Am. J. Respir. Cell Mol. Biol., Vol 11, No. 1, 07 1994, 42-48.
Chemical mutational analysis of the human glucocorticoid receptor cDNA in glucocorticoid-resistant bronchial asthma
SJ Lane, JP Arm, DZ Staynov and TH Lee
Department of Allergy and Allied Respiratory Disorders, U.M.D.S., Guy's Hospital, London, United Kingdom.
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.
