Charcot-Marie-Tooth type 2A in vivo models: Current updates.

Charcot-Marie-Tooth type 2A (CMT2A) is an inherited sensorimotor neuropathy associated with mutations within the Mitofusin 2 (MFN2) gene. These mutations impair normal mitochondrial functioning via different mechanisms, disturbing the equilibrium between mitochondrial fusion and fission, of mitophagy and mitochondrial axonal transport. Although CMT2A disease causes a significant disability, no resolutive treatment for CMT2A patients to date. In this context, reliable experimental models are essential to precisely dissect the molecular mechanisms of disease and to devise effective therapeutic strategies. The most commonly used models are either in vitro or in vivo, and among the latter murine models are by far the most versatile and popular. Here, we critically revised the most relevant literature focused on the experimental models, providing an update on the mammalian models of CMT2A developed to date. We highlighted the different phenotypic, histopathological and molecular characteristics, and their use in translational studies for bringing potential therapies from the bench to the bedside. In addition, we discussed limitations of these models and perspectives for future improvement.

Six-minute walk test as outcome measure of fatigability in adults with spinal muscular atrophy treated with nusinersen.

INTRODUCTION/AIMS: Fatigue (subjective perception) and fatigability (objective motor performance worsening) are relevant aspects of disability in individuals with spinal muscular atrophy (SMA). The effect of nusinersen on fatigability in SMA patients has been investigated with conflicting results. We aimed to evaluate this in adult with SMA3. METHODS: We conducted a multicenter retrospective cohort study, including adult ambulant patients with SMA3, data available on 6-minute walk test (6MWT) and Hammersmith Functional Motor Scale-Expanded (HFMSE) at baseline and at least at 6 months of treatment with nusinersen. We investigated fatigability, estimated as 10% or higher decrease in walked distance between the first and sixth minute of the 6MWT, at baseline and over the 14-month follow-up. RESULTS: Forty-eight patients (56% females) were included. The 6MWT improved after 6, 10, and 14 months of treatment (p < 0.05). Of the 27 patients who completed the entire follow-up, 37% improved (6MWT distance increase ≥30 m), 48.2% remained stable, and 14.8% worsened (6MWT distance decline ≥30 m). Fatigability was found at baseline in 26/38 (68%) patients and confirmed at subsequent time points (p < 0.05) without any significant change over the treatment period. There was no correlation between fatigability and SMN2 copy number, sex, age at disease onset, age at baseline, nor with 6MWT total distance and baseline HFMSE score. DISCUSSION: Fatigability was detected at baseline in approximately 2/3 of SMA3 walker patients, without any correlation with clinical features, included motor performance. No effect on fatigability was observed during the 14-month treatment period with nusinersen.

Verbal learning in frontal patients: area 9 is critical for employing semantic strategies.

INTRODUCTION: Learning is a long-term memory process heavily influenced by the control processes implemented by working memory, including recognition of semantic properties of items by which subjects generate a semantic structure of engrams. AIM: The aim of this study is to investigate the verbal learning strategies of patients affected by a tumor in the left frontal lobe to highlight the role of area 9. METHOD: Ten patients with frontal low-grade gliomas and ten healthy control subjects, matched for age, sex and education, were recruited and then evaluated with a two-part verbal learning test: multi-trial word list learning in free recall, and multi-trial word list learning preceded by an explicit semantic strategy cue. Frontal patients were divided into two groups: those either with frontal lesions involving or sparing area 9. RESULTS: In comparison to healthy control subjects, frontal patients with lesions involving area 9 memorized fewer words and displayed difficulty in using semantic strategies. When the strategy was suggested by the examiner, their performance improved, but to a lesser extent than the healthy control. Conversely, frontal patients with lesions sparing area 9 showed similar results to healthy control subjects. CONCLUSION: The results suggested that, while the identification of the categorical criterion requires the integrity of the entire dorsolateral prefrontal area, only area 9, and not the surrounding areas, could be responsible for the effective use of semantic strategies in learning tasks.

Association between ZASP/LDB3 Pro26Ser and Inclusion Body Myopathy.

Inclusion body myositis (IBM) is a slowly progressive disorder belonging to the idiopathic inflammatory myopathies, and it represents the most common adult-onset acquired myopathy. The main clinical features include proximal or distal muscular asymmetric weakness, with major involvement of long finger flexors and knee extensors. The main histological findings are the presence of fiber infiltrations, rimmed vacuoles, and amyloid inclusions. The etiopathogenesis is a challenge because both environmental and genetic factors are implicated in muscle degeneration and a distinction has been made previously between sporadic and hereditary forms. Here, we describe an Italian patient affected with a hereditary form of IBM with onset in his mid-forties. Next-generation sequencing analysis disclosed a heterozygous mutation c.76C>T (p.Pro26Ser) in the PDZ motif of the LDB3/ZASP gene, a mutation already described in a family with a late-onset myopathy and highly heterogenous degree of skeletal muscle weakness. In the proband's muscle biopsy, the expression of ZASP, myotilin, and desmin were increased. In our family, in addition to the earlier age of onset, the clinical picture is even more peculiar given the evidence, in one of the affected family members, of complete ophthalmoplegia in the vertical gaze. These findings help extend our knowledge of the clinical and genetic background associated with inclusion body myopathic disorders.

The Profiling of 179 miRNA Expression in Serum from Limb Girdle Muscular Dystrophy Patients and Healthy Controls.

Limb girdle muscular dystrophies (LGMDs) are a group of genetically inherited neuromuscular diseases with a very variable clinical presentation and overlapping traits. Over the last few years there has been an increasing interest in the use of non-invasive circulating biomarkers to monitor disease progression and to evaluate the efficacy of therapeutic approaches. Our aim was to identify the miRNA signature with potential value for LGMD patient screening and stratification. Using miRCURY LNA miRNA qPCR Serum/Plasma Panel, we analyzed 179 miRNAs from 16 patients, divided in four pools based on their genetic diagnosis, and from healthy controls. The miRNAs analysis showed a total of 107 dysregulated miRNAs in LGMD patients when compared to the healthy controls. After filtering via skeletal tissue expression and gene/pathways target analysis, the number of dysregulated miRNAs drastically reduced. Six selected miRNAs-let-7f-5p (in LGMDR1), miR-20a-5p (in LGMDR2), miR-130b-5p, miR-378a-5p (both in LGMDR3), miR-376c-3p and miR-382-5p (both in LGMDR4)-whose expression was significantly lower compared to controls in the different LGMD pools, were further investigated. The bioinformatic analysis of the target genes in each selected miRNA revealed ECM-receptor interaction and TGF-beta signaling as the most involved pathways. The correlation analysis showed a good correlation of let-7f-5p with fibrosis and with the cross sectional area of type I and type II fibers, while miR-130b-5p showed a good correlation with the age of onset of the disease. The receiver operating characteristic curves showed how single miRNAs were able to discriminate a specific group of LGMD patients and how the combination of six miRNAs was able to discriminate LGMD patients from controls.

Sleep and sleep-related breathing disorders in patients with spinal muscular atrophy: a changing perspective from novel treatments?

Spinal Muscular Atrophy (SMA) is an inherited neuromuscular disorder characterized by progressive muscle weakness and atrophy, resulting from the degeneration of motor neurons in the spinal cord. A critical aspect of SMA is its impact on respiratory function. As the disease progresses, respiratory muscles, in particular intercostal muscles, become increasingly affected, leading to breathing difficulties and respiratory failure. Without intervention, many children with SMA type 1 die from respiratory failure before their second year of life. While assisted ventilation has improved survival, it often results in ventilator dependence. The development of new SMN-augmenting therapies has renewed optimism, but their long-term impact on respiratory function is uncertain, and non-invasive respiratory support remains an important part of SMA management. Despite the importance of respiratory support in SMA, knowledge regarding sleep disorders in this population is limited. This review aims to synthesize existing literature on sleep and sleep-related breathing disorders in patients with SMA, with a focus on SMA type 1. We summarize evidence of sleep-disordered breathing and respiratory failure in SMA, as well as outcomes and survival benefits associated with non-invasive or invasive ventilation with or without pharmacological therapies. We also discuss current knowledge regarding the effects of novel disease-modifying therapies for SMA on respiratory function and sleep. In conclusion, optimal care for children with SMA requires a multidisciplinary approach that includes neurology and respiratory specialists. This review highlights the importance of monitoring sleep and respiratory function in SMA, as well as the potential benefits and challenges associated with assisted ventilation combined with new therapies.

Case report: Dihydropyridine receptor (CACNA1S) congenital myopathy, a novel phenotype with early onset periodic paralysis.

Introduction: CACNA1S related congenital myopathy is an emerging recently described entity. In this report we describe 2 sisters with mutations in the CACNA1S gene and the novel phenotype of congenital myopathy and infantile onset episodic weakness. Clinical description: Both sisters had neonatal onset hypotonia, muscle weakness, and delayed walking. Episodic weakness started in infancy and continued thereafter, provoked mostly by cold exposure. Muscle imaging revealed fat replacement of gluteus maximus muscles. Next generation sequencing found the missense p.Cys944Tyr variant and the novel splicing variant c.3526-2A>G in CACNA1S. Minigene assay revealed the splicing variant caused skipping of exon 28 from the transcript, potentially affecting protein folding and/or voltage dependent activation. Conclusion: This novel phenotype supports the notion that there are age related differences in the clinical expression of CACNA1S gene mutations. This expands our understanding of mutations located in regions of the CACNA1S outside the highly conserved S4 segment, where most mutations thus far have been identified.

Case report: A novel ACTA1 variant in a patient with nemaline rods and increased glycogen deposition.

Background: Congenital myopathies are a group of heterogeneous inherited disorders, mainly characterized by early-onset hypotonia and muscle weakness. The spectrum of clinical phenotype can be highly variable, going from very mild to severe presentations. The course also varies broadly resulting in a fatal outcome in the most severe cases but can either be benign or lead to an amelioration even in severe presentations. Muscle biopsy analysis is crucial for the identification of pathognomonic morphological features, such as core areas, nemaline bodies or rods, nuclear centralizations and congenital type 1 fibers disproportion. However, multiple abnormalities in the same muscle can be observed, making more complex the myopathological scenario. Case presentation: Here, we describe an Italian newborn presenting with severe hypotonia, respiratory insufficiency, inability to suck and swallow, requiring mechanical ventilation and gastrostomy feeding. Muscle biopsy analyzed by light microscopy showed the presence of vacuoles filled with glycogen, suggesting a metabolic myopathy, but also fuchsinophilic inclusions. Ultrastructural studies confirmed the presence of normally structured glycogen, and the presence of minirods, directing the diagnostic hypothesis toward a nemaline myopathy. An expanded Next Generation Sequencing analysis targeting congenital myopathies genes revealed the presence of a novel heterozygous c.965 T > A p. (Leu322Gln) variant in the ACTA1 gene, which encodes the skeletal muscle alpha-actin. Conclusion: Our case expands the repertoire of molecular and pathological features observed in actinopathies. We highlight the value of ultrastructural examination to investigate the abnormalities detected at the histological level. We also emphasized the use of expanded gene panels in the molecular analysis of neuromuscular patients, especially for those ones presenting multiple bioptic alterations.

Early spinal muscular atrophy treatment following newborn screening: A 20-month review of the first Italian regional experience.

OBJECTIVES: Mandatory newborn screening (NBS) for spinal muscular atrophy (SMA) was implemented for the first time in Italy at the end of 2021, allowing the identification and treatment of patients at an asymptomatic stage. METHODS: DNA samples extracted from dried blood spot (DBS) from newborns in Apulia region were analysed for SMA screening by using a real-time PCR-based assay. Infants harbouring homozygous deletion of SMN1 exon 7 confirmed by diagnostic molecular tests underwent clinical and neurophysiological assessment and received a timely treatment. RESULTS: Over the first 20 months since regional NBS introduction, four out of 42,492 (0.009%) screened children were found to carry a homozygous deletion in the exon 7 of SMN1 gene, with an annual incidence of 1:10,623. No false negatives were present. Median age at diagnosis was 7 days and median age at treatment was 20.5 days. Three of them had two copies of SMN2 and received gene therapy, while the one with three SMN2 copies was treated with nusinersen. All but one were asymptomatic at birth, showed no clinical signs of disease after a maximum follow-up of 16 months and reached motor milestones appropriate with their age. The minimum interval between diagnosis and the treatment initiation was 9 days. INTERPRETATION: The timely administration of disease-modifying therapies prevented presymptomatic subjects to develop disease symptoms. Mandatory NBS for SMA should be implemented on a national scale.

Shaping the Neurovascular Unit Exploiting Human Brain Organoids.

Brain organoids, three-dimensional cell structures derived from pluripotent stem cells, closely mimic key aspects of the human brain in vitro, providing a powerful tool for studying neurodevelopment and disease. The neuroectodermal induction protocol employed for brain organoid generation primarily gives rise to the neural cellular component but lacks the vital vascular system, which is crucial for the brain functions by regulating differentiation, migration, and circuit formation, as well as delivering oxygen and nutrients. Many neurological diseases are caused by dysfunctions of cerebral microcirculation, making vascularization of human brain organoids an important tool for pathogenetic and translational research. Experimentally, the creation of vascularized brain organoids has primarily focused on the fusion of vascular and brain organoids, on organoid transplantation in vivo, and on the use of microfluidic devices to replicate the intricate microenvironment of the human brain in vitro. This review summarizes these efforts and highlights the importance of studying the neurovascular unit in a forward-looking perspective of leveraging their use for understanding and treating neurological disorders.

Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Lafora Disease: A Case Report and Evolving Treatment Advancements.

Lafora disease is a rare genetic disorder characterized by a disruption in glycogen metabolism. It manifests as progressive myoclonus epilepsy and cognitive decline during adolescence. Pathognomonic is the presence of abnormal glycogen aggregates that, over time, produce large inclusions (Lafora bodies) in various tissues. This study aims to describe the clinical and histopathological aspects of a novel Lafora disease patient, and to provide an update on the therapeutical advancements for this disorder. A 20-year-old Libyan boy presented with generalized tonic-clonic seizures, sporadic muscular jerks, eyelid spasms, and mental impairment. Electroencephalography showed multiple discharges across both brain hemispheres. Brain magnetic resonance imaging was unremarkable. Muscle biopsy showed increased lipid content and a very mild increase of intermyofibrillar glycogen, without the polyglucosan accumulation typically observed in Lafora bodies. Despite undergoing three lines of antiepileptic treatment, the patient's condition showed minimal to no improvement. We identified the homozygous variant c.137G>A, p.(Cys46Tyr), in the EPM2B/NHLRC1 gene, confirming the diagnosis of Lafora disease. To our knowledge, the presence of lipid aggregates without Lafora bodies is atypical. Lafora disease should be considered during the differential diagnosis of progressive, myoclonic, and refractory epilepsies in both children and young adults, especially when accompanied by cognitive decline. Although there are no effective therapies yet, the development of promising new strategies prompts the need for an early and accurate diagnosis.

Case report: A novel patient presenting TRIM32-related limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy autosomal recessive 8 (LGMDR8) is a rare clinical manifestation caused by the presence of biallelic variants in the TRIM32 gene. We present the clinical, molecular, histopathological, and muscle magnetic resonance findings of a novel 63-years-old LGMDR8 patient of Italian origins, who went undiagnosed for 24 years. Clinical exome sequencing identified two TRIM32 missense variants, c.1181G > A p.(Arg394His) and c.1781G > A p.(Ser594Asp), located in the NHL1 and NHL4 structural domains, respectively, of the TRIM32 protein. We conducted a literature review of the clinical and instrumental data associated to the so far known 26 TRIM32 variants, carried biallelically by 53 LGMDR8 patients reported to date in 20 papers. Our proband's variants were previously identified only in three independent LGMDR8 patients in homozygosis, therefore our case is the first in literature to be described as compound heterozygous for such variants. Our report also provides additional data in support of their pathogenicity, since p.(Arg394His) is currently classified as a variant of uncertain significance, while p.(Ser594Asp) as likely pathogenic. Taken together, these findings might be useful to improve both the genetic counseling and the diagnostic accuracy of this rare neuromuscular condition.

Prominent muscle involvement in a familial form of mitochondrial disease due to a COA8 variant.

Isolated mitochondrial respiratory chain Complex IV (Cytochrome c Oxidase or COX) deficiency is the second most frequent isolated respiratory chain defect. Causative mutations are mainly identified in structural COX subunits or in proteins involved in the maturation and assembly of the COX holocomplex. We describe an Italian familial case of mitochondrial myopathy due to a variant in the COX assembly factor 8 gene (COA8). Patient 1 is a 52-year-old woman who presented generalized epilepsy and retinitis pigmentosa at 10 years of age. From her early adulthood she complained about cramps and myalgia after exercise, and bilateral hearing loss emerged. Last neurological examination (52 years of age) showed bilateral ptosis, muscle weakness, peripheral neuropathy, mild dysarthria and dysphonia, cognitive impairment. Muscle biopsy had shown the presence of ragged-red fibers. Patient 2 (Patient 1's sister) is a 53-year-old woman presenting fatigability, myalgia, and hearing loss. Neurological examination showed ptosis and muscle weakness. Muscle biopsy displayed a diffuse reduction of COX activity staining and ragged-red fibers. Both sisters presented secondary amenorrhea. After ruling out mtDNA mutations, Whole Exome Sequencing analysis identified the novel homozygous COA8 defect c.170_173dupGACC, p.(Pro59fs) in the probands. Loss-of-function COA8 mutations have been associated with cavitating leukoencephalopathy with COX deficiency in 9 reported individuals. Disease course shows an early-onset rapid clinical deterioration, affecting both cognitive and motor functions over months, followed by stabilization and slow improvement over several years. Our findings expand the clinical spectrum of COA8-related disease. We confirm the benign course of this rare disorder, highlighting its (intrafamilial) clinical variability.