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New Insights into the Genetic Landscape of DMD Gene Mutations in Iran and the Applicability of Exon Skipping Therapies

Clinical Genetics and Therapeutics
  • Primary Categories:
    • Clinical Genetics
  • Secondary Categories:
    • Clinical Genetics
Introduction:
The X-linked DMD gene (MIM:300377) is the largest known human gene encompassing 79 exons and encodes dystrophin, a protein that anchors the cytoskeleton to the plasma membrane. Pathogenic variants in the DMD gene cause Duchene muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), characterized by the growing deterioration of muscle tissue and consequent weakness, with a prevalence of 4.78 and 1.53 per 100,000 males respectively. Regarding the growing evidence of the applicability of molecular therapies for DMD, accurate genetic diagnosis is necessary for genetic counseling and the investigation of possible treatments. Therefore, we retrospectively analyzed data from a cohort of 2,161 subjects referred to the Kariminejad - Najmabadi Pathology & Genetics Center between 2009 and 2024 for genetic diagnosis, carrier detection, and prenatal diagnosis. Our aim was to determine the mutation spectrum of the DMD gene and examine the applicability of mutation-specific treatments among Iranian patients. 

Methods:
The subjects were primarily examined using Multiplex ligation-dependent probe amplification (MLPA) to screen large deletions/duplications and the negative cases were further analyzed to detect potential small mutations using next generation sequencing (NGS) or sanger sequencing. Data filtration and descriptive analysis was conducted using RStudio version 2024.04.2.

Results:
Of the 1,051 male patients referred for MLPA testing, 553 harbored large mutations, resulting in a diagnostic rate of 52.62%. Screening for small mutations among MLPA-negative patients identified 32 pathogenic or likely pathogenic variants using NGS and 18 variants using Sanger sequencing.  Of all 603 mutations detected among the patients, large deletions (77.94%), large duplications (13.43%), stop-gain (4.81%), and frameshift mutations (2.49%) were the most common. In total, 80% of the affected cases exhibited mutations in exons 44–55 (44.41%) and exons 3–27 (35.59%), whereas the remaining cases had mutations in other exons. Large deletions occurred in exons 44-55 and exons 3-27 in 52.98% and 29.73% of cases, respectively. Large duplications occurred in exons 3-27 in 58.59% of the cases (mainly exons 2-11 (31.46%)). In addition, 69.39% of point mutations occurred in the first 40 exons. Carrier testing of 911 females revealed 203 (22.28%) carriers. Of the 123 fetuses referred for prenatal diagnosis, 113 were screened using MLPA, identifying 31 affected cases (27.43%), and 10 were screened using Sanger sequencing, with six fetuses (60.00%) being affected. The study demonstrated that 178 patients with large deletions (29.52% of all patients) were eligible for FDA-approved exon-skipping treatments.

Conclusion:
To the best of our knowledge, this study is the largest reported cohort of patients with DMD mutations in Iran. Consistent with large DMD databases, such as UMD-DMD and TREAT-NMD, as well as previous studies from Iran, large deletions account for most cases, with stop-gain mutations being the most prevalent among small mutations. Based on previous reports, exons 2-20 and 45-55 are generally considered proximal and distal hotspots, respectively. While the distal hotspot in our study remained consistent with prior findings, the proximal hotspot appeared to be broader (exons 3-27), even when compared with previous studies in Iran. Notably, large deletions in our study showed a shift from distal hotspots (52.98%) to proximal hotspots (29.73%), compared to the 65-70% and approximately 15% reported in these regions in previous studies. In contrast, large duplications remained consistent with previous reports, and unlike the study by Zamani et al. from Iran, no hotspots were detected in the distal region for duplications. Our findings provide a comprehensive picture of the mutation spectrum of DMD in Iran with a larger sample size and help in genetic counseling, categorizing eligible cases for treatment, and future advances in DMD treatments.

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