Germline mutations in BRAF and other components of the RAS/MAPK pathway are found in RASopathies, whose features include short stature and pubertal delay. The underlying mechanism of endocrinopathies in RASopathies has not been fully elucidated. We report four BRAF mutations (two of which are novel) in four children with congenital hypopituitarism and RASopathy features. To demonstrate the functional role of the variants we performed phosphoproteomic analyses using mass spectrometry and identified that they significantly increase B-Raf kinase activity resulting in hyper-phosphorylation of members of the RAS/MAPK and JAK/STAT pathways. To validate our results we assessed the levels of phosphorylated ERK as a readout of RAS/MAPK pathway acitivity and confirmed that the variants increase levels of phosphorylated ERK demonstrating that the BRAF genetic variants are pathogenic 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 × BrafV600E/V600E genetic crosses showed a significant deviation from the expected Mendelian ratio, indicating embryonic lethality. Prop1:Cre;BrafV600E/+ pups exhibited dwarfism and died prematurely suggesting a functional compromise of the HP-axis. Pituitary specification markers including Lhx3, Pitx1 and Hesx1 were appropriately expressed during early embryogenesis. However, there was an impairment of cell lineage determination with increased expression of POMC1 and reduced expression of PIT1. Furthermore, 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 findings relate mutations in the RAS/MAPK pathway to defects in pituitary development and demonstrate that activating BRAF mutations present during pituitary development lead to congenital hypopituitarism. We propose that children with germline mutations in the RAS/MAPK pathway should undergo pituitary function monitoring and that a diagnosis of RASopathies needs to be kept in mind in children with hypopituitarism.
22 - 24 Nov 2017
British Society for Paediatric Endocrinology and Diabetes