Cardio-facio-cutaneous (CFC) syndrome is one of the 'RASopathies', a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS-MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here Braf(Q241R/+)) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. Braf(Q241R/+) mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. Braf(Q241R/+) embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in Braf(Q241R/+) embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome.

Activation of the RAS pathway has been implicated in oncogenesis and developmental disorders called RASopathies. Germline mutations in BRAF have been identified in 50-75% of patients with cardio-facio-cutaneous (CFC) syndrome, which is characterized by congenital heart defects, distinctive facial features, short stature and ectodermal abnormalities. We recently demonstrated that mice expressing a Braf Q241R mutation, which corresponds to the most frequent BRAF mutation (Q257R) in CFC syndrome, on a C57BL/6J background are embryonic/neonatal lethal, with multiple congenital defects, preventing us from analyzing the phenotypic consequences after birth. Here, to further explore the pathogenesis of CFC syndrome, we backcrossed these mice onto a BALB/c or ICR/CD-1 genetic background. On a mixed (BALB/c and C57BL/6J) background, all heterozygous Braf(Q241R/+) mice died between birth and 24 weeks and exhibited growth retardation, sparse and ruffled fur, liver necrosis and atrial septal defects (ASDs). In contrast, 31% of the heterozygous Braf(Q241R/+) ICR mice survived over 74 weeks. The surviving Braf(Q241R/+) ICR mice exhibited growth retardation, sparse and ruffled fur, a hunched appearance, craniofacial dysmorphism, long and/or dystrophic nails, extra digits and ovarian cysts. The Braf(Q241R/+) ICR mice also showed learning deficits in the contextual fear-conditioning test. Echocardiography indicated the presence of pulmonary stenosis and ASDs in the Braf(Q241R/+) ICR mice, which were confirmed by histological analysis. These data suggest that the heterozygous Braf(Q241R/+) ICR mice show similar phenotypes as CFC syndrome after birth and will be useful for elucidating the pathogenesis and potential therapeutic strategies for RASopathies.