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Endocrine Abstracts (2013) 31 OC2.6 | DOI: 10.1530/endoabs.31.OC2.6

SFEBES2013 Oral Communications Steroids and thyroid (8 abstracts)

An N-ethyl-N-nitrosourea induced Corticotrophin releasing hormone promoter mutation provides a mouse model of Cushing's syndrome

Liz Bentley 1 , Christopher T Esapa 1, , M Andrew Nesbit 1, , Rosie A Head 1, , Holly Evans 1, , Darren Lath 1, , Tertius A Hough 1, , Christine Podrini 1, , William D Fraser 1, , Peter I Croucher 1, , Matthew A Brown 1, , Steve D M Brown 1 , Roger D Cox 1 & Rajesh V Thakker 1,

1MGU, MRC Harwell, Oxford, UK; 2OCDEM, University of Oxford, Oxford, UK; 3Mellanby Centre for Bone Research, University of Sheffield, UK; 4MLC, MRC Harwell, Oxford, UK; 5Sanger Institute, Cambridge, UK; 6University of East Anglia, Norwich, UK; 7Garvan Institute, University of New South Wales, Australia; 8Diamantina Institute, University of Queensland, Australia.

Cushing’s syndrome, which is characterised by excessive circulating glucocorticoid (GC) concentrations, may be due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumours, respectively. In the course of our phenotype-driven screens of mouse mutants induced by the chemical mutagen N-ethyl-N-nitrosourea (ENU), we observed a mutant mouse with obesity, hyperglycaemia and low bone mineral density, features that are consistent with Cushing’s syndrome. This phenotype was inherited as an autosomal dominant trait and the disease locus was mapped to chromosome 3 and to a 6.6 Mbp interval that contained the gene encoding corticotrophin releasing hormone (Crh). DNA sequence analysis of the Crh gene did not identify any coding region mutations, in affected mice, but instead a T to C transition at −120 bp relative to the transcription start site, within the Crh promoter, was identified. Luciferase reporter assays demonstrated that this T to C transition resulted in a greater than twofold increase in transcription activity in Neuro2a cells (P<0.001) and was thus a gain-of-function mutation. Crh120/+ mice, when compared to wild-type littermates (Crh+/+mice), had obesity, muscle wasting, thin skin, hair loss, elevated plasma concentrations of corticosterone (mean±SEM: Crh120/+=721.8±88.8 ng/ml; Crh+/+=414.3±59.5 ng/ml, P=0.01) and 24 h urinary concentrations of corticosterone (Crh120/+=504.3±14.9 ng/ml; Crh+/+=141.9±30.5 ng/ml, P<0.0001). In vivo assessment of Crh120/+ mice revealed them to have elevated plasma concentrations of glucose (P=0.01), insulin (P<0.0001), leptin (P<0.01), cholesterol (P<0.001) and triglycerides (P<0.01). Crh120/+ mice also had low bone mineral density (P<0.0001), hypercalcaemia (P<0.001), hypercalciuria (P<0.0001) and decreased concentrations of plasma parathyroid hormone (P<0.05) and osteocalcin (P<0.0001). Thus, a mouse model for Cushing’s syndrome has been established and this will help in further elucidating the pathophysiological effects of GC excess.

Declaration of funding

This work was supported by the Medical Research Council UK (including grant numbers G0600702/1, G9825289 and G1000467); and European Union Framework 7 (grant number FP7-200800, TREAT-OA).

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