Somatic activating mutations in BRAF, encoding B-Raf, have been described in tumours and recently craniopharyngiomas. Germline mutations in BRAF and other components of the RAS/MAPK pathway are found in RASopathies, whose features include endocrine deficiencies but not craniopharyngiomas. We report three BRAF mutations (two of which are novel) in four children with congenital hypopituitarism. To demonstrate the functional role of the three variants we assessed the levels of phosphorylated ERK as a readout of RAS/MAPK pathway activity and performed phosphoproteomic analyses using Mass Spectrometry. We identified that the BRAF mutations increase levels of phosphorylated ERK and significantly increase B-Raf kinase activity resulting in hyper-phosphorylation of members of the RAS/MAPK and the JAK/STAT pathways demonstrating that the BRAF genetic variants are activating mutations. To further demonstrate the role of activated RAS/MAPK pathway in hypopituitarism, we used a murine transgenic approach to express the activating Braf V600E mutation using a pituitary-specific Cre reporter line, Prop1:Cre. Genotypes of offspring from Prop1:Cre x BrafV600E/V600E genetic crosses showed a significant deviation from the expected Mendelian ratio, indicating embryonic lethality. Prop1:Cre;BrafV600E/+ pups exhibited dwarfism and premature death suggesting a functional compromise of the HP-axis. Pituitary specification markers such as Lhx3, Pitx1 and Hesx1 were found to be appropriately expressed during early embryogenesis. However, pituitary glands exhibited hyperplasia with multiple clefts due to an increase in mitotic index at E11.5 and E13.5 (P<0.01). Immunohistochemistry at E16.5 revealed impaired terminal differentiation of hormone-producing cells with an increase in ACTH and prolactin but absence of all other hormone-producing cells. Our data demonstrate that activating BRAF mutations present during pituitary development lead to congenital hypopituitarism both in mouse and humans. Our findings relate mutations in the RAS/MAPK pathway to pituitary abnormalities and suggest that activating BRAF mutations in pituitary stem cells during embryogenesis do not cause tumours.
06 Nov 2017 - 08 Nov 2017