Common genetic variants in the chromogranin A promoter alter autonomic activity and blood pressure

Abstract

Chromogranin A (CHGA) is stored and released from the same secretory vesicles that contain catecholamines in chromaffin cells and noradrenergic neurons. We had previously identified common genetic variants at the CHGA locus in several human populations. Here we focus on whether inter-individual variants in the promoter region are of physiological significance. A common haplotype, CGATA (Hap-B), blunted the blood pressure response to cold stress and the effect exhibited molecular heterosis with the greatest blood pressure change found in Hap-A/Hap-B heterozygotes. Homozygosity for three minor alleles with peak effects within the haplotype predicted lower stress-induced blood pressure changes. The G-462A variant predicted resting blood pressure in the population with higher pressures occurring in heterozygotes (heterosis). Using cells transfected with CHGA promoter-luciferase reporter constructs, the Hap-B haplotype had decreased luciferase expression compared to the TTGTC (Hap-A) haplotype under both basal conditions and after activation by pre-ganglionic stimuli. The G-462A variant altered a COUP-TF transcriptional control motif. The two alleles in transfected promoters differed in basal activity and in the responses to COUP-II-TF transactivation and to retinoic acid. In vitro findings of molecular heterosis were also noted with the transfected CHGA promoter wherein the diploid combination of the two G-462A alleles gave rise to higher luciferase expression than either allele in isolation. Our results suggest that common genetic variants in the CHGA promoter may regulate heritable changes in blood pressure.

Chromogranin A (CHGA (MIM 118910)), a 48-kDa acidic polypeptide,^1,2 is the major protein co-stored and co-released with catecholamines from secretory vesicles in adrenal medulla and postganglionic sympathetic axons.³ CHGA is required for formation of catecholamine secretory vesicles in chromaffin cells and its expression may be sufficient to induce a regulated secretory system even in non-secretory cells.⁴ CHGA is also a pro-hormone that gives rise to biologically active peptides such as the dysglycemic peptide pancreastatin,^5,6 the antimicrobial peptide chromacin,⁷ the vasodilator vasostatin,⁸ and catestatin that acts to inhibit catecholamine release.^9,10 Phenotypic links between CHGA and essential (idiopathic, genetic) human^11–14 and rodent¹⁵ hypertension have been repeatedly observed.¹⁶ Plasma CHGA concentration correlates with catecholamine release rate,¹⁷ and increases in blood pressure (BP) caused by the action of catecholamines are coupled to the formation of dense-core secretory granules, whose biogenesis is regulated in vivo by CHGA.¹⁸

Recently, we systematically identified common genetic variation in human CHGA by resequencing the gene in several human populations.¹⁹ Here, we explore whether common interindividual genetic variation at the CHGA promoter contributes to heritable BP variation after environmental stress, an early pathogenic phenotype for later hypertension, as well as basal BP in the population. We then characterized the effects of an associated promoter variant on gene expression in transfected promoter/reporter plasmids in chromaffin cells. Our results suggest novel effects of particular CHGA promoter variants on autonomic circulatory control, with likely transcriptional mechanisms identified.

RESULTS

Structure of the human CHGA locus: patterns of linkage disequilibrium

After systematic variant discovery, we used 16 common single-nucleotide polymorphisms (SNPs) (each with minor allele frequency >5%), distributed across ~13 kb at the CHGA locus, to probe patterns of pairwise linkage disequilibrium (LD) in 2n=102 chromosomes (Figure 1a). The four-gamete algorithm²⁰ suggested three blocks of LD across the locus, with the most 5′ block extending across the promoter and into intron 2. Figure 1b shows the common variants discovered in the CHGA proximal promoter.

Figure 1. The human CHGA locus.

(a) Patterns of linkage disequilibrium. Data are shown for 16 common (minor allele frequency >5%) biallelic polymorphisms spanning the CHGA gene, discovered by systematic resequencing with amplicons encompassing each exon, exon/intron border, 5′- and 3′-UTR, and proximal promoter.¹⁹ Pairwise results are plotted on a pseudocolor scale for LD, with the Haploview algorithm,²⁰ for subjects self-identified as White (European ancestry, 2n=102 chromosomes). LD blocks are determined by the four-gamete rule. (b) Promoter polymorphism. Common polymorphisms in the CHGA proximal promoter are displayed in context with other consensus promoter elements. CRE, cyclic AMP response element; UTR, untranslated region.

CHGA promoter genotypes and the heritable response to environmental stress: studies in twin pairs

CHGA promoter haplotype effects on stress traits

As systemic hypertension may result from the cumulative effects of transient adverse BP responses to environmental stress in genetically predisposed individuals,²¹ we probed the BP response to environmental stress, using cold as the systematic stimulus²² in a series of predominantly normotensive twin pairs. The stress BP traits were significantly heritable as estimated by twin pair variance components:²² change in diastolic blood pressure (DBP) at 32±8% (P=0.0003) and final DBP at 37±8% (P<0.0001).

We then genotyped five tightly linked common promoter variants (at positions C−1014T, G−988T, G−462A, C−415T, A−89C; Figure 1b), and inferred haplotypes with the HAP algorithm.²³ The three most common haplotypes, TTGTC (Hap-A, 56.9%), CGATA (Hap-B, 23.0%), and TTGCC (Hap-C, 16.5%), accounted for >95% of all haplotypes.

Diploid haplotypic variation in the region affected both the change in DBP (P=0.0222) and the final DBP (P=0.0167) during stress (Figure 2a). Homozygosity for Hap-B (CGATA/CGATA) seemed to blunt both δ-DBP and final DBP, whereas Hap-A/Hap-B heterozygosity (TTGTC/CGATA) was associated with the greatest values of both δ-DBP and final DBP (Figure 2a). The more extreme trait values for heterozygotes (as compared to either homozygote class) suggested the phenomenon of molecular heterosis.²⁴

Figure 2. CHGA promoter polymorphism and autonomic control of the circulation: BP response to environmental stress.

(a) Common diploid haplotypic variation in the proximal promoter (C−1014T→G−988T→G−462A→C−415T→A−89C): predicting the BP response to environmental stress in twin pairs. Provocation of efferent sympathetic outflow was undertaken in each subject by immersion of one hand in ice water (at 0 °C) for 1 min, with continuous BP monitoring. Results are shown for final DBP and δ-DBP in 224 twins (112 twin pairs), and analyzed by generalized estimating equations, establishing an exchangeable correlation matrix to take into account intra-twin-pair correlations. (b) Individual CHGA promoter polymorphisms predict the DBP response to environmental (cold) stress in twin pairs. Results are shown for final DBP in twin pairs and analyzed by generalized estimating equations, establishing an exchangeable correlation matrix to take into account intra-twin-pair correlations. (c) Haplotype phylogeny in the CHGA promoter: T−1014C→T−988G→G−462A→C−415T→A−89C. Haplotype inference²³ based on genotyping data in twin pairs. The likely phylogeny of this block is plotted. Haplotype variants A, B, and C are found in the contemporary population at the frequencies indicated. Ancestral haplotype L is inferred rather than observed in the contemporary population (numbers in parentheses).

Although Hap-A and Hap-B each influenced the stress traits (Figure 2a), Hap-3 did not demonstrate an independent effect, perhaps reflecting the limited statistical power of this less common (16.5%) variant.

Individual SNP effects on the stress trait

We then analyzed each SNP within the promoter LD block individually; the minor alleles (in bold) at positions T−1014C, T−988G, and G−462A predicted lower final/post-stress DBP (P=0.010–0.036) (Figure 2c), with the most substantial and significant (P=0.010) effect at G−462A. Once again, there was the suggestion of molecular heterosis, with G/A heterozygotes exhibiting the highest final DBP. By Single Nucleotide Polymorphism Spectral Decomposition (SNPSpD),²⁵ the experiment-wide significance threshold required to keep the type I error rate at ≤5% within the promoter haplotype block was P=0.0187; this threshold was exceeded only by G−462A.

Promoter haplotype phylogeny

To explore lineages within the trait-associated human CHGA promoter variants, we constructed a likely phylogeny²³ based on these five SNPs genotyped in twins (Figure 2b). HAP indicated that haplotype CGATA (Hap-B), containing the −462A allele, is relatively ancestral within the human lineage. Hap-A (TTGTC) is likely to be descended from another ancestral haplotype, TTG?C, with uncertainty at position C−415T. Each allele of variant G−462A is found in a likely ancestral haplotype. Thus, both of the trait-associated haplotypes (Hap-A, TTGTC; Hap-B, CGATA), as well as both alleles at G−462A, are likely to be rather ancient in the human lineage. Similar results were obtained when we estimated the phylogeny using data across several human populations (ancestries: European, African American, Hispanic, and east Asian).

CHGA promoter polymorphism effects on basal BP in the population

To probe the significance of CHGA promoter variation for disease, we selected the variant with the peak/most significant effect on stress BP (Figure 2b), G−462A, and typed it in a series of normotensive and hypertensive individuals (n=920) from San Diego (UCSD and VA clinics). There were significant effects of G−462A genotype on both systolic blood pressure (SBP) (P=0.030) and DBP (P=0.013). Heterozygosity (G/A genotype) predicted higher SBP and DBP, by ~3/2mmHg over major allele homozygotes (Figure 3), once again suggesting molecular heterosis.²⁴

Figure 3. CHGA promoter common variant G−462A: effect on BP as a quantitative trait in hypertension population.

Data were analyzed by univariate analysis of variance. SBP (left) or DBP (right) values and number (n) are shown.

Analysis with the covariate of ethnicity did not affect the association results (SBP P=0.028/DBP P=0.011), nor did adjustment for the effects of antihypertensive medications²⁶ in the hypertensives (SBP P=0.049/DBP P=0.024).

Function of CHGA promoter variants (haplotypes and variants)

Basal activity of transfected promoters in chromaffin cells and corticotropes

To study the functional consequences of the two principal trait-associated human CHGA haplotypes, we amplified the CHGA promoter (−1142/+54 bp) from human samples, subcloned it into the promoter site of the pGL3-Basic luciferase reporter vector, created necessary variants by site-directed mutagenesis, then confirmed the haplotypes by sequencing. We tested promoter strength by transfection into the neuroendocrine cell lines PC12 (rat chromaffin/pheochromocytoma cells) and AtT20 (mouse pituitary corticotropes). In both PC12 and AtT20 cells, haplotype TTGTC (Hap-A, containing the G−462 allele) showed higher expression of the reporter gene than Hap-B (CGATA, with the −462A allele) (Figure 4).

Figure 4. Human CHGA promoter haplotypes (C−1014T→G−988T→G−462A→-C−415T→A−89C): differential activity in chromaffin cells and corticotropes.

(a) Basal activity. Transfection results for human CHGA promoter haplotype/luciferase reporters are shown for mouse AtT20 corticotropes versus rat PC12 chromaffin cells. Results are expressed as the ratio of firefly luciferase/Renilla luciferase (encoded by the transfection efficiency plasmid pRL-TK). Each experiment was performed in triplicate, and such experiments were repeated at least three times. (b) Human CHGA promoter polymorphisms: site-specific effects on transcription in chromaffin cells and corticotropes. Common variant effects on transcription of reporter gene are presented as significance (1/P-value, by analysis of variance, with SNPs as the independent variables) in PC12 chromaffin cells and AtT20 corticotropes. (c) Human CHGA promoter haplotypes (T−1014C→T−988G→G−462A→C−415T→A−89C): differential activity during secretory stimulation in chromaffin cells. Results are shown as stimulated activity minus control activity (i.e., without secretagogue). Each experiment was performed in triplicate, and such experiments were repeated at least three times. VIP, vasoactive intestinal peptide; PACAP, pituitary adenylyl cyclase-activating polypeptide.

Effect of secretory stimulation

We tested the reactions of the two major haplotypes to preganglionic secretory stimuli known to induce the transcription of CHGA: nicotinic cholinergic agonist (nicotine), and the neuropeptides vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide. Exposure to each of these secretagogues augmented transcription of both haplotypes (Hap-A, TTGTC; Hap-B, CGATA), but the TTGTC promoter Hap-A (containing the G−462 allele) displayed greater (P=0.011) responses than Hap-B (CGATA) to both nicotine and vasoactive intestinal peptide (Figure 5).

Figure 5. CHGA promoter common variant G−462A.

(a) Transcriptional motif prediction for the sequence surrounding CHGA G−462A. The COUP-TF motif in the sequence is shown as two direct repeats (AGGTCA) separated by a one nucleotide spacer (N). (b) Human CHGA promoter G−462A and nuclear receptor activation: effects of G−462A on transcriptional activity in chromaffin cells. Allele G−462 occurs naturally as part of Hap-A (TTGTC); on the Hap-A mutation background, G−462 was point mutated to −462A, creating non-natural/artificial haplotype TTATC. Results for TTGTC versus TTATC are compared with T-test. Chromaffin (PC12) cell transfection results are shown. Left: basal activity. CHGA promoter haplotypes were transfected into PC12 cells without stimulation (‘mock’). Basal activity results are presented as firefly luciferase activity normalized to cell protein. Middle: COUP-II-TF expression. COUP-II-TF (in cytomegalovirus promoter expression plasmid pCR3.1; Invitrogen) was co-transfected at 50 ng. The control transfection was performed with the empty vector pCR3.1. Results are presented as net activity (stimulation minus basal, for each haplotype). Right: retinoic acid stimulation. Results are presented as net activity (stimulation minus basal, for each haplotype), where stimulation was 1 µm retinoic acid.

Single-nucleotide variant effects on reporter gene activity

To probe the effect of each variant in the promoter, we constructed a series of haplotypes, both naturally occurring and artificial, in which we systematically varied the base at each variant (T−1014C, T−988G, G−462A, C−415T, A−89C) on different backgrounds ligated to luciferase reporters. Two-way analysis of variance then probed the effects of each variant on basal activity in PC12 or AtT20 cells. Variants at positions T−1014C, T−988G, and G−462A exerted effects on reporter gene expression in both PC12 and AtT20 cells. Variants T−415C and C−89A exhibited differential activity on gene expression: T−415C principally in PC12 cells, whereas C−89A was functional mainly in AtT20 (Figure 6).

Figure 6. Human CHGA promoter variants in diploid combinations in cella: transfection of CHGA promoter/luciferase reporter plasmids into PC12 chromaffin cells.

(a) Haplotype combinations: apparent recessive/dominant actions of Hap-A/Hap-B in cella. Hap-A (TTGTC) and Hap-B (CGATA), each fused to the luciferase reporter, were transfected in three diploid combinations: Hap-A/Hap-A (1000 ng of Hap-A alone, to test Hap-A homozygosity), Hap-A (500 ng)/Hap-B (500 ng) (to test heterozygosity), or Hap-B/Hap-B (1000 ng of Hap-B alone, to test Hap-B homozygosity). (b) G−462A combinations: evidence for molecular heterosis in cella. The parent haplotype was Hap-A (TTGTC) fused to luciferase, onto which the −462A variant was introduced by site-directed mutagenesis, yielding TTATC. Three diploid combinations were transfected: G/G (TTGTC alone, to test homozygosity, 1000 ng), G/A (TTGTC/TTATC, to test heterozygosity, 500 ng/500 ng), or A/A (TTATC alone, to test homozygosity, 1000 ng).

Role of promoter variant G−462A and transactivation

As G−462A exhibited the peak effect on stress-induced DBP changes in twins (Figure 2), as well as an effect (~3/2mmHg) on basal BP (Figure 3), we studied its effects more closely. Analysis of the sequence immediately surrounding G−462A (Figure 5a) by ConSite (http://www.phylofoot.org/consite)²⁷ revealed a partial (7/12 position, P=0.0046 by Fisher’s exact test) match to a consensus COUP-TF (chicken ovalbumin upstream promoter–transcription factor)²⁸ nuclear hormone binding motif (AGGTCANAGG TCA), consisting of two direct hexanucleotide repeats (AGGTCA, in bold) separated by a one base spacer (‘N’). The match improved to 8/12 for the −462A allele. Among sequenced mammals (human, chimp, cow, rat, and mouse), all had either G or A at the equivalent position of human G−462A; chimp and mouse displayed 6/12 base identity with the COUP-TF motif (Figure 5a).

Basal activity

To isolate the effect of G−462A as a single independent variable, we mutated the G−462 to the −462A allele on a background of Hap-A (that is, TTGTC to TTATC), and then transfected each haplotype into PC12 cells. Under basal circumstances, the promoter bearing the G−462 allele was ~25% more active than the −462A allele (P=0.036; Figure 5b).

COUP-II-TF stimulation

To probe the significance of the putative COUP-TF motif (Figure 5a), we tested the effect of COUP-II-TF by co-transfection with CHGA promoter/reporters bearing either the G−462 or the −462A allele (Figure 5b). Each promoter variant was stimulated by COUP-II-TF, although the increment was ~8-fold greater for the −462A variant (P=0.0316) that had the superior (8/12 position) COUP-TF match (Figure 5a).

Retinoic acid stimulation

The retinoid X receptor may heterodimerize with COUP-TF,²⁸ and retinoic acid reportedly alters CHGA promoter activity in neurons.²⁹ Upon retinoic acid stimulation (Figure 5b), both G−462A promoter variants were activated, although the increment was ~2.5-fold greater for the G−462 allele.

Human CHGA promoter variants in diploid combinations: results in cella

Having observed the phenomenon of molecular heterosis (the most extreme phenotype for the heterozygote class) in vivo for both promoter haplotypes on stress BP increments (Figure 2a) and G−462A on basal BP in hypertension (Figure 3), we wished to explore whether such heterosis could also be demonstrated in an isolated cellular transfection system (PC12 chromaffin cells).

Promoter haplotypes

We transfected luciferase-encoding Hap-A (TTGTC)/Hap-B (CGATA) on separate plasmids in three diploid combinations: Hap-A/Hap-A (that is, Hap-A alone), Hap-A/Hap-B (that is, a 50:50 mixture), and Hap-B/Hap-B. The results yielded substantial (P<0.001) differences in luciferase expression, and reproduced the previous findings that Hap-A was more active than Hap-B (Figure 4a). But in combination, the two haplotypes yielded an apparent recessive/dominant pattern of actions of Hap-A/Hap-B in cella (Figure 6a)

Promoter variant G−462A

When the two alleles at G−462A (G−462 and −462A) were isolated on the Hap-A background (TTGTC-TTATC), fused to luciferase, and transfected into chromaffin cells, the finding of G>A activity was reproduced (Figure 5b), but the two alleles in combination yielded a pattern of molecular heterosis reminiscent of the clinical effect on BP (Figure 3), in that the heterozygous combination programmed more luciferase expression than either of the two alleles in isolation (Figure 6b).

DISCUSSION

Overview

CHGA plays a pivotal role in the sympathochromaffin system, both in the formation of catecholamine secretory vesicles and in the regulation of transmitter release.^1,18 In this report, we approach the impact of common human variations in the CHGA promoter region for autonomic physiology and disease.

We found that common promoter variants constitute lie within an LD block (Figure 1). A common haplotype of the CHGA promoter region, CGATA (Hap-B; across T−1014C→T−988G→G−462A→C−415T→A−89C), blunted the heritable change in BP during environmental stress in twin pairs (Figure 2a). Homozygosity for the minor alleles at T−1014C (C/C), T−988G (G/G), or G−462A (A/A) was also associated with post-stress BP in normotensive individuals (Figure 2b), with the peak effect at G−462A, suggesting a mechanism for very early effects of these variants on a pathogenic series of events that may eventuate in sustained/fixed basal BP elevation (Figure 6). A/G heterozygosity at crucial variant G−462A predicted higher SBP and DBP in the population (Figure 3).

Studies with isolated/transfected CHGA promoters showed that common haplotype CGATA (Hap-B) exhibited lower expression of a reporter gene, when compared to the most common haplotype, TTGTC (Hap-A) (Figure 4). Under stimulation by triggers to chromaffin cell exocytosis (Figure 4c), such as nicotine and vasoactive intestinal peptide, both promoter haplotypes were activated, but the increase was blunted in CGATA (Hap-B) compared with TTGTC (Hap-A). Crucial SNP G−462A affected a consensus nuclear receptor transcriptional motif for COUP-TF (Figure 5a), and when created by site-directed mutagenesis, this variant affected not only basal expression but also the response to COUP-II-TF or to retinoic acid (Figure 5b). These results suggest a mechanistic chain of events (Figure 6) whereby common CHGA promoter variant G−462A plays a role in the determination of human BP.

Intermediate phenotypes

Essential hypertension is a complex trait,³⁰ with multiple contributory factors derived from both genes and environment. In the setting of late penetrance of the ultimate disease trait (such as hypertension), as well as likely genetic heterogeneity, the ‘intermediate phenotype’³¹ strategy may be a useful approach in the search for disease predisposition loci. Autonomic traits with heritable determination may be of particular value in investigation of the genetic underpinnings of hypertension. In accordance with this pathway concept, we pursued intermediate traits in this study. The hemodynamic response to environmental (cold) stress may be a predictor of the development of later cardiovascular events, such as hypertension.^32–36 Such a response, occurring even before the onset of disease, would be a useful physiological intermediate phenotype in probing the genetic determinants of hypertension. ^16,31,37 We report the cold stress response in our twins, indicating that both change in DBP and final (post-stress) DBP are heritable.²²

Molecular heterosis

CHGA promoter Hap-A (TTGTC) and Hap-B (CGATA), the two most common promoter haplotypes, influenced the BP response to environmental stress (Figure 2a), with Hap-B homozygosity (CGATA/CGATA) predicting both lower change and final BP after stress (Figure 2a). The peak effect for this process was noted for position G−462A (Figure 2b). Both the haplotype (Figure 2a) and individual SNP (Figure 2b) effects were more extreme for heterozygotes, suggesting ‘molecular heterosis’,²⁴ a phenomenon also observed for the effect of G−462A on basal/resting BP in the population (Figure 3).

The phenomenon of molecular heterosis has been defined as occurring when polymorphism at a single genetic locus displays a significantly greater or lesser effect on a quantitative trait than homozygosity at the same locus.²⁴ Although the phenomenon may initially seem counter-intuitive, it may be explained by one of several underlying mechanisms, including a U-shaped dose–response relationship for gene on trait, greater ‘fitness’ in heterozygotes, or hidden stratification in one homozygote class. One such stratification might be the effect of allelic variation at other (non-CHGA) trans-Quantitative Trait Loci (QTLs) on cold stress and basal BPs, such as the associations we have reported for polymorphisms at tyrosine hydroxylase^22,24 or rho kinase.³⁸

We also observed that heterozygous diploid combinations of G−462A (Figure 6b) created a more extreme phenotype (in this case, luciferase expression) than either homozygote class. Thus, the phenomenon of molecular heterosis at G−462A seems not to require systemic influences, or even effects of hidden stratification at other loci, as PC12 is a clonal chromaffin cell line.³⁹ Perhaps a clue to the origin of molecular heterosis in this setting lies in the motif harboring G−462A (Figure 5a): the nuclear receptor COUP typically forms heterodimers with other members of that transcription factor subfamily (such as the retinoid X receptor), creating the possibility of interactive (that is, non-additive) effects. Indeed, the retinoid receptor ligand retinoic acid differentially activated the G−462 and −462A alleles in the transfected CHGA promoter (Figure 5b, right).

Regardless of mechanism, the phenomenon of molecular heterosis points out the value of trait associations with diploid genotypes rather than simply with alleles, which could not capture such effects.²⁴

Biological role of promoter haplotypes and G−462A

Our results suggest that a discrete variant in the CHGA promoter (G−462A) may play a role in control of BP, with sequential effects (Figure 7) beginning at transcriptional control (Figure 4 and Figure 5), proceeding through an intermediate/risk phenotype (Figure 2), and eventuating in basal BP changes in the population (Figure 3). Both hypertensive patients and subjects with still-normal BP (but at genetic risk of hypertension) may display derangements of autonomic function, including changes in CHGA expression.^3,12 As CHGA is crucial in the formation of catecholamine secretory vesicles as well as the regulation of transmitter release,^1,18 transcriptional control of CHGA may thus be an early control point in development of cardiovascular risk.

Figure 7. Intermediate phenotype hypothesis for the cardiovascular consequences of CHGA promoter genetic variation.

Framework hypothesis for the effects of CHGA promoter variant G−462A on intermediate traits, and ultimately on BP in the population

Previously, we found that the proximal promoter region of CHGA governs its transcription, under both basal circumstances and secretory stimulation, consistent with the notion of ‘stimulus–secretion–synthesis coupling,’ or ‘stimulus–transcription coupling.’^40–44 The cyclic AMP response element site in the very proximal CHGA promoter (human CHGA promoter 5′-T₋₅₁GACGTCA₋₄₄-3′) was identified to be crucial in neuroendocrine-specific expression of CHGA^45,46 and its response to secretory stimulation.^40–42 When we undertook systematic polymorphism discovery at human CHGA,¹⁹ the cyclic AMP response element box was devoid of variation, but we did discover 13 single-nucleotide variants within the first ~1.2 kb of the CHGA promoter, with minor allele frequencies up to ~29%; indeed, the abundance of heterozygosity at the CHGA promoter suggested a signature for positive (perhaps ‘balancing’) selection.¹⁹

As the two most common CHGA promoter haplotypes, TTGTC and CGATA, showed differential effects on post-stress BP (Figure 2), we attempted to understand how these two haplotypes influence gene expression. Hap-A (TTGTC) showed higher expression of the reporter gene than Hap-B (CGATA), under not only basal (Figure 4a) but also secretion–stimulatory conditions (Figure 4c). Nicotine consumption has been identified as a risk factor for cardiovascular diseases, such as hypertension.⁴⁷ The transcription of CHGA is activated by nicotinic stimulation, and the response seems to map onto the proximal promoter.^40,42 Greater reactivity of Hap-A (TTGTC) to both stress in vivo (Figure 2a) and secretory stimulation in cultured chromaffin cells (Figure 4) suggests that augmented transcription of this common haplotype may play a role in human stress responses that are precursors to adverse cardiovascular outcomes, such as hypertension.^40,42

Among the five common variants (T−1014C→T−988G→G−462A→C−415T→A−89C) used to probe the CHGA promoter haplotype block, G−462A yielded the peak prediction of post-stress BP (Figure 2b), as well as BP in the population (Figure 3).

As this variant modified a known transcriptional control motif (Figure 5a), we explored the effect of this variant in transfected promoters. First, we created the variant, by site-directed mutagenesis (G→A) on the Hap-A background (that is, TTGTC-TTATC). Then, we tested the effect of this variant on transfected promoter/reporter gene expression in chromaffin cells, under basal circumstances as well as during perturbation by co-transfected/expressed COUP-II-TF or retinoic acid (selected as COUP may heterodimerize with other nuclear hormone receptors, such as the retinoid X receptor).²⁸ In each case, the G−462A purine→purine transition altered transcriptional efficiency of the promoter (Figure 5b).

The COUP-TF is an ‘orphan’ (that is, endogenous ligand unknown) within the nuclear receptor family.²⁸ Two loci, COUP-TFI (also termed EAR3) and COUP-TFII (also termed ARP-1), have been identified in mammals. These are closely related transcription factors that are widely expressed and involved in the regulation of several biological processes.²⁸ COUP-TFs are generally considered to be repressors of transcription for other nuclear hormone receptors;^28,48,49 however, COUP-TFs can also activate an ever-growing list of gene promoters.⁵⁰ COUP-TFs can exist in solution as either homo- or heterodimers, binding with high affinity to an imperfect (GTGTCANAGGTCA, or ‘DR1’) or perfect (AGGTCANAGGTCA) direct hexanucleotide repeat (in bold type) separated by a one nucleotide ‘spacer’ (or N).^51,52 COUP-TFs may also bind to a number of variably spaced direct hexanucleotide repeats.^51,52 Here, we found that COUP-II-TF exerted quite different effects on the G and A alleles (Figure 5b, center), with the A response exaggerated, perhaps expected in that the A allele provides a better COUP-TF consensus match (Figure 5a).

The basal expression pattern of G−462A was G>A (Figure 5b, left), whereas the COUP-activated pattern was A>G (Figure 5b, center), suggesting that COUP alone might not account for differences in basal expression of the G−462 versus −462A alleles. We thus turned to other potential transactivators of G−462A. To activate potential COUP-TF/retinoid X receptor heterodimers, we treated the transfected promoters with retinoic acid, a stimulus known to affect CHGA expression;²⁹ once again, the G and A alleles responded differently, this time with a greater response by the G variant (G>A; Figure 5b, right). The response to retinoic acid mirrored the basal promoter differences (in each case, G>A; Figure 5b, left and right panels), suggesting that the basal expression differences (G>A) might in part be mediated by retinoids.

Conclusion and perspectives

We began with an analysis of common haplotypes in the CHGA promoter, and first established an early, potentially pathogenic effect on autonomic control of the circulation. The effect was maximal for promoter variant G−462A, which also exhibited an effect on resting/basal BP in the population. Common promoter haplotypes differed in transcriptional strength in transfected promoter/reporter constructs. The G−462A site altered a consensus COUP-TF transcriptional motif, and site-directed mutagenesis of the site altered not only basal transcription but also the response to COUP-II-TF or retinoic acid. The findings are consistent with a cascade of events (Figure 7), beginning with the G−462A variant and eventuating in altered BP. The results suggest new approaches to the pathogenesis, prediction, and management of disorders of autonomic circulatory control.

MATERIALS AND METHODS

Molecular genetics

Genomic DNA was prepared from leukocytes in EDTA-anti-coagulated blood, using PureGene extraction columns (Gentra Biosystems, Minneapolis, MN, USA) as described.⁵³

Genotyping

SNP diploid genotypes were scored by either of two base-extension systems: the MALDI-TOF system of Sequenom (La Jolla, CA, USA)⁵⁴ or the luminescent system of Pyrosequencing (Uppsala, Sweden).⁵⁵ In each case, initial PCR amplification of the template was followed by primer-mediated base extension across the variant position. Pyrosequencing primers were designed using the dedicated software provided by Pyrosequencing. Target sequences were amplified by PCR from 15 ng genomic DNA in a final volume of 10 µl. To ensure accurate assignment, genotypes were verified by visual inspection and artifactual data were excluded from further statistical analysis. CHGA SNP genotype call rates in the twins (with the Hardy–Weinberg equilibrium, χ² and P-values) were as follows: C−1014T, 97.9% (χ²=10.98, P=0.001); G−988T, 98.1% (χ²=0.755, P=0.385); G−462A, 92.8% (χ²=0.699, P=0.403); C−415T, 90.0% (χ²=1.77, P=0.183); and A−89C, 97.2% (χ²=0.359, P=0.549). As C−1014T deviated from the Hardy–Weinberg equilibrium, we also imputed promoter haplotypes (see below) with and without this SNP, but the individual haplotype assignments did not change.

Polymorphism discovery across the CHGA locus

Human reference genome sequence was obtained from the University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu). We resequenced the CHGA locus (eight exons, intron/exon borders, untranslated regions, and proximal promoter) for systematic variant discovery in n=180 subjects (2n=360 chromosomes) of diverse ancestries, as previously described.¹⁹

Subjects and clinical characterization

Twin pairs

Twin subject characteristics are described in our previous reports,²² and each subject gave written informed consent to the protocol approved by the local institutional review board. Twin pairs, aged 15–84 years (median, 40 years), were 69% monozygotic and 31% dizygotic. Twin zygosity was confirmed by single-nucleotide and microsatellite polymorphisms, as previously described.^38,56,57 A total of 9.9% of the twins are hypertensive (8.8% treated with antihypertensive medications). Twin phenotyping is described below.

Environmental (cold) stress in twin pairs

A total of 112 (N) twin pairs (2n=224 individuals) were studied. For marker-on-trait analyses, each subject was self-identified as being of white (European) ancestry, as were both parents and all four grandparents. BP and heart rate were recorded continuously and non-invasively with a calibrated radial artery applanation device and dedicated sensor hardware (Colin Pilot; Colin Instruments, San Antonio, TX, USA) and software (ATLAS, WR Medical, Stillwater, MN, USA; TDA (Tonometric Data Analysis), Colin Instruments) during the CPT (cold pressor test; immersion of the left hand in ice water for 60 s, after a 10-min rest), as previously described.^16,22 Heart rate was similarly recorded with thoracic EKG electrodes. We identified at least three beats with consistent (within ±10%) values for BP and heart rate just before and at the end of the CPT, and resulting changes in BP and heart rate were recorded after 1 min of cold exposure.

Hypertension sample

A case–control study was performed in a University of California, San Diego (UCSD and VA clinics) sample of 920 individuals, of whom 22% (n=204 cases) were diagnosed with essential hypertension, whereas 78% (n=715 controls) were normotensive. BP was obtained in seated individuals by brachial oscillometric cuff, and triplicate values were averaged. These individuals were 49.4% (n=454) male and 50.6% (n=465) female, and aged 42.9±0.5 (range: 15–89) years. Hypertensive subjects with evidence of secondary hypertension (on physical examination or screening laboratory tests) were excluded; renal function was normal (serum creatinine ≤1.5 mg per 100 ml) in all individuals. BP values were age- and sex-adjusted for genetic analyses. All individuals were self-identified as of white race, with 90.5% identified as solely of European ancestry and 9.5% with Mexican ancestry. Diploid genotype frequencies at CHGA G−462A did not differ by ancestry (χ²=4.48, P=0.106), and ethnicity as a covariate did not affect the marker-on-trait analysis for hypertension. Seventy-two percent of the hypertensives were treated with one or more antihypertensive medications (including angiotensin-converting enzyme inhibitors, β-adrenergic antagonists, and diuretics); marker-on-trait analysis results were not affected by medication adjustment of BP values using the algorithm of Cui et al.²⁶ SBP (at 143.6±1.3 versus 126.5±0.6mmHg, P<0.001), DBP (at 79.0±0.8 versus 70.8±0.4mmHg, P<0.001), and body mass index (at 28.5±0.4 versus 25.6±0.2 kgm⁻², P<0.001) were elevated in the subjects with hypertension.

Function of CHGA promoter variants

Cell culture

Neuroendocrine cell lines were the rat adrenal medullary chromaffin cell line PC12³⁹ and the mouse anterior pituitary corticotrope cell line AtT20.⁵⁸ Cell lines were grown in high-glucose Dulbecco’s modification of Eagle’s medium with penicillin G (100Uml⁻¹) and streptomycin sulfate (100 mgml⁻¹). The medium for AtT20 cells was supplemented with 10% fetal bovine serum. The medium for PC12 cells was supplemented with 10% horse serum and 5% fetal bovine serum.

Promoter/luciferase reporter plasmids

Human CHGA promoter/reporter plasmids were constructed essentially as previously described.^19,46,59 Haplotype-specific promoter fragments corresponding to CHGA −1142/+54 bp (−/+ with respect to the cap site) were amplified from genomic DNA of known homozygotes (or heterozygotes for the two least common haplotypes), and subcloned into the upstream promoter site of the pGL3-Basic vector (Promega Inc., Madison, WI, USA). Synthetic replacements were made by site-directed mutagenesis (Quik-Change; Stratagene, La Jolla, CA, USA). All promoter fragments were sequence-verified before use. Promoter positions are numbered upstream (−) or downstream (+) of the cap site. Plasmids were purified on columns (Qiagen, Valencia, CA, USA) before transfection.

Transfection and reporter assay

Cell lines were transfected (at 50–60% confluence, 1 day after 1:4 splitting) with 1 µg of the constructs mentioned above and 10 ng of the Renilla luciferase expression plasmid pRL-TK (Promega Inc.), as an internal control per well, by the liposome method (Superfect; Qiagen). The firefly and Renilla luciferase activities in the cell lysates were measured 18 h after transfection, and the results were expressed as the ratio of firefly/Renilla luciferase activity (Stop & Glo; Promega Inc.). Each experiment was repeated a minimum of three times.

Secretory or transcriptional stimulation

Human CHGA promoter/reporter plasmids were transfected into PC12 cells as described above, and the secretory stimuli nicotine (1mm), vasoactive intestinal peptide (1 µm), or pituitary adenylyl cyclase-activating polypeptide (0.2 µm) were added to the medium. The firefly luciferase activities in the cell lysates were measured 18 h after transfection, and the results were expressed as the ratio of firefly luciferase activity/cell lysate protein. Retinoic acid (1 µm) was added to PC12 cell cultures after transfection of CHGA promoter vectors. The firefly luciferase activities in the cell lysates were measured 24 h after transfection, and the results were expressed as the ratio of firefly luciferase activity/cell protein. A 50 ng portion of a COUP-TFII (ARP-1)⁶⁰ expression plasmid (in the cytomegalovirus promoter vector pCR3.1; Invitrogen, Carlsbad, CA, USA) or the insert-less control vector (pCR3.1) were co-transfected with the CHGA promoter/reporter plasmid (1 µg) into PC12 cells. The firefly luciferase activities in the cell lysates were measured 18 h after transfection, and the results were expressed as the ratio of firefly luciferase activity/cell protein. The transactivator plasmids were the gift from Nathanael J Spann, UCSD.

Statistical analyses

Pairwise LD between each common SNP pair across the CHGA locus was visualized by Haploview.²⁰ We selected promoter SNPs for analysis based upon the 5′ haplotype block at CHGA (Figure 1), focusing on the core of that block within the proximal promoter (SNPs 3–7; C−1014T, G−988T, G−462A, C−415T, and A−89C) with especially high LD between SNPs 3 (T−1014C) and 7 (C−89A). HAP (version 3.0) was used to impute promoter haplotypes from diploid genotype data at these five individual SNP loci in twins.²³ Haplotype assignments were conducted with all twin individuals included or with just one twin per pair; the results were identical.

Descriptive statistics (mean±s.e.m.) were computed for genotype groups across both members of each twin pair, using generalized estimating equations, in SAS (Statistical Analysis System, Cary, NC, USA), establishing an exchangeable correlation matrix to take into account intra-twin-pair correlations.⁶¹ Data were stored in Microsoft Access, and analyses were conducted in SPSS (Statistical Package for the Social Sciences, Chicago, IL, USA; for the BP association study), SAS, or SOLAR (for trait heritability). If traits were not normally distributed, values were log₁₀-transformed to decrease skewness. One-way post hoc analysis of variance with Bonferroni correction and T-tests were performed to evaluate the effects of particular promoter SNPs during in vitro reporter gene expression activity. To safeguard against the consequences of multiple comparisons when testing the effect of five CHGA SNPs in the promoter haplotype block on autonomic traits, we used the method of SNPSpD proposed by Nyholt²⁵ and available at http://genepi.qimr.edu.au/general/daleN/SNPSpD/ to yield an ‘effective’ number of markers within a block of linkage disequilibrium. For this purpose, we used CHGA promoter SNP data from one member of each twinship (that is, one founder per family). This method takes into account intermarker correlations in calculating a new experiment-wide threshold to keep the type I error rate at ≤5%.