Further characterization of human glucocorticoid receptor mutants, R477H and G679S, associated with primary generalized glucocorticoid resistance

Primary generalized glucocorticoid resistance is a rare condition characterized by a generalized insensitivity to glucocorticoids, to some extent due to an impaired function of the glucocorticoid receptor. Our earlier genetic analysis of the human glucocorticoid receptor (hGR) in 12 unrelated patients with primary generalized glucocorticoid resistance revealed two new mutations, R477H in exon 4 and G679S in exon 8 in two patients. In order to further study the molecular mechanisms underlying the phenotype of these mutations we have investigated their effect on glucocorticoid signal transduction.We have studied the DNA-binding ability of the R477H mutant with an electrophoretic mobility shift assay (EMSA). The ability of the R477H and the G679S mutants to affect TNFα induced NF-κB activity and wild-type GR signalling was studied in transient transfection assays.In EMSA the R477H mutation showed a reduced ability to bind to a glucocorticoid-response element compared to the wild-type GR. In transient transfection assays both the R477H mutant and the G679S mutant showed a dominant negative effect on co-transfected wild-type GR in Cos 7 cells. However, both mutants showed full capacity to repress TNFα-induced NF-κB activity.The impaired DNA-binding of the hGR, R477H mutant may explain the severe phenotype of cortisol resistance seen with this mutation. The dominant negative effects of both mutants on wild-type GR signalling probably contribute to the patients' cortisol resistance.