Dataset Information

MLPA followed by target-NGS to detect mutations in the dystrophin gene of Peruvian patients suspected of DMD/DMB.

ABSTRACT:

Background

We report the molecular analysis of the DMD gene in a group of Peruvian patients with Duchenne/Becker dystrophinopathy. This is the first study to thoroughly characterize mutations in this population.

Methods

We used the combination of multiplex ligation-dependent probe amplification (MLPA) and sequencing analysis of the DMD gene. We recruited Peruvian patients in 2 years from reference national hospitals. We performed DNA tests in 152 patients, checking first exon deletion/duplication by MLPA, and subsequently, if negative, samples were sequenced to detect point mutations.

Results

The average age for diagnosis was 9.8 years, suggesting a delay for timely diagnosis and care. We found causal DMD mutations in 125 patients: 72 (57.6%) exon deletions/duplications (41.6% deletions, 16.0% duplications), and 53 (42.4%) point mutations (27.2% nonsense, 9.6% small indels, and 5.6% splice site).

Conclusion

Due to our genetic background, we expected a higher number of novel and recurrent causal mutations in our sample. Results showed 16% of novel mutations, similar to other well-studied populations.

SUBMITTER: Guevara-Fujita ML

PROVIDER: S-EPMC8457708 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining.ResultsWe generated a Dmd (EGFP) reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes.ConclusionsThe novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.