Genetic Variation at the Human α2B-Adrenergic Receptor Locus
Role in Blood Pressure Variation and Yohimbine Response
Exaggerated response to α2-adrenergic receptor (α2-AR) blockade by yohimbine in normotensive subjects is an intermediate phenotype that predicts increased risk for development of hypertension. Here, we assessed the 3 α2-AR loci (α2A, α2B, α2C) as candidate genes for their influence on baseline and yohimbine-mediated increase in mean arterial pressure. Because initial results with 173 individuals implicated a possible association of yohimbine response with genetic variation at a site in the α2B-AR gene, but not at sites in the other 2 α2-AR, we sequenced the α2B-AR gene (4.4 kb, including 1.2 kb upstream and 1.9 kb distal to the coding sequence) in those subjects and an additional 81 individuals to search for other α2B-AR variants. We identified 25 polymorphisms, of which 14 are previously unreported, and 2 major haplotypes that differ by the presence/absence of a 9-bp in-frame deletion that encodes Glu301 to Glu303. Frequency differences in haplotypes were observed between blacks and whites but did not predict response to yohimbine. Genotyping of 2 additional white cohorts, including 1269 individuals with extremes in blood pressure selected from >50 000 subjects, also failed to reveal an association of the 2 major α2B-AR haplotypes with differences in blood pressure. Thus, despite considerable polymorphism in α2-AR genes, such variation is not a major determinant of variability in yohimbine response and by inference, in susceptibility to essential hypertension.
The sympathetic nervous system regulates cardiovascular physiology primarily by the release of catecholamines and activation of adrenergic receptors (AR). Multiple subtypes of α1-, α2-, and β-AR regulate cardiovascular cells and contribute to the pathophysiology of cardiovascular disease.1 For example, heritable differences in α2-AR subtypes have been implicated in human2 and rodent3 hypertension. The 3 α2-AR subtypes in the human genome, α2A, α2B, and α2C, are located on separate chromosomes, expressed in different locations, and regulate different functional responses.1,4⇓
α2-AR agonists have an antihypertensive effect that is primarily attributed to stimulation of central presynaptic receptors and a decrease in sympathetic activity5 as a consequence predominantly of central α2A- AR.6,7⇓ α2C-AR also contributes to this response, especially at low levels of stimulation of nerve activity.6 Although central inhibition of sympathetic outflow not does involve α2B-AR, the receptor may have a role in blood pressure (BP) through peripheral effects. Mice with a knockout of 1 copy of the α2B-AR are resistant to the development of salt-induced hypertension.8 Moreover, the latter form of hypertension is partially dependent on neural mechanisms and expression of α2B-AR, because it can be treated by injection of antisense α2B receptor DNA into the cerebrospinal fluid.9 It is unclear whether this salt-induced hypertensive effect is mediated by central α2B-AR or receptors in the periphery. Activation of α2B -AR in peripheral vessels can produce vasoconstriction10 and α2-ARs in the kidney, where α2B is the predominant subtype,11 have been implicated in salt retention.12
Common genetic variants in the α2-ARs have been associated with cardiovascular pathology.13 Homozygosity for a 9-bp in-frame deletion (Del) of 3 glutamic acid residues (Glu301 to Glu303) in the third intracellular loop of the α2B receptor, which leads to decreased agonist-stimulated desensitization,14 has been associated with increased risk of myocardial infarction and sudden cardiac death,15,16⇓ vasoconstriction of the coronary vasculature,17 endothelial dysfunction (as defined by decreased flow-mediated dilation)18 and obesity.19 Although some studies have found no association between this genotype and hypertension,15,20⇓ a Swedish population of individuals homozygous for the deletion were reported to have increased risk for early-onset hypertension.21 Akin to the α2B-AR, the α2C-AR has a 12-bp deletion variant in its third intracellular loop, which leads to decreased coupling of the receptor to Gαi22; blacks homozygous for the deletion allele are at increased risk for developing congestive heart failure.23
In this study, we identified novel genetic variants of the α2B-ARs and assessed the contribution of common genetic variants of α2A, α2B, and α2C-ARs to interindividual differences in BP using 3 independent cohorts of subjects phenotyped for BP traits. In addition, we have assessed the role of an “intermediate phenotype” for hypertension and yohimbine response. The α2-AR antagonist yohimbine is a useful probe of involvement of α2-AR in hypertension:24,25⇓ a subset of young normotensive individuals at genetic risk for hypertension display exaggerated increments in catecholamine release and BP after yohimbine administration, implying that this response is an “intermediate phenotype.”24,26⇓ The effect of yohimbine to increase BP is predominantly mediated by norepinephrine and cardiac output.24 Because differences in yohimbine metabolism are not responsible for this observed interindividual variation in yohimbine-promoted increase in BP,27 genetic variants of presynaptic or postsynaptic α2-AR may mediate the altered response to yohimbine. We thus assessed the contribution of genetic variants of α2-ARs to receptor blockade by yohimbine, predicting that individuals with exaggerated yohimbine response would have a combination of α2-ARs alleles different from subjects with normal response.
Materials and Methods
Subject Selection and Clinical Characterization
A cohort of 173 individuals was phenotyped for cardiovascular response to yohimbine as part of ongoing studies into the genetic risk factors for hypertension, by methods previously described.24 See Table I (http://hyper.ahajournals.org) for demographics of subjects and detailed methods for yohimbine response assessment. Criteria for exclusion were cardiovascular pathology (eg, myocardial infarction, cardiac failure, arrhythmia, severe hypertension, or stroke). One third of the subjects had a clinical diagnosis of hypertension and 95.4% had a family history of hypertension. Subjects were from urban southern California and gave informed consent according to a protocol approved by the University of California San Diego institutional review board.
An additional 81 unrelated individuals (48 whites, 11 blacks, 5 Hispanics, 11 Asians/Pacific Islanders, and 6 individuals with mixed ethnicity) were sequenced at the α2B-AR locus to enhance ethnicity-specific SNP discovery.
We genotyped 2 additional cohorts for the α2B-AR Gly394Gly SNP: (1) 441 unrelated whites phenotyped for BP status and2 611 male and 658 female white subjects chosen from 25 599 males and 27 479 females, respectively, within a database developed by Kaiser-Permanente of Southern California and on the basis of having diastolic BP (DBP) in the upper and lower extreme percentiles of distribution.28 See online supplement for detailed information on cohort.
Initial screening of candidate α2-AR SNPs for yohimbine response variability was conducted using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Sequenom).
Polymerase chain reaction primers to amplify and sequence the α2B-AR gene were designed based on sequence obtained from GenBank (ADRA2B NM_000682), and polymerase chain reaction conditions are as previously described.29 Eight overlapping polymerase chain reaction fragments were generated to sequence 1 kb upstream of the translation initiation site, the coding region, and 1.9 kb of the 3′ untranslated region (see online supplement for details on sequencing methods and analysis).
Two-way repeated measures analysis of variance (ANOVA) was performed using the SPSS software package to evaluate in vivo yohimbine-evoked physiological response and α2B-AR genotypes and haplotypes. Contingency table χ2 analysis was used to assess significance of differences between individuals in the cohort of individuals with BP extremes. ANOVA, used to determine significance of differences in the DBP measurements between such individuals, was separately conducted on males and females with age as a covariate. HAP-Haplotype Resolution v3.0 (http://diego.ucsd.edu/hap/html/)30 was used to infer phylogenic relationship of haplotypes.
Five previously reported SNPs at the 3 α2-ARs with high minor allele frequency were chosen from dbSNP (Table 1) and genotyped in 173 individuals to assess the contribution of α2-AR loci to yohimbine response. We did not observe statistically significant association with the SNPs at α2A-AR and α2C-AR; however, we observed a trend for association with the α2B-AR SNP. This result, coupled with evidence for association of a variant in that receptor with early-onset hypertension,21 led us to undertake more thorough sequencing and genotyping studies on α2B-AR.
Genomics of the α2B-AR
We sequenced the entire α2B-AR gene (4444 kb) in the 173 subjects and in an additional 81 unrelated ethnically diverse individuals to increase our power to detect rare variations within the locus and assess population differences in SNP frequency. We discovered 25 SNPs (Table 2), of which 14 are novel, previously unreported in public databases (the common 5′ untranslated region variant at −98 was recently described31); we also identified the previously reported 9-bp insertion–deletion14 (Ins/Del) coding polymorphism (Figure 1). Three SNPs and the Ins/Del polymorphism occurred with minor allele frequency >30%; 5 polymorphisms were identified within the coding region of the gene. All polymorphisms were in Hardy–Weinberg equilibrium. We observed ethnic variation in allele frequency among the SNPs in the α2B-AR (Tables 2 and 3⇓): blacks expressed the Del allele at a much lower frequency than did other ethnicities. The SNPs A +36G and T +2203G are relatively uncommon in whites and blacks, but more common in Hispanics and Asians, albeit only small numbers of subjects with those ethnicities were genotyped. Several SNPs appeared to be specific for individuals of particular ethnicities (Table 2).
Pair-wise linkage disequilibrium (LD) calculation among the 4 common polymorphisms (G-98C, Glu301–303, Gly394Gly, and C +1776A) revealed high levels of LD throughout the gene, with SNPs located at −98 and +1182 in almost complete LD with the Ins/Del polymorphism (D′=1.0, Δ2=0.948 and 0.909, respectively) (Figure 2A). In whites, the Ins/Del polymorphism was observed to be in near-absolute LD with both the C +1182A (Gly394Gly) and G-98C SNP (D′=1.0, Δ2=0.978 for both markers); therefore, either SNP can serve as a marker for the Ins/Del polymorphism in the white population.
Haplotype inference revealed 7 distinct haplotypes (Table 4) with haplotypes 2 and 6, which have opposite alleles at all 4 loci, accounting for 89% of all observed chromosomes. Among whites, the strong LD between the Ins/Del polymorphism and Gly394Gly is demonstrated by the fact that only 0.5% of chromosomes lack concordance between the 2 loci (+1182 cytosine with the Ins allele, adenine with the Del allele).
Phylogenetic analysis to infer the evolution of the recent haplotypes from the common ancestral haplotypes (Figure 2B) showed that nearly all their evolution likely occurred through point mutations rather than recombination events, consistent with the strong block of LD spanning the locus. Only haplotype 3 appears to result from a recombination between the 2 common haplotypes.
Interaction of α2B-Receptor Genotype With BP and Yohimbine Responses
Yohimbine administration significantly (P≤0.001) increased mean arterial pressure (MAP) in all subjects. To evaluate the effect of the major α2B-AR haplotypes on this response, 2-way-repeated-measures ANOVA was used to assess the impact of haplotype 2, the common haplotype containing the Ins allele, and haplotype 6, the common haplotype with the Del allele, on MAP determined at 5-minute intervals during and immediately after yohimbine infusion. Individuals with zero copies of haplotype 2 had lower MAP initially and throughout the infusion with yohimbine (P=0.015), whereas those with 1 or 2 copies of haplotype 2 had similar initial MAP and responses to yohimbine (Figure 3). We observed no significant interaction between yohimbine response and copy number of haplotype 2, implying that haplotype 2 predisposes to higher MAP but does not modify the response to yohimbine. Reciprocal results were obtained when testing for association of MAP with haplotype 6 copy number (Figure IA). Subjects with 2 copies of haplotype 6 had lower MAP initially and during yohimbine administration (P=0.031), but no association with yohimbine response. Examination of common haplotype combinations (diplotypes) and their relationship to MAP (Figure IB) showed that individuals homozygous for haplotype 2 and heterozygous for haplotype 2 and 6 have higher MAP than homozygotes of haplotype 6 (P=0.05), implying that the presence of a single copy of haplotype 2 contributes to an increase in MAP.
Although these analyses examined the effect of the common α2B-AR haplotypes on MAP, they excluded individuals with rare haplotypes. To analyze all 173 subjects treated with yohimbine, we evaluated the effect of the Ins/Del polymorphism genotypes on MAP and observed that Del/Del homozygous individuals had a significantly lower MAP (F=3.73, P=0.026) but no association with yohimbine response. In addition, including age and gender as covariates caused the association of the Del/Del genotype and a lower MAP to lose significance at the level of P=0.05.
Because differences in SNP frequencies between populations can lead to false-positive or false-negative associations, we repeated the analysis within the black and white populations and found no statistically significant (P<0.05) association between yohimbine response and Ins/Del polymorphism genotypes in either population, although black Del/Del individuals tended to have lower MAP during yohimbine administration (F=2.65; P=0.078) when including age and gender as covariates. Further analysis revealed that white individuals who were homozygous for the Del allele had a significantly lower mean age than individuals with either the Ins/Del or Ins/Ins genotypes: Del/Del (n=14), mean age=29; Ins/Ins (n=48), mean age=39; Ins/Del (n=47), mean age=38 (P=0.05 by 1-way ANOVA). In these white subjects, the frequency of Del/Del individuals younger than the median age of 35 years was 19.2%, whereas those older than the median age had a Del/Del frequency of 7.0%.
Because the latter data suggested that healthy older white subjects have the Del/Del genotype less frequently, we analyzed 2 larger, independent white cohorts for the +1182(C/A) (ie, Gly394Gly) α2B-AR SNP, which, as described, serves as a marker for the Ins/Del polymorphism. We genotyped 441 unrelated white individuals and found for subjects aged 40 and younger the following genotype frequencies: A/A=10%, A/C=40%, and C/C=49%, and for subjects aged older than 40 the following: A/A=14%, A/C=40%, and C/C=46% (P=not significant by χ2). In addition, we observed no association with +1182 genotypes and systolic BP, DBP, or diagnosis of hypertension in this cohort.
As a further test of the α2B-AR Ins/Del polymorphism on BP, we examined a third cohort, individuals from a community-derived (Kaiser Permanente) Southern California white population of >25 000 men and >27 000 women. Although this cohort has the statistical power to detect association of a genetic variant with BP that contributes as little as 1% to the total variation, we found no association of the +1182 genotypes with systolic BP, DBP, or diagnosis of hypertension. When separately analyzed based on age, the men aged 40 or younger showed genotype frequencies of A/A=7%, A/C=50%, and C/C=43%, whereas in those older than age 40, the frequencies were A/A=11%, A/C=44%, and C/C=45%(not significant by χ2). Similarly, the women showed no significant differences in age between genotypes and no association with systolic BP, DBP, or BP status (data not shown). These results, consistent with those for the second cohort, do not support the hypothesis that the Ins/Del polymorphism in α2B-AR contributes to BP variation.
This study includes the most comprehensive analysis to date of the genetic diversity of the α2B-AR locus. We identified multiple previously unknown SNPs, defined 7 haplotypes in an ethnically diverse population, found that 2 of these haplotypes represent the overwhelming majority of haplotypes, and characterized their effect on BP and BP variation. The 2 major haplotypes differ most notably by an Ins/Del variant in the third intracellular loop domain, which has been previously shown to affect agonist-induced desensitization of the receptor.14 Impairment of α2B-AR desensitization by the Del variant would be predicted to result in greater vasoconstriction and elevated BP by postsynaptic α2B-ARs or alternatively, and we believe less likely (based on distribution of α2B-ARs), opposite effects via actions at presynaptic α2B-ARs. Overall, our data fail to reveal an important contribution of genetic variants of the α2B-AR locus to BP, particularly in whites, including in terms of altered response to yohimbine. Although in vitro studies are needed to assess the differential response of yohimbine by Del and wild-type α2B-ARs, our study indicates that there is no observable difference in yohimbine response between individuals with and without the Del variant.
These latter data regarding BP contrast with several previous reports that imply such variation contributes to cardiovascular morbidity and mortality. Evidence has been reported in a Finnish population that the deletion polymorphism, particularly Del/Del homozygosity, increases the risk for acute coronary events and sudden cardiac death.15,16⇓ Other data have provided possible mechanistic explanations for the impact of the Del/Del genotype on cardiovascular risk through increased vasoconstriction of the coronary vasculature,17 endothelial dysfunction as defined by decreased flow mediated dilatation,18 and association with obesity.19 Because our study was designed to assess sympathetic activation through α2-AR blockade, individuals with cardiac morbidity, such as myocardial infarction, were excluded.
Consistent with the results of Snapir et al,15,16⇓ our initial analysis of the 173 individuals who underwent testing with yohimbine revealed a decrease in Del/Del individuals aged 40 and older, suggesting a selection “bias” caused by the lack of healthy older individuals with that genotype, because individuals with Del/Del might have experienced premature mortality or been excluded because of previous cardiovascular disease. However, subsequent analysis in 2 larger groups of subjects, including those drawn from a large population-based cohort with extreme values for BP, failed to confirm our initial conclusions from the smaller-sized population. White Americans might differ from Finnish individuals in terms of the role of the Del/Del α2B-AR on cardiovascular morbidity and mortality, but further studies will be necessary to test this. The results emphasize the need to assess multiple large groups of individuals when attempting to draw inferences regarding genetic association.
With respect to hypertension, our data in the large cohorts are consistent with an earlier study with a smaller sample20 but contrast with those of Von Wowern et al (2004),21 who showed an increased risk (OR=2.01) for early-onset (younger than 50 years old) hypertension in Swedish α2B-AR Del/Del homozygotes, an effect that lost significance in an all-age population. Even when we stratified our data and compared younger versus older subjects (using age 40, as reported, or when we used age 50 as a cutoff, data not shown), we failed to find evidence to support the conclusions of Von Wowern et al (2004), perhaps because of differences between our white population and their more ethnically homogenous Swedish population. Especially because the odds ratio in the Swedish study was modest and highly age-dependent, we conclude that the α2B-AR gene makes, at best, a minimal contribution to development of essential hypertension.
The lack of association between genetic variants in the α2-ARs and intersubject variation in response to yohimbine, previously identified as defining an intermediate phenotype for essential hypertension, implies that this phenotype must result from an influence of genes other than those encoding the 3 α2-ARs or from nongenetic factors. Although our studies were performed on subjects with intact baroreceptor reflexes, the initial identification of a relationship between yohimbine response and hypertension risk was performed using similar methods.24 In addition, we recently reported on the relationship between dorsal hand vein venoconstrictor response, which avoids the baroreceptor reflex, to the α2B-AR agonist azepexol and failed to identify a relationship between venoconstriction and α2B-AR genotypes, supporting the results shown here regarding the lack of contribution of α2B-AR genetic variability to interindividual is cardiovascular variation.29 Perhaps greater pressor response to yohimbine is determined by reduced baroreflex buffering, which involves a different set of targets than α2-ARs.
The current study defines genetic variation of the α2B-AR locus, identifying 2 major haplotypes, and used multiple cohorts to test the contribution of variation in the α2B-AR to interindividual BP variability in whites. The data fail to define such a contribution. In addition, genetic variations of the 3 α2-ARs fail to predict intersubject variation in response to yohimbine, an intermediate phenotype for essential hypertension.
Funded in part by an award to J.P.E. from the American Heart Association Western States Affiliate Medical Student Research Program and by grants from the National Institutes of Health. The authors thank Dr Bruce A. Hamilton for use of an ABI 3100 sequencer, Eleazar Eskin for his software on haplotype phylogeny, Jennifer Wessel for assistance in statistical interpretation, and Kenton Murthy for his help in preparation of this manuscript.
J.P.E. and B.K.R. contributed equally to this work.
- Received January 12, 2005.
- Revision received February 3, 2005.
- Accepted April 6, 2005.
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