Allele-specific binding of airway nuclear extracts to polymorphic beta2-adrenergic receptor 5' sequence

Abstract

Like other intronless G protein-coupled receptor genes, the beta(2)-adrenergic receptor (beta(2)AR) 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 beta(2)AR 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 approximately 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 beta(2)AR 5'-flanking polymorphisms affect obstructive lung phenotypes.

Figure 1.

Localization of polymorphisms of the β₂AR 5′-flanking region. Shown are the nucleotides listed as major/minor followed by the allele frequency of the minor allele in parenthesis. Where there were differences between allele frequencies between whites and African Americans, the higher frequency is given (information compiled from References 11 and 14). *This polymorphism is within a small open reading frame, the 5′-leader cistron (10), and was not studied.

Figure 2.

Electrophoretic mobility shift assays (EMSAs) using β₂AR polymorphic oligonucleotide duplexes and human airway smooth muscle cell nuclear extracts. Shown are representative results where the major allele has greater binding than the minor ([A] position −3,459), and where the minor allele has greater binding than the major allele ([B] position −2,051). Also shown is a representative experiment showing equivalent binding between the allelic oligonucleotide duplexes ([C] position −2,387) and one where there was no detectable binding ([D] position −468). See Tables 1 and 2 for mean data and statistical analysis. NE, nuclear extract; UC, unlabeled competitor oligonucleotide.

Figure 3.

Allele-specific binding of epithelial and smooth muscle nuclear extracts to polymorphic β₂AR oligonucleotides. Shown are the results from EMSAs performed with oligonucleotide duplexes designed around the human polymorphic sites shown (see Materials and Methods). Results in (A) are reported as the ratio of the minor allele to the major allele signal. The dashed lines show ±75% changes from unity (solid line). (B) Results are summarized as shown by the color code to indicate differences between EMSA ratios by cell-type nuclear extract (green, minor < major; yellow, minor > major; blue, minor = major; white, no binding). A relevant difference was defined as a ±15% or greater difference (P < 0.05). P values and the number of experiments are given in Tables 1 and 2.

Figure 4.

Absence of a relationship between allele frequency and nuclear extract binding ratio. Shown are results from BEAS-2B experiments, plotted against the minor allele frequency for each polymorphism (higher of the two when different between whites and African Americans).