Published online before print January 23, 2009, doi: 10.1161/CIRCGENETICS.108.785659
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Original Articles |
Autonomic Function in Hypertension
Role of Genetic Variation at the Catecholamine Storage Vesicle Protein Chromogranin B
From the Departments of Medicine, Pharmacology, and the Center for Human Genetics and Genomics (K.Z., F.R., B.K.R., J.R.G., M.M., S.V., V.M., R.M.S., J.L.R.-F., M.M.F., D.W.S., N.J.S., M.G.Z., L.T., S.K.M., D.T.O.), University of California and the VA San Diego Healthcare System, San Diego, Calif; Max Planck Institute of Molecular Cell Biology and Genetics (F.C., A.K., W.B.H.), Dresden, Germany; Department of Medical Sciences (M.S.), Uppsala University, Uppsala, Sweden; and Department of Medical Pharmacology (P.R.), CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, University of Milan, Italy.
Correspondence to Sushil K. Mahata, PhD or Daniel T. O'Connor, MD, Department of Medicine and CHGG, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0838. E-mail: smahata{at}ucsd.edu or doconnor@ucsd.edu
Received April 15, 2008; accepted November 20, 2008.
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Abstract |
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Background— Hypertension is a complex trait, with deranged autonomic control of circulation. Chromogranin B (CHGB) is the most abundant core protein in human catecholamine secretory vesicles, playing an important role in their biogenesis. Does common interindividual variation at the CHGB locus contribute to phenotypic variation in CHGB and catecholamine secretion, autonomic stability of circulation, or blood pressure (BP) in the population?
Methods and Results— To probe interindividual variability in CHGB, we systematically studied polymorphism across the locus by resequencing CHGB (
6 kbp footprint spanning the promoter, 5 exons, exon/intron borders, untranslated regions) in 160 subjects (2n=320 chromosomes) of diverse biogeographic ancestries. We identified 53 single-nucleotide polymorphisms, of which 22 were common. We then studied 1182 subjects drawn from the most extreme BP values in the population (highest and lowest 5th percentiles), typing 4 common polymorphisms spanning the 14 kbp locus. Sliding-window haplotype analysis indicated BP associations peaking in the 5'/promoter region, most prominent in men, and a peak effect in the proximal promoter at variant A-261T (A>T), accounting for 8/6 mmHg BP in males. The promoter allele (A-261) that was a predictor of higher diastolic BP and systolic BP was also associated with lower circulating/plasma CHGB concentration (CHGB439 to 451 epitope) in twin pairs. In twins, the same CHGB variants that were predictors of lower basal CHGB secretion were also associated with exaggerated catecholamine secretion and BP response to environmental (cold) stress; likewise, women displayed increased plasma CHGB439 to 451 but decreased catecholamine secretion as well as BP response to environmental stress. The effect of A-261T on CHGB expression was confirmed in chromaffin cells by site-directed mutagenesis on transfected CHGB promoter/luciferase reporter activity, and the allelic effects of A-261T on gene expression were directionally coordinate in cella and in vivo. To confirm these clinical associations experimentally, we undertook targeted homozygous (–/–) ablation of the mouse CHGB gene; knockout mice displayed substantially increased BP, by 20/18 mmHg, confirming the mechanistic basis of our findings in humans.
Conclusion— Common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression and later catecholamine secretion and the early heritable responses to environmental stress, eventuating in changes in resting/basal BP in the population. Both the early (gene expression) and late (population BP) consequences of CHGB variation are sex dependent. These results point to new molecular strategies for probing autonomic control of circulation and, ultimately, the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension.
Key Words: genetics • hypertension • gene expression • catecholamine • epidemiology • nervous system • autonomic
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Introduction |
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Hypertension is a complex trait in which deranged autonomic control of circulation may be an early etiologic culprit. 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 The chromogranins/secretogranins comprise a family of acidic, soluble proteins that are widely stored in secretory granules with hormones, transmitters, and neuropeptides throughout the endocrine and nervous systems.2 Chromogranin B (CHGB), first described in the 1980s,3,4 appears to be the quantitatively most abundant matrix protein in the core of human catecholamine storage vesicles.5,6 Overexpression and underexpression studies in chromaffin cells indicate that CHGB plays an important role in secretory vesicle biogenesis.7
Clinical Perspective see p 46
CHGB has both extracellular and intracellular roles in the neuroendocrine system. Extracellular roles for CHGB are dependent on its extensive proteolytic processing within chromaffin granules at dibasic cleavage sites8 to form smaller peptides; such peptides may have a role in the neuroendocrine/sympathoadrenal stress response to systemic infection.2 Within chromaffin cells and sympathetic axons, CHGB functions in sorting and trafficking of peptide hormone and neuropeptide precursors to secretory granules,9 perhaps as triggers to secretory granulogenesis.7
Because excess sympathetic activity is implicated in causing hypertension,10 and alterations in sympathetic responses may occur even in the normotensive relatives of patients at genetic risk for later development hypertension, we wondered whether the sympathochromaffin mechanisms, such as the catecholaminergic CHGB system, might be altered in hypertension or in individuals at risk for development of hypertension. In this study, we undertook a systematic study of polymorphism at the human locus, by resequencing CHGB, discovering and associating a series of naturally occurring CHGB variants with gene expression in vivo, and series of sympathochromaffin traits eventuating in the disease state of essential hypertension.
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Methods |
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Subjects and Clinical Characterization
Subjects were volunteers from urban southern California (San Diego), and each subject gave informed, written consent; the protocol was approved by the institutional review board. Recruitment procedures, definitions, and confirmation of subject diagnoses are according to previous reports.11 Genomic DNA of each individual was prepared from leukocytes in EDTA-anticoagulated blood, using PureGene extraction columns (Gentra Biosystems).
Polymorphism Discovery
Because allele frequencies and haplotypes may differ substantially across biogeographic ancestries, a series of 160 individuals (ie, 2n=320 chromosomes; Supplemental Table I) was selected to span a diverse range of 4 biogeographic ancestry groups: white (European ancestry), black (sub-Saharan African), east Asian, and Hispanic (Mexican American) ethnicities, for systematic/comprehensive discovery by resequencing. Characterization of 320 chromosomes afforded us the >99% power for discovery of polymorphisms with as low as 1.4% minor allele frequency. Ethnicity was established by self-identification. None of the subjects had a history of secondary hypertension, renal failure, or diabetes mellitus.
Primary Care Population with Extremes of High and Low Blood Pressure
From a database of more than 53000 people (27478 females and 25528 males) in southern California, we ascertained 1182 European-ancestry individuals, of both sexes, from the highest and lowest 5th percentiles of a primary care population12 in diastolic blood pressure (DBP) distribution. This population sample afforded us >90% power to detect genotype association with a trait when the genotype contributes as little as 3% to the total variation in males; the power is even higher in the females.13 Evaluation included physical examination, blood chemistries, hemogram, and extensive medical history questionnaire. A total of 1.98% of subjects were excluded because of elevated serum creatinine (>1.5 mg/dL).
Twin Pairs
Twins enable estimation of heritability for any trait. In studies of CHGB heritability, as well as the influence of CHGB polymorphism on CHGB expression in vivo and the cold stress test in vivo, 171 twin pairs (342 individuals) were evaluated. Zygosity (69% monozygotic and 31% dizygotic pairs) was confirmed by extensive microsatellite and single-nucleotide polymorphism (SNP) genotyping, as described.14 Twins ranged from 15 to 84 years; 10% were hypertensive. All of the twins in these allelic/haplotype association studies were self-identified as of European (white) ancestry, to guard against the potentially artifactual effects of population stratification.
Molecular Genetics
Details on resequencing of CHGB locus and genotyping of CHGB variants are available in the Data Supplement.
Phenotyping
Details on biochemical phenotyping and physiological phenotyping: environmental (cold) stress test in twin pairs are available in the Data Supplement.
Statistical Analyses
Estimates are stated as mean value±1 standard error. Heritability (h2) is the fraction of phenotypic variance accounted for by genetic variance (h2=VG/VP). Estimates of h2 were obtained by using the variance component method implemented in the Sequential Oligogenic Linkage Analysis Routines package <www.sfbr.org/solar/>.15
Haplotypes were inferred from common SNPs (minor allele frequency 
10%) of CHGB by using either the HAP program,16 which can also generate a likely phylogeny for each variant, or by PHASE.17 Pairwise linkage disequilibrium (LD) between common SNPs was quantified as D' by the Graphical Observation of Linkage Disequilibrium software package.18 Two-way ANOVA or multivariate general linear modeling, using post hoc Bonferroni corrections, was performed in SPSS (Chicago, Ill) to evaluate the significance of single variants and interaction of variants during in vivo association studies as well as in vitro haplotype-specific CHGB promoter/reporter activity. Haplotypes in the blood pressure (BP) extreme population were estimated from unphased diploid genotypes by use of the SNP-Expectation Maximization (SNP-EM) program, which includes omnibus likelihood ratio permutation tests.19 Comparison of haplotype frequencies between population BP extremes (hypertensive cases versus controls) was performed using the SNP-EM algorithm.20 SNP-EM estimates haplotype frequencies for each group using the EM algorithm, taking into account the probability of all possible haplotype pairs, and calculates an omnibus likelihood ratio statistic to compare haplotype frequencies between 2 groups (cases versus controls), and a permutation test to determine significance in the face of multiple comparisons (set at 10000 permutations). SNP-EM was used to perform a "sliding window" analysis to identify associated haplotype lengths (from 1 to 4 SNPs) across the locus,21 thus evaluating all possible haplotypes across the 4 SNPs, thereby interrogating genetic variation at the locus in an unbiased, hypothesis-free way. Additional permutation tests22 on 3x2 contingency tables (diploid genotype versus BP status), implemented at http://www.physics.csbsju.edu/stats/exact.html, were used to confirm genotype effect on the dichotomous BP trait.
For twin pair analyses, descriptive (genotype-specific mean and standard error) and inferential (
2, probability value) statistics were computed across all of the twins, using generalized estimating equations (GEEs; PROC GENMOD) in SAS (Statistical Analysis System; Cary, NC), to account for correlated trait values within each twins, using an exchangeable correlation matrix.23 Details on CHGB promoter/luciferase reporter activity assays and generation and phenotyping of mouse CHGB-targeted gene ablation (knockout) animals are available in the Data Supplement. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agreed to the manuscript as written.
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Results |
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SNP Discovery
In 160 individuals of diverse biogeographic ancestries, we identified 52 SNPs and 1 single base insertion/deletions in 12 amplicons spanning a 5935 bp footprint,
50% of which were not in the previous public databases (eg, dbSNP) (Supplemental Table II; Figure 1). Of these, 22 SNPs were common (at minor allele frequency 5%); 8 SNPs with exceedingly high minor allele frequencies (>40%) were noted in the proximal promoter, exon 4, and intron 4 (Figure 1). There were 14 SNPs in the promoter region, 1 in the 5'-untranslated region (UTR), 21 in the coding exons, 14 in introns, and 3 in the 3'-UTR (Supplemental Table II). Global minor allele frequencies ranged from 0.6% to 48.7%.
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When comparing results across ethnicities, some SNPs were common in each of the 4 groups sampled. On the other hand, the frequencies of some SNPs differed substantially across ethnicities: eg, A is the major allele of promoter SNP A-261T in whites, but the minor allele in blacks, Asians, and Hispanics. We also resequenced CHGB from 3 nonhuman primates (chimp, gorilla, orangutan) to determine the likely ancestral alleles at polymorphic sites (Figure 1); the most common human haplotype matched the chimp at 15 of 18 sites; at the remaining 3 sites (A11727G, G13383A, and A13612C), the chimp allele was found in less common human haplotypes.
SNP Distribution: Haplotypes
To identify variants that are linked in the population, we inferred haplotypes from diploid genotypes at 18 common (minor allele frequency >5%) SNPs at CHGB (Figure 1), stretching 14 kbp from the proximal promoter (C-1239T) to the 3'-UTR (C13612A). Initially using PHASE,17 we identified 7 major haplotypes spanning the locus (Figure 1), accounting for 65% of chromosomes examined. These 7 haplotypes include common variation in coding and regulatory regions and act by LD to span other regions that might influence CHGB gene function. The frequencies of these 7 haplotypes varied across ethnicities (Figure 1), with haplotype 1 the most common in whites/blacks/Hispanics, but haplotype 7 the most common in Asians.
LD Across the CHGB Locus
We scored 11 variants (Supplemental Table III) spanning the locus in n=468 subjects (2n=936 chromosomes) of European ancestry. To visualize patterns of SNP associations, pair-wise correlations among the 11 common SNPs were quantified as LD parameter D' by Graphical Observation of Linkage Disequilibrium18 across the CHGB locus. In these subjects, a single block of LD was maintained across much of the 14 kbp locus (D'>0.8) (Supplemental Figure I).
CHGB Genetic Variation in 1182 Subjects with the Most Extreme BP Values in the Population
Given the relatively high degree of LD across the locus, we selected 4 SNPs with high minor allele frequencies (each at >25%), each in Hardy Weinberg equilibrium, to span 4 structural/functional domains across the gene (promoter, intron, exon, 3'-UTR) in this case/control study. Characteristics of the 4 SNPs and their major haplotypes are shown in Figure 2 and Supplemental Table IV.
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Initially, association of BP status with CHGB polymorphism was performed by a haplotype "sliding window" analysis in SNP-EM (Figure 2A). When all subjects (male and female) were included, none of the 4 variants alone was predictive of BP status; but the most 5' 2-SNP haplotype (across A-261T, C10501T) associated with BP status (P<0.001); haplotypes formed from 3 SNPs (A-261T, C10501T, G11873A; P<0.001) or all 4 SNPs (P<0.001) were also associated with BP status.
When the SNP-EM analysis was confined to male subjects, single SNPs at A-261T, C10501T, or G11873A were each predictive of BP status (P<0.01), with the most significant association by promoter variant A-261T (P=0.003; omnibus likelihood ratio statistic 11.36). Haplotypes composed of 2, 3, or 4 contiguous SNPs could were associated with BP status (each at P<0.05; omnibus likelihood ratio statistics of 22.6, 29.1, and 41.1, respectively).
When the SNP-EM analysis was confined to female subjects, no single SNP was predictive of BP status by itself (P>0.05); when 2 nearby SNPs were included, only the haplotype using the 5'-most SNPs (A-261T, C10501T) was associated with BP status (P=0.05); and when 3 nearby SNPs were tested, no haplotype was predictive of BP status. Haplotypes spanning all 4 SNPs were associated with BP status (P<0.05).
Because the peak SNP and haplotype effects seemed to occur toward the 5'-end of the gene (Figure 2A), we also analyzed the association of promoter A-261T genotype and BP as a quantitative trait (Figure 2B), revealing effects apparently confined to the male sex. In the promoter region (A-261T), the effect of the A-261 allele is to raise both systolic blood pressure (SBP; P<0.001) and DBP (P=0.001) in males.
Coding Region
We identified 21 SNPs in the coding region (Supplemental Figure III), among which 15 nonsynonymous SNPs encoded amino acid substitutions, as well as 1 SNP in the 5'-UTR and 3 in the 3'-UTR. Glu247Arg (0.6%) disrupts an acidic domain (Glu241-Asp249) likely to be important for trafficking of CHGB into secretory granules.2 Gly266Arg (0.6%) creates a dibasic site (Arg266Arg267), likely a recognition site for prohormone processing enzymes.24
Most of the coding region SNPs lie toward the amino-terminal region of the protein, rather than in the carboxy-terminal region encoding the 5 best-studied peptides, ie, GAWK, PE-11, secretolytin, CCB, or chrombacin, suggesting relative conservation of sequence in this region (Supplemental Figure III).
Heritability and the Effect of CHGB Variation on Gene Expression in 171 Twin Pairs In Vivo
The plasma concentrations of 3 CHGB regions assayed seemed to be under substantial genetic control, with h2 ranging from 50% to 90% (Supplemental Figure IV), and maximal h2 for CHGB439 to 451 (at 91±9%, P<0.0001), encoded by exon 4 (Supplemental Figure III).
To probe the functional significance of common variation at CHGB, we examined the influence of common polymorphism on the expression of CHGB gene products in vivo. Because CHGB439 to 451 bears the highest heritability in CHGB fragments, we assayed plasma CHGB439 to 451 in n=171 twin pairs (342 individuals) typed for 11 common SNPs spanning the locus (Supplemental Table III); these 11 variants were chosen for very dense spacing across the locus (every 1.3 kbp across 14 kbp), high minor allele frequency (>20% for each), and each in Hardy Weinberg equilibrium. We then inferred haplotypes in the twins, using HAP16 and the 11 SNPs (Supplemental Table V). Copy number of the most common haplotype spanning the locus (haplotype 1, at 34% of chromosomes; AATGAGCAGGA) was predictive of plasma CHGB439 to 451 (P=0.019, Figure 3A): increasing copy number (0
12 copies/genome) progressively decreased the peptide by 33%. When we considered haplotype pairs (diplotypes), combinations of haplotypes 1 and 2 (haplotype 2 is CTTAAATGAAC, at 23% of chromosomes) revealed a progressive increase in plasma CHGB439 to 451, by 55% (P=0.0033), from haplotype 1 homozygotes through haplotype 2 homozygotes (Figure 3B).
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Twin pairs also allowed us to estimate the h2 of other "intermediate" traits for future development of hypertension,14 such as urine epinephrine excretion (at h2=67.6±4.9%, n=316, P<0.0001) and the DBP response to cold stress (at h2=32±8%, n=326, P=0.0003).
Sex Influence on CHGB Secretion and "Intermediate" Phenotypes for Later Development of Hypertension (in 171 Twin Pairs)
Because sex had a profound effect on the association between CHGB genotype and resting BP in the population (Figure 2A and 2B), we used our set of predominantly healthy, normotensive twin pairs and siblings to explore the effect of sex on both CHGB secretion and other early, "intermediate" phenotypes for later development of hypertension25: the BP response to environmental (cold) stress (Figure 4A) and epinephrine secretion (Figure 4B). Plasma CHGB439 to 451 concentration was lower (P=0.015) in males (0.313±0.012 nM, n=203) than females (0.349±0.008 nM, n=544). By contrast, the 
DBP was higher (P=0.014) in males (14.3±1.6 mmHg, n=62) than females (11.3±0.8 mmHg, n=236); similarly, urine epinephrine secretion was lower (P=0.028) in females (9879±686 ng/g, n=267) than males (13526±1337 ng/g, n=77). Thus women, a group at decreased risk for development of hypertension,13 displayed not only diminished pressor responses and catecholamine secretion, but also elevated CHGB biosynthesis/secretion.
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CHGB Polymorphism: Effects on Biochemical and Physiological Intermediate Traits (in 171 Twin Pairs)
In the twin sample, genetic variation across CHGB (Figure 4A), as captured by common haplotype 2 spanning 11 variants (CTTAAATGAAC, at 23%), was associated with not only CHGB secretion but also the BP response to stress. Plasma CHGB439 to 451 concentration was lower (P=0.0243) in subjects without haplotype 2 (0.335±0.011 nM, n=186) than in those who carried 1 or 2 copies of that haplotype (0.376±0.014 nM, n=142). By contrast, the
DBP was higher (P=0.0073) in haplotype 2 noncarriers (12.4±1.1 mmHg, n=186) than in carriers (8.9±1.0 mmHg, n=142). Similarly, increasing CHGB haplotype 2 copy number was predictive of decreased catecholamine secretion (Figure 4B). Thus, haplotype 2 appeared to affect CHGB expression and adrenergic/pressor responses inversely, suggesting that subjects with a genetically programmed (ie, cis-QTL) increase in CHGB synthesis exhibit greater autonomic stability and thereby decreased cardiovascular risk.
CHGB Promoter Variation: Coordinate Directional Function both In Cella and In Vivo (Twins)
Because common promoter variant A-261T associated with BP (Figure 2A and 2B), we studied the effect of A-261T alleles on transfected CHGB promoter strength in chromaffin cells (Figure 4C), using a 1365 bp proximal promoter fragment driving expression of a luciferase reporter in vector pGL3-Basic, and varying the allele at position –261 from T to A by site-directed mutagenesis, followed by sequence verification. Because plasmids carry only 1 genotype (either T or A), we plotted both phenotype values for homozygotes (A/A or T/T) only. Transversion from A to T at –261 resulted in an increase in luciferase expression (P=0.0086), which paralleled an increase (P=0.035) in plasma CHGB439 to 451 expression in vivo in twins selected for –261 homozygosity: 0.333±0.015 nM in A/A homozygotes (n=126) versus 0.372±0.021 nM in T/T homozygotes (n=58). Thus, A-261T variation exerted parallel effects in cella and in vivo.
CHGB Promoter Variation: Reciprocal Effects In Vivo on Gene Expression and Basal BP in the Population
To probe the consequences of CHGB expression in vivo for long-term control of BP (Figure 4C), we plotted plasma CHGB (in twin pairs) versus resting/basal DBP (in the population BP extremes), simplifying the plot by focusing on homozygotes. The same allele (T) that raised CHGB expression also lowered population DBP (from 78.3±1.0 to 74.5±1.6 mmHg, P=0.011).
BP After Targeted Ablation of the CHGB Gene in Mice
Chgb–/– mice displayed substantially higher SBP/DBP (by 20/18 mmHg) than Chgb+/+ mice. SBP was 112.0±1.7 mmHg for Chgb+/+ mice (n=12), rising to 131.8±2.3 mmHg for Chgb–/– mice (n=12, F=47.8, P<0.001). DBP was 91.3±1.8 mmHg for Chgb+/+ mice (n=12) and 109.3±2.6 mmHg for Chgb–/– mice (n=12, F=32.7, P<0.001, Figure 5). This finding is consistent with the relationship between plasma Chgb439 to 451 concentration and BP that we found in humans (Figure 4C).
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Discussion |
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Overview
Patients with hypertension often exhibit increased sympathetic activity,26,27 and people with sympathetic overactivity tend to develop hypertension.28,29 Suppression of CHGB expression in neuroendocrine PC12 cells leads to a reduction in the number of catecholamine secretory granules, whereas ectopic expression of CHGB in nonneuroendocrine cells, which normally do not contain any secretory machinery, leads to granule biogenesis.7 In light of the emerging secretory biology of CHGB, we undertook the present study, using the tools of genome technology and statistical genetics 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.
Polymorphic Profile at the CHGB Locus
Systematic identification of genetic variants at a candidate locus is a strategy for disease association.30 At CHGB, we therefore resequenced all 5 exons, adjacent intronic regions, and the proximal promoter in n=160 human subjects from 4 biogeographic ancestry groups, thereby identifying 53 polymorphisms over a 5935 bp footprint, or just under 1 variant every 100 bp. The genetic diversity that we report at CHGB is somewhat more comprehensive than that previously reported for 32 individuals from the Han Chinese population (15 SNPs),31 or 24 individuals of Japanese ancestry (24 SNPs),32 as a consequence of the greater number of individuals studied, the multiethnic samples, and the greater resequencing footprint, but no inconsistencies were noted. In the previous Asian reports,31,32 CHGB polymorphisms (mainly toward the 3' end of the gene) were associated with schizophrenia; because the population prevalence of schizophrenia is only 0.4% to 0.6%,33 our resequenced sample of n=160 normotensive and hypertensive individuals from southern California does not have statistical power to detect such an effect on neuropsychiatric disease. Although the pattern of LD across the CHGB locus suggested a extended single block of LD across the 14 kbp locus in individuals of European ancestry (Supplemental Figure I), allele frequencies did differ substantially across the 4 biogeographic ancestries sampled (Supplemental Table II).
Strategies for Hypertension Risk: Heritability and CHGB Variant Effects on Multiple "Intermediate Phenotypes" in Twin Pairs
We developed series of twin pairs of southern California, typed for traits likely to contribute to later development of hypertension.11 The twin data offer the advantage of determining trait heritability (h2), the fraction of phenotypic variance accounted for by genetic variance, a logical estimator of the tractability of any trait to genetic investigation; indeed, the twin traits were substantially heritable (Supplemental Figure IV).
Multiple autonomic phenotypes in the twins, both biochemical and physiological, allowed construction of an integrated picture of the effects of genetic variation at CHGB on a very proximate biochemical phenotype, plasma CHGB concentration (Figure 4A); a later biochemical consequence, epinephrine excretion (Figure 4B); a more distant physiological consequence, change in BP during environmental stress (Figure 4A); and finally basal/resting BP in the population (Figure 2B).
Intriguingly, the same CHGB genetic variants that increased CHGB storage and secretion (Figure 4A) also decreased catecholamine secretion (Figure 4B), the sensitivity of BP to environmental stress (Figure 4A), and finally basal BP in the population (Figure 2B). A unifying hypothesis is presented in Figure 6. Such a hypothetical framework is supported by the experimental findings of Huh et al,7 who found by overexpression and underexpression of CHGB in catecholaminergic cells that CHGB regulates the biogenesis of hormone storage granules of the regulated secretory pathway. Absence of CHGB would therefore be predicted to disrupt the pathway, perhaps leading to constitutive (unregulated or autonomous) transmitter release. Indeed, we observed excess catecholamine secretion (Figure 4B) and autonomic BP instability (Figure 4A) in subjects with genetically programmed decrease in CHGB biosynthesis and secretion (Figure 4A). Evidence in cella34 and in vivo35 also supports a critical role for the CHGB paralog CHGA in the biogenesis of catecholamine storage vesicles; indeed, targeted ablation of CHGA35 results in dysregulated catecholamine storage and secretion, accompanied by systemic hypertension.
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In this report, we focused primarily on 2 indices of sympathoadrenal function: catecholamine secretion (Figure 4B) and the BP response to environmental stress (Figure 4A). Because CHGB has such a widespread occurrence in amine and peptide storage vesicles,2,3,9,10,36,37 it is likely that additional consequences of CHGB genetic variation might be uncovered in other branches (eg, parasympathetic) of the autonomic system, or the wider neuroendocrine system. However, such alterations are beyond the scope of this initial report.
Established Hypertension and CHGB Genetic Variation: Population BP Extremes
To pursue the genetic involvement in established essential hypertension, we developed a powerful resource in a sample set consisting of individuals with extremely high and low BPs. From a population sample of more than 53000 people, we ascertained >1100 age-, gender-, and ethnicity-matched individuals from the upper and lower 5th percentiles of the population BP distribution.13
Using CHGB polymorphisms spanning the locus (Supplemental Figure II), we took a haplotype "sliding window" approach in SNP-EM19 to test the effects of CHGB regions on the hypertension trait. We found that variation across the locus was predictive of BP category (Figure 2A), and the SBP and DBP quantitative traits (haplotypes, Figure 2B), but the effects were substantially more impressive in males than females, and the genetic effects in males seemed to peak toward the 5' (promoter) end of the gene, with the most significant single effect at promoter variant A-261T.
When we studied the effect of A-261T variation on the SBP/DBP quantitative traits (Figure 2B), once again the effects were most impressive for males (P<0.001/P=0.001) than females (P=0.182, P=0.459). The profound difference in genetic effects between sexes led us to explore the effects of sex at earlier stages in the hypothetical phenotypic chain between gene and the ultimate disease trait (Figure 6; see below). Of note, sex itself had a profound effect on BP in the population (Figure 2B), consistent with epidemiological findings over several decades.38
Although we undertook CHGB polymorphism discovery systematically across several biogeographic ancestry groups (Supplemental Tables I and II; Figure 1), we conducted CHGB marker-on-trait BP studies in subjects of a single ancestry: European (Figure 2A and 2B). We restricted our initial analyses to subjects of 1 ancestry because allelic association studies can be susceptible to artifactual conclusions resulting from even inapparent population admixture.39 Studies of additional ethnic or population groups will be required to evaluate whether our CHGB results are of more general importance in the overall population.
Sex and Genetic Risk of Hypertension: "Intermediate" and Ultimate Disease Traits
Sex exerted profound effects on not only the BP trait in general (Figure 2B)38 but also the BP response to CHGB genetic variation (Figure 2A and 2B). Previously we have noted significant gene-by-sex interactions on BP in the population, and sex differences in the response to adrenergic drug provocations.40,41 To understand how sex might influence the genetic predisposition to hypertension, we studied the effect of sex on each of the "intermediate phenotypes" influenced by CHGB genetic variation. We found that sex systematically influenced each such trait, in ways predicted to reduce risk of future development of hypertension: females exhibited increased CHGB expression (Figure 4A), reduced epinephrine secretion (Figure 4B), and reduced pressor responses to environmental stress (Figure 4A). We noted effects of CHGB genetic variation on BP in males though not females (Figure 2A and 2B), and observed that autonomic traits, both biochemical (Figure 4A) and physiological (Figure 4A and 4B), differed in males and females; nonetheless, we did not statistically document significant CHGB gene-by-sex interaction on BP (Figure 2B), suggesting caution during interpretation of sex-specific roles of genes in cardiovascular trait determination.
Although we measured BP before and after environmental stress (Figure 4A), we measured catecholamines only before stress. However, previous longitudinal studies indicate that the pressor (BP) response to cold is an effective predictor of future development of hypertension,42 although the predictive value of such early stress tests seems to be more apparent in males.43 Thus, at every stage of the putative pathogenic chain (Figure 6) between CHGB genetic variation and the development of hypertension, sex may be involved as a potential modifier of gene effects.
Role of the CHGB Promoter: Coordinate CHGB Genetic Effects In Cella and In Vivo
Because BP in the population was best associated with genetic variation in the CHGB promoter region (Figure 2A and 2B), we ligated a 1.4 kb proximal promoter fragment to a luciferase reporter and tested the effect of the A-261T variant on promoter activity in transfected chromaffin cells (Figure 4C). We found that the minor (T) was significantly more active than the A allele in programming transcription; of further note was the coordinate effect of the 2 alleles in cella and in vivo: the T allele not only increased transcription in chromaffin cells, but also increased plasma CHGB in twin pairs. These results are consistent with genetic variation in the CHGB promoter initiating the entire cascade of phenotypic events illustrated in our hypothetical schema (Figure 6). Loss-of-function CHGB promoter variants (such as allele A at A-261T) would give rise to decreased CHGB expression, thereby unleashing sympathochromaffin activity, leading to exaggerated pressor responses to environmental stressors, and ultimately fixed systemic hypertension.
BP in Mice with Targeted Ablation of the CHGB Locus
Because we found inverse effects of CHGB expression on catecholamine secretion (Figure 4B) pressor responses (Figure 4A), and because the CHGB genotype associated with lower CHGB expression in cella (Figure 4C) and in vivo (Figure 4C) was also predictive of higher resting SBP/DBP in the population (Figure 4C), we hypothesized that experimental disruption of CHGB expression would elevate BP. We tested this hypothesis by targeted ablation of the CHGB locus, and as predicted found substantial elevations in both SBP (by 20 mmHg) and DBP (18 mmHg) in Chgb(–/–) mice. Thus, experimental evidence further strengthens our clinical conclusions (Figure 6) that CHGB genetic variation initiates a cascade of events ultimately resulting in BP disturbances in the population.
Conclusions and Perspectives
Common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression as well as catecholamine secretion in vivo, and later the early heritable responses to environmental stress, and finally resting/basal BP in the population (Figure 6 hypothesis). These changes are modified at each pathophysiologic level by the influence of sex. Although causal inferences in this proposed chain of events are not yet established, the pathway illustrated in Figure 6 does yield testable predictions for experimental verification. These results point to new molecular strategies for probing autonomic control of circulation and, ultimately, the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension.
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Acknowledgments |
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Sources of Funding
This work was supported by the Department of Veterans Affairs, National Institutes of Health.
Disclosures
None.
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The online-only Data Supplement is available at http://circgenetics.ahajournals.org/cgi/content/full/CIRCGENETICS.108.785659/DC1.
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