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. Author manuscript; available in PMC: 2011 Jul 20.
Published in final edited form as: N Engl J Med. 2011 Jan 20;364(3):215–225. doi: 10.1056/NEJMoa0911064

Figure 2. Loss-of-Function Mutations in Patients with Hypothalamic Amenorrhea.

Figure 2

Panels A and B show the structures of FGFR1 and PROKR2, respectively, as well as the mutations of interest. Panels C, D, and E show that the FGFR1 G260 and R756 amino acids and the PROKR2 R85 amino acid are highly conserved across vertebrate species. Panels F, G, and H show that the overall expression levels of FGFR1 G260E and R756H were normal, whereas PROKR2 R85H expression levels were decreased (P<0.01), as compared with wild-type levels. Heat-shock protein 90 (HSP90) was a positive control for gel loading. An empty vector (EV) was used as a negative control for protein expression. Panels I, J, and K show that the receptor cell-surface expression levels in COS-7 cells were similar to the wild-type levels for both FGFR1 mutants but were significantly decreased for PROKR2 R85H (P<0.001). Panels L, M, and N show that the FGFR1 G260E mutant has decreased fibro-blast growth factor 8 (FGF8)-induced osteocalcin FGF response element (OCFRE) activity as compared with the wild type (P<0.001), that the FGFR1 R756H mutant has decreased FGF2-induced OCFRE activity as compared with the wild type (P<0.001), and that the PROKR2 R85H mutant has decreased PROK2-induced early growth response 1 (Egr-1) activity as compared with the wild type (P<0.001). D1, D2, and D3 denote the ligand-binding domains of FGFR1; Luc the lucifer-ase reporter vector; and TM the transmembrane domain of FGFR1. T and I bars indicate standard errors of the means of two experiments performed in triplicate for the gene-reporter assays or the means of three experiments performed in quadruplicate for the antibody-binding assays.