Effects of Triheptanoin on Mitochondrial Respiration and Glycolysis in Cultured Fibroblasts from Neutral Lipid Storage Disease Type M (NLSD-M) Patients.

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.

Using Cluster Analysis to Overcome the Limits of Traditional Phenotype-Genotype Correlations: The Example of RYR1-Related Myopathies.

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype-phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype-phenotype correlations, we found clustering to overcome the limits of the "single-dimension" paradigm traditionally used to describe genotype-phenotype relationships.

Post-COVID simultaneous onset of Graves' disease and ocular myasthenia gravis in a patient with a complex ocular motility impairment.

A 74-years-old man experienced severe diplopia one month after recovery from an uncomplicated SARS-CoV-2 infection. Neurological examination was normal whereas ophthalmological examination showed bilateral exophthalmos with a complex ocular motility disorder characterized by a pseudo-internuclear ophthalmoplegia after fatigue associated to impairment of elevation and infraduction. Antibodies against TSH and acetylcholine receptors were positive; subsequent hormonal tests, ultrasonography of thyroid gland, single fiber electromyography and orbit MRI confirmed the diagnosis of concomitant Graves Disease (GD) and Myasthenia Gravis (MG). The coexistence between MG and GD is not rare but simultaneous onset after viral infection is very unsual. The complex ocular disorder simulated a deficit of the oculomotor nerve nuclei, and on clinical examination it posed some problems in the diagnosis. We suggest that recent SARS-COV-2 infection may have triggered a complex autoimmune response.

Real-life experience with inotersen in hereditary transthyretin amyloidosis with late-onset phenotype: Data from an early-access program in Italy.

BACKGROUND AND PURPOSE: Hereditary transthyretin (TTR) amyloidosis (ATTRv) is a dominantly inherited, adult-onset, progressive, and fatal disease caused by mutations in the transthyretin gene. Therapeutic agents approved for this disease include the TTR stabilizer tafamidis and the gene-silencing drugs patisiran and inotersen. Inotersen is an antisense oligonucleotide that suppresses the hepatic production of transthyretin. After European Medical Agency approval in 2018, an early-access program was opened in Italy, and in this article, we present the long-term outcome of a cohort of Italian ATTRv patients who received inotersen within this program. METHODS: This is a multicenter, observational, retrospective study of patients affected by ATTRv that started inotersen during the early-access program. The primary end point was safety. Secondary end points included change from baseline in familial amyloid polyneuropathy (FAP) stage, Polyneuropathy Disability, Neuropathy Impairment Scale, Compound Autonomic Dysfunction Test, Norfolk Quality of Life-Diabetic Neuropathy, troponin, N-terminal pro-brain natriuretic peptide, interventricular septum thickness, and body mass index. RESULTS: In total, 23 patients were enrolled. No patient permanently discontinued the treatment because of thrombocytopenia, and no cases of severe thrombocytopenia were observed. Five patients discontinued the treatment permanently because of voluntary withdrawal (two patients), renal failure after infective pyelonephritis, not related to inotersen, drug-related hypotension, and amyloid-negative crescentic glomerulonephritis. In seven patients, dosing frequency was reduced to every 2 weeks due to recurrent thrombocytopenia. Considering the FAP stage, only two patients worsened, whereas the other 21 patients remained stable until the last follow-up available. CONCLUSIONS: The long-term safety profile of inotersen is favorable. Neurologic disease severity at baseline is the main factor associated with progression.

Muscle magnetic resonance imaging in myotonic dystrophy type 1 (DM1): Refining muscle involvement and implications for clinical trials.

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.

ATTRv in Lazio-Italy: A High-Prevalence Region in a Non-Endemic Country.

Hereditary transthyretin amyloidosis (ATTRv, v for variant) prevalence in Italy, a non-endemic region, has been established by ATTRv amyloidosis Italian Registry. However, values of prevalence were extremely heterogeneous, considering different regions. To properly establish the prevalence of the disease in the Lazio region, a survey was sent to university regional hospitals and to main regional hospitals, in order to collect all affected patients regularly followed. We identified 100 ATTRv patients and, considering a Lazio population of 5.8/million, we estimated a ATTRv prevalence of 17.2/million. The ATTRv amyloidosis Italian Registry reported a prevalence of 8.0/million in Lazio, while our survey showed a value of double this. Our survey documented a high-prevalence for a non-endemic country. The increased awareness of the disease among general practitioners and medical specialists is a fundamental step to reduce the diagnostic delay and start an effective treatment of this disease.

Novel ACTA1 mutation causes late-presenting nemaline myopathy with unusual dark cores.

ACTA1 gene encodes the skeletal muscle alpha-actin, the core of thin filaments of the sarcomere. ACTA1 mutations are responsible of several muscle disorders including nemaline, cores, actin aggregate myopathies and fiber-type disproportion. We report clinical, muscle imaging, histopatological and genetic data of an Italian family carrying a novel ACTA1 mutation. All affected members showed a late-presenting, diffuse muscle weakness with sternocleidomastoideus and temporalis atrophy. Mild dysmorphic features were also detected. The most affected muscles by muscle MRI were rectus abdominis, gluteus minimus, vastus intermedius and both gastrocnemii. Muscle biopsy showed the presence of nemaline bodies with several unusual dark areas at Gomori Trichrome, corresponding to unstructured cores with abundant electrodense material by electron microscopy. The molecular analysis revealed missense variant c.148G>A; p.(Gly50Ser) in the exon 3 of ACTA1, segregating with affected members in the family. We performed a functional essay of fibre contractility showing a higher pCa50 (a measure of the calcium sensitivity of force) of type 1 fibers compared to control subjects' type 1 muscle fibers. Our findings expand the clinico-pathological spectrum of ACTA1-related congenital myopathies and the genetic spectrum of core-rod myopathies.

Aminopiridines in the treatment of multiple sclerosis and other neurological disorders.

Symptomatic treatment has a great relevance for the management of patients with neurologic diseases, since it reduces disease burden and improves quality of life. Aminopyridines (APs) are a group of potassium (K+) channel blocking agents that exert their activity both at central nervous system level and on neuromuscular junction. This review describes the use of APs for the symptomatic treatment of neurological conditions. We will describe trials leading to the approval of the extended-release 4-aminopyridine for MS and evidence in support of the use in other neurological diseases.

Estimating the impact of COVID-19 pandemic on services provided by Italian Neuromuscular Centers: an Italian Association of Myology survey of the acute phase.

INTRODUCTION: Since February 2020, the outbreak of COVID-19 in Italy has forced the health care system to undergo profound rearrangements in its services and facilities, especially in the worst-hit areas in Northern Italy. In this setting, inpatient and outpatient services had to rethink and reorganize their activities to meet the needs of patients during the "lockdown". The Italian Association of Myology developed a survey to estimate the impact of these changes on patients affected by neuromuscular disorders and on specialized neuromuscular centers during the acute phase of COVID-19 pandemic. METHODS: We developed an electronic survey that was sent to neuromuscular centers affiliated with the Italian Association of Myology, assessing changes in pharmacological therapies provision, outpatient clinical and instrumental services, support services (physiotherapy, nursing care, psychological support) and clinical trials. RESULTS: 40% of surveyed neuromuscular centers reported a reduction in outpatient visit and examinations (44.5% of centers in Northern regions; 25% of centers in Central regions; 50% of centers in Southern regions). Twenty-two% of centers postponed in-hospital administration of therapies for neuromuscular diseases (23.4% in Northern regions; 13.0% in Central regions; 20% in Southern regions). Diagnostic and support services (physiotherapy, nursing care, psychological support) were suspended in 57% of centers (66/43/44% in Northern, Central and Southern centers respectively) Overall, the most affected services were rehabilitative services and on-site outpatient visits, which were suspended in 93% of centers. Strategies adopted by neuromuscular centers to overcome these changes included maintaining urgent on-site visits, addressing patients to available services and promoting remote contact and telemedicine. CONCLUSIONS: Overall, COVID-19 pandemic resulted in a significant disruption of clinical and support services for patients with neuromuscular diseases. Despite the efforts to provide telemedicine consults to patients, this option could be promoted and improved further. A close collaboration between the different neuromuscular centers and service providers as well as further implementation of telehealth platforms are necessary to ensure quality care to NMD patients in the near future and in case of recurrent pandemic waves.

Muscle involvement in myasthenia gravis: Expanding the clinical spectrum of Myasthenia-Myositis association from a large cohort of patients.

Myastenia-Inflammatory Myopathy (MG-IM) association has been described in less than 50 cases, as isolated reports or in few case series. In most cases, MG and IM onset occur simultaneously even if the overlapping clinical manifestations could lead to delay the diagnosis in the early stage of disease. In these cases, thymic pathology is present in more than 50% of cases. Pathological findings can be consistent of polymyositis (63%), dermatomyositis (25%) or granulomatosis (12%). Accurate clinical manifestations and severity of IM in MG, including muscle specific antibodies (MSA) and muscle MRI, have not been systematically investigated and focal or mild subclinical myositis have not been reported. We observed that focal myositis or asymptomatic CK elevation can also occur in MG. In this review we have also retrospectively re-analyzed the clinical, serological, pathological and muscle imaging data from 13 patients with MG- IM from our cohort of 441 MG patients (2,9%). Clinical onset occurred simultaneously in 10/13 patients, whereas in 2 patients the IM appeared later in MG disease course (range 10-14 years) and conversely in 1 patient MG symptoms occurred later in IM disease course (4 years). Median age at disease onset was 51 year (range 24-73 years) regardless of clinical onset (MG or IM). Median clinical follow-up was 88 months (range 31-237 months). IM was suspected by CK elevation in all patients (ranging 800-3000 UI/L at first detection) and non-fatigable muscle weakness unresponsive to acetylcholinesterase inhibitors. All the patients presented mild to moderate MG symptoms. Three main categories of muscle involvement, sometimes overlapping, were recognizable: distal, proximal and subclinical myositits, leading to three main clinical groups (A,B,C) and two overlapping subgroups (A/B and B/C). Thymus pathology was present in 10/13 patients. Anti-AChR was detected in al all patients associated with anti-Titin and -RyR1 in those patients with thymoma. No MSA, nor MAA antibodies were detected. Muscle biopsy confirmed IM in all patients. In conclusion we redefined the clinical spectrum of muscle involvement in MG-IM association, which represent a continuum among 3 main clinical groups: distal, proximal and subclinical muscle involvement. Minimal muscle involvement and focal myositis could be underestimated among myasthenic patients and early aggressive immunotherapy could be required in focal group.

Expanding the spectrum of genes responsible for hereditary motor neuropathies.

BACKGROUND: Inherited peripheral neuropathies (IPNs) represent a broad group of genetically and clinically heterogeneous disorders, including axonal Charcot-Marie-Tooth type 2 (CMT2) and hereditary motor neuropathy (HMN). Approximately 60%-70% of cases with HMN/CMT2 still remain without a genetic diagnosis. Interestingly, mutations in HMN/CMT2 genes may also be responsible for motor neuron disorders or other neuromuscular diseases, suggesting a broad phenotypic spectrum of clinically and genetically related conditions. Thus, it is of paramount importance to identify novel causative variants in HMN/CMT2 patients to better predict clinical outcome and progression. METHODS: We designed a collaborative study for the identification of variants responsible for HMN/CMT2. We collected 15 HMN/CMT2 families with evidence for autosomal recessive inheritance, who had tested negative for mutations in 94 known IPN genes, who underwent whole-exome sequencing (WES) analyses. Candidate genes identified by WES were sequenced in an additional cohort of 167 familial or sporadic HMN/CMT2 patients using next-generation sequencing (NGS) panel analysis. RESULTS: Bioinformatic analyses led to the identification of novel or very rare variants in genes, which have not been previously associated with HMN/CMT2 (ARHGEF28, KBTBD13, AGRN and GNE); in genes previously associated with HMN/CMT2 but in combination with different clinical phenotypes (VRK1 and PNKP), and in the SIGMAR1 gene, which has been linked to HMN/CMT2 in only a few cases. These findings were further validated by Sanger sequencing, segregation analyses and functional studies. CONCLUSIONS: These results demonstrate the broad spectrum of clinical phenotypes that can be associated with a specific disease gene, as well as the complexity of the pathogenesis of neuromuscular disorders.

Metabolic lipid muscle disorders: biomarkers and treatment.

Lipid storage myopathies (LSMs) are metabolic disorders of the utilization of fat in muscles due to several different defects. In this review, a molecular update of LSMs is presented and recent attempts of finding treatment options are discussed. The main topics discussed are: primary carnitine deficiency, riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency, neutral lipid storage disorders and carnitine palmitoyl transferase deficiency. The most frequent presentations and genetic abnormalities are summarized. We present their diagnosis utilizing biomedical and morphological biomarkers and possible therapeutic interventions. The treatment of these metabolic disorders is a subject of active translational research but appears, in some cases, still elusive.

A novel family with axonal Charcot-Marie-Tooth disease caused by a mutation in the EGR2 gene.

EGR2 (Early Growth Response 2) is one of the most important transcription factors involved in myelination in the peripheral nervous system. EGR2 mutations typically cause different forms of demyelinating neuropathy, that is, Charcot-Marie-Tooth type 1D (CMT1D), Dejerine-Sottas Syndrome (DSS), and Congenital Hypomyelinating Neuropathy (CHN). However, the EGR2 gene has been recently associated with an axonal phenotype (CMT2) in a large CMT family. Here, we report another CMT family exhibiting an axonal phenotype associated with a missense change (c.1235A>G, p.E412G) in the EGR2 gene. Neurological evaluation of five affected members of the family showed a classical CMT phenotype including distal muscle atrophy and weakness, absence of deep tendon reflexes, pes cavus, and scoliosis. Electrophysiological examination was consistent with a motor-sensory axonal neuropathy. Sural nerve biopsy performed in one patient showed a loss of myelinated and unmyelinated nerve fibers without de-remyelinating signs and onion bulbs. This study confirms the phenotypical heterogeneity of EGR2-related neuropathy, indicating a role for EGR2 in primary axonal degeneration.

Lipid Myopathies.

Disorders of lipid metabolism affect several tissues, including skeletal and cardiac muscle tissues. Lipid myopathies (LM) are rare multi-systemic diseases, which most often are due to genetic defects. Clinically, LM can have acute or chronic clinical presentation. Disease onset can occur in all ages, from early stages of life to late-adult onset, showing with a wide spectrum of clinical symptoms. Muscular involvement can be fluctuant or stable and can manifest as fatigue, exercise intolerance and muscular weakness. Muscular atrophy is rarely present. Acute muscular exacerbations, resulting in rhabdomyolysis crisis are triggered by several factors. Several classifications of lipid myopathies have been proposed, based on clinical involvement, biochemical defect or histopathological findings. Herein, we propose a full revision of all the main clinical entities of lipid metabolism disorders with a muscle involvement, also including some those disorders of fatty acid oxidation (FAO) with muscular symptoms not included among previous lipid myopathies classifications.

Muscle MRI in neutral lipid storage disease (NLSD).

Neutral lipid storage disease (NLSD) is a rare inherited disorder of lipid metabolism resulting in lipid droplets accumulation in different tissues. Skeletal muscle could be affected in both two different form of disease: NLSD with myopathy (NLSD-M) and NLSD with ichthyosis (NLSD-I). We present the muscle imaging data of 12 patients from the Italian Network for NLSD: ten patients presenting NLSD-M and two patients with NLSD-I. In NLSD-M gluteus minimus, semimembranosus, soleus and gastrocnemius medialis in the lower limbs and infraspinatus in the upper limbs were the most affected muscles. Gracilis, sartorius, subscapularis, pectoralis, triceps brachii and sternocleidomastoid were spared. Muscle involvement was not homogenous and characteristic "patchy" replacement was observed in at least one muscle in all the patients. Half of the patients showed one or more STIR positive muscles. In both NLSD-I cases muscle involvement was not observed by T1-TSE sequences, but one of them showed positive STIR images in more than one muscle in the leg. Our data provides evidence that muscle imaging can identify characteristic alterations in NLSD-M, characterized by a specific pattern of muscle involvement with "patchy" areas of fatty replacement. Larger cohorts are needed to assess if a distinct pattern of muscle involvement exists also for NLSD-I.

Neutral Lipid Storage Diseases: clinical/genetic features and natural history in a large cohort of Italian patients.

BACKGROUND: A small number of patients affected by Neutral Lipid Storage Diseases (NLSDs: NLSD type M with Myopathy and NLSD type I with Ichthyosis) have been described in various ethnic groups worldwide. However, relatively little is known about the progression and phenotypic variability of the disease in large specific populations. The aim of our study was to assess the natural history, disability and genotype-phenotype correlations in Italian patients with NLSDs. Twenty-one patients who satisfied the criteria for NLSDs were enrolled in a retrospective cross-sectional study to evaluate the genetic aspects, clinical signs at onset, disability progression and comorbidities associated with this group of diseases. RESULTS: During the clinical follow-up (range: 2-44 years, median: 17.8 years), two patients (9.5%, both with NLSD-I) died of hepatic failure, and a further five (24%) lost their ability to walk or needed help when walking after a mean period of 30.6 years of disease. None of the patients required mechanical ventilation. No patient required a heart transplant, one patient with NLSD-M was implanted with a cardioverter defibrillator for severe arrhythmias. CONCLUSION: The genotype/phenotype correlation analysis in our population showed that the same gene mutations were associated with a varying clinical onset and course. This study highlights peculiar aspects of Italian NLSD patients that differ from those observed in Japanese patients, who were found to be affected by a marked hypertrophic cardiopathy. Owing to the varying phenotypic expression of the same mutations, it is conceivable that some additional genetic or epigenetic factors affect the symptoms and progression in this group of diseases.

An Age-Standardized Prevalence Estimate and a Sex and Age Distribution of Myotonic Dystrophy Types 1 and 2 in the Rome Province, Italy.

BACKGROUND: Prevalence estimates for the 2 forms of myotonic dystrophy types 1 and 2 (DM1 and DM2) are not exhaustive or non-available. Our aim was to estimate the minimum prevalence of DM1 and DM2 in Italy in the Rome province, applying standards of descriptive epidemiology. METHODS: All patients with a molecular diagnosis of DM1/DM2 and residents in the Rome province in 2013 have been enrolled, and the age-standardized prevalence has been calculated, assuming a Poisson distribution and adjusting for age. RESULTS: We identified 395 DM1 patients: the age-standardized prevalence for total, females and males was 9.65, 8.35 and 11.07/100,000, respectively. The mean age of subjects differed considerably according to CTG repeat length (p = 0.001). Forty DM2 patients were identified. The age-standardized prevalence for total, females and males was 0.99, 1.07 and 0.90/100,000, respectively. The mean age was 57.05. CONCLUSIONS: We estimated for the first time the age-standardized prevalence and the sex and age distribution of DM1 and DM2 in a general population. A higher prevalence of males in DM1 and females in DM2 and a higher mean age of DM2 patients (+8 years) were ascertained. Prevalence of DM2 was 10% that of DM1. These prevalence values are probably lower than mutational rates due to the incomplete penetrance of DM1 mutations and to the clinical elusiveness of DM2. Our findings will be useful in designing cohort studies and for developing a disease registry.