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Open AccessArticle

Consequences of Lmna Exon 4 Mutations in Myoblast Function

1
Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo km2.2, E-28029 Madrid, Spain
2
Fundación Andrés Marcio, niños contra la laminopatía, C/Núñez de Balboa, 11, E-28001 Madrid, Spain
3
Universidad Europea de Madrid, C/ Tajo, s/n, E-28670 Villaviciosa de Odón, Spain
4
Institute for Bioengineering of Catalonia (IBEC), C/Baldiri Reixac, 10-12, E-08028 Barcelona, Spain
5
Fundación de Investigación HM Hospitales, Plaza del Conde Valle Suchil, 2, E-28015 Madrid, Spain
6
Sorbonne Université, INSERM UMRS 974, Center of Research in Myology, Institut de Myologie, 75013 Paris, France
7
ICREA-Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain
*
Author to whom correspondence should be addressed.
Cells 2020, 9(5), 1286; https://doi.org/10.3390/cells9051286
Received: 13 April 2020 / Revised: 6 May 2020 / Accepted: 16 May 2020 / Published: 21 May 2020
Laminopathies are causally associated with mutations on the Lamin A/C 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 generated a collection of Lmna exon 4 mutants in mouse C2C12 myoblasts. These cell models included 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 (L-CMD). We characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and levels of p-ERK and p-AKT (phosphorylated Mitogen-Activated Protein Kinase 1/3 and AKT serine/threonine kinase 1). Our results indicated that Lmna exon 4 mutants showed abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC (LInker of Nucleoskeleton and Cytoskeleton) complex were 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.
Keywords: LMNA; laminopathy; CRISPR; nuclear envelope LMNA; laminopathy; CRISPR; nuclear envelope
MDPI and ACS Style

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.

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