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BRAF mutations disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mouse and humans

ABSTRACT: Germline mutations in BRAF and other components of the Mitogen-Activated Protein Kinase (MAPK) pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harboring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAFp.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression in cell cycle inhibitors, cell growth arrest and apoptosis but not tumour formation. Our findings show a critical role for BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Kidney Cell Line, Kidney

DISEASE(S): Mutational_hotspot

SUBMITTER: Vinothini Rajeeve

LAB HEAD: Vinothini Rajeeve, Pedro Maria Casado-Izquierdo, Pedro Cutillas

PROVIDER: PXD018190 | Pride | 2020-11-17

REPOSITORIES: Pride

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Project description:The oncogenic mutation BRAFV600E is a common event in nevi and melanomas, which are aggressive skin tumors characterized by MAPK signaling pathway activation. It has been observed that mutated benign melanocytic lesions can remain unchanged for decades, but also proliferate or undergo oncogene-induced senescence. The purpose of this study was to investigate the presence of a gene expression signature in BRAFV600E mutated nevi compared to wild-type ones, all derived from sun exposed sites. Microdissected tissues from excisional biopsies of acquired nevi were analyzed to detect the presence of BRAF and NRAS mutations and to profile whole genome expression by means of oligonucleotide microarrays. BRAFV600E mutation was evidenced in 64% of nevi, while no NRAS mutations were detected. Functional analysis of genes differentially expressed between wild-type and mutated lesions pointed out the role of oxidative stress in causing the oncogenic BRAF mutation and that the direct consequence of constitutive activation of BRAF-MEK-ERK pathway, in a benign contest, is the activation of apoptosis, autophagy and senescence. Our data also indicate that in mutated lesions, p53 plays a crucial role in the maintenance of the benign status. In our study, in order to understand the role of BRAF mutation in acquired nevi, we performed gene expression profiling analysis on fourteen lesions derived from intermittently exposed sites, using whole genome oligonucleotide microarrays. Expression data were coupled with sequencing results on BRAF and NRAS genes and class comparison algorithms were applied to detect sequences differentially expressed between BRAFV600E and BRAFwt-NRASwt lesions. Some of the obtained results were further validated by Real-Time RT-PCR on an independent cohort of samples. Mutation analysis evidenced BRAF mutations in 64% of the common acquired nevi. All the mutations consisted in the substitution V600E (T1799A). No alteration was found on exon 11 of the BRAF gene nor on exons 1 and 2 of the NRAS gene, as expected since no lesion came from chronically exposed sites. Transcriptome analysis was carried out using oligo-microarrays and differential expression was tested applying the Linear Models package LIMMA available at www.bioconductor.org that uses Bayesian statistics to compute the probability of a gene being differentially expressed. Only 25 transcripts had B value greater then 2. We then performed a one-way ANOVA, dividing samples into three classes according to their BRAF mutational status (wt, ++, +++) as assessed by pyrosequencing. We found 2882 known transcripts by selecting those with ANOVA p-value<0.01 and increasing or decreasing expression trends along the three classes. Functional analysis was performed using the MetaCoreTM software package to identify overrepresented functional processes. BRAF signature, acquired nevi, gene expression profiling "++ and +++" refer to the percentage of alleles carrying the mutation in the *BRAF*V600E lesions. This has been assessed by pyrosequencing analysis (Venesio et al, 2008. PMID= 18408659).

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BRAF mutations disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mouse and humans

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