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. Author manuscript; available in PMC: 2011 Apr 6.
Published in final edited form as: J Am Coll Cardiol. 2010 Apr 6;55(14):1463–1475. doi: 10.1016/j.jacc.2009.11.064

Common Functional Genetic Variants in Catecholamine Storage Vesicle Protein Promoter Motifs Interact to Trigger Systemic Hypertension

Kuixing Zhang 1, Fangwen Rao 1, Lei Wang 1, Brinda K Rana 1, Sajalendu Ghosh 1, Manjula Mahata 1, Rany M Salem 1, Juan L Rodriguez-Flores 1, Maple M Fung 1, Jill Waalen 1, Bamidele Tayo 1, Laurent Taupenot 1, Sushil K Mahata 1, Daniel T O'Connor 1
PMCID: PMC2889490  NIHMSID: NIHMS193643  PMID: 20359597

Abstract

Objectives

The purpose of this study was to explore transcriptional mechanisms whereby genetic variation in the CHGB promoter influence BP and hypertension.

Background

Hypertension is a complex trait in which deranged autonomic control of the circulation may be an etiological culprit. Chromogranin B (CHGB) is a major soluble protein in the core of catecholamine storage vesicles, playing a necessary (catalytic) role in the biogenesis of secretory vesicles. Previously we found that genetic variation at CHGB influenced plasma CHGB expression as well as autonomic function, and that BP association was maximal towards the 5′ end of the gene.

Methods

After polymorphism discovery, we functionally characterized the 2 common variants in the proximal CHGB promoter, A-296C and A-261T, which lay within the same haplotype block in black and white populations. CHGB promoter activity was studied by haplotype/luciferase reporter transfection. Transcriptional mechanisms were probed by EMSA and ChIP.

Results

The A-296C variant disrupted a c-FOS motif, and exhibited differential mobility shifting to chromaffin cell nuclear proteins during EMSA, differential binding of endogenous c-FOS on ChIP, and differential transcriptional response to exogenous c-FOS. A-261T disrupted motifs for SRY and YY1, with similar consequences for gel mobility during EMSA, endogenous factor binding during ChIP, and transcriptional responses to the exogenous factors. 2-SNP haplotype analyses demonstrated a profound (p∼3×10-20) effect of CHGB promoter variation on BP in the European ancestry population, with a rank order of CT<AA≪CA<AT on both SBP and DBP, accounting for ∼2.3% of SBP variance and ∼3.4% of DBP variance; the haplotype effects on BP in vivo paralleled those on promoter activity in cella. Site-by-site interactions at A-296C and A-261T yielded highly non-additive effects on SBP and DBP. CHGB haplotype effects on BP were also noted in an independent (African ancestry) sample. In a predominantly normotensive twin sample, parallel haplotype effects were noted for a pre-hypertensive phenotype, the BP response to environmental (cold) stress.

Conclusions

Common CHGB promoter variants A-296C and A-261T, and their consequent haplotypes, alter the binding of specific transcription factors so as to influence gene expression in cella as well as BP in vivo. Such variation contributes substantially to the risk for human hypertension. Involvement of the sex-specific factor SRY suggests a novel mechanism for development of sexual dimorphism in BP.

Keywords: Hypertension, catecholamine, chromaffin, chromogranin, exocytosis

Introduction

The sympathoadrenal system exerts minute-to-minute control over cardiac output and vascular tone. Genes governing catecholaminergic processes may play a role in the development of hypertension(1). Sympathoadrenal catecholamine secretion is exocytotic (all-or-none), releasing not just catecholamines also the acidic proteins with which catecholamines are stored. The chromogranins/secretogranins a family of acidic, soluble proteins that are stored in secretory granules with different hormones, transmitters, and neuropeptides throughout the endocrine and nervous system (2). Chromogranin B (CHGB), first described in the 1980s (3,4) (5), is a major catecholamine storage vesicle core protein and seems to play a necessary role in the biogenesis of catecholamine secretory vesicles (6).

CHGB is differentially expressed in neuroendocrine diseases, and its measurement may serve in the diagnosis and staging of such conditions (7-15). Interaction of CHGB with signaling molecules such as the inositol-1,4,5-trisphosphate-activated calcium channel (16) may influence cytosolic calcium and ultimately risk for such disease states as Alzheimer's disease, epilepsy, or schizophrenia (17). In addition, polymorphisms in the CHGB gene may be associated with schizophrenia in Chinese and Japanese populations (18,19).

Expression of CHGB may mark the action of still poorly characterized trans-QTLs influencing exocytotic sympathoadrenal activity (20,21). CHGB is over-expressed in rodent models of genetic (22,23) as well as acquired (24) hypertension, thus suggesting augmented sympathoadrenal activity in the pathogenesis of these syndromes. Therefore CHGB might give rise to early, pathogenic “intermediate phenotypes” (25) for exploration of sympathoadrenal activity in human essential hypertension.

Previously we described genetic variation at the CHGB locus, and concluded that sex and CHGB interact to influence BP(26). Since the association of CHGB genetic variation to BP was maximal towards the 5′ end of the gene, and such variation predicted quantitative changes in CHGB expression, we turned to potential transcriptional mechanisms. Here we characterize the proximal promoter region of CHGB, discovering two common polymorphisms that disrupt transcription factor binding, giving rise to systemic hypertension. One of these sites recognizes the sex-specific factor SRY, providing insight into sexual dimorphism of BP.

Materials and Methods

Subjects and clinical characterization

Subjects were volunteers, and each gave informed, written consent to protocols approved by local institutional review boards. Recruitment procedures, definitions and confirmation of subject diagnoses are according to previous reports.

Systematic polymorphism discovery across the CHGB locus

As previously described (26), we resequenced each of CHGB's 5 exons, exon/intron borders, UTRs, and proximal promoter in n=160 subjects (2n=320 chromosomes) of 4 self-identified biogeographic ancestries: white/European (n=56), black/sub-Saharan African (n=56), Hispanic/Mexican Amerian (n=24), and east Asian (n=24). We used an ABI-3100 capillary system (Applied Biosystems, Foster City, CA) to accomplish dideoxy sequencing.

Primary care population with extremes of high and low blood pressure (European ancestry)

As previously described (26), we ascertained 951 European-ancestry individuals, ∼1/2 male and ∼1/2 female, from the highest and lowest 5th DBP percentiles of a large primary care population in the Kaiser-Permanente Medical Group of southern California (27). The DBP criterion was chosen because of the heritability of DBP (28). The statistical power of association between biallelic DNA markers and human quantitative trait loci can be substantially augmented by the sampling individuals from opposite (upper and lower) ends of the trait distribution (29,30) (31), and analyses of the quantitative trait in extreme subjects (as opposed to dichotomization of the trait) further enhances power (32). This population sample afforded us >90% power (29,30) to detect genotype association with a trait when the genotype contributes as little as 2.5% to the total variation in males (even at p<10-8); the power is even higher in the females(31). Evaluation included physical examination, blood chemistries, hemogram, and extensive medical history questionnaire. 40.6% of the hypertensive group were taking anti-hypertensive medications, while no one in the normotensive group was on such drugs. The subjects are described in on-line Table 1. 1.98% of subjects were excluded because of elevated serum creatinine (>1.5 mg/dl).

Black population with blood pressure extremes (sub-Saharan African ancestry)

357 adult Nigerians (∼1/2 male, ∼1/2 female) selected from the highest (n=191) and lowest (n=190) 25th %iles of population BP were included as a replication sample for CHGB promoter variant effects on BP. This population has been described (33).

Twin pairs (European ancestry)

In studies of the influence of CHGB polymorphism on the pressor response to environmental stress in vivo, 156 twin pairs and 80 siblings (312 individuals) were evaluated. The response of BP to cold stress (by immersion of one hand in ice water for one minute) was evaluated as previously described (34); responses wherein DBP increased after cold stress were analyzed. Zygosity (69% monozygotic and 31% dizygotic pairs) was confirmed by extensive microsatellite and SNP genotyping, as described (35). Twins ranged in age from 15-84 years; 10% were hypertensive. Twins in these allelic/haplotype association studies were self-identified as of European (white) ancestry, to guard against potential artifactual effects of population stratification.

Statistical analyses

Haplotype blocks were visualized in Haploview (36), while haplotype assignments in individuals were performed by the HAP algorithm (37) in individuals with both A-296C and A-261T genotypes. Chi-square tests were performed to test for deviations from HWE. When testing for associations of haplotypes with continuous/quantitative BP traits, sex, age and body mass index (BMI) were included as covariates in the univariate tests of the general linear model in SPSS 11.5 (Chicago, IL). Both the final measured BP, and that BP adjusted for the effects of antihypertensive medication (38), were analyzed. Each factor was then assessed for significance using standard ANOVA F-tests (39). Haplotype analyses were both (diploid) individual-based, as well as chromosome-based: here each haplotype allele (as opposed to a haplotype allele pair) was considered and analyzed separately, using the outcomes and characteristics of the subject carrying that allele (40). Associations between BP status and allele, genotype or haplotype were analyzed in n×2 tables by either ANOVA or by SHEsis (41) at <http://analysis.bio-x.cn/myAnalysis.php>. Twin analyses were conducted in two ways. Twin trait heritability (h2) was estimated in SOLAR (42). Twin descriptive and inferential statistics were computed by generalized estimating equations (GEE) in SAS, to account for intra-pair correlations (35). A p-value of ≤0.05 was considered significant.

Genomics

Genomic DNA was prepared from leukocytes in EDTA-anticoagulated blood, using PureGene extraction columns (Gentra Systems, Minnesota).

Molecular biology

Transfected CHGB promoter haplotype/luciferase reporter activity (on-line supplement).

Genotyping of CHGB variants (on-line supplement).

Electrophoretic gel Mobility Shift Assays (EMSA) (on-line supplement).

Chromatin ImmunoPrecipitation (ChIP) (on-line supplement).

Results

Patterns of linkage disequilibrium (LD) across the CHGB locus

To visualize association patterns, 16 SNPs (each in Hardy Weinberg equilibrium) were scored and plotted by Haploview as pair-wise LD parameter r2 across the ∼14 kbp locus. The proximal promoter (including common variants A-296C and A-261T) was maintained within a single block in both white and black subjects. The allele and genotype frequencies differed between white and black populations (on-line supplementary Table 2). Although pair-wise r2 values were generally higher in white than black subjects, just 2 LD blocks spanned the locus in each group (Figure 1A). The original resequencing strategy and SNP discovery have been described (43).

Figure 1. Human CHGB promoter genetic variation.

Figure 1

Figure 1

Figure 1

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1A: Human CHGB common SNP LD blocks. LD blocks were derived by the solid spine algorithm in Haploview. Resequencing was accomplished across the 5 exons, exon/intron borders, UTRs, and proximal promoter. Results are shown for n=56 subjects of white/European ancestry, and n=56 of sub-Saharan African ancestry.

1B: Human CHGB promoter common variants and domains. Positions are numbered with respect to the mRNA cap (transcriptional initiation) site. The CHGB promoter has a functional TATA box at -44/-38 bp, and a cAMP response element at -119/-112 bp. There are two common variants in proximal promoter (A-296C and A-261T), with and one common variant in the more distal promoter (C-1239T).

1C: Luciferase reporter activity of CHGB promoter 2-SNP haplotype constructs: Shown are basal activity as well as the transcriptional responses to classical chromaffin cell secretory stimuli: nicotine (acting at nicotinic cholinergic receptors) and PACAP. ANOVA was computed on unadjusted data for each state, rather than fold-change.

Domains and motifs in the CHGB promoter

Figure 1B diagrams known motifs in the CHGB promoter, and superimposes common variants. Functional domains in the core/proximal promoter (such as the TATA box, cAMP response element, and G/C-rich regions) were invariant in ∼180 people (∼360 chromosomes) subjected to systematic polymorphism discovery by resequencing.

Two very common SNPs (MAF >30%) occur in the proximal promoter: A-296C and A-261T, whose allele, diploid genotype, and haplotype frequencies differed by ethnicity (on-line/supplementary Table 2). The A-296C variant lies in a c-FOS transcriptional control motif (-298/-291), while A-261T lies in recognition motifs for both YY1 (-264/-259) and SRY (-265/-260).

CHGB promoter haplotype/reporter activity assays

To probe the functional significance of the two common promoter variants for transcriptional efficiency, we inserted each of the 4 haplotypes of the 2 promoter SNPs (A-296C and A-261A) into the luciferase reporter plasmid pGL3-Basic (supplementary Figure 1). After transfection into rat chromaffin (PC12) cells, these 4 haplotypes yielded substantially different luciferase reporter activity (Table 1). Site-specific effect analysis of reporter activity showed that both A-296 and A-261 have context-dependent actions. The strengths of combinations of A-296C and A-261T were: AT>CT>AA>CT (p=8.46E-07, Table 1). By 2-way ANOVA: overall F=107.2, p=8.46E-07; A-296C, p=2.09E-05; A-261T, p=0.064; 296-by-261 interaction, p=3.07E-07.

Table 1. CHGB promoter variants A-296C and A-261T: Context-dependent allelic effects on luciferase reporter activity.

The relative activities of haplotypic combinations of A-296C and A-261T are: AT>CA>AA>CT. 2-way ANOVA: overall F=107.2, p=8.46E-07; A-296C, p=2.09E-05; A-261T, p=0.064; 296-by-261 interaction, p=3.07E-07.

Variant Context Allele Luciferase Change %
(Maj→Min)		 Two tailed
T-test (P)
Mean SEM
A-296C -261T A-296 4.144 0.173 -73.36 0.0006
-296C 1.104 0.087
A-261 A-296 1.942 0.103 +42.47 0.007
296C 2.767 0.121
A-261T A-296 A-261 1.942 0.103 +113.4 0.0011
-261T 4.144 0.173
-296C A-261 2.767 0.121 -60.10 0.0006
-261T 1.104 0.087
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Secretory stimulation

We evaluated responses of CHGB promoter haplotypes to agents simulating natural secretory stimuli (Figure 1C). PACAP increased promoter activity by ∼4.1-7.7-fold (p=4.86E-14), while nicotine increased activity by ∼0.6-1.5-fold (p=1.34E-8). There were differences among the 4 haplotypes in response to PACAP (p=1.9E-5), though nicotine responses were similar (p=0.31).

CHGB promoter variant A-296C

The A-296C variant lies in an evolutionarily conserved region among humans, non-human primates, and other mammals (supplementary Figure 2). During EMSA, a labeled oligonucleotide representing the C allele was shifted by PC12 nuclear proteins; specificity was suggested by displacement with the same (C) allele when unlabeled (supplementary Figure 3).

A-296C occurred in a potential recognition site for transcription factor c-FOS, with a 7/8 base consensus match for the A allele, declining to 6/8 for the C allele (Figure 2A). When interrogated by ChIP, endogenous c-FOS binding to the motif was detected in all four haplotypes, though unexpectedly more intense for the -296C than the A-296 allele (Figure 2A).

Figure 2. Functional characterization of CHGB promoter common variant A-296C.

Figure 2

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2A: Endogenous c-FOS: Motif and ChIP. A-296 creates a 7/8 bp match with c-FOS. Experimentally, endogenous c-FOS can be captured by ChIP on all four 2-SNP haplotypes, though more intense at -296C than A-296. The sequence match for the c-FOS binding site is on the reverse complement (i.e., minus) strand, is. The c-FOS core motif, TGASTCAC, is found in the JASPAR (60) transcription factor binding database at <http://jaspar.cgb.ki.se/>. IUPAC symbols are according to: <http://www.bioinformatics.org/sms/iupac.html>.

2B: Exogenous c-FOS: Effect of co-transfected c-FOS on transcriptional activity of CHGB promoter haplotypes in PC12 chromaffin cells. c-FOS consistently increases CHGB promoter expression, though the magnitude is haplotype-dependent, especially in the -261T context.

When a plasmid expressing c-FOS was co-transfected into PC12 cells with CHGB promoter/reporters, all 4 CHGB haplotypes responded (p=2.36E-9), though unequally (p=7.23E-5). On a haplotypic background of the A-261 allele, c-FOS stimulated A-296 and -296C similarly, though on a background of the -261T allele, the transcriptional response of A-296 was far greater than -296C (Figure 2B). Thus, the A-296C response to exogenous c-FOS seemed to be context-dependent.

CHGB promoter variant A-261T

A-261T variant also occurs in an evolutionarily conserved region (supplementary Figure 4). During EMSA the T allele was more effectively shifted by PC12 nuclear proteins than the A allele (supplementary Figure 5). During EMSA, an oligonucleotide spanning the A allele was shifted by PC12 nuclear proteins, while the T allele was shifted to a lesser degree; specificity was suggested, especially for the A allele, by displacement with the same allele when unlabeled (supplementary Figure 5).

A-261T occurred in potential recognition motifs for the transcription factors SRY and YY1: the A allele displayed a superior match to both SRY (5/6 bases) and YY1 (6/6 bases) motifs, as compared to the T allele (Figure 3B). Involvement of endogenous SRY and YY1 was probed by ChIP (Figure 3A): for both SRY and YY1, the A allele was more effectively bound than the T allele, on either A-296C haplotypic background.

Figure 3. Functional characterization of CHGB promoter common SNP A-261T.

Figure 3

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3A: Endogenous SRY and YY1: Motifs and ChIP. A-261 creates a better sequence match with both SRY (5/6 bp) and YY1 (6/6 bp). ChIP confirmed increased binding of the endogenous factors by A>T alleles, on either haplotypic background, for both SRY and YY1. The SRY motif WACAAW is derived from Lovell-Badge reviews (61,62). The YY1 motif RCCATC is found in the JASPAR (60) transcription factor binding database at <http://jaspar.cgb.ki.se/>. IUPAC symbols are according to: <http://www.bioinformatics.org/sms/iupac.html>.

3B: Exogenous SRY and YY1: Effect on transcriptional efficiency of CHGB promoter haplotypes in PC12 chromaffin cells. SRY consistently decreases transcriptional efficiency, though the effect is context-dependent. YY1 increases transcription efficiency, preferentially for the A-261 allele, on both A-296C backgrounds.

When a plasmid expressing SRY was co-transfected into PC12 cells with CHGB promoter/reporters, all four haplotypes showed decreased reporter activity (p=1.15E-10, Figure 3B), though the degree of inhibition depended on A-296C background (p=4.29E-8).

When co-transfected with a YY1 expression plasmid, CHGB promoter reporter activity increased for each haplotype (p=7.6E-10), and the effect was more prominent for the A-261 allele than the -261T allele, regardless of A-296C context (p=1.64E-4, Figure 3B).

CHGB promoter common variants A-296C and A-261T: Implications for hypertension in the population

Here we studied BP trait-extreme individuals of European ancestry, in order to enhance statistical power (29,30). Chromosome-based haplotype analysis on subjects dichotomized into two groups (higher versus lower BP) indicated that individuals with the less common AT or CA haplotypes had a strong tendency to be hypertensive (odds ratio=4.898 for AT haplotype, p=3.19E-11; odds ratio=3.84 for CA haplotype, p=3.64E-10). People with the more common haplotype CT had a strong tendency to be normotensive (odds ratio=0.637, 95% CI=0.524-0.773, p=4.64E-6). The most common haplotype AA had no effect on blood pressure status. The overall effect of CHGB haplotypes on BP status was substantial (Figure 4A and Table 2), whether analyzed by chromosome/haplotype (global χ2=93.9, p=3.16E-20); or by diploid haplotype pairs (global χ2=75.0, p=4.92E-13).

Figure 4. CHGB promoter haplotypes and blood pressure in a population sample with extreme trait values.

Figure 4

Figure 4

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Haplotypes are constructed across promoter polymorphisms A-296C/A-261T.

4A: Prediction of blood pressure status by CHGB promoter haplotypes in white (European ancestry) BP extremes. Haplotypes (or haplotype pairs) are ordered by rank of association with elevated blood pressure. Left: Haplotype pair (diploid haplotype) effects. Right: Haplotype (chromosome) effects. The two most common haplotypes (AA and CT) are more frequent in subjects with lower BP, while the two less common haplotypes (CA and AT) are more frequent in subjects with higher BP.

4B: CHGB promoter haplotypes in white BP extreme subjects: Effect of copy number. Three haplotypes demonstrated copy-number (0,1,2) dependent effects on both DBP and SBP. Haplotypes AT and CA increased blood pressure, while CT decreased blood pressure. Age, sex and BMI had significant effects on SBP and DBP; thus, analyses included age, sex and BMI as covariates.

4C: CHGB promoter common polymorphisms A-296C/A-261T: Effect of SNP-by-SNP interaction on BP in white trait extremes. For A-296C polymorphism, on the background of A/A (major allele) homozygosity, both SBP and DBP were elevated progressively by A/A<A/T<T/T genotypes at A-261T (accounting for ∼27 mmHg SBP and ∼22 mmHg DBP); on a background of C/C (minor allele) homozygosity, both SBP and DBP declined progressively as a function of A/A<A/T<T/T genotypes at A-261T (accounting for ∼22 mmHg of SBP and ∼18 mmHg of DBP).

Left panel: Interaction effect on SBP. 2-way ANOVA, overall: F=33.67, p=9.34E-61; interaction: F=8.4, p=1.15E-6.

Right panel: Interaction effect on DBP. 2-way ANOVA, overall: F=36.27, p=6.16E-65; interaction: F=13.49, p=1.07E-10.

4D: CHGB promoter common polymorphisms A-296C/A-261T: Parallel effects on BP in vivo and gene expression in cella.

Left panel: CHGB promoter haplotypes as predictors of BP in the white trait extreme sample. Covariates for SBP: Age: F=229.98, p=3.83E-49, Sex: F=16.64, p=4.718E-5, BMI:F=380.19, p=2.27E-77. Covariates for DBP: Age: F=39.34, p=4.4E-10; Sex: F=28.64, p=9.78E-8; BMI: F=550.08, p=5.5E-107.

Right panel: Parallel effects of CHGB promoter haplotypes on both BP in vivo and gene expression in cella.

4E: CHGB promoter polymorphisms A-296C/A-261T: Haplotype effects on BP in a black (Nigeria) sample with trait extreme values.

Left panel: CHGB promoter haplotype CA in a Nigerian sample: Copy number effect. Covariates for SBP: Sex: F=0.684, p=0.4092; Age: F=5.63, p=0.018; BMI: F=3.07, p=0.081. Covariates for DBP: Sex: F=2.589, p=0.108; Age: F=2.48, p=0.116; BMI: F=4.65, p=0.032.

Right panel: CHGB promoter haplotype effects on BP in a Nigerian BP extreme sample. Covariates for SBP: sex: F=1.375, p=0.241; Age: F=11.26, p=0.001; BMI: F=6.34, p=0.012. Covariates for DBP: Sex: F=5.12, p=0.024; Age: F=4.94, p=0.027, BMI: F=9.48, p=0.002.

Table 2. CHGB common promoter variant effects on blood pressure in a European ancestry population sample with extreme BP values.

This case/control study of CHGB promoter common SNPs and haplotypes with BP status in white population with BP extremes was conducted by the program SHEsis1. Bold: significant p values (p<0.05). (Parentheses): fraction of individuals in that group with the indicated haplotype or genotype. HT: hypertensive. NT: normotensive.

2A: Haplotype analysis.
Haplotype HT NT Odds Ratio [95%CI] χ2 Fisher's P
AA 437 (0.534) 610 (0.562) 0.892 [0.743∼1.070] 1.52 0.218
AT 67 (0.082) 19 (0.018) 4.898 [2.933∼8.180] 44.3 3.19E-11
CA 74 (0.091) 27 (0.025) 3.840 [2.455∼6.008] 39.5 3.64E-10
CT 240 (0.293) 428 (0.394) 0.637 [0.524∼0.773] 21.0 4.64E-06
Total n 818 1084
Global χ2 93.9
P value 3.16E-20
2B: Association study based on allele frequencies: n (fraction).
A-296C BP A-296 -296C χ2 P
HT 577 (0.622) 351 (0.378) 4.3 0.038
NT 648 (0.577) 476 (0.423)
A-261T A-261 -261T χ2 P
HT 539 (0.625) 323 (0.375) 1.2 0.109
NT 651 (0.59) 453 (0.41)
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1

Shi YY, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphic loci. Cell Res. 2005;15:97-98.

Single SNP-based allele tests showed far less power to detect BP associations (Table 2): A-296C (though not A-261T) had an effect on blood pressure status (p=0.038), with the A-296 allele tending towards hypertension. Of note, when subjects were stratified by sex, A-261T displayed significant (p<0.001/p=0.011) effects on SBP/DBP in males, though not females.

We also pursued association of the quantitative traits (SBP and DBP in mmHg) with CHGB haplotypes (Figure 4B), and here we found substantial predictions for both SBP and DBP. With increasing copy number (0,1,2) of haplotype AT, SBP increased by ∼29 mmHg (p=0.0002), while DBP increased by ∼21 mmHg (p=1.59E-5), each in additive fashion. With increasing copy number of haplotype CA, SBP increased by ∼13 mmHg (p=0.005) while DBP increased by ∼12 mmHg (p=8.49E-6). With increasing copy number of haplotype CT, SBP decreased by ∼11 mmHg (p=0.0045), with a parallel decrease in DBP by ∼7 mmHg (p=0.011). When we adjusted BP values for the effects of antihypertensive medications in treated hypertensives (38), the haplotype effects on SBP/DBP persisted or increased: AT, p=4.1E-5/p=6.83E-6; CA, p=0.008/p=2.04E-5; CT, p=0.002/p=0.008. Haplotypes AA and AT displayed more prominent effects on BP in females, while CT had a greater effect in males.

Promoter polymorphisms A-296C/A-261T interacted non-additively to influence SBP and DBP (p=1.15E-6/p=1.07E-10, Figure 4C). On a background of A-296C major allele homozygosity (A/A), the A-261T major (A) allele lowered SBP/DBP by ∼27/∼22 mmHg, while on a background of A-296C minor allele homozygosity (C/C), the A-261T major (A) allele elevated SBP/DBP by ∼21/∼18 mmHg. When treatment-adjusted (38) BPs were analyzed, the interactions persisted or increased: SBP/DBP, p=4.41E-7/p=1.08E-10. The A-296C-by-A-261T (SNP-by-SNP) interaction was confirmed on analyses of the dichotomous BP trait (higher versus lower): p=3.71E-15.

SBP/DBP values predicted by CHGB promoter haplotypes were, in rank order (Figure 4D, left): CT<AA≪CA<AT (SBP/DBP, p=6.16E-8/p=5.18E-12). On ANOVA, CHGB promoter genetic variation accounted for ∼2.3% of SBP variance and ∼3.4% of DBP variance in this primary care population.

Parallel effects of human CHGB promoter hapotypes in vivo and in cella

Of note, the 4 promoter haplotypes display the same rank order for effects on BP in vivo and luciferase reporter activity in chromaffin cells in cella (Figure 4D, right).

Extension to a second population: sub-Saharan African hypertension

An association of CHGB promoter haplotypes and hypertension was also found in a Nigerian population selected for extreme BP values (top and bottom 25th %iles)(p=0.007 for BP status, on-line Table 3). Here haplotype -296C/A-261 increased SBP by ∼34 mmHg (p=0.002) and DBP by ∼22 mmHg (p=3.52E-4, Figure 4E, left). The rank order of overall haplotypic variation on blood pressure (Figure 4E, right) was: AA, AT<CT≪CA (SBP/DBP, p=0.007/p=0.002). While haplotype CA was associated with higher BP in both black and white subjects, haplotype AT predicted higher BP only in whites. However, CHGB promoter allele and haplotype frequencies differed substantially between black and white populations (on-line supplementary Table 2), perhaps contributing to different haplotype effects on BP; for example haplotype AT is relatively unusual in whites but far more common in blacks (Figure 4E and on-line Table 2). CHGB promoter haplotypes did not clearly differ in effects on BP between sexes.

“Intermediate” trait: Association of heritable BP response to environmental stress with CHGB promoter variants A-296C and A-261T

In predominantly (∼90%) normotensive white twin pairs (n=163 pairs), the DBP response to environmental (cold) stress was substantially heritable (h2=32±8%, p=0.0003)(44). CHGB promoter individual genotypes A-296C (A>C, p=0.0237) and A-261T (A>T, p=0.037) predicted ΔDBP in the cold stress test. Because of the modest sample size (2n=326 chromosomes), we observed only 5 examples of CA haplotypes and one AT haplotype, so analyses could only be performed for haplotypes AA and CT. Consistent with basal BP in white BP extremes (Figure 4A), the CT haplotype decreased the stress ΔDBP, while the AA haplotype increased ΔDBP (Figure 5); thus, the rank order of effects of the haplotypes on BP was preserved in twins (AA>CT), though the effects of the more prominent BP-increasing haplotypes (CA, AT) could not be quantified in this predominantly normotensive sample.

Figure 5. CHGB promoter common polymorphisms A-296C/A-261T: Haplotype effects on ΔDBP in the environmental (cold) stress test in twin pairs.

Figure 5

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Diploid haplotype pair (diplotype) effects for the two most common haplotypes (A→A and C→T) are shown. Results are analyzed by GEE.

Discussion

Overview

Patients with hypertension often exhibit increased sympathetic activity,(45,46) and people with sympathetic overactivity tend to develop hypertension(47,48). Suppression of Chgb expression in chromaffin cells leads to a reduction in the number of catecholamine secretory granules, whereas ectopic expression of Chgb in non-neuroendocrine cells, which normally do not contain regulated secretory machinery, leads to granule biogenesis(6). In light of the emerging secretory biology of CHGB, we undertook the present study in order to probe how heredity shapes human functional responses in the sympathetic neuroeffector junction, using CHGB as a likely focal point in the pathogenesis of essential hypertension. Recently we reported CHGB haplotype effects on BP across the CHGB locus, suggesting that the major effect was located in the 5′/promoter region (43), and the effect of CHGB on intermediate traits seems to be quantitative rather than qualitative; thus we focused here on promoter variation at the CHGB locus. We also noted a sex-dependent effect of CHGB polymorphism on BP (43); here we defined the effect of genetic variation on gene expression, and found evidence that the response of the gene to the male-determination factor SRY was altered by one promoter variant (A-261T), raising a potential mechanism for the well-known sexual dimorphism in BP.

CHGB promoter common variants: Transcriptional mechanisms of action

We identified two common variants in the CHGB proximal promoter: A-296C and A-261T. Here we established that these two variants occur in evolutionary conserved regions, and can create or interrupt particular transcriptional control motifs. We probed these processes in two ways: by exposing the variants to the exogenous factors, and by testing whether the endogenous factors recognize the motifs.

A-296C: c-FOS motif

A c-FOS motif was activated by the exogenous factor (Figure 2), and bound by the endogenous factor (Figure 2). c-FOS, a b-ZIP (leucine zipper) factor of the immediate/early class, may heterodimerize with a variety of other such family members (e.g., c-JUN) to trigger transcription, especially by activating AP-1 sites.

A-261T: YY1 and SRY motifs

This variant spanned motifs for both YY1 and SRY (Figure 3). Exogenous YY1 increased CHGB promoter expression (especially for the A-261 allele), while exogenous SRY decreased expression; endogenous factors YY1 and SRY each bound the motif, preferentially for the A-261 (major) allele.

YY1 (Yin-Yang-1) is a widely expressed C2H2 zinc finger transcription factor whose ability to direct local histone modifications within chromatin yields fundamental roles in embryogenesis, differentiation, replication, and proliferation(49).

SRY (sex-determining region Y protein, testis-determining factor) is an HMG (high mobility group box) factor best known as an initiator of male development, in which its major transcriptional targets may include SOX9 (50). Of note for hypertension, we previously found that promoter A-261T exerted a sex-specific effect on population BP, with the effect confined to males (43). Here we provide a basis for that sex-specific effect, since only males express the SRY factor, encoded by the SRY locus on human chromosome Yp11.31. While we focused on the SRY motif match in the A-261T region of the CHGB promoter (Figure 3A), and demonstrated its binding (Figure 3A) and trans-activation (Figure 3B) by SRY, other members of the SOX transcription factor family, including the SRY target SOX9, may share similar consensus DNA target motifs (51) (i.e., SRY as WACAAW; SOX9 as AACAAT), and hence both constitute potential CHGB trans-activators.

Interactions (A-296C-by-A-261T)

The likelihood of site-by-site interactions within the CHGB promoter (Figure 4C) is suggested by two previous observations. First, the YY1 promoter responds transcriptionally to c-FOS (52). Second, the multifunctional factor YY1 interacts non-covalently with a variety of other transcription factors, including members of the b-ZIP family such as CREB (53). While we have documented an interaction in cis between A-296C and A-261T in transfected CHGB promoter haplotype/luciferase reporter plasmids (296-by-261 interaction p=3.07E-07, Table 1), we have not yet explored factor interactions in trans during such transfections.

CHGB promoter common variants and hypertension across populations

LD analysis across the CHGB locus indicated that the proximal CHGB promoter, including common variants A-296C and A-261T, is maintained within one block (Figure 1A) in both white and black populations.

We enhanced power to detect genetic associations by using population trait-extreme values (29,30). We then found that haplotype effects upon BP in the population were highly significant (Figure 4A-4E), indeed far more significant than the effects of single SNPs alone (Table 2). Substantially greater effects on BP by the A-296C/A-261T haplotypes (Figure 4A) than by either SNP alone (Table 2), also speaks toward functional SNP-by-SNP interactions in the CHGB promoter (see transcription factor section just above).

In the European ancestry population, the rank order of haplotype effects on SBP or DBP was AT>CA≫AA>CT (Figure 4D, left), which is the same pattern of CHGB promoter haplotype activity in cella (Figure 1C), lending weight to the viewpoint that altered CHGB transcription underlies the BP differences between haplotypic groups.

In an independent (African) population, allele and haplotype frequencies differed substantially from those in subjects of European ancestry (on-line/supplementary Table 2). Even so, CA haplotype copy number (0,1,2) influenced BP in the Nigeria sample (Figure 4E), with the same directional effect found in white subjects (Figure 4B). Although haplotype AT was found in substantial numbers in Nigerians (134 chromosomes), AT carriers did not display elevated BP, suggesting other factors (such as differences in environment or genetic background) influencing BP in this population, or the inclusion of other variants in the promoter LD block in subjects of African ancestry (Figure 1A).

A previous large, genome-wide association case/control study (GWAS) (the Wellcome Trust Case Control Consortium, or WTCCC) did not find association of the CHGB locus to hypertension(54). How is our study different? First of all, the WTCCC used the Affymetrix 500K gene chip, whose average marker spacing of ∼3×109/500×103, or ∼6 kbp, did not include the CHGB promoter variants considered here; indeed, the closest CHGB promoter variant studied on the Affymetrix 500K chip in WTCCC was rs236129, which is 4729 bp upstream of the cap site. Second, the WTCCC employed single point associations, rather studying haplotype or SNP-by-SNP interaction effects (which were crucial in our analyses). Third, because of relatively sparse marker spacing, the HapMap approach employed in the WTCCC does not fully capture the full spectrum of potentially causal allelic variation at candidate loci (55,56). Finally, the WTCCC used unselected/unphenotyped population controls (57); the high population prevalence (∼24%) of hypertension thus greatly diminishes the power of the WTCCC for associations to hypertension. By contrast, our approach (31), using population trait (BP) extremes, offers substantially greater statistical power to detect genetic associations; indeed, we estimate that our sample has >80% power to detect loci contributing as little as ∼2.5% of BP variance.

“Intermediate” trait: CHGB promoter variants and the heritable BP response to environmental stress in twin pairs

In longitudinal studies, the pressor response to environmental (cold) stress is an effective predictor of future development of hypertension (58,59). We therefore evaluated whether CHGB genetic variation in the transcriptional control region might influence this risk predictor.

In a series of twin pairs, CHGB promoter common haplotypes influenced ΔDBP in the cold stress test (Figure 5), with A-296/A-261 elevating and -296C/-261T diminishing the pressor response, findings that are in rank order with basal BP effects in the population (AA<CT; Figure 4A). Haplotypes associated with much higher BP in the population extreme subjects (AT, CA; Figure 4A) were too infrequent in this predominantly normotensive twin sample for meaningful conclusions to be drawn.

Conclusions and perspectives

Common genetic variants in the CHGB proximal promoter seem to exert a powerful, interactive effect on BP. CHGB promoter variants A-296C and A-261T differed substantially in transcriptional efficiency during luciferase reporter activity assays (Figure 1C), and such activity paralleled SBP and DBP in the population (Figure 4D, right). Particular transcription factors (c-FOS at A-296C; YY1 and SRY at A-261T) differed in activity at the variant sites (Figure 2,3); such effects were captured by both co-transfection and ChIP. Differential SRY effects at A-261T suggest a mechanism that might ultimately contribute to the sexual dimorphism of BP in the population. Substantially greater effects on BP of the variants in combination (rather than as individual SNPs) suggests intra-promoter A-296C-by-A-261T interactions. Finally, CHGB promoter variants predict change in BP in response to environmental stress even in predominantly normotensive individuals, suggesting an early pathway by which hypertension may ultimately be mediated.

Thus common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression, and later the early heritable responses to environmental stress, and finally resting/basal BP in the population (Figure 6). These results point to new molecular strategies for probing autonomic control of the circulation, and ultimately the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension.

Figure 6. CHGB genetic variation: Consequences for autonomic physiology and cardiovascular disease.

Figure 6

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The schematic presents a hypothetical framework integrating the experimental results, and suggests future questions for exploration.

Supplementary Material

Online Figures
Online Tables

Acknowledgments

We appreciate the support of NIH/NHLBI (HL58120), the NIH/NCMHD-sponsored (MD000220) EXPORT/CRCHD minority health center, as well as the NIH/NCRR-sponsored (RR00827) General Clinical Research Center.

Support: Department of Veterans Affairs, National Institutes of Health.

Abbreviations and acronyms

BMI

Body mass index

BP

Blood Pressure

c-FOS

Transcription factor (b-ZIP family)

ChIP

Chromatin ImmunoPrecipitation

EMSA

Electrophoretic Mobility Shift Assay

HWE

Hardy-Weinberg Equilibrium

MAF

Minor Allele Frequency

SNP

Single Nucleotide Polymorphism

SRY

Transcription factor, Sex-determining Region Y (HMG-box family)

QTL

Quantitative Trait Locus

YY1

Transcription factor, Yin-Yang 1 (zinc finger family)

Footnotes

Disclosures. No conflicts of interest to disclose.

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