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Functional restoration of mouse Nf1 nonsense alleles in differentiated cultured neurons

Abstract

Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant genetic disorders, is caused by mutations in the NF1 gene. NF1 patients have a wide variety of manifestations with a subset at high risk for the development of tumors in the central nervous system (CNS). Nonsense mutations that result in the synthesis of truncated NF1 protein (neurofibromin) are strongly associated with CNS tumors. Therapeutic nonsense suppression with small molecule drugs is a potentially powerful approach to restore the expression of genes harboring nonsense mutations. Ataluren is one such drug that has been shown to restore full-length functional protein in several models of nonsense mutation diseases, as well as in patients with nonsense mutation Duchenne muscular dystrophy. To test ataluren’s potential applicability to NF1 nonsense mutations associated with CNS tumors, we generated a homozygous Nf1R683X/R683X-3X-FLAG mouse embryonic stem (mES) cell line which recapitulates an NF1 patient nonsense mutation (c.2041 C > T; p.Arg681X). We differentiated Nf1R683X/R683X-3X-FLAG mES cells into cortical neurons in vitro, treated the cells with ataluren, and demonstrated that ataluren can promote readthrough of the nonsense mutation at codon 683 of Nf1 mRNA in neural cells. The resulting full-length protein is able to reduce the cellular level of hyperactive phosphorylated ERK (pERK), a RAS effector normally suppressed by the NF1 protein.

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Acknowledgements

This work was supported by grants to AJ (616394) and SAW (563749) from the Gilbert Family Foundation. We thank Pengpeng Liu and Sneha Suresh for helpful discussions, and PTC Therapeutics Inc. for providing ataluren.

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CW and AJ conceived and designed the work. CW performed experiments. SI and SAW provided technical support. CW and AJ wrote the manuscript with input from all authors.

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Correspondence to Allan Jacobson.

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AJ is co-founder, director, and SAB chair of PTC Therapeutics Inc. All other authors declare no competing interests.

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Wu, C., Iyer, S., Wolfe, S.A. et al. Functional restoration of mouse Nf1 nonsense alleles in differentiated cultured neurons. J Hum Genet (2022). https://doi.org/10.1038/s10038-022-01072-7

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