Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness

Abstract

The human beta(2)-adrenergic receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5' upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common beta(2)-adrenergic receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to beta agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. beta(2)-adrenergic receptor mRNA levels and receptor density in cells transfected with the haplotype associated with the greater physiologic response were approximately 50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.

Figure 1

Phylogeny of β₂AR haplotypes. Each haplotype is represented by a circle whose area represents the overall frequency of that haplotype in the sample. Lines connecting haplotypes are solid black for single-site differences, solid blue for two-site differences, and dashed for more than two differences. Each circle is subdivided to show the proportion of the individual haplotype frequency found in each of the four population groups as represented by the indicated colors.

Figure 2

Linkage disequilibrium between β₂AR SNPs. Genotypes from the Caucasian samples were determined at the 13 loci shown in Table 1 and the degree of linkage disequilibrium (Δ) between SNPs was calculated. Results are color coded as shown. The site at −406 was monomorphic in the Caucasian sample.

Figure 3

In vivo responses to β₂AR agonist depend on β₂AR haplotypes. Shown are data from 121 subjects whose FEV₁s were measured before and after inhalation of the agonist albuterol as described in Methods. The response was significantly related to β₂AR genotype (P = 0.007 by ANCOVA). See text for additional statistical analysis.

Figure 4

β₂AR haplotype determines receptor transcript and protein expression. HEK293 cells were transiently transfected with the indicated β₂AR haplotypic vector and a luciferase vector (to control for transfection efficiency) as described in Methods. β₂AR protein density was determined by ¹²⁵I-cyanopindolol radioligand binding and normalized to membrane protein (A) or luciferase expression (B). β₂AR mRNA levels were determined by quantitative ribonuclease protection assays. Shown are results from four to seven experiments. Haplotype 4 expression was less than haplotype 2 at P < 0.005.