Consistent with the prediction that -2232 C>G would lead to loss of a GRE half-site, gel shift assays performed using hypothalamic nuclear extract (generated from IVB cells, which expresses glucocorticoid receptors and in which corticosteroid effects on CRH expression have been extensively studied) demonstrated an attenuation in protein binding to -2232G probes (Figure 1C). Similar results were obtained using a nuclear extract enriched for GR (data not shown). To test whether corticosteroid-sensitivity of the CRH regulatory region was altered as a consequence of this SNP, reporter assays were performed in HT22 cells transfected with -2232C or G pDsRed constructs. There were main effects of treatment (F(2,27) = 11.8, P = 0.0002) and genotype (F(1,27) = 4.7, P = 0.04). A significant decrease in reporter activity following cortisol treatment was observed in forskolin-stimulated cells expressing the -2232 C allele constructs (Figure 1D, Tukey-Kramer, P < 0.05), but this effect was not observed in those transfected with G allele constructs (Figure 1D, n.s.) (No Treatment: C, N=5, G, N=4; Forskolin: C, N=6, G, N=5; Forskolin + Cort: C, N=6, G, N=7).
