Abstract
Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.
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Acknowledgements
We thank the NF2 patient reported here and his relatives who participated in this study. We thank Juana Fernández-Rodríguez for advice and reagents, Josep Biayna and Bernat Gel for support, Eugeni Aragall and Pilar Armengol for their help with sequencing and also the HUGTiP-IGTP Biobank. This work has been supported by the Institut de Medicina Predictiva i Personalitzada del Càncer (IMPPC); the Government of Catalonia (2009SGR290); and the Spanish Ministry of Science and Innovation, Carlos III Health Institute (ISCIII-RTICC RD06/0020/1051); (PI11/1609). CL, IB and ES thank the continuous support from the Spanish association of Patients with Neurofibromatosis (Asociación de Afectados de Neurofibromatosis) and the Asociación Española Contra el Cáncer (AECC).
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Castellanos, E., Rosas, I., Solanes, A. et al. In vitro antisense therapeutics for a deep intronic mutation causing Neurofibromatosis type 2. Eur J Hum Genet 21, 769–773 (2013). https://doi.org/10.1038/ejhg.2012.261
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DOI: https://doi.org/10.1038/ejhg.2012.261
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