Elena Pegoraro

Amyotrophic lateral sclerosis is an incurable neurodegenerative disease involving motor neuron degeneration and metabolic and immune dysfunction. We combined clinical data, cerebrospinal fluid biomarkers and fluorodeoxyglucose positron emission tomography with magnetic resonance imaging to investigate the role of reactive microglia in disease pathogenesis. Patients showed increased cerebrospinal fluid levels of neurofilament light chain and chitinases, along with hypermetabolism in the medulla oblongata. Chitinase levels correlated with brainstem metabolism and clinical severity. These findings suggest a link between microglial activation, brainstem hypermetabolism and disease progression in amyotrophic lateral sclerosis, supporting a central role for neuroinflammation in disease pathogenesis.

Genetic modifiers of Duchenne muscular dystrophy (DMD) that alter disease severity or response to therapy have been reported using natural history or registry data sets of older corticosteroid‐treated patients. We tested associations of genetic modifiers on motor function outcomes in young (4 to < 7 years) steroid naïve clinical trial participants. Participants in clinical trials (VBP15‐002/003 [ n = 48]; VBP15‐004 [ n = 121]; DNA available for n = 110) were genotyped for eight published genetic modifier loci, and associations of genotypes with baseline motor function defined via an age‐adjusted linear model. Corticosteroid drug response was modeled by genotype‐stratified placebo vs. steroid treatment at 12‐ and 24‐weeks posttreatment (mixed model for repeated measures). Serum proteome profiles (SomaScan) were stratified by modifier genotype, and modifier‐associated biomarkers mapped to muscle cell types using snSeq datasets. Two loci showed association with baseline motor outcomes ( LTBP4 , DYNLT5 ). LTBP4 genotype (rs1131620) was associated with baseline (pretreatment) motor function for all five motor tests studied (time to stand from supine velocity, time to run/walk 10 m velocity, time to climb 4 stairs velocity, 6‐min walk distance, and NorthStar Ambulatory Assessment). DYNLT5 genotype (rs1060575) was associated with time to stand from supine velocity, time to climb 4 stairs velocity, and time to run/walk 10 m velocity. Stratification of baseline proteome profiles by LTBP4 genotype and mapping of genotype‐associated serum proteins to specific cell types in muscle (snRNAseq) suggested that IL‐23, IL‐6, and IL‐17D interacting pathways in muscle capillaries are contributors to disease progression. In contrast, the DYNLT5 genotype was associated with proteosome and chaperonin pathways. Trial Registration: clinicaltrials.gov identifier: NCT02760264, NCT02760277, NCT03439670.

Piezo2 channels are mechanosensors expressed in dorsal root ganglia and Merkel cells, and involved in proprioception and touch sensation. PIEZO2 pathogenic variants cause rare autosomal dominant and recessive disorders. Dominant forms include distal arthrogryposis type 3, 5, and Marden-Walker syndrome, whereas the recessive form corresponds to distal arthrogryposis with impaired proprioception and touch (DAIPT). Given the rarity of these conditions, additional reports are essential to refine their phenotypic characterization. We conducted a multicenter, retrospective study to identify patients with genetically confirmed PIEZO2-related disorders. We collected clinical data through a standardized assessment. Previously published cases were included when more detailed phenotypic information was available. We identified 44 individuals with a clinical diagnosis of PIEZO2-related disorders. Among them, we selected 42 patients with a confirmed molecular diagnosis. Sixteen of them (10 males and 6 females; median age at diagnosis: 8 years; range 0.9-14) carried biallelic pathogenic PIEZO2 variants. They typically presented with neonatal hypotonia and respiratory distress with feeding difficulties. Distal arthrogryposis was associated with a variable multisystemic involvement, including the musculoskeletal system with scoliosis, hip dislocation and laryngomalacia. Twenty-five patients (11 males and 14 females; median age at diagnosis: 7 years; range 0.3-39 years) carried monoallelic variants, exhibiting a broad phenotypic spectrum. An additional prenatal diagnosis (at 23 weeks of gestation) was reported among heterozygous cases. In our cohort, ten previously unpublished causative variants were identified. A systematic assessment of skeletal muscle involvement revealed a severe myofibrillar disarray in one case. This study provides a comprehensive and systematic phenotypic characterization of patients across the spectrum of PIEZO2-related disorders. Our findings support a clear clinical distinction between recessive and dominant forms. The clinical manifestations extend beyond the peripheral nervous system and the Merkel cells, suggesting a broader PIEZO2 tissue expression during early developmental stages. Dominant PIEZO2-related disorders are heterogeneous, ranging from mild to severe forms. Our study also suggests that many patients exhibit a respiratory involvement, emphasizing the need for regular respiratory follow-up in the long-term clinical management of both dominant and recessive forms.

Reducing body myopathy (RBM) is a rare inherited disorder, due to pathogenic variant in the FHL1 gene. The disease is characterized by protein aggregates in myocytes strongly stained with menadione-nitro blue tetrazolium with α-glycerophosphate and immunoreactive for FHL1 as myopathologic hallmarks. RBM is an X-linked dominant disorder, with the most severe cases occurring in hemizygous sporadic male patients, who usually present with early onset and rapid progression. This study aimed to comprehensively analyse a cohort of FHL1 female patients presenting with a severe and rapidly progressive phenotype clinically mimicking an inflammatory idiopathic myopathy. This is retrospective study of a cohort of female patients with RBM harboring pathogenic variants in FHL1 gene from France and Italy. Data regarding clinical onset and progression, myopathologic features, muscle imaging, and genetic testing have been collected retrospectively and analyzed from medical records. We present 5 young girls and women with FHL1 pathogenic variants presenting a mean age at onset of 13 years. All patients showed an asymmetric pattern of muscle weakness evolving in bilateral proximo-distal involvement and restrictive respiratory syndrome, rapidly leading to severe tetraparesis and loss of ambulation after a mean period of 6.2 years in 4 of them. Serum CK level at onset was slightly elevated with a mean value of 539 UI/L. Muscle MRI revealed severe asymmetric proximo-distal involvement with STIR positive sequences. Muscle biopsies showed reducing bodies and prominent autophagic material accumulation. FHL1-related reducing body myopathy can present with a rapidly progressive muscle weakness mimicking an inflammatory myopathy also in female patient. Muscle biopsy is an useful tool in the contest of rapidly progressive myopathy to distinguish between genetic myopathies and treatable inflammatory myopathy.

We present a comprehensive molecular and histopathological characterization of nine patients with mitochondrial myopathy, predominantly manifesting progressive external ophthalmoplegia (PEO), associated with heteroplasmic variants in mitochondrial tRNA genes (mt-tRNA). Among the ten variants identified, four were novel and previously unreported in MITOMAP. Using laser capture microdissection and deep next-generation sequencing, we quantified heteroplasmy at the single-muscle-fibre level, demonstrating that cytochrome c oxidase (COX)–deficient fibres consistently reached near-homoplasmic mutant loads, whereas COX-positive fibres remained heteroplasmic with lower variant fractions. These findings firmly support the pathogenic role of all variants. Furthermore, digital droplet PCR revealed an increased mitochondrial DNA (mtDNA) content in COX-deficient fibres, indicating compensatory mitochondrial biogenesis. Of particular note, one patient harboured two novel heteroplasmic variants, m.10009G > A and m.15961G > A, for which long-read sequencing identified mitogenomes carrying both variants also in cis, suggesting the occurrence of mtDNA recombination in human tissue. By applying refined American College of Medical Genetics and Genomics (ACMG) criteria specific for mt-tRNA, we reclassified several variants as pathogenic or likely pathogenic, including three previously deemed of uncertain significance. Overall, our integrative approach—combining single-fibre molecular dissection, mtDNA quantification, and long-read sequencing—broadens the mutational spectrum of pathogenic mt-tRNA variants, highlights the diagnostic value of single-fibre analyses in confirming pathogenicity, and provides new insights into mitochondrial genome dynamics and compensatory responses in mitochondrial disease.

Objective: The aim of our study was to establish the prevalence of adverse events in a real‐world setting in boys living with Duchenne muscular dystrophy (DMD) treated with givinostat as part of an Expanded Access Program (EAP) in Italy. Methods: The cohort included 90 ambulant boys, with age when treatment started between 6 and 23 years (mean 10.1 years, SD: 3.2 years) and with a follow up between 6.0 and 14.6 months (mean 10.6 months; SD 2.6 months). Results: Platelets count decrease and triglyceride levels increase were the most common adverse events, followed by diarrhea. A dose reduction was needed in 38 of the 90 boys following thrombocytopenia ( n = 34), diarrhea ( n = 2), and hypertriglyceridemia ( n = 4), with two of the boys presenting both thrombocytopenia and hypertriglyceridemia. Eleven of the 38 boys with initial dose reduction (10 with thrombocytopenia and 1 with hypertriglyceridemia) required an additional dose reduction for persistence of values outside the threshold, with one of them discontinuing treatment because of persistent hypertriglyceridemia even with the lowest dose. Interpretation: Our results confirm the safety profile observed in the pivotal study providing further evidence of the management of the drug in a real‐world setting. In our experience, treatment with givinostat was well managed by maintaining a strict monitoring. This was facilitated by allowing families to perform blood tests in local labs, reducing the stress and burden of frequent visits to the hospital.

Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an extremely rare autosomal recessive disease caused by variants in the thymidine phosphorylase gene ( TYMP ), primarily characterized by severe gastrointestinal and neurological symptoms. The complete phenotype of MNGIE has not been linked to any gene other than TYMP . Methods: We describe two identical twins who exhibited delayed psychomotor development, infantile bilateral cataract, congenital demyelinating polyneuropathy, and severe progressive gastrointestinal dysmotility with recurrent pseudo‐obstruction episodes, along with diffuse supratentorial leukoencephalopathy that mainly overlaps with classic TYMP ‐related MNGIE. During the course of the disease, one patient developed Wernicke encephalopathy, triggered by chronic malnutrition related to recurrent gastrointestinal pseudo‐obstruction. This patient later suffered from a catastrophic stroke‐like episode, resulting in massive cerebral edema and brain death at the age of 38. Results: Next‐generation sequencing (NGS) using a custom‐targeted mitochondrial gene panel identified two compound heterozygous variants in the POLG gene: the paternal variants p.Thr251Ile and p.Pro587Leu, occurring in cis , and the novel maternal variant p.Arg853Gly. Quantification of mtDNA by real‐time PCR on skeletal muscle DNA detected significant depletion, but no multiple deletions were detected with mtDNA analysis by long‐range PCR and Nanopore sequencing. Conclusions: These cases showed a very distinctive POLG phenotype, with some MNGIE‐like features, expanding the clinical and genetic spectrum of the POLG ‐related diseases. Additionally, they highlighted the importance of monitoring for thiamine deficiency in mitochondrial patients with severe gastrointestinal dysmotility who experience sudden clinical deterioration.

Background Becker muscular dystrophy (BMD) is a rare X-linked neuromuscular disorder predominantly affecting males. While respiratory function has been characterised in small cohorts over limited time frames, comprehensive long-term longitudinal data remain lacking, as do established care guidelines to inform respiratory surveillance in BMD. Methods In this retrospective analysis, we present a large longitudinal analysis of respiratory function in patients with BMD, based on 1360 spirometry measurements from a single-centre cohort of 152 patients. Analysed assessments spanned paediatric to adult ages (3.4–86.3 years), with a mean follow-up duration of 11 years per patient. Linear mixed-effects models were used to study longitudinal changes in respiratory function. Results Respiratory decline in BMD appears gradual and variable, typically not affecting children (Conclusion These findings provide important evidence to inform clinical management, supporting the recommendation of individualised respiratory monitoring strategies and contributing to the design and interpretation of clinical trials in BMD.

Twinkle, encoded by the TWNK gene, is a mitochondrial DNA helicase that unwinds the double helix of DNA during replication, playing a pivotal role in mitochondrial function. Twinkle-related disorders encompass a variety of genetic disorders characterized by mitochondrial dysfunction. Although several phenotypes have been described, the full clinical and molecular spectrum remains poorly defined. The aim of this study was to characterize the phenotypic and genotypic variability among multinational patients diagnosed with Twinkle-related disorders. A retrospective cohort study was conducted in patients with Twinkle-related disorders at several specialized centers in Italy, France, Germany, Spain, Denmark, Hungary, and the United States, establishing the Twinkle-Related Disorders International Consortium for Trial Readiness (TReDIC). Data were collected from medical records, including clinical features, age at onset, disease progression, and results from genetic testing. Phenotypic categories included infantile-onset cerebellar ataxia, parkinsonism, primary mitochondrial myopathy (PMM), multisystem involvement, asymptomatic carriers, undetermined phenotypes, and other phenotypes. All patients' diagnoses were confirmed by genetic analysis, and their genetic variants were noted. Outcomes included prevalence of phenotypes, symptom chronology, and mutational patterns. The study included a total of 189 patients (116 female), with a mean age at symptom onset of 40.3 years. At the time of analysis, 70.4% were alive. PMM was the predominant syndrome (85.2%), and most common features were progressive external ophthalmoplegia (84.7%) and skeletal myopathy (55.6%), followed by hearing loss (17.5%) and psychiatric symptoms (15.3%). Most patients (76.8%) presented with neuromuscular symptoms, with fewer showing CNS (19.6%) or multiorgan (3.6%) features at onset; by more than 8 years from onset, these proportions shifted to 54.4%, 23.3%, and 23.3%, respectively. A total of 73 TWNK variants (16 novel) were found, mostly missense, clustered in functionally critical regions. This large multinational cohort analysis advances our understanding of Twinkle-related disorders by identifying mutational hotspots with clinical relevance and illustrating the broad phenotypic spectrum and progression patterns. In the context of such rare diseases, the formation of international collaborations, such as TReDIC, can enhance our understanding and support the design of upcoming clinical trials.