Complex haplotypes derived from noncoding polymorphisms of the intronless alpha2A-adrenergic gene diversify receptor expression

Abstract

Alpha(2A)-adrenergic receptors (alpha(2A)AR) regulate multiple central nervous system, cardiovascular, and metabolic processes including neurotransmitter release, platelet aggregation, blood pressure, insulin secretion, and lipolysis. Complex diseases associated with alpha(2A)AR dysfunction display familial clustering, phenotypic heterogeneity, and interindividual variability in response to therapy targeted to alpha(2A)ARs, suggesting common, functional polymorphisms. In a multiethnic discovery cohort we identified 16 single-nucleotide polymorphisms (SNPs) in the alpha(2A)AR gene organized into 17 haplotypes of two major phylogenetic clades. In contrast to other adrenergic genes, variability of the alpha(2A)AR was primarily due to SNPs in the promoter, 5' UTR and 3' UTR, as opposed to the coding block. Marked ethnic variability in the frequency of SNPs and haplotypes was observed: one haplotype represented 70% of Caucasians, whereas Africans and Asians had a wide distribution of less common haplotypes, with the highest haplotype frequencies being 16% and 35%, respectively. Despite the compact nature of this intronless gene, local linkage disequilibrium between a number of SNPs was low and ethnic-dependent. Whole-gene transfections into BE(2)-C human neuronal cells using vectors containing the entire approximately 5.3-kb gene without exogenous promoters were used to ascertain the effects of haplotypes on alpha(2A)AR expression. Substantial differences (P < 0.001) in transcript and cell-surface protein expression, by as much as approximately 5-fold, was observed between haplotypes, including those with common frequencies. Thus, signaling by this virtually ubiquitous receptor is under major genetic influence, which may be the basis for highly divergent phenotypes in complex diseases such as systemic and pulmonary hypertension, heart failure, diabetes, and obesity.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Organization and localization of polymorphisms of the intronless human α2AAR gene. Indicated are the promoter, 5′ UTR, coding region, and 3′ UTR boundaries and the nucleotide position of the polymorphism. +1 is the adenine of the initiator methionine. ∗, Synonymous coding.

Fig. 2.

The frequencies of α2AAR polymorphisms display marked ethnic variation. The polymorphism positions and frequencies are given on the x and y axes, respectively, stratified by ethnic group (z axis). CA, Caucasian; AA, African American; AS, Asian.

Fig. 3.

LD between polymorphisms of the α2AAR gene. LD was calculated as r2 (see Materials and Methods), and data are presented as a matrix such that relative values for any pair of polymorphisms can be determined based on the indicated scale. White squares indicate a monomorphic position in the indicated group.

Fig. 4.

Phylogenetic analysis of α2AAR haplotypes. The neighbor-joining method was used to reconstruct phylogeny from the full-length nucleotide sequences. Polarity was set by midpoint rooting (see Materials and Methods). Two major clades (A and B) were identified.

Fig. 5.

α2AAR haplotypes display unique profiles of mRNA and protein expression. BE(2)-C cells were transfected with constructs (lacking exogenous promoters) consisting of the ≈5.3-kb contiguous sequence of the α2AAR gene. mRNA was quantitated by RT-PCR by using GAPDH primers as controls for transfection efficiency. α2AAR protein was determined by quantitative radioligand binding with [3H]yohimbine. Both transcript and protein expression were related to haplotype (P < 0.001 by ANOVA). Data shown are from five experiments. See Results and Discussion for selected individual comparisons.