Muscle health varies continuously from optimal function to severe pathology, yet no unified genetic framework quantifies this spectrum objectively. Here we develop MyoScore, a transcriptomic scoring system derived from transcriptome-wide association studies of 27 muscle-related phenotypes in over one million participants. TWAS selects genes whose genetically regulated expression in skeletal muscle associates with muscle-related traits, providing the genetic anchoring of the scoring system, while MyoScore itself is computed from measured bulk RNA-seq expression in new samples. From 1,116 transcriptome-wide association study (TWAS)-significant genes, 417 are expressed in skeletal muscle and form the basis of the scoring system. These genes are organized into five dimensions of muscle biology (Strength, Mass, LeanMuscle, Youth and Resilience), each scored from 0 to 100. Across 1,722 human skeletal muscle transcriptomes from four independent cohorts, MyoScore defines a continuous four-stage muscle health spectrum, discriminates healthy from diseased muscle (area under the curve 0.751-0.873), and correlates with histopathological severity, quantitative MRI and clinical outcomes. Functional validation through iPSC-to-myotube differentiation supports predicted expression changes for novel MyoScore genes. UK Biobank analysis of blood biomarker proxies in 467,123 participants demonstrates concordant associations with muscle phenotypes, and two-sample Mendelian randomization using skeletal muscle cis-eQTL supports causal directionality for 78% of gene-outcome pairs tested. Single-cell validation across 475,584 cells from two independent muscle ageing atlases shows that pseudobulk MyoScore declines with age, with type II myofibre nuclei most affected. Together, MyoScore establishes the first genetically anchored, dimension-resolved quantification of human muscle health, enabling objective assessment, patient stratification and biomarker discovery across the full spectrum from optimal function to severe disease. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by the National Natural Science Foundation of China (82471426 to C.Z.; 82571592 to S.L.), the Open Research Fund of Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases (gect-2025-Z01 to H.Z.), the Folkhalsan Research Center (FHRC; to M.S. and M.J.), the European Commission (project CoMPaSS-NMD funded by HORIZON-HLTH-2022-TOOL-12-two-stage, GA n101080874 to M.S.), the Research Council of Finland (grants #339437, #346209 and #361979 to M.S.), Samfundet Folkhalsan i Svenska Finland (to M.S. and B.U.), the Sigrid Juselius Foundation (grant #230217 to M.S. and B.U.; grant #260226 to M.S.), the Jane and Aatos Erkko Foundation (to P.H.), the Magnus Ehrnrooth Foundation (to A.O.) and AFM-Telethon (to M.J.). The funders had no role in the study design, data collection, analysis or interpretation, manuscript preparation, or the decision to submit the manuscript for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the relevant institutional review boards. The Helsinki Myofin cohort was approved by the Ethics Committee of Helsinki University Hospital (HUS; approval number 195/13/03/00/11), and written informed consent was obtained from each participant. The HuashanMuscle cohort was collected at Huashan Hospital, Fudan University, under institutional review board approval (approval numbers 2022-913 and 2019-409), and written informed consent was obtained from all participants. iPSC lines were generated from peripheral blood mononuclear cells of four healthy donors who provided written informed consent under the same Huashan Hospital ethical approvals. The GTEx dataset was accessed through the Genotype-Tissue Expression Project (dbGaP accession phs000424); all GTEx tissue samples were collected from deceased donors under informed consent from next of kin. UK Biobank data were accessed under approved application number 19542. GEO datasets were obtained from publicly available repositories; ethics approvals for these cohorts were obtained by the original investigators as described in the respective publications. All procedures conformed to the principles of the Declaration of Helsinki. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The integrated and processed RNA-seq datasets, MyoScore results, gene weights and calculation scripts used in this study are available at https://github.com/Hirriririir/MyoScore. The following public data sources were used in this study: Transcriptomic data. GTEx v8 skeletal muscle RNA-seq (n = 803) is available from the GTEx Portal (gtexportal.org). GEO datasets are publicly available at ncbi.nlm.nih.gov/geo (accession numbers listed in Supplementary Table 2). GWAS summary statistics. GWAS data for 27 muscle-related phenotypes were obtained from the OpenGWAS database (gwas.mrcieu.ac.uk; IEU identifiers listed in Supplementary Table 1), the UK Biobank Neale Lab round 2 (nealelab.is/uk-biobank) and FinnGen release 9 (finngen.gitbook.io/documentation). eQTL and TWAS resources. GTEx v8 skeletal muscle cis-eQTL summary statistics and pre-computed FUSION TWAS weights are available from the GTEx Portal and the FUSION TWAS website (gusevlab.org/projects/fusion). The 1000 Genomes Phase 3 European LD reference panel is available from the International Genome Sample Resource (internationalgenome.org). Blood eQTL data from eQTLGen (n = 31,684) are available at eqtlgen.org. Single-cell/single-nucleus RNA-seq atlases. The HLMA (Human Limb Muscle Ageing) atlas (292,423 cells/nuclei, 23 donors) is available from the CNGB database (db.cngb.org/cdcp/hlma). The Sanger Skeletal Muscle atlas is available from the CellxGene portal (cellxgene.cziscience.com). UK Biobank. Individual-level data for biomarker analyses (plasma acetate, serum GGT, grip strength, appendicular lean mass, walking pace, whole-body fat-free mass) were accessed under application 19542. Histopathological data. H&E whole slide images from the HuashanMuscle cohort, together with processed GTEx slides, are publicly accessible via the MyoToolkit portal (myotoolkit.huashanmuscle.com); the GTEx slides are additionally available from the GTEx Portal (gtexportal.org). Other data not publicly deposited are available from the corresponding authors upon reasonable request.

Abstract The eukaryotic transcriptome diversity arises largely from alternative splicing. One of the widely used high-throughput methods to study this diversity is RNA sequencing. RNA sequencing has become a cornerstone of both basic biology and precision medicine, facilitating the quantification of gene and transcript expression, as well as the characterization of alternative splicing events and regulatory biological pathways in these studies. As there is a wide interest in studying non-ribosomal RNAs, which constitute about 20% of cellular RNAs, it is common to either select for poly(A)+ RNAs or to deplete ribosomal RNAs during the library preparation stage of RNA sequencing. At the time of library preparation, poly(A)+ selected RNA-Seq captures the polyadenylated transcripts, whereas rRNA-depleted RNA-Seq pools a broader spectrum of RNA species, including non-polyadenylated and premature transcripts. Using blood and skeletal muscle transcriptomics datasets, we examined how these two library enrichment techniques influence transcript representation, transcript-body coverage, and splice junction detection. We observed that poly(A)+ selected libraries display length-dependent differences, reduced splice junction representation and pronounced 3’ end coverage bias for transcripts of total transcription length over 5 kb. In contrast, rRNA depletion provides a more uniform 5′-3′ coverage, an improved detection of splice junctions, and a robust detection of long disease-relevant transcripts. These differences are evident in the detection of extremely large transcripts, such as the sarcomeric genes OBSCN (~ 39 kb) and TTN (> 100 kb). This study discusses how RNA-Seq library preparation techniques capture different RNA types and emphasizes the importance of interpreting poly(A)+ selected and rRNA depleted data in the appropriate biological and clinical contexts.

Skeletal muscle is composed of type I and type II fibers, each characterized by specific metabolic machinery. LACTB is a conserved mitochondrial protein implicated in lipid utilization and tumorigenesis, but its precise function in the cellular metabolism remains unclear. To gain novel insight into the functional role of LACTB, we investigated skeletal muscle to determine whether LACTB is segregated by fiber type. The expression of LACTB was determined by immunohistochemistry (IHC) and immunoblotting in skeletal muscle from healthy human subjects and the laboratory rat. The specificity of the antibody was assessed using recombinant human LACTB protein and endogenous LACTB in isolated mitochondria. IHC results were validated in a cellular model of myoblast differentiation using the C2C12 and L6 cell lines. The results demonstrated that LACTB is highly enriched in adult type I muscle fibers. During development, LACTB expression commences in type I primary myotubes at the time of their formation around week 20 of gestational age. LACTB expression in myoblasts is low but increases rapidly upon the induction of myotube differentiation. We conclude that LACTB plays a distinct role in mitochondria of type I fibers, most likely acting in oxidative metabolism related to energy use from lipids. This defines LACTB as a mitochondrial marker for type I fibers.

The small heat shock protein HSPB6 (a.k.a. Hsp20) is highly expressed in striated and smooth muscles. It modulates the oligomerization of its paralogs HSPB1 and CRYAB (HSPB5) and is involved e.g. in cytoskeletal regulation and autophagy. While HSPB6 variants have been implicated in cardiomyopathy, they have not been previously linked to neuromuscular disease. We report here a patient with late-onset myopathy and cataract, carrying in cis the novel HSPB6 variant c.464delC and the common polymorphism c.488G > C, together resulting in the extended protein p.Pro155Argfs*25;p.Gly163Arg. The family history was consistent with dominant inheritance. The mutant protein showed decreased solubility due to phase separation propensity, and caused mislocalization of CRYAB and BAG3, and a decrease of HSPB1 in transfected cells. The patient's muscle biopsy showed rimmed vacuoles and, in line with the functional studies, accumulation of HSPB6 and its interaction partners. The identified HSPB6 variants are most likely the cause of the muscle disease in this family, thus identifying HSPB6 mutations as a novel cause of vacuolar myopathy. Other reported HSPB6 variants causing a late frameshift or extension may cause disease in a similar fashion.

BACKGROUND:Highly expressed in skeletal muscles, the gene Obscurin (i.e. OBSCN) has 121 non-overlapping exons and codes for some of the largest known mRNAs in the human genome. Furthermore, it plays an essential role in muscle development and function. Mutations in OBSCN are associated with several hypertrophic cardiomyopathies and muscular disorders. OBSCN undergoes extensive and complex alternative splicing, which is the main reason that its splicing regulation associated with skeletal and cardiac muscle development has not previously been thoroughly studied. METHODS:We analyzed RNA-Seq data from skeletal and cardiac muscles extracted from 44 postnatal individuals and six fetuses. We applied the intron/exon level splicing analysis software IntEREst to study the splicing of OBSCN in the studied samples. The differential splicing analysis was adjusted for batch effects. Our comparisons revealed the splicing variations in OBSCN between the human skeletal and cardiac muscle, as well as between post-natal muscle (skeletal and cardiac) and the pre-natal equivalent muscle. RESULTS:We detected several splicing regulations located in the 5'end, 3' end, and the middle of OBSCN that are associated with human cardiac or skeletal muscle development. Many of these alternative splicing events have not previously been reported. Our results also suggest that many of these muscle-development associated splicing events may be regulated by BUB3. CONCLUSIONS:We conclude that the splicing of OBSCN is extensively regulated during the human skeletal/cardiac muscle development. We developed an interactive visualization tool that can be used by clinicians and researchers to study the inclusion of specific OBSCN exons in pre- and postnatal cardiac and skeletal muscles and access the statistics for the differential inclusion of the exons across the studied sample groups. The OBSCN exon inclusion map related to the human cardiac and skeletal muscle development is available at http://psivis.it.helsinki.fi:3838/OBSCN_PSIVIS/ . These findings are essential for an accurate pre- and postnatal clinical interpretation of the OBSCN exonic variants.

Aim: Tibial muscular dystrophy (TMD; MIM#600334, ORPHA:609) is an adult-onset, slowly progressive distal myopathy resulting from dominant variants in exon 364 of the TTN gene. The Finnish founder variant (FINmaj), characterized by an 11-bp insertion/deletion, causes autosomal dominant (AD) TMD in heterozygous individuals. Our aim was to assess the prevalence and origin of the FINmaj variant within the Estonian population. Methods: We reanalyzed next-generation sequencing panels and whole-exome sequencing data from 2014 to 2025 to identify individuals carrying the FINmaj variant. The study included three cohorts: Tartu University Hospital (n = 15,178), West Tallinn Central Hospital (n = 52), and the Estonian Genome Center (n = 4,776). Most carriers of the FINmaj variant underwent muscle magnetic resonance imaging (MRI) and haplotype analysis. Results: We identified 13 individuals from five families with the heterozygous FINmaj variant, including two individuals with autosomal recessive limb-girdle muscular dystrophy-10 and eleven with AD TMD. By the age of 50, all patients diagnosed with TMD showed symptoms of distal myopathy and characteristic MRI findings. The carrier frequency of the FINmaj variant in the Estonian cohort was one in 3,036, with no carriers in the Estonian Genome Center cohort. The average haplotype length was estimated to be ~4.1 Mb in Estonians, compared to ~5 Mb in Finns. Conclusion: AD TMD is one of the most prevalent but underdiagnosed hereditary muscle diseases in the Estonian population. Since Estonian patients exhibit an estimated shorter haplotype length than Finnish patients, the FINmaj variant likely originated in Estonia before spreading to Finland.

Background: Titin, the largest human protein, is essential for sarcomere structure and function. The TTN gene, spanning 364 exons, undergoes extensive alternative splicing thus producing multiple isoforms. The M-band region, encoded by exons 359-364, plays a critical role in sarcomere integrity and mechanical stability. Exon 363 is of interest due to its involvement in titinopathies. Pathogenic truncating variants in this exon have been linked to recessive myopathies, including and mainly young-onset recessive distal titinopathy. Methods: A multicenter study was conducted on six patients from five unrelated families with confirmed recessive titinopathy and truncating variants in exon 363. Clinical evaluations were performed. Genetic testing and segregation analysis confirmed the phase of the variants. Results: A novel truncating variant c.107578C>T, p.(Gln35860Ter) was identified in four unrelated patients of Eastern European ancestry, all carrying a second pathogenic variant in a canonical TTN exon. These patients exhibited juvenile/young-adult onset recessive distal titinopathy with progressive lower limb weakness, frequently asymmetric muscle involvement, and no cardiac or respiratory complications. A Belgian family presented with a congenital myopathy caused by a novel frameshift deletion c.107430delA, p.(Ser35811AlafsTer32) in exon 363, in compound heterozygosity with a truncating variant in exon 208. These patients showed a more severe phenotype. Conclusions: This study expands the spectrum of TTN-related myopathies, emphasizing exon 363's pathogenic significance. Truncating exon 363 variants contribute to young onset recessive distal and sometimes early onset titinopathy with contractures, and the phenotype severity is influenced by the second variant's location and exon usage.

Optimizing in vitro differentiation protocols for skeletal muscle cells is essential for producing mature, functional myotubes suitable for disease modeling and therapeutic screening. While C2C12 murine myoblasts are a widely used model, achieving consistent and advanced differentiation remains challenging. In this study, we systematically evaluated conditions that improve myotube formation and maturation in a 2D culture system using ultra-compliant gelatin hydrogels. We compared standard and commercial differentiation media and identified that supplementation of DMEM with 2% horse serum and 10% Opti-MEM (DMO) supported robust myotube formation, with thin and aligned fibers. Insulin supplementation significantly increased expression of myosin heavy chain (MyHC) and calsequestrin, while pyruvate provided additional benefit by further enhancing myotube maturation. Media change frequency was also critical: daily replacement was necessary to maintain optimal differentiation, although the addition of insulin and pyruvate partly mitigated the effects of less frequent changes. Application of electrical pulse stimulation (EPS) improved sarcomeric α-actinin organization without significantly altering MyHC isoform expression. RNA sequencing confirmed transcriptional reprogramming consistent with myogenic progression, including early upregulation of key muscle-specific genes. Our findings present a cost-effective, reproducible protocol that supports advanced C2C12 differentiation in a scalable 2D system, offering practical guidance for generating mature, functional myotubes in vitro for both basic and translational muscle research.

BACKGROUND:Inherited rare skeletal muscle diseases cause muscle weakness and wasting of variable severity. Without a molecular diagnosis, patients often endure prolonged diagnostic journeys, leading to delays in appropriate management of the disease. This occurs in approximately 60% of patients with rare diseases. METHODS:To facilitate reanalysis of 278 unsolved patients, we used a gene prioritisation tool Exomiser, which standardises analysis by ranking causative variants based on phenotype relevance and variant pathogenicity. Before analysis, we benchmarked Exomiser for variant prioritisation with solved cases and for novel disease gene discovery with mock cases with variants in candidate disease genes. Additionally, we studied the significance of the specificity of the phenotype descriptions. RESULTS:In our study, Exomiser ranked genes in the top 10 correctly in 97.4% of controls with previously detected causative variants. Moreover, 57.1% of candidate genes in mock cases were similarly prioritised in the top 10. We also showed that three parental muscle disease human phenotype ontologies describing the patient phenotype performed as well as patient-specific ones, with a p value of 0.68 for difference in performance. The provided automation and standardisation of variant interpretation resulted in two novel diagnoses and in findings, either in known muscle disease genes or in novel candidate genes, which need further investigation. CONCLUSIONS:Exomiser is recommended for initial and periodic reanalyses of exomes in unsolved patients with myopathy, as it benefits from literature updates and minimises effort. This approach could also extend to whole genome sequencing data, aiding the interpretation of variants beyond coding regions.

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.

PURPOSE:Heterozygous pathogenic variants in SPTAN1 cause a diverse spectrum of neurogenetic disorders ranging from peripheral and central nervous system involvement to complex syndromic presentations. We set out to investigate the role of SPTAN1 in genetically unsolved hereditary myopathies. METHODS:Through international collaboration we identified 14 families with distal weakness and heterozygous SPTAN1 loss-of-function variants. Clinical data, electrophysiology, muscle computed tomography or magnetic resonance imaging, and muscle biopsy findings were collected and standardized. SPTAN1 protein, messenger RNA expression analysis and copy DNA sequencing was performed on muscle tissue from 2 participants. RESULTS:Five families showed autosomal dominant mode of inheritance, whereas in 9 patients the variant was shown to be de novo, including 2 pairs of monozygotic twins. In 2 families, further segregation analysis was not possible. All affected participants presented with early childhood-onset distal weakness and foot abnormalities. Muscle magnetic resonance imaging or computed tomography in 10 patients showed fatty infiltration of the distal lower limb anterior compartment and/or selective involvement of the extensor hallucis longus muscle. Muscle biopsy revealed myopathic changes in 7 patients. Finally, we provide proof for nonsense-mediated decay in muscle tissue derived from 2 patients. CONCLUSION:We present evidence linking heterozygous SPTAN1 loss-of-function variants to childhood-onset distal myopathy in 14 unrelated families.

BACKGROUND:Variants in CASQ1, encoding a calcium-binding protein in the fast-twitch fibers of skeletal muscle, cause sarcoplasmic reticulum aberrations such as large vacuoles with CASQ1 inclusions or, less commonly, tubular aggregates. To date, seven pathogenic variants have been described, all dominant missense variants. The typical symptoms of the disease include muscle weakness, cramps, myalgia, and fatigue. METHODS:We used genome and exome sequencing to identify the disease-causing variants in two families with dominant myopathy. The candidate variants were further characterized by cell-transfection studies and western blotting. RESULTS:In Family 1, three patients presented with exercise intolerance, cramps, and myalgia. Additionally, the proband had muscle weakness and her muscle biopsy showed nemaline bodies. In electron microscopy, there were morphological changes in the triads and the SR-feet in all patients. A variant in CASQ1, p.(Glu89Lys), was found in all patients, whereas the proband had also two compound heterozygous variants in NEB. In Family 2, three patients presented with progressive muscle weakness. The proband's muscle biopsy showed marked atrophy. The frameshift variant p.(Gly383Alafs*39) in CASQ1 was found in all three patients. In silico analysis indicated that the variant results in protein extension, which was confirmed by western blotting of patient muscle. Cell-transfection studies showed that the variant protein forms aggregates. CONCLUSION:This study expands the spectrum of pathogenic CASQ1 variants. The morphological changes in the SR-feet indicate a novel pathogenetic mechanism.

Background:Neurogenetic disorders caused by pathogenic variants in four genes encoding non-erythrocytic spectrins ( SPTAN1, SPTBN1, SPTBN2, SPTBN4) range from peripheral and central nervous system involvement to complex syndromic presentations. Heterozygous pathogenic variants in SPTAN1 are exemplary for this diversity with phenotypes spanning almost the entire spectrum. Methods:Through international collaboration we identified 14 families with genetically unsolved distal weakness and unreported heterozygous SPTAN1 loss-of-function variants including frameshift, nonsense and splice-acceptor variants. Clinical data, electrophysiology, muscle CT or MRI and muscle biopsy findings were collected and standardized. SPTAN1 protein, mRNA expression analysis and cDNA sequencing was performed on muscle tissue from two patients. Results:All 20 patients presented with early childhood onset distal weakness. The severity varied both within families and between different families. Foot abnormalities ranged from hammer toes and pes cavus to distal arthrogryposis. Electrophysiology showed mixed myogenic and neurogenic features. Muscle MRI or CT in 10 patients showed fatty infiltration of the distal lower limb anterior compartment and/or selective involvement of the extensor hallucis longus muscle. Muscle biopsy revealed myopathic changes with mild dystrophic and chronic neurogenic changes in 7 patients. Finally, we provide proof for nonsense mediated decay in tissues derived from two patients. Conclusions:We provide evidence for the association of SPTAN1 loss-of-function variants with childhood onset distal myopathy in 14 families. This finding extends the phenotypic spectrum of SPTAN1 loss-of-function variants ranging from intellectual disability to distal weakness with a predominant myogenic cause. KEY MESSAGES:SPTAN1 loss-of-function variants, including frameshift, nonsense and splice site variants cause a novel childhood onset distal weakness syndrome with primarily skeletal muscle involvement. Hereditary motor neuropathies and distal myopathic disorders present a well-known diagnostic challenge as they demonstrate substantial clinical and genetic overlap. The emergence of SPTAN1 loss-of-function variants serves as a noteworthy example, highlighting a growing convergence in the spectrum of genotypes linked to both hereditary motor neuropathies and distal myopathies.

Myopathy refers to a large group of heterogeneous, rare muscle diseases. Bulk RNA-sequencing has been utilized for the diagnosis and research of these diseases for many years. However, the existing valuable sequencing data often lack integration and clinical interpretation. In this study, we integrated bulk RNA-sequencing data from 1221 human skeletal muscles (292 with myopathies, 929 controls) from both databases and our local samples. By applying a method similar to single-cell analysis, we revealed a general spectrum of muscle diseases, ranging from healthy to mild disease, moderate muscle wasting, and severe muscle disease. This spectrum was further partly validated in three specific myopathies (97 muscles) through clinical features including trinucleotide repeat expansion, magnetic resonance imaging fat fraction, pathology, and clinical severity scores. This spectrum helped us identify 234 genuinely healthy muscles as unprecedented controls, providing a new perspective for deciphering the hallmark genes and pathways among different myopathies. The newly identified featured genes of general myopathy, inclusion body myositis, and titinopathy were highly expressed in our local muscles, as validated by quantitative polymerase chain reaction.

OBJECTIVE:Biallelic titin truncating variants (TTNtv) have been associated with a wide phenotypic spectrum, ranging from complex prenatal muscle diseases with dysmorphic features to adult-onset limb-girdle muscular dystrophy, with or without cardiac involvement. Given the size and complexity of TTN, reaching an unequivocal molecular diagnosis and precise disease prognosis remains challenging. METHODS:In this case series, 12 unpublished cases and one already published case with biallelic TTNtv were collected from multiple international medical centers between November 2022 and September 2023. TTN mutations were detected through exome or genome sequencing. Information about familial and personal clinical history was collected in a standardized form. RNA-sequencing and analysis of TTN exon usage were performed on an internal sample cohort including postnatal skeletal muscles, fetal skeletal muscles, postnatal heart muscles, and fetal heart muscles. In addition, publicly available RNA-sequencing data was retrieved from ENCODE. RESULTS:We generated new RNA-seq data on TTN exons and identified genotype-phenotype correlations with prognostic implications for each titinopathy patient (whether worsening or improving in prenatal and postnatal life) using percentage spliced in (PSI) data for the involved exons. Interestingly, thanks to exon usage, we were also able to rule out a titinopathy diagnosis in one prenatal case. INTERPRETATION:This study demonstrates that exon usage provides valuable insights for a more exhaustive clinical interpretation of TTNtv; additionally, it may serve as a model for implementing personalized medicine in many other genetic diseases, since most genes undergo alternative splicing.

BACKGROUND AND PURPOSE:Tibial muscular dystrophy (TMD) is a dominant late onset distal titinopathy. It was first described in Finnish patients 3 decades ago. TMD patients with several other TTN mutations occur in many European populations. In this retrospective study, we were able to obtain longitudinal follow-up data of the disease progression over 15 years in 137 TMD patients.METHODS:We retrieved clinical data retrospectively from three examinations spanning a period of 15 years. The data were analyzed in R. Frequencies, percentages, and median values were used to describe data. Probability values were determined with the chi-squared test.RESULTS:In the cohort, the first symptoms were walking difficulties (97.8%) and weakness in distal lower limbs (98.5%). The progression of the weakness in distal lower limbs was moderate, and in the proximal lower limbs and proximal upper limbs it was mild. The distal upper limbs were not affected. Magnetic resonance imaging results indicated fatty degeneration preferentially in lower leg anterior muscles, gluteus minimus, and hamstring muscles. Serum creatine kinase values in the cohort were mostly normal (40.7%) or mildly elevated (53.7%). The data suggest that 50% of patients need walking aids by the age of 88 years.CONCLUSIONS:Despite individual variability of severity, the overall disability due to walking difficulties and upper limb weakness remained moderate even at very advanced ages, and cardiomyopathy did not develop due to the titin defect alone. The acquired results promote the correct identification of TMD, and the obtained trajectories of disease evolution can be used as natural history data for any therapeutic intervention.

Recessive mutations in the DNAJB2 gene, encoding the J-domain co-chaperones DNAJB2a and DNAJB2b, have previously been reported as the genetic cause of progressive peripheral neuropathies, rarely involving pyramidal signs, parkinsonism and myopathy. We describe here a family with the first dominantly acting DNAJB2 mutation resulting in a late-onset neuromyopathy phenotype. The c.832 T > G p.(*278Glyext*83) mutation abolishes the stop codon of the DNAJB2a isoform resulting in a C-terminal extension of the protein, with no direct effect predicted on the DNAJB2b isoform of the protein. Analysis of the muscle biopsy showed reduction of both protein isoforms. In functional studies, the mutant protein mislocalized to the endoplasmic reticulum due to a transmembrane helix in the C-terminal extension. The mutant protein underwent rapid proteasomal degradation and also increased the turnover of co-expressed wild-type DNAJB2a, potentially explaining the reduced protein amount in the patient muscle tissue. In line with this dominant negative effect, both wild-type and mutant DNAJB2a were shown to form polydisperse oligomers.