal in FSHD patients, comparing muscles displaying early lesions on short-tau inversion recovery (STIR) sequences with normal ones. Patients with one T1-weighted normal and STIR hyperintense (STIR+) and contralateral T1-weighted and STIR normal (STIR-) lower limb muscle were asked to enter the study. Twelve consecutive patients, five controls, and one non-penetrant gene carrier underwent prolonged muscle microdialysis with high cut-off membranes. Microdialysates were analyzed using xMAP technology with a wide panel for cytokines, chemokines, and growth factors. A small number of inflammatory mediators were dysregulated in STIR+ versus STIR- and control muscles: CXCL13, upregulated in STIR+ muscles compared with controls (p

Sporadic late-onset nemaline myopathy (SLONM) is a rare acquired myopathy characterized by rapid-onset proximal weakness in late adulthood, and the presence of nemaline bodies on muscle biopsy. In recent years, several therapeutic interventions, including immunomodulating agents and autologous stem cell transplantation, have shown variable degrees of efficacy in different patients, but no consensus has been reached to allow an effective tailoring of treatments in this severe disease. We performed a retrospective evaluation of clinical, pathological, laboratory, muscle MRI, and follow-up data of SLONM patients diagnosed in the period 2010–2015 in our neuromuscular center. Six patients (three males and three females) were identified. Average time elapsed from the onset of symptoms to referral to the neuromuscular specialist was 23.7 months. Monoclonal gammopathy was detectable in five patients. Nemaline bodies were detected in all the patients, and their abundance correlated with clinical severity. Signs of cardiac involvement were present in all the patients to different extents. Muscle MRI showed a preferential involvement of neck extensors, paraspinal, gluteal, hamstring and soleus muscles. All patients were treated with prednisone and repeated courses of intravenous immunoglobulins, and a favorable outcome was reached in five patients. Our experience confirms that SLONM is clinically characterized by subacute proximal and axial muscle weakness. Time to referral was relatively long and should be reduced with increasing awareness of the disease. Muscle MRI could be of help as a diagnostic tool to identify this potentially treatable myopathy. Cardiac evaluation should be warranted in all SLONM patients to detect subclinical heart involvement.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motor neurons followed by muscle weakness, paralysis and death. The disease progression is extremely variable among patients, and reliable prognostic markers have not been identified. The aim of the study was to functionally characterize selected genes and microRNAs acting in the skeletal muscle of ALS patients, taking into account the duration and evolution of the disease, in order to obtain information regarding the muscle response to ALS progression. This prospective, longitudinal study enrolled 14 ALS patients and 24 age- and sex-matched healthy controls. Gene expression and histological analysis indicated an increase of MIR208B and MIR499 levels and the predominance of slow fibres, respectively, in the muscles of patients with a slower disease progression. A decreased expression of MIR206 and increased levels of HDAC4, during the progression of the disease were also observed. Taken together, our data suggest that the molecular signalling that regulates re-innervation and muscle regeneration is hampered during the progression of skeletal muscle impairment in ALS. This could provide precious hints towards defining prognostic protocols, and designing novel tailored therapeutic approaches, to improve ALS patients’ care and delay disease progression.

Aims The pathogenesis of myotonic dystrophy type 1 (DM1) and type 2 (DM2) has been related to the aberrant splicing of several genes, including those encoding for ryanodine receptor 1 (RYR1), sarcoplasmatic/endoplasmatic Ca2+-ATPase (SERCA) and α1S subunit of voltage-gated Ca2+ channels (Cav1.1). The aim of this study is to determine whether alterations of these genes are associated with changes in the regulation of intracellular Ca2+ homeostasis and signalling. Methods We analysed the expression of RYR1, SERCA and Cav1.1 and the intracellular Ca2+ handling in cultured myotubes isolated from DM1, DM2 and control muscle biopsies by semiquantitative RT-PCR and confocal Ca2+ imaging respectively. Results (i) The alternative splicing of RYR1, SERCA and Cav1.1 was more severely affected in DM1 than in DM2 myotubes; (ii) DM1 myotubes exhibited higher resting intracellular Ca2+ levels than DM2; (iii) the amplitude of intracellular Ca2+ transients induced by sustained membrane depolarization was higher in DM1 myotubes than in controls, whereas DM2 showed opposite behaviour; and (iv) in both DM myotubes, Ca2+ release from sarcoplasmic reticulum through RYR1 was lower than in controls. Conclusion The aberrant splicing of RYR1, SERCA1 and Cav1.1 may alter intracellular Ca2+ signalling in DM1 and DM2 myotubes. The differing dysregulation of intracellular Ca2+ handling in DM1 and DM2 may explain their distinct sarcolemmal hyperexcitabilities.