Decreased dystrophin expression and elevated dystrophin-targeting miRNAs in anti-HMGCR immune-mediated necrotizing myopathy

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by loss of function mutations in the Dystrophin gene resulting in loss of dystrophin protein. Current DMD therapeutics use phosphorordiamidate morpholino oligomers (PMO) to skip over the frame-shifting exon during the splicing of the dystrophin pre-mRNA, resulting in translation of a truncated dystrophin protein product. While exon skipping therapies are promising, their potential has not been fully realized as increases in dystrophin protein have been minimal and highly variable in clinical trials. We previously described microRNAs that are upregulated in DMD and BMD muscle biopsies, bind to the dystrophin 3′UTR and inhibit dystrophin protein production. One of these dystrophin-targeting microRNAs, miR-146a, is regulated by the pro-inflammatory transcription factor NF-κB, is highly elevated in the muscles of dystrophin-deficient mice and is reduced by anti-inflammatory drugs. Here, we show that inflammation induces miR-146a expression in dystrophic myotubes. Using bioinformatics analysis, we validate previous findings that the dystrophin 3′UTR harbors a miR-146a binding site. Anin vitro3′UTR luciferase reporter assay further confirms that miR-146a inhibits dystrophin translation, while mutating the miR-146a binding site attenuates inhibition. In dystrophin-deficient mice we find that co-injection of an exon skipping PMO with miR-146a but not a control sequence reduces the extent of dystrophin positive fibers. To test the hypothesis that miR-146a is inhibitory to exon skipping dystrophin restoration, we generated a novel DMD double knockout mouse model with body-wide miR-146a deletion (146aXmice) and administered an exon 51 skipping PMO into the tibialis anterior (TA) muscles ofmdx52and146aXmice. Excitingly,146aXTAs showed increased dystrophin protein versusmdx52as measured by capillary Western immunoassay and dystrophin-positive fiber quantification. Additionally, systemic PMO delivery increased dystrophin protein levels and increased the number of dystrophin-positive fibers in146aXversusmdx52muscles despite similar levels of skipped dystrophin transcripts in both groups. These data demonstrate genetic deletion of miR-146a is sufficient to increase dystrophin rescue via exon skipping. Our data suggests that antagomiR-mediated inhibition of miR-146a or other dystrophin targeting miRNAs could be a viable exon skipping DMD co-therapy and warrants further research.

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Asymptomatic HyperCKemia in the Pediatric Population

Marti,

Pitarch-Castellano,

Muelas

et al. 2025

Neurology

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Background and ObjectivesPersistent elevation of serum creatine kinase levels (hyperCKemia) as an isolated manifestation presents a diagnostic challenge. Genetic myopathies are frequently involved; however, studies using next-generation sequencing (NGS) in pediatric patients are lacking, and the significance of genetic aberrations remains poorly understood. This study, therefore, aimed to investigate the relevance of NGS and the support of contemporary diagnostic tools in the diagnosis of pediatric asymptomatic hyperCKemia.MethodsThis was a prospective cohort study enrolling pediatric (0–18 years old) patients meeting the predefined criteria for asymptomatic/paucisymptomatic hyperCKemia, excluding DMD gene deletion/duplication, recruited from a referral center. NGS, muscle MRI, EMG, and muscle biopsies with immunolabeling and inflammatory markers were performed according to a prespecified protocol. Data analysis was performed using descriptive/univariate statistics and Bayesian logistic regression.ResultsThe series comprised 65 patients (78% male). NGS diagnosis was achieved in 55% of the cohort, with 70% of the pathogenic variants involving 7 genes (DMD, CAPN3, ANO5, DYSF, RYR1, GAA, and CAV3). The diagnostic rate was similar across all age groups; however, the gene profiles varied between the childhood and juvenile groups. EMG yielded myopathic features in 48% of the investigated cases, being predictive for diagnosis (p < 0.05; odds ratio [OR] 13.484, 95% CI 1.358–705.297). MRI showed normal (64%), focal fatty change (26%), or short-tau inversion recovery hyperintensity (10%) profiles, which were not predictive of diagnosis but supported muscle biopsy indications. Muscle biopsy provided a significant diagnostic effect (p < 0.05; OR 0.028, 95% CI 0.001–0.238), contributing to myopathologic features clarifying the variant pathogenicity and identifying inflammatory myopathies. The diagnoses remained inconclusive and unresolved in 14% and 29% of the cohorts, respectively. The diagnostic rate for patients with CK levels below the threshold of 3× was 42%. In multivariate analysis, NGS was the only variable achieving a significant diagnostic effect (β = 9.85, 95% CI 4.65–16.09).DiscussionNGS, as the primary diagnostic tool for investigating hyperCKemia in the pediatric population, yielded a higher diagnostic rate. However, muscle biopsies are necessary to define variants of uncertain pathogenicity and aid in identifying inflammatory myopathies. EMG and MRI may play a role in hyperCKemia characterization, guiding the decision to perform muscle biopsy. The primary limitation of this study was that not all ancillary tests were performed in all recruited patients owing to ethical restrictions, which lowered the power of the predictive analysis.

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Decreased dystrophin expression and elevated dystrophin-targeting miRNAs in anti-HMGCR immune-mediated necrotizing myopathy

Cited by 3 publications

References 10 publications

Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping

Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping

Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping

Asymptomatic HyperCKemia in the Pediatric Population

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