Identification of novel polymorphisms within the promoter region of the human beta2 adrenergic receptor gene

Abstract

By screening the 1470 bp 5' to the start codon of the human beta2 adrenergic receptor gene, we have identified a total of eight polymorphisms (-20 T-->C, -47 T-->C, -367 T-->C, -468 C-->G, -654 G-->A, -1023 G-->A, -1343 A-->G and -1429 T-->A c.f. beta2 adrenergic receptor start codon). Transient transfection of 5' flanking deletion luciferase reporter constructs demonstrated the majority of activity of the human beta2 adrenergic gene 5' flanking region to be present within a 549 bp fragment immediately upstream from the start codon. Because of linkage disequilibrium, some combinations of polymorphisms were particularly frequent. We transiently transfected COS-7 cells with luciferase constructs under the control of the 549 bp of 5' flanking DNA containing the two most frequent extended haplotypes in this region. Luciferase activity was significantly reduced in cells transfected with the 'mutant' construct (-20C, -47C, -367C, -468G) c.f. the 'wild-type' construct (-20T, -47T, -367T, -468C). These data suggest that polymorphisms have the potential to alter human beta2 adrenergic receptor gene expression.

Figure 1

Relative promoter activity of the human β₂ adrenergic receptor gene in COS-7 cells. Schematic drawings of the constructs, indicating the restriction enzyme sites used for subcloning into pGL3 Enhancer, are shown on the left. Nomenclature of the constructs refers to the number of nucleotides (in kbp) of 5′ flanking region relative to the translational start codon. Firefly luciferase activity was normalized to Renilla luciferase activity of the co-transfected plasmid pRL.CMV to correct for variation in transfection efficiency. The results are expressed relative to the backbone vector pGL3 Enhancer. pGL3 Control, which contains the luciferase cDNA under the control of both SV40 promoter and enhancer elements, was used as a positive control. Each bar represents the mean±s.e.mean from triplicate determinations in individual experiments, n=7–19. Where error bars are not shown they lie within the bar.