Duchenne and Becker muscular dystrophies are X-linked disorders caused by mutations in the dystrophin gene, one of the largest protein-coding genes in mammals. Duchenne affects roughly 4.8 individuals per 100,000 people and typically arises from deletions that disrupt the reading frame, abolishing dystrophin production entirely. The result is progressive muscle degeneration and premature death from cardiorespiratory failure. Becker disease, affecting about 1.5 per 100,000, stems from in-frame mutations that preserve a shortened but partially functional dystrophin protein, producing a far gentler clinical course. A particularly important pathogenic mutation is the deletion of exon 52, which is amenable to exon-skipping therapies that target exon 51 or 53 to restore the reading frame.

Mouse models have long dominated dystrophy research, but they poorly predict human disease progression. The mdx mouse shows only a mild phenotype with robust regeneration and minimal cardiac involvement, while the Golden Retriever muscular dystrophy dog, though clinically closer to humans, suffers from phenotypic variability, long generation intervals and small litter sizes. Pigs occupy a valuable middle ground: their muscle structure and biology closely resemble human muscle, their body size permits realistic dosing and delivery studies, and modern genome editing allows the creation of large, genetically homogeneous cohorts. Using somatic cell nuclear transfer, the researchers previously generated pigs lacking dystrophin exon 52, which develop dystrophin deficiency, impaired mobility and progressive muscle pathology with lifespans of three to nine months. They then deleted exon 51 in those same cells, restoring the reading frame and creating a Becker-like line that expresses dystrophin in skeletal muscle and myocardium with markedly improved pathology.

For the new study, the team analysed triceps brachii biopsies from thirteen animals at 3.5 months of age: four Duchenne-model pigs, four Becker-model pigs and five wild-type controls. They prepared stranded total RNA sequencing libraries and small RNA libraries, sequenced them on an Illumina NextSeq 1000 platform, and aligned the reads to the pig reference genome. Differential expression analysis flagged genes and microRNAs with at least a twofold change and an adjusted p-value of 0.05 or below. To move from correlation to regulation, the researchers predicted microRNA targets across 3′ untranslated regions, 5′ untranslated regions and coding sequences using stringent hybridization criteria, then retained only pairs showing significant inverse correlations between microRNA abundance and messenger RNA expression, consistent with the canonical repressive role of microRNAs.

The messenger RNA results were striking. In Duchenne muscle, 1,440 genes were upregulated and 487 downregulated relative to wild type, while Becker muscle showed 1,804 upregulated and only 155 downregulated genes. Crucially, most changes were condition-specific: 1,282 genes were upregulated only in Duchenne, 1,646 only in Becker, and just 158 were shared. Genes uniquely downregulated in Duchenne muscle were enriched for muscle cell differentiation, sarcomere structure, contractile fibre components and metabolic regulation, including insulin signalling and lipolysis. Genes uniquely upregulated in Duchenne pointed to a fierce immune activation, with enrichment in complement cascades, cytokine signalling, chemokine pathways and phagocytosis. Quantitative PCR confirmed key inflammatory changes, with IL10 and TGFB elevated in Duchenne but not Becker muscle.

On the microRNA front, the team detected 457 microRNAs, of which 34 were significantly dysregulated in Duchenne versus wild type, 57 in Becker versus wild type and 55 in the direct Becker-versus-Duchenne comparison. Hierarchical clustering again placed Duchenne samples in a distinct cluster while Becker samples often grouped with wild type, mirroring the milder phenotype. The resulting microRNA-messenger RNA networks were vast: in the Becker-versus-Duchenne comparison alone, 24 upregulated microRNAs were linked to 875 downregulated target genes through 3,875 unique interactions, while 31 downregulated microRNAs targeted 568 upregulated genes through 2,675 pairs. Enrichment analysis of these targets implicated chemokine signalling, phagosome function, neurogenesis, apoptosis, insulin and glucagon signalling, and synaptic signalling, suggesting that exon skipping reshapes developmental, metabolic and inflammatory regulatory circuits.

Two microRNAs emerged as headline candidates. ssc-miR-296-3p was upregulated exclusively in Duchenne muscle and predicted to target 228 downregulated genes enriched in muscle structure development, skeletal system development, fatty acid metabolism and hormone signalling, positioning it as a potential driver of Duchenne pathology and a pig-specific biomarker. ssc-miR-423-5p, by contrast, was upregulated only in Becker muscle, targeting 67 genes involved in endothelial cell proliferation, tissue development, calcium signalling and cytokine receptor interaction, a profile more consistent with tissue and vascular homeostasis than direct muscle regulation. Neither microRNA had previously been linked directly to human muscular dystrophies, making both novel findings.

Familiar suspects also appeared. miR-199a-5p, previously implicated in dystrophic muscle pathogenesis and elevated in exosomes from Duchenne muscle fibroblasts, was strongly upregulated in Duchenne pigs and predicted to target 77 downregulated genes, while miR-199b, known to repress porcine muscle satellite cell proliferation through a JAG1-NOTCH1 feedback loop, targeted 22 genes. A cross-species comparison with published human datasets covering ten neuromuscular disorders identified 35 microRNAs dysregulated in both the porcine data and human data, underscoring shared molecular mechanisms. Intriguingly, several microRNAs known for protective or anti-atrophic effects, including miR-199a-3p, miR-23a, miR-223, miR-146b and miR-224, were also upregulated in Duchenne muscle, possibly reflecting compensatory feedback against wasting and fibrosis. In Becker muscle, the greater number of dysregulated microRNAs may reflect active regeneration and remodelling, whereas advanced degeneration in Duchenne may simplify the regulatory landscape.

The functional implications align with earlier work showing severely reduced muscle force in Duchenne pigs and partial, incomplete recovery in Becker animals. The constitutive genomic deletion in the Becker-like pigs provides uniform reframing across all muscle fibres, representing an upper bound of molecular rescue that FDA-approved exon-skipping therapies, which achieve low and variable dystrophin restoration in patients, do not reach. Exon 51 skipping is applicable to roughly 14 percent of Duchenne patients, particularly those with deletions involving exon 50 or 52, so the pig model offers a realistic ceiling for what such interventions could accomplish at the molecular level. The authors caution that whole-muscle biopsies may partly reflect shifts in cellular composition, such as inflammatory infiltration, and that the predicted microRNA-messenger RNA interactions require functional validation to establish causality; future single-nucleus RNA sequencing should help disentangle cell-type-specific contributions.

Even with those caveats, the study delivers a resource with immediate translational value. Sixteen microRNAs were specifically upregulated in Duchenne relative to both Becker and wild type, and eighteen were upregulated specifically in Becker, each with distinct target gene sets and pathway signatures. These condition-specific signatures could support non-invasive disease stratification, monitoring of exon-skipping therapy efficacy and the design of microRNA-based interventions. Combined with earlier demonstrations that these pig models support non-invasive imaging of muscle fibrosis and in vivo CRISPR/Cas9 genome editing, the new microRNA-messenger RNA atlas strengthens the case that tailored porcine dystrophinopathy models can bridge the persistent gap between encouraging rodent studies and the harsh realities of human therapeutic development.

Subject of Research: MicroRNA-mRNA regulatory networks in skeletal muscle of genetically engineered pig models of Duchenne and Becker muscular dystrophy

Article Title: MicroRNA–mRNA Networks in Skeletal Muscle of Tailored Pig Models for Dystrophinopathies

Article References: Reschke, S., Graf, A., Hadlich, F., Jaudas, F., Fröhlich, T., Stirm, M., Klymiuk, N., Krebs, S., Wolf, E., & Ali, A. (2026). MicroRNA–mRNA Networks in Skeletal Muscle of Tailored Pig Models for Dystrophinopathies. Journal of Cachexia, Sarcopenia and Muscle, 17(5), Article e70337. https://doi.org/10.1002/jcsm.70337

Image Credits: AI Generated

DOI: 10.1002/jcsm.70337

Keywords: Duchenne muscular dystrophy, Becker muscular dystrophy, microRNA, dystrophin, exon skipping, pig models, skeletal muscle, RNA sequencing, biomarkers, gene regulation, muscle degeneration, porcine models

The research, led by Çağrı Doğan of the Department of Medical Genetics at Ordu University Training and Research Hospital together with pediatric neurologist Seren Aydın of Ondokuz Mayıs University Hospital, examined every consecutive individual tested for DMD variants at their centre between January 2022 and February 2026. Forty-seven individuals passed through the diagnostic pipeline, 29 female and 18 male, with a median age of 12.8 years. The team applied two complementary molecular techniques: multiplex ligation-dependent probe amplification, or MLPA, a copy-number detection method available for 45 of the 47 participants, and whole-gene sequencing, performed in 16 individuals and run in parallel with MLPA in 13. All variants were described against the reference transcript NM_004006.3 and classified according to the 2015 ACMG/AMP standards for sequence variants and the 2020 ACMG/ClinGen standards for copy-number variants.

The headline result is deceptively simple: pathogenic variants were identified in 19 of the 47 individuals. Ten of these were probands, the first affected or index individuals in their families to receive a molecular diagnosis, and nine were heterozygous female carriers discovered through cascade screening, the systematic testing of relatives once a familial variant is known. In index testing, the diagnostic yield was 30.3 percent, or ten of 33 individuals tested, with a 95 percent confidence interval of 17.4 to 47.3 percent. Once a familial variant was established, cascade screening detected carriers in nine of 14 relatives tested, a carrier-detection rate of 64.3 percent with a confidence interval of 38.8 to 83.7 percent. Those figures underline a point that geneticists have long emphasised: identifying one patient can protect an entire extended family.

The variant spectrum itself conformed closely to what has been reported across Türkiye and worldwide. Among the nine families with characterised variants, seven carried deletions and two carried single-nucleotide sequence variants: a nonsense change, NM_004006.3:c.701C>G producing p.(Ser234Ter), and a canonical splice-site variant, c.2803+1G>T, which disrupts the invariant guanine at the first position of an intron and is predicted to derail RNA splicing. Strikingly, six of the nine families carried deletions within the so-called distal hotspot spanning exons 45 to 55, a known deletion-prone region of this extraordinarily large gene. Three of the deletions were in-frame, meaning the reading frame of the dystrophin protein was preserved, and four were out-of-frame, consistent with the classical reading-frame rule that links out-of-frame disruptions to the severe Duchenne phenotype and in-frame disruptions to the milder Becker form. Each deletion was cross-verified against public resources, the Leiden Open Variation Database and the DOVE deletion-out-of-frame evaluation tool.

The study also captured a subtler and less frequently discussed facet of dystrophinopathy: female carriers with biochemical evidence of muscle damage. Four heterozygous women, three of whom were probands in their own right rather than relatives of an affected boy, presented with unexplained hyperCKemia, with creatine kinase values ranging from 776 to 2,816 U/L, but no documented muscle weakness. The one carrier who underwent complete evaluation had normal strength and a normal echocardiogram. This pattern reflects the stochastic nature of X-chromosome inactivation, the process by which female cells randomly silence one X chromosome. When inactivation happens to favour the chromosome carrying the mutant DMD allele, enough dystrophin-deficient muscle fibres can exist to leak creatine kinase into the blood, occasionally producing overt disease in women, including cardiomyopathy, decades before anyone suspects a condition classically associated with boys.

Two of the deletions were apparently de novo, arising fresh in the affected child rather than being inherited from a carrier mother, a well-recognised phenomenon in DMD given the enormous size of the gene and its high spontaneous mutation rate. Equally consequential for treatment planning, none of the probands in this series carried a variant amenable to an approved exon-skipping oligonucleotide. These antisense drugs, including eteplirsen, golodirsen, viltolarsen and casimersen, work by masking specific exons during RNA splicing so that the reading frame is restored and a shorter but partly functional dystrophin protein can be made. Eligibility depends entirely on the exact exon boundaries of the deletion or point mutation, which is why a precise molecular diagnosis is not merely academic but the gateway to genotype-matched therapy.

Technically, the study illustrates the standard two-step architecture of DMD molecular diagnostics. MLPA, first described by Schouten and colleagues in 2002, uses pairs of probes that ligate only when bound to their target sequences and are then amplified, allowing relative quantification of all 79 exons of the DMD gene in a single reaction; it remains the most efficient way to detect the exon deletions and duplications that account for the majority of pathogenic DMD variants. When MLPA is negative, sequencing of the gene captures the point mutations, small insertions and deletions, and splice-site changes that copy-number assays cannot see. Running both methods in parallel, as this centre did in 13 cases, accelerates turnaround. The authors’ adherence to Human Genome Variation Society nomenclature and to consensus ACMG classification frameworks ensures that their variants can be compared directly with entries in international databases such as the TREAT-NMD global mutation registry, which has catalogued more than 7,000 DMD mutations.

The regional significance of the work lies in establishing a baseline. Previous Turkish series, including a 2019 analysis of 260 dystrophinopathy patients and later single-centre surveys, had sketched the national mutation spectrum, but the Central-Eastern Black Sea provinces had remained a blank spot on that map. The new data suggest that the local spectrum is broadly congruent with national and global patterns, dominated by deletions concentrated in the distal hotspot. That congruence matters for genetic counselling: it means clinicians in the region can reasonably apply the established genotype-phenotype correlations and exon-skipping eligibility frameworks without needing region-specific correction, while still sequencing each family’s variant to confirm its exact boundaries.

The authors are careful about the limits of what such a small series can prove. With only ten probands, the reported proportions are descriptive rather than prevalence estimates, and genotype-based phenotype assignment in individual patients requires prospective confirmation with longitudinal follow-up. The retrospective, single-centre design also means the cohort reflects referral patterns and test availability at one hospital rather than systematic population screening. Still, the cascade-screening outcome, nine additional heterozygotes identified from fourteen relatives, is a concrete demonstration of why molecular diagnosis should trigger immediate family testing. Every carrier identified can be offered cardiac surveillance, reproductive counselling including prenatal and preimplantation genetic options, and the knowledge that ultimately saves the next generation of boys in the family from a delayed diagnosis.

For a disease in which early corticosteroid treatment, standardised cardiac and respiratory care, and emerging gene-targeted therapies all depend on catching patients early, the message from Türkiye’s Black Sea coast is clear: find the variant in one child, and the laboratory work has only just begun. The first regional map of DMD variants is now drawn, and its most important lines may be the family trees radiating outward from each proband.

Subject of Research: Mutational spectrum of DMD gene variants and cascade screening for Duchenne and Becker muscular dystrophy in Türkiye

Article Title: Mutational Spectrum of DMD Gene Variants and Cascade Screening Outcomes in the Central-Eastern Black Sea Region of Türkiye: A Single-Centre Retrospective Study

Article References: Doğan, Ç., & Aydın, S. (2026). Mutational Spectrum of DMD Gene Variants and Cascade Screening Outcomes in the Central-Eastern Black Sea Region of Türkiye: A Single-Centre Retrospective Study. Molecular Biology Reports, 53(1), Article 1600. https://doi.org/10.1007/s11033-026-12785-9

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12785-9

Keywords: Duchenne muscular dystrophy, DMD gene, Becker muscular dystrophy, cascade screening, MLPA, female carriers, exon deletions, genetic testing, dystrophinopathy, hyperCKemia, Türkiye, X-chromosome inactivation