Pathogenic DPAGT1 variants in limb-girdle congenital myasthenic syndrome (LG-CMS) associated with tubular aggregates and ORAI1 hypoglycosylation.

AIMS: Limb-girdle congenital myasthenic syndrome (LG-CMS) is a genetically heterogeneous disorder characterized by muscle weakness and fatigability. The LG-CMS gene DPAGT1 codes for an essential enzyme of the glycosylation pathway, a posttranslational modification mechanism shaping the structure and function of proteins. In DPAGT1-related LG-CMS, reduced glycosylation of the acetylcholine receptor (AChR) reduces its localization at the neuromuscular junction (NMJ), and results in diminished neuromuscular transmission. LG-CMS patients also show tubular aggregates on muscle biopsy, but the origin and potential contribution of the aggregates to disease development are not understood. Here, we describe two LG-CMS patients with the aim of providing a molecular diagnosis and to shed light on the pathways implicated in tubular aggregate formation. METHODS: Following clinical examination of the patients, we performed next-generation sequencing (NGS) to identify the genetic causes, analysed the biopsies at the histological and ultrastructural levels, investigated the composition of the tubular aggregates, and performed experiments on protein glycosylation. RESULTS: We identified novel pathogenic DPAGT1 variants in both patients, and pyridostigmine treatment quantitatively improved muscle force and function. The tubular aggregates contained proteins of the sarcoplasmic reticulum (SR) and structurally conformed to the aggregates observed in tubular aggregate myopathy (TAM). TAM arises from overactivation of the plasma membrane calcium channel ORAI1, and functional studies on muscle extracts from our LG-CMS patients evidenced abnormal ORAI1 glycosylation. CONCLUSIONS: We expand the genetic variant spectrum of LG-CMS and provide a genotype/phenotype correlation for pathogenic DPAGT1 variants. The discovery of ORAI1 hypoglycosylation in our patients highlights a physiopathological link between LG-CMS and TAM.

ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.

Exome sequencing in undiagnosed congenital myopathy reveals new genes and refines genes-phenotypes correlations.

BACKGROUND: Congenital myopathies are severe genetic diseases with a strong impact on patient autonomy and often on survival. A large number of patients do not have a genetic diagnosis, precluding genetic counseling and appropriate clinical management. Our objective was to find novel pathogenic variants and genes associated with congenital myopathies and to decrease diagnostic odysseys and dead-end. METHODS: To identify pathogenic variants and genes implicated in congenital myopathies, we established and conducted the MYOCAPTURE project from 2009 to 2018 to perform exome sequencing in a large cohort of 310 families partially excluded for the main known genes. RESULTS: Pathogenic variants were identified in 156 families (50%), among which 123 families (40%) had a conclusive diagnosis. Only 44 (36%) of the resolved cases were linked to a known myopathy gene with the corresponding phenotype, while 55 (44%) were linked to pathogenic variants in a known myopathy gene with atypical signs, highlighting that most genetic diagnosis could not be anticipated based on clinical-histological assessments in this cohort. An important phenotypic and genetic heterogeneity was observed for the different genes and for the different congenital myopathy subtypes, respectively. In addition, we identified 14 new myopathy genes not previously associated with muscle diseases (20% of all diagnosed cases) that we previously reported in the literature, revealing novel pathomechanisms and potential therapeutic targets. CONCLUSIONS: Overall, this approach illustrates the importance of massive parallel gene sequencing as a comprehensive tool for establishing a molecular diagnosis for families with congenital myopathies. It also emphasizes the contribution of clinical data, histological findings on muscle biopsies, and the availability of DNA samples from additional family members to the diagnostic success rate. This study facilitated and accelerated the genetic diagnosis of congenital myopathies, improved health care for several patients, and opened novel perspectives for either repurposing of existing molecules or the development of novel treatments.

A Homozygous NDUFS6 Variant Associated with Neuropathy and Optic Atrophy.

Background: The NADH dehydrogenase [ubiquinone] iron-sulfur protein 6 (NDUFS6) gene encodes for an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). Bi-allelic NDUFS6 variants have been linked with a severe disorder mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS). Objective: Here, we identified a homozygous variant (c.309 + 5 G > A) in NDUFS6 in one male patient with axonal neuropathy accompanied by loss of small fibers in skin biopsy and further complicated by optic atrophy and borderline intellectual disability. Methods: To address the pathogenicity of the variant, biochemical studies (mtDNA copy number quantification, ELISA, Proteomic profiling) of patient-derived leukocytes were performed. Results: The analyses revealed loss of NDUFS6 protein associated with a decrease of three further mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4 and NDUFV1). Mitochondrial copy number is not altered in leukocytes and the mitochondrial biomarker GDF15 is not significantly changed in serum. Conclusions: Hence, our combined clinical and biochemical data strengthen the concept of NDUFS6 being causative for a very rare form of axonal neuropathy associated with optic atrophy and borderline intellectual disability, and thus expand (i) the molecular genetic landscape of neuropathies and (ii) the clinical spectrum of NDUFS6-associated phenotypes.

Lessons Learned From Clinical Studies in Centronuclear Myopathies: The Patient Perspective-A Qualitative Study.

BACKGROUND: Since 2014, several clinical studies focusing on centronuclear myopathies have been conducted, including a prospective natural history study, a gene transfer clinical trial and a clinical trial using an antisense oligonucleotide. Dedicated patient organizations have played an important role in this process. The experience of members of these organizations, either as a study participant, parent or as a patient organization member communicating with the sponsors are potentially very informative for future trial design. METHODS: We investigated the burden of and the lessons learned from the first natural history studies and clinical trials from a patient perspective using a qualitative approach. We arranged 4 focus groups with a total of 37 participants from 3 large international patient organizations: ZNM-ZusammenStark!, the Myotubular Trust, and the MTM-CNM Family Connection. 4 themes, based on a systematic literature search were discussed: Expectations and preparation, Clinical study participation, Communication and Recommendations for future clinical trials. The focus group recordings were transcribed, anonymized, and uploaded to Atlas-ti version 8.1 software. The data were analyzed using a thematic content analysis. RESULTS: Overall, participants were realistic in their expectations, hoping for small improvements of function and quality of life. The realization that trial participation does not equate to a treatment was challenging. Participating in a clinical study had a huge impact on many aspects of daily life, both for patients and their immediate families. First-hand insights into the burden of the design and its possible effect on performance were provided, resulting in numerous compelling recommendations for future clinical studies. Furthermore, participants stressed the importance of clear communication, which was considered to be especially vital in cases of severe adverse events. Finally, while patients were understanding of the importance of adhering to the regulations of good clinical practice, they indicated that they would strongly appreciate a greater understanding and/or acknowledgment of the patient perspective and a reflection of this perspective in future clinical trial design. CONCLUSION: The acknowledgment and inclusion of patients' perspectives and efficient and effective communication is expected to improve patient recruitment and retention in future clinical studies, as well as more accurate assessment of the patient performance related to suitable planning of the study visits.

The experience of clinical study and trial participation in rare diseases: A scoping review of centronuclear myopathy and other neuromuscular disorders.

The design of a clinical trial for a rare disease can be challenging. An optimal study design is required to effectively study the clinical outcomes for possible therapies for these types of disorders. Understanding the study participants' experiences as well as barriers and facilitators of participation are important to optimize future research and to inform clinical trial management. Centronuclear myopathies (CNMs) including X-linked myotubular myopathy (XLMTM) are a group of rare congenital myopathies for which there is no cure currently. Since 2014, a number of natural history studies and clinical trials have been conducted in CNMs. Two trials have been prematurely terminated because of severe adverse events. Since no research has been conducted regarding trial experience in CNM, we performed a scoping literature research on clinical trial experience of patients with neuromuscular disorders in general. The most common barriers to trial participation of patients comprise concerns about potential harmful effects, opportunity loss and the expected burden on daily life. The most common facilitators were an expected benefit on the disease course, altruism and collateral benefit. While several results are in line with trial experiences of other types of patients, for example oncological patients, distinctions can be made for patients with CNM and other neuromuscular disorders. However, the limited availability of relevant literature suggests that future (qualitative) research should focus on trial experiences in CNM patients.

Individuals and Families Affected by RYR1-Related Diseases - The Patient/Caregiver Perspective.

Background and objective: Pathogenic variants of RYR1, the gene encoding the principal sarcoplasmic reticulum calcium release channel (RyR1) with a crucial role in excitation-contraction coupling, are among the most common genetic causes of non-dystrophic neuromuscular disorders. We recently conducted a questionnaire study focusing on functional impairments, fatigue, and quality of life (QoL) in patients with RYR1-related diseases (RYR1-RD) throughout the recognized disease spectrum. In this previous questionnaire study the medical perspective was taken, reflective of a study protocol designed by neurologists and psychologists. With this present study we wanted to specifically address the patient perspective. Methods: Together with affected individuals, family members, and advocates concerned with RYR1-RD, we developed an online patient survey that was completed by 227 patients or their parents/other caretakers (143 females and 84 males, 0-85 years). We invited 12 individuals, representing most of the patient group based on age, sex, race, and type and severity of diagnosis, to share their personal experiences on living with a RYR1-RD during an international workshop in July 2022. Data were analyzed through a mixed-methods approach, employing both a quantitative analysis of the survey results and a qualitative analysis of the testimonials. Results: Data obtained from the combined quantitative and qualitative analyses provide important insights on six topics: 1) Diagnosis; 2) Symptoms and impact of the condition; 3) Physical activity; 4) Treatment; 5) Clinical research and studies; and 6) Expectations. Conclusions: Together, this study provides a unique patient perspective on the RYR1-RD spectrum, associated disease impact, suitable physical activities and expectations of future treatments and trials, and thus, offers an essential contribution to future research.