Gómez-Domínguez, D.; Epifano, C.; Miguel, F.; Castaño, A.G.; Vilaplana-Martí, B.; Martín, A.; Amarilla-Quintana, S.; Bertrand, A.T.; Bonne, G.; Ramón-Azcón, J.; Rodríguez-Milla, M.A.; Pérez de Castro, I. Consequences of Lmna Exon 4 Mutations in Myoblast Function. Cells2020, 9, 1286.
Gómez-Domínguez, D.; Epifano, C.; Miguel, F.; Castaño, A.G.; Vilaplana-Martí, B.; Martín, A.; Amarilla-Quintana, S.; Bertrand, A.T.; Bonne, G.; Ramón-Azcón, J.; Rodríguez-Milla, M.A.; Pérez de Castro, I. Consequences of Lmna Exon 4 Mutations in Myoblast Function. Cells 2020, 9, 1286.
Laminopathies are causally associated with mutations on Lamin A gene (LMNA). To date, more than 400 mutations in LMNA have been reported in patients. These mutations are widely distributed throughout the entire gene and are associated with a wide range of phenotypes. Unfortunately, little is known about the mechanisms underlying the effect of the majority of these mutations. This is the case of more than 40 mutations that are located at exon 4. Using CRISPR/Cas9 technology, we have generated a collection of Lmna exon 4 mutants in mouse C2C12 myoblasts. These cell models include different types of exon 4 deletions and the presence of R249W mutation, one of the human variants associated with a severe type of laminopathy (LMNA-associated congenital muscular dystrophy). We have characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and p-ERK and p-AKT levels. Our results indicate that Lmna exon 4 mutants show abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC complex are altered in all these mutants. Whereas no significant differences were observed for ERK and AKT activities, the accumulation of DNA damage was associated to the Lmna p.R249W mutant myoblasts. Finally, significant myogenic differentiation defects were detected in the Lmna exon 4 mutants. These results have key implications in the development of future therapeutic strategies for the treatment of laminopathies.
LMNA; laminopathy; CRISPR; nuclear envelope
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