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Chemical mutational analysis of the human glucocorticoid receptor cDNA in glucocorticoid-resistant bronchial asthma
Ist Teil von
American journal of respiratory cell and molecular biology, 1994-07, Vol.11 (1), p.42
Ort / Verlag
United States
Erscheinungsjahr
1994
Link zum Volltext
Quelle
MEDLINE
Beschreibungen/Notizen
Corticosteroid-resistant (CR) asthma is not caused by altered bioavailability of the administered drug, altered ligand-binding characteristics, or altered nuclear translocation of the activated human glucocorticoid receptor (hGR) complex. We have tested the hypothesis that CR asthma results from a consistent polymorphism in the functionally diverse hGR cDNA using the sensitive screening technique of polymerase chain reaction (PCR) amplification and chemical mutational analysis. Total RNA was extracted from peripheral blood monocytes derived from six corticosteroid-sensitive (CS) and six CR asthmatic subjects. The RNA was reverse transcribed, and overlapping hGR cDNA fragments were amplified by nested PCR. Double-stranded hGR cDNA fragments were hybridized to corresponding 32P-5'-labeled wild-type fragments, chemically modified with osmium and hydroxylamine, and cleaved with piperidine. The resultant cleaved strands were detected by autoradiography. As controls, single base pair mutated hGR cDNA fragments sensitive to hydroxylamine and osmium modification were used. Using this technique, we did not detect any base pair mismatch between the six CS and six CR patients and the corresponding wild-type hGR, despite a 100% detection of control mutations. We conclude that the defect in CR asthma does not lie in the structure of the hGR.