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Allele-Specific Binding of Airway Nuclear Extracts to Polymorphic ₂-Adrenergic Receptor 5' Sequence

Department of Medicine, Cardiopulmonary Genomics Program, University of Maryland School of Medicine, Baltimore, Maryland

Correspondence and requests for reprints should be addressed to Stephen B. Liggett, M.D., Department of Medicine, Cardiopulmonary Genomics Program, University of Maryland School of Medicine, 20 Penn Street, HSF-II, Room S-114, Baltimore, MD 21201-1075. E-mail: sligg001{at}umaryland.edu

Like other intronless G protein–coupled receptor genes, the ₂-adrenergic receptor (₂AR) has minimal genetic space for population variability, and has attained such via multiple coding and noncoding polymorphisms. Yet most clinical studies use the two nonsynonymous polymorphisms of the coding region for association analysis despite low levels of linkage disequilibrium with some promoter and 5'UTR polymorphisms. To assess the potential for allele-specific transcription factor binding to ₂AR 5'-flanking sequence, 3'-biotin–labeled oligonucleotide duplexes were synthesized. Each was centered on variable sites representing major or minor alleles found in the human population with frequencies of 5% or greater (20 polymorphic sites). Electrophoretic mobility shift assays were performed using human airway smooth muscle or airway epithelial cell nuclear extracts. Many of these polymorphisms resulted in an alteration in binding, and both major allele and minor allele dominance were observed. For example, in airway smooth muscle nuclear extracts, 10 polymorphisms decreased and 2 increased binding, whereas 5 showed no differences. Concordance between airway smooth muscle and epithelial cell nuclear extract binding to polymorphic alleles was found in only 50% of cases. There was no tendency for the rare variants to be more likely to have altered nuclear extract binding compared to the more common variants. Taken together, these results provide potential mechanisms by which ₂AR 5'-flanking polymorphisms affect obstructive lung phenotypes.

Key Words: asthma • • -agonist • polymorphism

CLINICAL RELEVANCE The variability in the response to -agonists in obstructive lung disease is not fully understood. We show that polymorphisms in the promoter 5'-flanking region of the receptor alter transcription factor binding.