Mice with missense and nonsense NF1 mutations display divergent phenotypes compared with human neurofibromatosis type I

Abstract

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models available for assessing patient-specific mutations. In order to test therapeutic approaches that might restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681*) and a missense mutation (c.2542G>C; p.Gly848Arg). The latter is associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1(Arg681*) and missense NF1(Gly848Arg) mutations have different effects on neurofibromin expression in the mouse and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1(Gly848Arg) mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1(Arg681*) mutation are not viable. Mice with one Nf1(Arg681*) allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf1(4F/Arg681*); DhhCre) display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for preclinical testing of novel nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in individuals with NF1.

Keywords: Missense mutation; Neurofibromatosis type 1; Nonsense mutation; Patient-derived mouse models.

Fig. 1.

Nf1^{Arg681*/Arg681*} mice fail to develop after E9.5. (A,B) Morphology of representative wild-type (A) and Nf1^{Arg681*/Arg681*} (B) embryos at E10.5. The red arrowhead in B indicates effusion into the pericardial space. The magnification is noted in the lower right corner. (C-F) Western blot of neurofibromin (C) and phosphorylated ERK (pERK) (E) in E9.5 MEFs of indicated genotypes. Results are representative of three independent experiments or samples. β-tubulin is shown as loading control. The western blot analysis of neurofibromin (D) and pERK (F) was quantified, and the results are expressed in histograms. *P<0.05, **P<0.01, ***P<0.001 vs wild type by one-way ANOVA. Data are presented as mean±s.e.m. MEFs, mouse embryonic fibroblasts. N.D., none detected.

Fig. 2.

Nf1^4F/Arg681*; Dhh-cre mice develop neurofibromas along the spinal column and display disrupted ultrastructure. (A) Schematic illustration of the strategy used to breed the Nf1^4F/Arg681*; Dhh-Cre mice. The circle indicates the targeted mutation, triangles represent LoxP sites. (B-D) Gross dissection of spinal neurofibromas (B) and H&E staining (C) of neurofibromas along spinal cord of Nf1^{Arg681*/Arg681*}; Dhh-Cre mice. Red dashed lines demarcate the neurofibroma boundaries. (D) A higher magnification demonstrating classic neurofibroma histology. (E,F) The aberrant ultrastructure of the neurofibromas as shown by electron microscopy of the Cre-negative control in (E) and the mutant in (F). The white arrow and red arrowhead indicate normal myelinated axons or disrupted nonmyelinating axons, respectively. The black arrowhead indicates collagen deposits. The magnifications are as indicated. Scale bars: 40 μm in D, 1 μm in E,F.

Fig. 3.

Nf1^4F/4F; DhhCre mice develop neurofibromas and sciatic nerve lesions. (A,B) Gross dissection (A) and H&E staining (B) of spinal tumors in Nf1^4F/4F; DhhCre mice. Red dashed lines demarcate the neurofibroma boundaries. Red arrows indicate neurofibromas. (C,D) The ultrastructure, as shown by electron microscopy, shows similar lesions in the Nf1^4F/Arg681*; DhhCre mice in both neurofibromas (C) and sciatic nerves (D). Black arrows indicate abnormal nonmyelinating axons. The magnifications are as indicated. Scale bars: 4 μm in C,D.

Fig. 4.

The missense Nf1^Gly848Arg allele impairs neurofibromin expression and function. (A-D) Western blots of neurofibromin (A) and pERK (C) in E13.5 MEFs from three independent experiments. β-tubulin is shown as loading control. The western blot analysis of neurofibromin (B) and pERK (D) was quantified and the results expressed in histograms. (E,F) Electron micrographs from spinal nerve roots of control (E) and Nf1^{Gly848Arg/Gly848Arg} (F) mice. White arrows represent normal nonmyelinating axons. *P<0.05 vs wild type by one-way ANOVA. Data are presented as mean±s.e.m. Scale bars: 4 μm in E,F.