Diagnosing rare muscle diseases can be challenging due to their genetic heterogeneity. The French National Network for Rare Neuromuscular Diseases (FILNEMUS) has previously established a pioneering nationwide strategy based on gene lists organized in 13 phenotype-specific gene panels. We now revise these lists and add recently described genes. Using data collected from all FILNEMUS diagnostic laboratories, we also establish a "Major Muscle Genes" panel that includes genes responsible for the most frequent genetic muscle diseases. The updated diagnostic strategy of the FILNEMUS network will help reduce the turn-around time for genetic results and facilitate rapid access to the French national genome sequencing platforms.

High-throughput genome and exome sequencing have uncovered numerous intronic variants in disease genes, yet predicting their impact on pre-mRNA splicing is still challenging. While deep learning tools have improved variant prioritization, their ability to detect atypical regulatory mechanisms remains unknown. We combined predictions from multiple complementary splicing prediction tools with functional RNA studies to evaluate the splicing consequences of intronic variants identified in patients with neurodegenerative diseases. Nine intronic variants with uncertain impact on splicing were selected, including six deep-intronic variants, two non-canonical splice-site variants, and one canonical splice-site variant associated with an atypical phenotype. Three variants were predicted to disrupt a donor splice site, five to create a cryptic donor splice site, and one was located close to a putative acceptor site. Transcript analysis in blood revealed splicing abnormalities in eight of the nine cases. All algorithms failed at least once: deep learning tools missed specific donor site losses or cryptic splice-site activation events, while motif-based approaches failed to predict some donor site activation or generated false-positive predictions. The only variant not directly located in a splice site was predicted to activate a deep intronic splicing enhancer. Motif-based tools suggested the creation of an SRSF2 protein-dependent enhancer. Functional studies based on minigene assays support a role for SRSF2 in promoting pseudoexon inclusion. These findings highlight the complementarity of splicing prediction tools and the need to integrate them into diagnostic pipelines, with transcript-level confirmation remaining critical for accurate pathogenicity assessment.

Titinopathies are complex neuromuscular disorders with multiple phenotypes. The gene's size, comprising 364 exons, as well as the protein's size of 3.8 MDa and its extensive network of protein interactors, are key factors underlying this complexity. Various phenotypes characterize titinopathies, and this study focuses on two of them: arthrogryposis and myofibrillar myopathies. The protein deregulations associated with these two phenotypes remain unknown or have been minimally explored; however, understanding these consequences is essential for better characterizing the pathophysiological aspects of these titinopathies.The objective was to analyze protein deregulations in two cohorts of French patients with titinopathies exhibiting the arthrogryposis and myofibrillar myopathy phenotypes, and to compare them with control individuals. Protein extracts were obtained from muscle biopsies of patients, and changes in protein levels within these two groups were analyzed by mass spectrometry. The results indicate specific deregulations in each group. The networks analyzed revealed deregulation of proteins involved in fibrosis mechanisms or in the actomyosin complex for the arthrogryposis phenotype. Regulation of the muscle contraction system through deregulation of proteins involved in the cytoskeleton is impacted in patients with myofibrillar myopathy. The proteins that are quantitatively abnormal in these two groups also provide insights into the major signaling networks disrupted in titinopathies. These findings will contribute to a more precise characterization of titinopathies, enabling the identification of phenotype-specific biomarkers and potentially guiding the search for targeted therapies for these neuromuscular disorders.

Background The interpretation of non-coding variants (NCVs) from genome sequencing represents a major bottleneck in the diagnosis of rare diseases. Existing variant effect predictors (VEPs) show variable performance across different genomic contexts, and a lack of region-specific clinical guidance hinders accurate variant prioritization. This study aimed to rigorously benchmark state-of-the-art VEPs to define region-specific thresholds and to develop MobiDeep, a novel meta-score designed to improve NCV prioritization. Methods We curated a high-confidence dataset of 448 pathogenic NCVs (ClinVar, HGMD, literature) and 38,146 presumed benign NCVs. Critically, variants affecting splicing were excluded to focus on strictly regulatory mechanisms. We benchmarked the performance of ReMM, CADD, GPN-MSA, Cactus241way, and phyloP, both globally and stratified by genomic region (e.g., 5'UTR, 3'UTR). Subsequently, we developed MobiDeep, a neural network integrating these five scores, optimized using Optuna and validated on an independent holdout set of pathogenic NCVs. Results Benchmarking confirmed that no single tool is universally optimal, with performance varying significantly by genomic context; while ReMM excelled in non-coding exons (AUROC = 0.987), GPN-MSA demonstrated superior performance for 3'UTRs (AUROC = 0.901). We established data-driven clinical thresholds, identifying an optimal global cutoff of 10.37 for CADD v1.7, validating previous works of CADD ≥ 10 for regulatory variants and 0.80 for ReMM. Building on these insights, MobiDeep significantly outperformed all individual predictors on an independent test set, achieving an AUROC of 0.973 and an AUPRC of 0.888. In large-scale simulations mimicking a diagnostic, MobiDeep prioritized causal variants effectively, placing 52.0% and 75% within the top 5 and top 20 ranks respectively. Furthermore, the model correctly prioritized all Clinvar pathogenic variants in the recently discovered RNU4-2 non-coding gene. Conclusions Our findings confirm that individual predictors and uniform thresholds are insufficient for interpreting the diverse landscape of non-coding variants. We demonstrate that region-specific calibration is essential for accurate prioritization.. Our meta-score MobiDeep improves classification performance compared to existing tools. This meta-score serves as a robust filter to streamline the identification of high-confidence variants, thereby facilitating focused manual review and subsequent biological validation in diagnostic settings.

Tubulinopathies encompass a spectrum of disorders resulting from variants in genes encoding α- and β-tubulins, the key components of microtubules. While previous studies have linked de novo or dominantly inherited TUBA4A missense variants to neurodegenerative phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, spastic ataxia, and recently, an isolated congenital myopathy, the full phenotypic and genotypic spectrum of TUBA4A-related disorders remains incompletely characterised. In this multi-centre study, we identified one previously reported and 12 novel TUBA4A missense variants in 31 individuals from 19 unrelated families. Remarkably, individuals in 17 families presented with a myopathy without any CNS involvement or history of such disease. In the remaining two families, we observed probands with cerebellar ataxia and epilepsy accompanying proximal and axial muscle weakness along with protein aggregation. The coexistence of neuromuscular and neurodegenerative features with protein aggregation defines a multisystem proteinopathy. These two families thus establish the first association between TUBA4A and multisystem proteinopathy. Our cohort exhibited diverse genotypes and inheritance patterns: four families demonstrated autosomal dominant transmission through heterozygous variants in TUBA4A, three probands had recessive inheritance due to homozygous variants, while the respective heterozygous carriers were asymptomatic; five probands carried de novo variants, and nine probands with heterozygous variants were classified as sporadic cases. Clinical phenotypes ranged from mild to severe myopathy, predominantly affecting the axial and paraspinal muscles. We observed a range of disease onset, from congenital to late adulthood. Creatine kinase levels were variable, ranging from normal to highly elevated. Cardiac function remained preserved across the cohort. Muscle biopsies showed heterogenous myopathic changes, including myofibre size variation, nemaline bodies, core-like regions, and internal nuclei. Immunohistochemical analysis revealed protein accumulations positive for TDP-43 (n=2), p62 (n=5), and TUBA4A (n=6). Complementary in silico and in vitro investigations suggested that the identified TUBA4A variants cause significant protein abnormalities and may differentially impact microtubule dynamics. Correlation analyses integrating clinical severity, variant location, and mechanistic readouts further demonstrated that domain specificity within TUBA4A influences both the pattern of muscle involvement and the extent of microtubule disruption. Our findings establish myo-tubulinopathies as distinct clinical entities, encompassing both primary myopathies and multisystem proteinopathies with muscle involvement. This study broadens the phenotypic and genotypic spectrum of TUBA4A-related disorders beyond autosomal dominant or de novo mechanisms and neurodegenerative presentations. These results underscore the importance of considering TUBA4A variants in the differential diagnosis of axial myopathies and multisystem proteinopathies, regardless of central nervous system (CNS) involvement.

Females heterozygous for a variant in the DMD gene may develop dilated cardiomyopathy; however, progression to severe heart failure is uncommon, and heart transplantation in this population has rarely been reported. We describe nine females with dystrophinopathy and severe cardiac involvement: five underwent heart transplantation, one received a left ventricular assist device, one died from end-stage heart failure, and two remain on medical therapy. Overall, heart transplantation was well tolerated and effective, with 8-11 years of follow-up available for four patients. Immunostaining of explanted hearts from two patients showed mosaic dystrophin expression without evidence of skewed X-chromosome inactivation, suggesting additional genetic or environmental modifiers. These findings highlight the importance of regular cardiac surveillance in all females heterozygous for a DMD variant-particularly during pregnancy and the postpartum period and in families with severe cardiac involvement-and support heart transplantation as a viable option in end-stage heart failure.

MobiCT is a bioinformatics pipeline designed to detect ultra-low-frequency variants present in cell-free DNA samples using unique molecular identifier (UMI). The pipeline is composed of three main stages: (i) UMI deduplication, (ii) alignment to reference genome, and (iii) variant calling. It has been validated using a range of cancer patients and control samples, demonstrating sensitivity, precision, and F1-score around 90%. Implemented in Nextflow, following the nf-core guidelines, MobiCT ensures reliability and reproducibility, making it suitable for both research and clinical applications.

CACNA1S gene variants are associated with congenital myopathies (CMyo) with triad dysfunction (triadopathies), malignant hyperthermia susceptibility, hypokalemic periodic paralysis and thyrotoxic periodic paralysis. Here, we generated three iPSC lines derived from patients with CMyo linked to both autosomal dominant and recessive CACNA1S variants (CACNA1S-CMyo). The three lines displayed typical iPSC morphology, uniform expression of markers of the undifferentiated state, trilineage differentiation potential and normal karyotypes. As CACNA1S-CMyo are ultra-rare disorders, these lines enable a better in vitro characterization of CACNA1S pathophysiology and can be used to test different treatment approaches.

Tubulinopathies encompass a wide spectrum of disorders resulting from variants in genes encoding α- and β-tubulins, the key components of microtubules. While previous studies have linked de novo or dominantly inherited TUBA4A missense variants to neurodegenerative phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, hereditary spastic ataxia, and more recently, an isolated report of congenital myopathy, the full phenotypic and genotypic spectrum of TUBA4A-related disorders remains incompletely characterised. In this multi-centre study, we identified 13 novel TUBA4A missense variants in 31 individuals from 19 unrelated families. Remarkably, affected individuals in 17 families presented with a primary axial myopathy without any identified CNS involvement or history of such disease. In the remaining two families, we observed probands with cerebellar ataxia and epilepsy accompanying proximal and axial muscle weakness, establishing the first documented association between TUBA4A variants and multisystem proteinopathy. Our cohort exhibited diverse genotypes and associated inheritance patterns: four families demonstrated autosomal dominant transmission through heterozygous variants in TUBA4A, three probands had homozygous TUBA4A variants, where the biallelic genotype was found to be associated with the disease, and the heterozygous carriers were asymptomatic; five probands carried de novo variants, and nine probands with heterozygous TUBA4A variants were classified as "isolated-sporadic cases" where parental samples were unavailable. Clinical phenotypes ranged from mild to severe myopathy, predominantly affecting the axial and paraspinal muscles. We observed a range of disease onset, from congenital to late adulthood. Creatine kinase levels were also variable, ranging from normal to highly elevated. Cardiac function remained preserved across the cohort. Muscle biopsies revealed a range of pathologies, including myofibre size variation, myofibre atrophy, nemaline bodies, core-like regions, internal nuclei, and endomysial fibrosis. Immunohistochemical staining showed evidence of proteinopathy, with autophagic features and TUBA4A accumulation in patient myofibres. Complementary in silico and in vitro investigations suggested that the identified TUBA4A substitutions cause significant protein abnormalities and may differentially impact microtubule dynamics. Our findings establish myo-tubulinopathies as distinct clinical entities, encompassing both primary myopathies and multisystem proteinopathies with muscle involvement. This study broadens the phenotypic and genotypic spectrum of TUBA4A-related disorders beyond autosomal dominant or de novo mechanisms and neurodegenerative presentations. These results underscore the importance of considering TUBA4A variants in the differential diagnosis of axial myopathies and multisystem proteinopathies, regardless of central nervous system (CNS) involvement.

PURPOSE:In Friedreich ataxia (FRDA) the size of the smaller GAA expansion is a major determinant of disease severity; interruption motifs were identified after the discovery of the pathogenic expansions; however, their impact is only recently investigated. METHODS:164 patients with FRDA with biallelic expansions and 15 patients without FRDA were analyzed for interruption(s) number, position, and motif. Expansion size and age at onset of ataxia (AAO) were determined for patients with FRDA. RESULTS:Three groups of patients with FRDA were identified by the simultaneous analysis of the precise distance ("depth") between the interruptions (mostly nontriplet) and the 3' end of the expansion (P < .001), the smaller expansion size (P < .001), and AAO (P < .001). Classical FRDA corresponds to absence of interruption or interruption depth < 8 repeats, with AAO often <15 years (area under the curve [AUC] = 0.90; 95% CI, 0.84-0.96); LOFA to interruption depth of 8 to 18 repeats (AUC = 0.97; 95% CI, 0.94-1), with AAO 15 to 34 years (AUC = 1; 95% CI, 1-1); and vLOFA to interruption depth > 18 (AUC = 0.97; 95% CI, 0.92-1), with AAO > 34 years. Multiple (>5) triplet interruptions hamper further expansion. CONCLUSION:This study provides the molecular basis for a novel classification of FRDA that should be recommended for correct diagnosis.

OBJECTIVE:To report the incidental detection of maternal somatic mosaicism during the development of exclusion-based non-invasive prenatal diagnosis for monogenic disorders (NIPD-MD) initially indicated for apparently de novo pathogenic or likely pathogenic variants. METHOD:A droplet digital PCR (ddPCR)-based exclusion NIPD_MD assay was developed for four couples, each with a prior pregnancy affected by a rare autosomal dominant or X-linked condition due to a de novo pathogenic or likely pathogenic variant. Assays were designed to detect fetal-specific variants in maternal plasma, with validation performed on parental and proband samples. RESULTS:In four cases, maternal somatic mosaicism (3%-9%) among 70 personalized NIPD_MD (5.7%) was identified during assay validation, rendering NIPD_MD infeasible due to interference from maternal alleles. Each couple was informed of the elevated recurrence risk. Depending on their preferences, invasive prenatal testing or intensive ultrasound follow-up was undertaken. Retrospective analysis of maternal sequencing data confirmed low-level mosaicism that had been filtered out during routine analysis. CONCLUSION:These cases underscore a key limitation of exclusion NIPD_MD when maternal mosaicism is present. Its identification is essential for accurate recurrence risk estimation and genetic counseling. Sensitive detection methods, careful pre-test evaluation, and transparent communication are critical to ensure informed reproductive decision-making.

PURPOSE:Titin, the largest protein in the human body, has been associated with several disease phenotypes caused by variants in the TTN gene. With around 20% of the population carrying a rare TTN variant and over 60 million genomes expected to have been sequenced worldwide by 2025, interpreting these findings presents major challenges. This study analyzed TTN variants in the Solve-RD cohort, the European network for unsolved rare disease cases. METHODS:We collected data from 11,072 individuals with suspected rare diseases and 7390 healthy relatives from the Solve-RD consortium, checking and manually reviewing TTN variants. We then used a filtering approach focused on clinical relevance, and we provided updated recommendations based on recent literature. RESULTS:Among the cohort, 240 individuals (1.3%) carried at least one heterozygous TTN truncating variant (TTNtv), with a 3.8% prevalence in the neuromuscular subgroup, primarily composed of unsolved cases. Four individuals received a titinopathy diagnosis. Additionally, 99 participants (0.5%) had a TTNtv in a high cardiac percent spliced in exon (>80%), and 4 had an overt cardiomyopathy. CONCLUSION:This study highlights the need for standardized approach to TTN variants, and investigation of missing heritability in individuals with skeletal myopathy with het TTNtv. Establishing consensus on percent spliced in-based thresholds will be essential for assessing cardiac risk and guiding the management of asymptomatic individuals.

Papular epidermal naevus with ‘skyline’ basal cell layer (PENS) is a type of keratinocytic epidermal naevus for which the molecular genetic mechanism has not yet been identified. EGFR ex20ins could represent a new and specific genetic somatic marker of PENS.

Diagnosing rare muscular diseases can be challenging due to their genetic heterogeneity, with more than 200 implicated genes reported to date. As for other monogenic diseases, the implementation of gene panel strategies using high-throughput sequencing allowed for optimization of costs and turnaround times of genetic testing. In 2018, the French National Network for Rare Neuromuscular Diseases (Filière Nationale des Maladies Rares Neuromusculaires - FILNEMUS) established a new high-throughput sequencing diagnostic strategy for muscular diseases, based on 13 national consensus gene lists according to phenotypic and/or histological entry-diagnosis groups. This diagnostic approach has been implemented at the national level and is currently used by all French molecular diagnosis laboratories in the field. Since the initial consensus, several novel muscular disease genes have been discovered and gene-disease associations have evolved for the genes on the original panels, requiring an update.In concert with all FILNEMUS molecular diagnosis laboratories, we first carried out a literature search to identify genes described since the first version of the recommendations. The current gene-disease associations were then examined for the new genes as well as for genes previously included in the national FILNEMUS muscle diseases gene lists using data from the GeneCC consortium. The national FILNEMUS muscle diseases gene lists were updated by adding new genes with an established gene-disease association and by removing genes without sufficient evidence of association. Using data collected from all FILNEMUS molecular diagnosis laboratories, we then established an additional gene list that includes the genes most frequently identified as responsible for muscle diseases ("major genes list") in France. In addition to the current diagnostic strategy, the use of this new “major genes list” will enable a rapid response to exclude or confirm the most frequent genetic muscle diseases. In the absence of a positive diagnosis after the analysis of the “major genes list” or a “phenotype-list”, clinicians will have the option to continue with a gene analysis on an extended list (grouping together all genes from the different national FILNEMUS muscle diseases gene lists) or to proceed directly to genome analysis on the French national whole-genome sequencing platforms (Plan France Médecine Génomique 2025 - PFMG2025). The updated version of the National French consensus gene lists for the diagnosis of muscular diseases will help avoid unnecessary analysis of variants of uncertain significance, reduce the turn-around time for genetic results and facilitate rapid access to the French national whole-genome sequencing platforms.

BACKGROUND:Titinopathies are caused by mutations in the titin gene (TTN). Titin is the largest known human protein; its gene has the longest coding phase with 364 exons. Titinopathies are very complex neuromuscular pathologies due to the variable age of onset of symptoms, the great diversity of pathological and muscular impairment patterns (cardiac, skeletal muscle or mixed) and both autosomal dominant and recessive modes of transmission. Until now, only few CNVs in TTN have been reported without clear genotype-phenotype associations. METHODS:Our study includes eight families with dominant titinopathies. We performed next-generation sequencing or comparative genomic hybridisation array analyses and found CNVs in the TTN gene. We characterised these CNVs by RNA sequencing (RNAseq) analyses in six patients' muscles and performed genotype-phenotype inheritance association study by combining the clinical and biological data of these eight families. RESULTS:Seven deletion-type CNVs in the TTN gene were identified among these families. Genotype and RNAseq results showed that five deletions do not alter the reading frame and one is out-of-reading frame. The main phenotype identified was distal myopathy associated with contractures. The analysis of morphological, clinical and genetic data and imaging let us draw new genotype-phenotype associations of titinopathies. CONCLUSION:Identifying TTN CNVs will further increase diagnostic sensitivity in these complex neuromuscular pathologies. Our cohort of patients enabled us to identify new deletion-type CNVs in the TTN gene, with unexpected autosomal dominant transmission. This is valuable in establishing new genotype-phenotype associations of titinopathies, mainly distal myopathy in most of the patients.

OBJECTIVE:The objective of this study was to evaluate the implementation of NGS within the French mitochondrial network, MitoDiag, from targeted gene panels to whole exome sequencing (WES) or whole genome sequencing (WGS) focusing on mitochondrial nuclear-encoded genes.METHODS:Over 2000 patients suspected of Primary Mitochondrial Diseases (PMD) were sequenced by either targeted gene panels, WES or WGS within MitoDiag. We described the clinical, biochemical, and molecular data of 397 genetically confirmed patients, comprising 294 children and 103 adults, carrying pathogenic or likely pathogenic variants in nuclear-encoded genes.RESULTS:The cohort exhibited a large genetic heterogeneity, with the identification of 172 distinct genes and 253 novel variants. Among children, a notable prevalence of pathogenic variants in genes associated with oxidative phosphorylation (OXPHOS) functions and mitochondrial translation was observed. In adults, pathogenic variants were primarily identified in genes linked to mtDNA maintenance. Additionally, a substantial proportion of patients (54% (42/78) and 48% (13/27) in children and adults, respectively), undergoing WES or WGS testing displayed PMD mimics, representing pathologies that clinically resemble mitochondrial diseases.INTERPRETATION:We reported the largest French cohort of patients suspected of PMD with pathogenic variants in nuclear genes. We have emphasized the clinical complexity of PMD and the challenges associated with recognizing and distinguishing them from other pathologies, particularly neuromuscular disorders. We confirmed that WES/WGS, instead of panel approach, was more valuable to identify the genetic basis in patients with "possible" PMD and we provided a genetic testing flowchart to guide physicians in their diagnostic strategy.

The implementation of high-throughput diagnostic sequencing has led to the generation of large amounts of mutational data, making their interpretation more complex and responsible for long delays. It has been important to prioritize certain analyses, particularly those of "actionable" genes in diagnostic situations, involving specific treatment and/or management. In our project, we carried out an objective assessment of the clinical actionability of genes involved in myopathies, for which only few data obtained methodologically exist to date. Using the ClinGen Actionability criteria, we scored the clinical actionability of all 199 genes implicated in myopathies published by FILNEMUS for the "National French consensus on gene Lists for the diagnosis of myopathies using next generation sequencing". We objectified that 63 myopathy genes were actionable with the currently available data. Among the 36 myopathy genes with the highest actionability scores, only 8 had been scored to date by ClinGen. The data obtained through these methodological tools are an important resource for strategic choices in diagnostic approaches and the management of genetic myopathies. The clinical actionability of genes has to be considered as an evolving concept, in relation to progresses in disease knowledge and therapeutic approaches.

Filamin C-related disorders include myopathies and cardiomyopathies linked to variants in the FLNC gene. Filamin C belongs to a family of actin-binding proteins involved in sarcomere stability. This study investigates the pathogenic impact of the FLNC c.3557C > T (p.Ala1186Val) pathogenic variant associated with an early-onset cytoplasmic body myopathy and cardiomyopathy in three unrelated patients. We performed clinical imaging and myopathologic and genetic characterization of three patients with an early-onset myopathy and cardiomyopathy. Bioinformatics analysis, variant interpretation, and protein structure analysis were performed to validate and assess the effects of the filamin C variant. All patients presented with a homogeneous clinical phenotype marked by a severe contractural myopathy, leading to loss of gait. There was prominent respiratory involvement and restrictive or hypertrophic cardiomyopathies. The Ala1186Val variant is located in the interstrand loop involved in intradomain stabilization and/or interdomain interactions with neighbor Ig-like domains. 3D modeling highlights local structural changes involving nearby residues and probably impacts the protein stability, causing protein aggregation in the form of cytoplasmic bodies. Myopathologic studies have disclosed the prominent aggregation and upregulation of the aggrephagy-associated proteins LC3B and p62. As a whole, the Ala1186Val variant in the FLNC gene provokes a severe myopathy with contractures, respiratory involvement, and cardiomyopathy due to protein aggregation in patients’ muscles.

Congenital titinopathies reported to date show autosomal recessive inheritance and are caused by a variety of genomic variants, most of them located in metatranscript (MTT)-only exons. The aim of this study was to describe additional patients and establish robust genotype-phenotype associations in titinopathies. This study involved analyzing molecular, clinical, pathological, and muscle imaging features in 20 patients who had at least one pathogenic or likely pathogenic TTN variant in MTT-only exons, with onset occurring antenatally or in the early postnatal stages. The 20 patients with recessive inheritance exhibited a heterogeneous range of phenotypes. These included fetal lethality, progressive weakness, cardiac or respiratory complications, hyper-CKemia, or dystrophic muscle biopsies. MRI revealed variable abnormalities in different muscles. All patients presented severe congenital myopathy at birth, characterized by arthrogryposis (either multiplex or axial-distal) or neonatal hypotonia in most cases. This study provides detailed genotype-phenotype correlations in congenital titinopathies caused by mutations in MTT-only exons. The findings highlight the variability in clinical presentation and the severity of phenotypes associated with these specific genetic alterations. RNA-seq analyses provided valuable insights into the molecular consequences of TTN variants, particularly in relation to splicing defects and nonsense-mediated RNA decay. In conclusion, this study reinforces the genotype-phenotype correlations between congenital myopathies and variants in TTN MTT-only exons, improves their molecular diagnosis, and provides a better understanding of their pathophysiology.