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