CLINICAL PERSPECTIVE

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(Circulation. 2007;115:2271-2281.)
© 2007 American Heart Association, Inc.

Genetics

Catecholamine Release–Inhibitory Peptide Catestatin (Chromogranin A_352–372)

Naturally Occurring Amino Acid Variant Gly364Ser Causes Profound Changes in Human Autonomic Activity and Alters Risk for Hypertension

Fangwen Rao, MD; Gen Wen, MD, PhD; Jiaur R. Gayen, PhD; Madhusudan Das, PhD; Sucheta M. Vaingankar, PhD; Brinda K. Rana, PhD; Manjula Mahata, PhD; Brian P. Kennedy, PhD; Rany M. Salem, MPH; Mats Stridsberg, PhD; Kenneth Abel, PhD; Douglas W. Smith, PhD; Eleazar Eskin, PhD; Nicholas J. Schork, PhD; Bruce A. Hamilton, PhD; Michael G. Ziegler, MD; Sushil K. Mahata, PhD; Daniel T. O’Connor, MD

From the Departments of Medicine (F.R., G.W., J.R.G., M.D., S.M.V., M.M., B.P.K., R.M.S., K.A., B.A.H., M.G.Z., S.K.M., D.T.O.), Psychiatry (B.K.R., N.J.S.), Computer Science (E.E.), Pharmacology (D.T.O.), and Biology (D.W.S.), Polymorphism Research Laboratory (B.K.R., N.J.S.), and Center for Human Genetics and Genomics (N.J.S., D.T.O.), University of California at San Diego; the VA San Diego Healthcare System (S.K.M., D.T.O.), San Diego, Calif; and the Department of Medical Sciences (M.S.), University of Uppsala, Uppsala, Sweden.

Correspondence to Daniel T. O’Connor, MD, or Sushil K. Mahata, PhD, Department of Medicine and Center for Human Genetics and Genomics (0838), UCSD School of Medicine and VASDHS, 9500 Gilman Drive, La Jolla, CA 92093–0838. E-mail doconnor{at}ucsd.edu or smahata@ucsd.edu

Received March 31, 2006; accepted November 13, 2006.

	Abstract

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Background— Chromogranin A, coreleased with catecholamines by exocytosis, is cleaved to the catecholamine release–inhibitory fragment catestatin. We identified a natural nonsynonymous variant of catestatin, Gly364Ser, that alters human autonomic function and blood pressure.

Methods and Results— Gly364Ser heterozygotes and controls underwent physiological and biochemical phenotyping, including catecholamine production, chromogranin A precursor, and its catestatin product. Case-control studies replicated effects of the gene on blood pressure in the population. Gly364Ser displayed diminished inhibition of catecholamine secretion from cultured neurons. Gly/Ser heterozygotes displayed increased baroreceptor slope during upward deflections (by 47%) and downward deflections (by 44%), increased cardiac parasympathetic index (by 2.4-fold), and decreased cardiac sympathetic index (by 26%). Renal norepinephrine excretion was diminished by 26% and epinephrine excretion by 34% in Gly/Ser heterozygotes. The coalescent dated emergence of the variant to 70 000 years ago. Gly364Ser was in linkage disequilibrium with 1 major Chromogranin A promoter haplotype, although promoter haplotypes did not predict autonomic phenotypes. The 364Ser variant was associated with lower diastolic blood pressure in 2 independent/confirmatory groups of patients with hypertension; genotype groups differed by 5 to 6 mm Hg, and the polymorphism accounted for 1.8% of population diastolic blood pressure variance, although a significant gene-by-sex interaction existed, with an enhanced effect in men.

Conclusions— The catestatin Gly364Ser variant causes profound changes in human autonomic activity, both parasympathetic and sympathetic, and seems to reduce risk of developing hypertension, especially in men. A model for catestatin action in the baroreceptor center of the nucleus of the tractus solitarius accounts for these actions.

Key Words: acetylcholine • blood pressure • catecholamines • genetics • heart rate • hypertension • nervous system, sympathetic

	Introduction

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The catecholamine storage vesicle protein chromogranin A (CHGA)¹ plays a necessary role in formation of catecholamine secretory granules.^2,3 CHGA is also precursor of the catecholamine release–inhibitory peptide catestatin (human CHGA_352–372: S₃₅₂SMKLSFRARGYGFRGPGPQL₃₇₂), which acts as a nicotinic cholinergic antagonist to block transmitter release from chromaffin cells and noradrenergic nerves^1,4 in both isolated cells⁴ and intact organisms.⁵

Clinical Perspective p 2281

CHGA is overexpressed in genetic hypertension,^6,7 but catestatin is diminished in not only established hypertension but also the still-normotensive offspring of patients with hypertension,⁸ suggesting an early, pathogenic "intermediate phenotype" in the development of hypertension. Targeted ablation of the CHGA gene alters catecholamine storage and release, eventuating in hypertension that can be "rescued" by exogenous catestatin replacement.⁹

In probing human genetic variation at CHGA, we discovered 3 naturally occurring amino acid substitution variants in the catestatin region.¹⁰ Although 2 of the variants were relatively rare, 1 variant, Gly364Ser (S₃₅₂SMKLSFRARGYS₃₆₄FRGPGPQL₃₇₂; position in the mature CHGA protein after excision of the signal peptide) had an allele frequency of 3% to 4%, enabling us to study its consequences for autonomic function. Here we found that Gly364Ser displayed altered activity toward neuronal nicotinic cholinergic receptors in vitro. In vivo, human carriers of 364Ser had profound alterations in autonomic activity in both the parasympathetic and sympathetic branches and may be protected against future development of hypertension.

	Methods

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Portions of methodological procedures are available in the online Data Supplement.

Human Subjects
Characterization of human subjects by ethnicity is given in the online Data Supplement.

Hypertension
Initial Study (University of California San Diego)
First, we studied a University of California San Diego (UCSD) population of 519 individuals of white (European) ancestry: 166 hypertension cases (diastolic blood pressure [DBP] >95 mm Hg or a history of hypertension and antihypertensive treatment [in 65.6%]; 146 men, 20 women) and 353 unmedicated normotensive controls (DBP <85 mm Hg; 186 men, 167 women). The diagnosis of essential hypertension and family histories for hypertension (in a first-degree relative <60 years of age) were made as previously described.⁸ Blood pressure (BP) was obtained in seated subjects from prolonged (5 minute) noninvasive recordings as described.¹¹ Treatment in 65.6% of the hypertensives included angiotensin-converting enzyme inhibitors, thiazide diuretics, ß-adrenergic antagonists, and calcium channel antagonists. None of the subjects had a history of renal failure (serum creatinine 1.5 mg/dL). Definitions of subject characteristics are according to previous reports from our laboratory.⁸

Replication Study (Kaiser Permanente)
To validate the initial hypertension study of Gly364Ser in a larger population, we studied 1361 (643 male, 718 female) white (European ancestry) subjects drawn from 53 078 individuals (27 475 women, 25 538) recruited from a large primary care (Kaiser Permanente) population in San Diego as previously described.¹² In this primary care population, 81% attended the clinic, and 46% consented to participate in the study, with collection of blood for preparation of genomic DNA. The subjects in this study were selected from the consenting participants on the basis of measurement of DBP to represent the highest and lowest DBP percentiles in that population. Then, 662 subjects (339 men, 323 women) were chosen for the highest DBPs, whereas 699 subjects (304 men, 395 women) were chosen for the lowest DBPs. The ages of the high- and low-BP groups were not different (low BP, 59±0.5 years; high BP, 59±0.4 years; P=0.94). To accomplish this, low-BP individuals (systolic BP [SBP]/DBP=111±0.6/55±0.3 mm Hg) were selected from the bottom 4.8th percentile of DBP, and the high-BP group (SBP/DBP=153±0.6/100±0.8 mm Hg) was selected from the top 4.9th percentile of DBP. Both SBP and DBP differed significantly between the extreme BP groups (P<0.0001/P<0.0001). Forty-one percent of patients in the Kaiser high-BP group were taking 1 antihypertensive medications (including 15% on diuretics, and 19% on angiotensin-converting enzyme inhibitors), whereas no subjects in the low-BP group were on such treatment. BP was obtained by standard cuff aneroid sphygmomanometry in seated subjects; if elevated, repeat measurement was obtained.

When BP was treated with antihypertensive medications, we also used the method of Cui et al¹³ to adjust values for the effects of medications.

CHGA Gly364Ser (G9559A) Polymorphism Discovery
See the online Data Supplement.

Single Nucleotide Polymorphism Assays
See the online Data Supplement.

Biochemical Phenotyping
Catecholamines, CHGA, and Catestatin
See the online Data Supplement.

Physiological/Autonomic Phenotyping In Vivo
Autonomic phenotypes were obtained in subjects (17 cases, 48 controls) studied prospectively during the same time period before knowledge of genotypes was obtained. These 2 groups were comparable in age and sex.

Prolonged Recording of BP and Heart Rate
BP (in mm Hg) and pulse interval (R-R interval or heart period, in ms per beat) were recorded continuously and noninvasively for 5 minutes in seated subjects with a radial artery applanation device and dedicated sensor hardware (Colin Pilot, Colin Instruments, San Antonio, Tex) and software (ATLAS, WR Medical, Stillwater, Minn; Autonomic Nervous System, Tonometric Data Analysis [ANS-TDA], Colin Instruments, San Antonio, Tex) calibrated every 5 minutes against ipsilateral brachial arterial pressure with a cuff sphygmomanometer. Heart rate was recorded continuously with thoracic ECG electrodes to the Colin Pilot. Average, maximum, and minimum values, as well as coefficients of variation, were calculated for BP and pulse interval using the ANS-TDA software.

Environmental Stress: The Cold Pressor Test
BP and heart rate were recorded continuously and noninvasively with a calibrated radial artery applanation device and dedicated sensor hardware (Colin Pilot, Colin Instruments) and software (ATLAS, WR Medical; TDA, Colin Instruments) during the cold pressor test (immersion of the left hand in ice water for 60 seconds after a 10-minute rest) as previously described.⁸ We identified at least 3 beats with stable (within ±10%) values for BP and heart rate just before and at the end of the cold pressor test.⁸

Fourier Transformations (Time Domain Frequency Domain)
Power spectra for SBP, DBP, and pulse (R-R) interval were obtained during the 5-minute recording with the ANS-TDA software by Fourier transformation of data from the time domain into the frequency domain.¹⁴ Spectral "power" is the variance (ie, SD²) of the parameter in a particular frequency domain (eg, for R-R interval in ms, spectral power is in ms²). The frequency domains were as follows: high frequency (0.15 to 0.4 Hz, reflecting predominantly parasympathetic activity), low frequency (0.05 to 0.15 Hz, reflecting a mixture of parasympathetic and sympathetic activity), and very low frequency (0.01 to 0.05 Hz, reflecting predominantly sympathetic activity). In each of these domains, coupling of R-R interval power to SBP power (ms²/mm Hg²) was computed as (R-R power, ms²)/(SBP power/mm Hg²)=baroreflex sensitivity (ms/mm Hg) in the "frequency domain."

Sympathovagal Balance
Sympathovagal balance¹⁵ was computed as low-frequency R-R power (ms²/Hz, from 0.05 to 0.15 Hz)/high-frequency R-R power (ms²/Hz, from 0.15 to 0.4 Hz).

Lorenz Analyses of Heart Rate
Lorenz parameters¹⁶ of heart rate variability were evaluated with the ANS-TDA software to estimate cardiac parasympathetic and sympathetic indexes. Lorenz log₁₀[L*T] is the cardiac vagal (parasympathetic) index, and Lorenz L/T ratio is the cardiac sympathetic index.

Baroreceptor Sensitivity in the Time Domain
BP and heart rate also were recorded continuously with the same devices during spontaneous excursions of BP with reciprocal heart rate changes: upward excursions of BP with reflex bradycardia and downward excursions of BP with reflex tachycardia. In each case, baroreceptor slope in the "time domain"¹⁷ was calculated with the ANS-TDA software, with beat-by-beat regression of change in pulse interval (R-R interval; ms/beat) as a function of change in SBP (SBP, mm Hg) on the preceding beat (phase lag=1 beat). Time windows of >4 beats were used, with SBP of >1 mm Hg and R-R of >6 ms. Baroreceptor slope (ms/mm Hg) values were recorded for regressions with correlation coefficients of r>0.9. The slopes for 3 such regressions, if each was within ±10% of the mean value, were averaged to yield the final value for baroreceptor slope. Baroreceptor slopes were separately determined for upward and downward spontaneous excursions of BP.

Cardiac contractility, stroke volume index, and cardiac index were evaluated noninvasively with a brachial oscillometric device as previously described.¹⁸

CHGA Gly364Ser 3-Dimensional Structure: Homology Modeling
See the online Data Supplement.

CHGA Gly364Ser Cell Biology: Effects on Nicotinic Cholinergic Stimulation of Catecholamine Secretion in Primary Cultures of Hippocampal Neurons
See the online Data Supplement.

Statistical Analysis and Haplotypes
See the online Data Supplement for statistical and phylogenetic methods.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

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CHGA Gly364Ser: Discovery, Structure, and Functional Effects on Nicotinic Cholinergic Secretion of Catecholamines
During resequencing, 3 variants were discovered in the catestatin (human CHGA_352–372) region, the most common of which was Gly364Ser, at 3.1% of the initial 360 chromosomes resequenced.¹⁰ At this codon or its orthologs, the amino acid Gly is invariant in all mammals so far sequenced.

Figure I in the online Data Supplement shows Gly364Ser single nucleotide polymorphism (SNP) discovery by capillary DNA sequencing in a Gly/Gly (wild-type) homozygote and a Gly/Ser heterozygote.

A 3-dimensional nuclear magnetic resonance structure of wild-type human catestatin,¹⁹ coupled with a homology-modeled structure for Gly364Ser, showed the position of the variant residue (Ser₃₆₄), with its larger Van der Waals interaction radius, to be in the coiled loop portion of the peptide, a region functionally important in nicotinic cholinergic inhibition.²⁰

In cultured hippocampal neurons, wild-type human catestatin (SSMKLSFRARAYG₃₆₄FRGPGPQL) inhibited nicotinic cholinergic-stimulated catecholamine release completely (to 0.3±2.8% of control) at 10 µmol/L (Figure 1). The same dose of the 364Ser variant (SSMKLSFRARAYS₃₆₄FRGPGPQL) inhibited catecholamine release with substantially reduced potency: 35.7±3.1% of secretion persisted.

View larger version (18K): [in this window] [in a new window]   Figure 1. The CHGA Gly364Ser variant: inhibitory potency on nicotinic cholinergic-stimulated catecholamine secretion. Hippocampal neuron catecholamine storage vesicles were prelabeled with [3H]-L-norepinephrine, and exocytosis was triggered by 200 µmol/L nicotine in the presence or absence of wild-type catestatin or the Gly364Ser variant (each at 10 µmol/L). Results are shown as mean±SEM for 10 to 11 replicates per condition.

CHGA Gly364Ser Genomics: Population Structure, Hardy-Weinberg Equilibrium, and Ethnicity
Figure II shows Gly364Ser high-throughput biallelic SNP assay in 3 individuals (Figure IIa) and a signal-to-noise-ratio plot (low mass allele versus high mass allele) in a larger group (Figure IIb).

We initially genotyped CHGA Gly364Ser in 389 individuals. Overall allele frequencies were as follows: Gly364, 96.8%; 364Ser, 3.2%. The 364Ser allele occurred in each ethnic group (online Table I) except blacks. We cannot conclude that the 364Ser allele is absent in the black population because we sampled only 75 black subjects (ie, 150 chromosomes); the 95% confidence interval for the minor allele frequency in blacks is thus 0% to 2.0%. In Hispanics, only 1 364Ser allele was found among 52 individuals (104 chromosomes). Genotype frequencies thus differed by ethnicity (²=15.9, P=0.015), although the nonblack groups did not differ from each other (eg, whites versus Asians: Fisher exact test, P=0.52).

Genotype frequencies were in Hardy-Weinberg equilibrium (online Table I) for both the entire group (²=0.951, P=0.329) and each ethnic group (Because Gly364 was monomorphic in blacks, Hardy-Weinberg calculations were not applicable).

CHGA Gly364Ser and Physiological Evaluation of Autonomic Function: Parasympathetic and Sympathetic
To explore the consequences of the Gly364Ser variant in vivo, we extensively phenotyped 17 Gly/Ser heterozygotes and 48 Gly/Gly (wild-type) homozygotes (as controls; online Table II). The genotype groups (Table II) did not differ significantly in age, sex, BP status (only normotensives), family history of hypertension, ethnicity, weight, height, or body mass index. Ethnicity displayed a nonsignificant trend toward difference (P=0.052), but Gly364Ser genotype frequencies did not differ across the 2 ethnic groups represented, whites and Asians (Fisher exact test, P=0.52).

Resting BP and heart rate did not differ significantly between the groups, although the Gly/Ser heterozygotes showed 14% greater SBP variability (P=0.009). Gly/Ser heterozygotes also showed trends toward lower DBP (by 9 mm Hg; P=0.06) and higher pulse interval/lower heart rate (by 8%; P=0.07). After environmental (cold) stress, the maximum BPs achieved were lower in the Gly/Ser heterozygotes by 16/8 mm Hg (P=0.022/P=0.047; Table II). In the time domain of autonomic monitoring (Table II), Gly364Ser heterozygotes displayed several substantial differences: increased baroreceptor slope during upward deflections (by 47%; P=0.0036) and downward deflections (by 44%; P=0.016), increased cardiac parasympathetic index (Lorenz log₁₀[L*T], by 2.4-fold; P=0.032), and lower cardiac sympathetic index (Lorenz L/T ratio, by 26%; P=0.044).

Fourier transform of pulse interval and BP data into the frequency domain yielded 3 customary frequency intervals: a high-frequency band (HFB; 0.15 to 0.4 Hz) representing mainly parasympathetically mediated variability,¹⁴ a low-frequency band (LFB, 0.05 to 0.15 Hz) representing a mixture of parasympathetic and sympathetic activity corresponding to classic baroreflex-mediated changes,¹⁴ and a very-low-frequency band (VLFB; 0.01 to 0.05 Hz) that likely represents sympathetically mediated variation.

In the parasympathetic HFB (Table II), Gly364Ser heterozygotes showed increased pulse interval variability (by 96%; P=0.026) and increased baroreflex coupling (by 44%; P=0.049). In the mixed parasympathetic/sympathetic LFB, Gly364Ser heterozygotes showed increased baroreceptor coupling (by 35%; P=0.044) but no difference in pulse interval power. In the sympathetic VLFB, the 2 groups did not differ in either pulse interval power or baroreceptor coupling.

Three widely accepted indexes of baroreflex function are presented in Figure 2: baroreflex slope in the time domain from spontaneous upward or downward BP deflections or baroreceptor coupling in the customary LFB (0.05 to 0.15 Hz). In each case, the 364Ser carriers displayed increased baroreceptor sensitivity. An index of "sympathovagal balance,"¹⁵ derived as the LFB/HFB ratio of pulse interval power, was substantially reduced (by 44%; P=0.035) in Gly364Ser heterozygotes (Table II).

View larger version (21K): [in this window] [in a new window]   Figure 2. Baroreceptor function in individuals stratified by genotype at CHGA Gly364Ser. Results are shown for the time domain and after Fourier transform into the frequency domain. Baroreceptor slope (ms/mm Hg) in the time domain is shown during spontaneous upward and downward BP deflections with corresponding decreases or increases in pulse interval. Baroreceptor coupling (ms/mm Hg) in the frequency domain is shown for results in the customary LFB (0.05 to 0.15 Hz).

Physiological variables displayed significant interindividual correlations (online Table III), consistent with the participation of the autonomic system and its efferent parasympathetic and sympathetic branches, in virtually all aspects of reflex control of the circulation. As an example of such integration, the cardiac sympathetic index (Lorenz L/T ratio; Table II) correlated inversely with baroreceptor slope in the time domain (r=–0.324; n=62; P=0.01; Table III), documenting functional (albeit inverse) links between sympathetic and parasympathetic activity.

CHGA Gly364Ser Minor Allele (Ser/Ser) Homozygote
One Ser/Ser homozygote (No. 4416), a healthy 22-year-old normotensive unmedicated white man, consented to extensive autonomic phenotyping. His basal BP was 119/69 mm Hg; during cold stress, his BP fell to 104/56 mm Hg. Three different measures of baroreceptor sensitivity were enhanced in this subject: time-domain upward deflection slope (27.7 ms/mm Hg), time-domain downward deflection slope (13.1 ms/mm Hg), and LFB (0.05 to 0.15 Hz) coupling (24.7 ms/mm Hg).

CHGA Gly364Ser and Catecholamine Secretion In Vivo
The 364Ser carriers had diminished renal norepinephrine (by 26%; P=0.047) and epinephrine (by 34%; P=0.003) excretion (Figure 3 and Table II). Plasma norepinephrine also declined by 36% (P=0.019), whereas plasma epinephrine was unchanged.

View larger version (18K): [in this window] [in a new window]   Figure 3. Catecholamine secretion in individuals stratified by CHGA Gly364Ser genotype: Gly/Gly homozygotes (wild type) vs Gly/Ser heterozygotes.

CHGA Gly364Ser and Autonomic Phenotypic Pleiotropy
To test the proposition that the 364Ser allele jointly influenced multiple autonomic phenotypes, we undertook multivariate ANOVA tests of the effects of 1 independent variable (genotype) on >1 dependent variable. Gly364Ser genotype jointly influenced several phenotypic combinations, both parasympathetic and sympathetic: (1) time-domain baroreceptor slope (ms/mm Hg) with both upward and downward BP deflections (Pillai’s trace F=5.64, P=0.006); (2) time-domain baroreceptor slope (ms/mm Hg) with both upward and downward BP deflections, as well as frequency-domain LFB (0.05 to 0.15 Hz) baroreceptor coupling (Pillai’s trace F=3.89, P=0.013); (3) frequency-domain baroreceptor coupling (ms/mm Hg) in all 3 frequency domains—HFB (0.15 to 0.4 Hz), LFB (0.05 to 0.15 Hz), and VLFB (0.01 to 0.05 Hz) (Pillai’s trace F=5.43, P=0.002); (4) time-domain baroreceptor slope (ms/mm Hg) with both upward and downward BP deflections, as well as frequency-domain LFB (0.05 to 0.15 Hz) baroreceptor coupling and cardiac sympathetic index (Lorenz L/T ratio) (Pillai’s trace F=3.31, P=0.017); (5) time-domain baroreceptor slope with both upward and downward BP deflections, as well as sympathovagal balance (R-R power ratio, LFB/HFB) (Pillai’s trace F=3.86, P=0.014); and (6) all of the above autonomic phenotypes considered simultaneously (Pillai’s trace F=2.491, P=0.042).

Thus, the 364Ser allele caused coordinate autonomic changes in both the parasympathetic and sympathetic branches, suggesting pleiotropic effects of a single allele on multiple autonomic traits. Of further note, such multivariate analyses provide an effective approach to potential type I (false-positive) statistical errors in testing the effect of a single genotype on multiple potential phenotypic consequences (eg, Table II). Because these autonomic traits are highly correlated (eg, urine epinephrine with norepinephrine, r=0.50, P<0.01), simple Bonferroni corrections would be inappropriately conservative.

CHGA Gly364Ser: Association With Hypertension
Initial Study
In the initial (UCSD) case-control study of hypertension, Gly364Ser genotype significantly affected DBP in 489 subjects (Figure 4a; P=0.029), wherein genotype groups differed by 5.5 mm Hg DBP. Because BP was treated with antihypertensive medications in 65.6% of subjects, we also used the method of Cui et al¹³ to adjust BP for medication effects; the genotype effect on DBP remained significant (P=0.027; genotype groups differing by 5.7 mm Hg). Finally, we repeated the analysis excluding drug-treated hypertensives; the Gly364Ser effect on DBP persisted (P=0.048; 4.7 mm Hg).

View larger version (10K): [in this window] [in a new window]   Figure 4. Association of the CHGA Gly364Ser (A9559G) polymorphism with hypertension. Characteristics of the subjects (hypertensive cases or normotensive controls) are given in Methods. Subjects are stratified by diploid genotype at Gly364Ser, either Gly/Gly major allele homozygotes or Gly/Ser heterozygotes. Results are shown as DBP (mm Hg) (mean±SEM). A, Initial (UCSD) and replication (Kaiser) studies. UCSD subjects were drawn from UCSD or VA San Diego Healthcare System hospitals, clinics, or hypertension research program. Kaiser subjects were drawn from the DBP extremes (top 4.9th and bottom 4.8th percentiles) of a 53 078-patient primary care population. B, Genotype, sex, and DBP. Equivalent representation of both men and women in the Kaiser sample allowed test of gene-by-sex interaction in determination of DBP. Results of 2-way ANOVA are shown, factoring for the effects of genotype, sex, and genotype-by-sex interaction on DBP (mm Hg). Fisher exact test on the dichotomous BP trait: male, P=0.005; female, P=0.497.

Replication Study
To confirm the general significance of the association of Gly364Ser with hypertension, we studied the polymorphism in 1361 individuals selected from extremes of DBP in a large primary care population (see Methods).¹² The polymorphism showed an effect on DBP comparable to that found in the initial study (P=0.023; 5.0 mm Hg effect; Figure 4a). Because many of the hypertensives were treated with antihypertensive drugs, we repeated the analysis after exclusion of treated individuals; statistical power declined, but an effect of Gly364Ser genotype on DBP still existed (P=0.034; 4.7 mm Hg). Finally, we used the method of Cui et al¹³ to adjust for antihypertensive drug effects in the treated hypertensives; after such adjustment, the genotype effect on DBP persisted (P=0.031; 5.2 mm Hg).

Sex, Gly364Ser Genotype, and DBP
Although the initial (UCSD) hypertension study was unbalanced for sex, with relatively few female hypertensives (Fisher exact test, P<0.001), the replication (Kaiser) study was deliberately planned to contain comparable numbers of men and women with high versus low DBP (Fisher exact test, P=0.380). Because of the large sample size and equal sex representation, the Kaiser study offered the opportunity to test the interaction of Gly364Ser with sex in the determination of BP in the population. Indeed, when Kaiser subjects were stratified by Gly364Ser genotype and sex (Figure 4b), 2-way ANOVA revealed a significant effect of genotype on DBP (P=0.033) and genotype-by-sex interaction (P=0.027). Within men, the polymorphism seemed to confer a 13-mm Hg effect on DBP (P=0.008). After adjustment for antihypertensive medications,¹³ the DBP effects of genotype (F=4.48, P=0.034) and genotype-by-sex interaction (F=5.37, P=0.021) persisted. When we tested this model in the presence or absence of the genotype, we found that allelic variation at Gly364Ser accounted for 1.8% of the DBP variance (R²=0.018) in the population. To explore the same findings in a model-free fashion without relying on standard asymptotic distribution assumptions, we also used the more computationally intensive Fisher exact test (in effect, permutation tests on 2x2 tables), whereby the effect of CHGA Gly364Ser genotype on DBP in men was highly significant (P=0.005) and that in women was not (P=0.497).

SBP Results
In the balanced-sex (Kaiser) replication study, Gly364Ser displayed a significant gene-by-sex interaction on SBP (P=0.033), although the effect of genotype alone was of marginal significance in the entire group (P=0.061). In men alone, Gly364Ser conferred an 13-mm Hg fall in SBP (P=0.018); no effect was conferred on SBP in women. In the original (UCSD) study, the effect of genotype on SBP did not achieve significance (P=0.100).

Linkage Disequilibrium With Promoter Haplotypes or Nearby Coding Variants Does Not Explain Gly364Ser Effects
Because common genetic variants in the human CHGA promoter differ in transcriptional activity,¹⁰ we tested whether the Gly364Ser variant was in substantial linkage disequilibrium with any of the 8 common CHGA promoter haplotypes (Table IV) using the PHASE algorithm.²¹ The 8 polymorphic bases are located from –57 to –1106 bp upstream of the cap site.¹⁰

Three hundred fifty-four individuals had complete data for Gly364Ser and each of 8 variants in the proximal CHGA promoter (Table IV). In 11 Gly364Ser heterozygotes, the 364Ser variant was found in association with CHGA promoter haplotype 1 (AATTGTCC) in 10 instances and haplotype 2 (GATTGTCC) in 1 instance. The 364Ser variant was not found in association with the other 6 CHGA promoter haplotypes. Association of Gly364Ser with particular promoter haplotypes (especially haplotype 1) was thus highly significant (²=34.2, P<0.0001).

Because CHGA promoter haplotype 1 has relatively high transcriptional activity,¹⁰ we tested whether variation within the promoter influenced the autonomic phenotypes (Table II). CHGA promoter SNP G-462A (rs9658634) is a common variant (G=79%, A=21%) in the proximal promoter 462 bp upstream from the cap site but in only modest linkage disequilibrium with Gly364Ser (r²=0.090).¹⁰ The G-462A transition yields either haplotype 3 (GACGATAC; altered base in bold) or haplotype 6 (GACGATCC), each of which is reduced 30% to 50% in activity.¹⁰ Thus, G-462A is a suitable promoter SNP for "tagging" CHGA promoter haplotypes of very different transcriptional activity. In 115 unrelated individuals, the G-462A genotype frequencies were as follows: G/G=72, G/A=37, and A/A=6 (Hardy-Weinberg equilibrium; ²=0.188, P=0.664).

Because –462A was the minor allele (21% overall), we divided G-462A genotypes into 2 categories: without –462A (G/G homozygotes; 72 individuals) and with –462A (G/A or A/A; 43 individuals). These 2 G-462A groups did not differ in age, sex, BP status, hypertension family history, weight, height, body mass index, or autonomic physiological function (P=0.15 to 0.9) in either the time or frequency domain. Thus, linkage disequilibrium of Gly364Ser with functional promoter haplotypes (Table IV) cannot explain autonomic phenotype associations with the Gly364Ser variant (Table II and Figure 2).

Gly364Ser was also in only modest linkage disequilibrium with nearby, more common CHGA coding variants Glu248Asp (rs9658655; r²=0.08; minor allele frequency, 8.1%) and Arg381Trp (rs729940; r²=0.025; minor allele frequency, 13.8%). Neither of these nonsynonymous variants accounted for the change in the autonomic phenotypes shown in Table II (P>0.1).

CHGA Gly364Ser: Haplotype and Human Evolutionary Lineage
Phylogeny
The Gly364Ser variant does not occur in other mammals.¹⁰ Online Figure IIIa shows a deduced haplotype phylogeny for the CHGA coding region (exons 1 through 8). Nine coding region SNPs with minor allele frequency >10% are shown with Gly364Ser.¹⁰ Variation in the coding region grouped into 5 ancestral haplotypes (B, D, F, P, and Q; defined in Figure IIIa). Gly364Ser occurs only in a single haplotype "I." Phylogenetic analysis suggests that haplotype I occurred as a single base mutation from haplotype A, the most common haplotype in the current human population (at 93 of 360 occurrences, 25.8%), which in turn proceeds from ancestral group P, which is common in all populations. The population distribution of haplotypes A/C/I suggests that the Gly364Ser variant (on haplotype I) may have occurred shortly after the human "out of Africa" migration.²²

Coalescent
To assign a date to the emergence of Gly364Ser, we turned to the coalescent method.^23,24 Because the coalescent cannot accommodate ancestral recombination, we confined the input haplotypes to the local CHGA region spanning Gly364Ser: T9179CG9559A(Gly364Ser)C9610T, in which the TAC haplotype bears the Gly364Ser variant (Figure IIIb in the online Data Supplement). The coalescent estimated an age of 69 600 years for this variant, a time approximately contemporaneous with estimates of the most recent ancestral human migration out of Africa.²⁵ The likelihood for this coalescent tree was 1.50x10^–7.

CHGA Gly364Ser Genotype Does Not Alter Plasma Concentrations of CHGA or Catestatin In Vivo
We asked whether expression of the CHGA precursor or its catestatin fragment (CHGA_{352 to 372}) differed quantitatively in individuals stratified by Gly364Ser genotype using radioimmunoassays directed against catestatin (epitope, CHGA residues 361 to 372) or against a large CHGA fragment encompassing much of the primary structure of this precursor (CHGA_116–439). Gly364Ser genotype groups did not differ in plasma concentrations of catestatin (P=0.71) or CHGA (P=0.398) (Table II). To estimate the degree of processing of CHGA to catestatin in the circulating plasma immunoreactivity, we derived the quotient of catestatin/(catestatin+CHGA) in each individual. Each Gly364Ser genotype group demonstrated 30% processing of CHGA to catestatin in plasma, and the groups did not differ in this regard (P=0.73).

Because catestatin and CHGA immunoreactivities vary inversely in concentration in human plasma,^8,26 we tested the relationship between concentrations of precursor (CHGA) and product (catestatin) in plasma (Figure 5). The relationship was inversely exponential (y=2.6x^[–0.46]; R=0.42; P=0.0002), but Gly364Ser genotype groups (Gly/Gly versus Gly/Ser) did not differ significantly. Visual inspection of Figure 5 reveals substantial overlap among individual values for Gly/Gly and Gly/Ser subjects.

View larger version (15K): [in this window] [in a new window]   Figure 5. CHGA and catestatin processing: radioimmunoassays in plasma. The catestatin region (SSMKLSFRARGYGFRGPGPQL; human CHGA352–372) was accessed by a synthetic peptide epitope radioimmunoassay corresponding to human CHGA amino acids 361 to 372. The CHGA precursor was accessed by an assay using the large fragment human CHGA116–439. Radioimmunoassay results are shown (nmol/L). Fractional catestatin processing from CHGA was estimated from immunoreactivity as follows: catestatin/(catestatin+CHGA). Plasma catestatin (in nmol/L) is shown as a function of the concentration of the CHGA molecule (precursor, in nmol/L). The typical inverse relationship between precursor and product is illustrated (y=2.6x[–0.46]; R=–0.42; P=0.0002).

Thus, allelic variation at the Gly364Ser genotype did not confer detectable quantitative alterations in CHGA or catestatin expression in human plasma (Table II and Figure 5). The inference is that qualitative variation (amino acid substitution) in the gene product (at codon 364) gives rise to the associated autonomic traits (Table II).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Catestatin (CHGA_352–372) Naturally Occurring Variant Gly364Ser: Autonomic Effects in the Parasympathetic and Sympathetic Branches
CHGA precursor¹ is cleaved to catestatin (human CHGA_352–372), a fragment with potent catecholamine release–inhibitory activity.⁴ Catestatin is formed endogenously^27,28 and acts on the neuronal/autonomic nicotinic cholinergic receptor, the physiological trigger responding to efferent autonomic outflow, to block its signal transduction toward secretion.⁴

To probe autonomic effects in vivo of the 364Ser allele whose potency was altered in vitro (Figure 1b), we carefully phenotyped 17 Gly/Ser heterozygotes and a group of comparable Gly/Gly homozygote controls (48) (Table II). We found consistent changes in both parasympathetic and sympathetic function.

Furthermore, biochemical indexes also suggested a decrease in sympathetic activity in Gly/Ser heterozygotes (Table II and Figure 3); norepinephrine renal excretion was diminished by 26%, and epinephrine renal excretion was down by 34%. Plasma norepinephrine declined (by 36%), whereas plasma epinephrine was unchanged. Urinary excretion of catecholamines may provide an integrated measure of secretion over time.²⁹

CHGA Gly364Ser: Association With Hypertension
Gly364Ser genotype exerted a significant 5- to 6-mm Hg effect on DBP in the population in both the initial (UCSD) and independent replication (Kaiser) studies (Figure 4a). Thus, the effect seems to be robust and reproducible. The effect seemed to account for 1.8% of DBP variance in the population. An effect of the genotype on SBP was also apparent, especially in men of the replication study (P=0.018). However, DBP and SBP are highly correlated, and in each of these studies (initial and replication), hypertensive subjects were ascertained on the basis of elevated DBP; future studies on isolated systolic hypertension would be required to establish the specificity of a genotype effect on SBP.

The second (Kaiser) sample was deliberately selected not only for large numbers and extreme DBP values but also for comparable representation of both sexes. This sample thus provided an ideal setting to explore the interaction of genes and sex in control of BP. This sample displayed not only a significant DBP effect of the Gly364Ser genotype (F=4.57, P=0.033) but also a significant gene-by-sex interaction (F=4.92, P=0.027) (Figure 4b). Indeed, the effect seemed to be confined to men, with a 13-mm Hg effect that was apparently absent in women (Figure 4b).

Why did women not demonstrate the genotype effect on BP? Fundamental molecular and cellular mechanisms of BP control may differ in men and women,³⁰ although the ultimate implications of such differences for disease states such as hypertension are not clearly understood. Indeed, we have noted that aging-dependent changes in sympathetic activity differ between the sexes,¹¹ as do vascular responses to adrenergic agonists.³¹ The Gly364Ser genotype-by-sex interaction results (Figure 4b), however, have potential implications. Because sex is a necessary modifier of the Gly364Ser effect on BP, it will be an essential part of any Gly364Ser role in the diagnosis, pathogenesis, or treatment of hypertension.

CHGA Gly364Ser: Qualitative Versus Quantitative Effects
Could the Gly364Ser polymorphism be in linkage disequilibrium with another variant at the CHGA locus actually causing the observed autonomic phenotype associations (Table II; ie, could Gly364Ser simply be an "innocent passenger"?

One possibility would be linkage disequilibrium, with a regulatory polymorphism governing expression of the gene. However, the extent of linkage disequilibrium between Gly364Ser and the crucial promoter variant G-462A was only modest (r²=0.090), and stratification by G-462A genotype did not predict any of the autonomic phenotypes.

This conclusion is borne out by the results of CHGA biochemical phenotyping (Table II and Figure 3); plasma CHGA concentration, plasma catestatin concentration, and extent of processing of CHGA into catestatin immunoreactivity did not differ in 364Ser carriers.

Perspectives: Significance of These Results for Human Autonomic Function: "Intermediate Phenotypes" for Hypertension
Baroreceptors
The alterations in baroreceptor function by 364Ser were particularly striking and consistent across methods in both the time and frequency domains (Figure II). Because baroreceptor activity is diminished not only in hypertension but also in the still-normotensive offspring of patients with hypertension,³² these baroreceptor results in normotensive individuals raise the possibility that the 364Ser allele might be protective against the future development of hypertension. In this respect, the 364Ser allele appears dominant over the Gly364 allele because a single copy of 364Ser triggers alterations in autonomic function (Table II and Figures 2 and 3) and apparent protection against the development of hypertension (Figure 4).

Intermediate Phenotypes
Heritable autonomic traits may be of particular value in investigations of the genetic underpinnings of hypertension, a complex trait with both hereditary and environmental determinants and likely even multiple genetic (or "polygenic") determinants.^7,8 Human twin studies indicate that baroreceptor activity is a substantially heritable trait.³³ Such traits may be valuable "intermediate phenotypes"^7,8 for hypertension, especially if they display earlier penetrance than the ultimate disease trait (DBP), can be elicited in still-normotensive individuals, and suggest testable candidate genetic loci (such as CHGA). Catestatin itself may be such a phenotype.⁸ Several of the autonomic phenotypes studied here (Table II and Figures 2 and 3) meet these criteria.

Indeed, Gly364Ser genotypes were associated with hypertension (Figure 4a and 4b), especially in men (Figure 4b). Thus, the 364Ser allele may play a protective role against future the development of hypertension.

How might this happen? Because baroreflex sensitivity is diminished in both established hypertension and the still-normotensive offspring of patients with hypertension,^32,34 alterations of this trait in hypertension cannot be simply a response to hypertension but might be involved early in the pathogenesis or development of the disease trait. In experimental animals, lesions of the baroreceptor afferent nerves or central nuclei^35–37 may result in sustained hypertension. More recently, Lohmeier et al³⁸ showed that chronic activation of the baroreflex produces sustained reductions in BP and sympathetic activity in animals. Although baroreflex sensitivity was augmented in Gly364Ser heterozygotes (Table II and Figure 2), our measurements of baroreceptor slope reflect coupling of afferent BP stimuli to efferent heart rate responses (in ms/mm Hg) (ie, to cardiac autonomic tone); in contrast, the hemodynamic "lesion" in established systemic hypertension is elevated systemic vascular resistance. However, the coupling of reduced catecholamine secretion (Table II and Figure 3) to augmented baroreceptor activity (Table II and Figure 2) in Gly364Ser heterozygotes suggests that enhancement of autonomic reflexes does translate to reduced sympathetic outflow in these subjects.

CHGA 364Ser alleles seem to increase multiple measures of parasympathetic activity (Table II and Figure 2) and decrease indexes of sympathetic activity, including diminished sympathovagal balance (by 44%; P=0.045; Table II), decreased cardiac sympathetic tone (Lorenz L/T ratio; by 26%; P=0.044; Table II), and diminished renal catecholamine excretion (by >25%; Table II and Figure 3). In addition, cardiac sympathetic index (Lorenz L/T ratio; Table II) correlated inversely with baroreceptor slope in the time domain (r=–0.324; n=62; P=0.01; Table III), further documenting functional (albeit inverse) links between sympathetic and parasympathetic activity. Finally, multivariate ANOVA demonstrated joint effects of Gly364Ser genotype on both parasympathetic and sympathetic tone (Pillai’s trace F=3.31, P=0.017). Thus, the 364Ser allele seemed to exert pleiotropic effects on both branches of the autonomic system.

Central Mechanism
As a nicotinic cholinergic antagonist, the best-documented effect of catestatin is to inhibit catecholamine release.^4,5 Catecholamines have important central effects to influence efferent parasympathetic and sympathetic outflow. For example, the antihypertensive clonidine acts as a central ₂-adrenergic agonist to enhance baroreflex sensitivity through efferent parasympathetic activation.³⁹ Thus, altered potency of Gly364Ser to influence catecholamine release (Figure 1b) could have parasympathetic consequences. Alternatively, because ganglionic nicotinic cholinergic receptors control parasympathetic and sympathetic neurotransmission,^39,40 differential actions of wild-type versus Gly364Ser variant catestatin directly on parasympathetic tone might also contribute to the heart rate responses observed here (Table II and Figure 2).

A unitary site of catestatin action to explain all of the Gly364Ser observations in this report would be the nicotinic cholinergic synapses in the nucleus of the tractus solitarius (NTS) in the cardiovascular/baroreceptor control region of the brain stem (Figure 6). Several lines of experimental evidence are consistent with (or predictions of) this hypothesis.

View larger version (21K): [in this window] [in a new window]   Figure 6. Perspectives and hypotheses on the role of CHGA Gly364Ser in autonomic physiology and disease. Catestatin Gly364Ser and human autonomic function: mechanistic proposal. A schematic is shown wherein the experimental results described in this research can be unified to explain how Gly364Ser differentially/reciprocally alters central and peripheral noradrenergic activity. indicates stimulation; , inhibition; VLM, ventrolateral medulla; DMNX, dorsal motor nucleus of the vagus (X).

Central Nicotinic Cholinergic Anatomy and BP
Central nicotinic-cholinergic synapses⁴¹ in the NTS act to enhance baroreceptor activity, increasing parasympathetic while decreasing sympathetic activity. Gly364Ser is associated with such reciprocal changes in autonomic efferent activity (Table II and Figures 2 and 3). CHGA is found in the brain stem by both radioimmunoassay⁴² and immunohistology.⁴³

Catecholamine Secretion: Central and Peripheral
In the peripheral autonomic system, Gly364Ser is associated with decreased catecholamine secretion (Figure 3), whereas in central cholinergic neurons, Gly364Ser is relatively ineffective at inhibiting catecholamine release (Figure 1b). A central action of catestatin in the NTS (Figure 6) explains these initially paradoxical results.

Baroreceptor Activity
Gly364Ser is associated with augmented baroreceptor activity by multiple indexes in both the time and frequency domains (Table II and Figure 2). A central action of catestatin in the NTS provides a rationale for this effect (Figure 6).

Parasympathetic Activity
Gly364Ser is associated with increased indexes of parasympathetic activity (Table II), including increased cardiac parasympathetic index and decreased sympathovagal balance. These effects are explained by catestatin at the NTS (Figure 6).

Sympathetic Activity
Gly364Ser heterozygotes display decreased sympathetic activity by both physiological (Table II) and biochemical (Table II and Figure 3) indexes. Once again, an action of catestatin in the NTS explains these findings.

Human History
Haplotype lineage analysis (online Figure III) suggested that Gly364Ser occurred as a point mutation on the most common coding region haplotype, likely shortly after the emergence of humankind from Africa. Thus, an ancient mutation in the human lineage seems to alter cardiovascular disease risk. At another cardiovascular risk candidate gene, angiotensin-converting enzyme, Rieder et al⁴⁴ used phylogeny to conclude that disease-associated angiotensin-converting enzyme variants were ancient within the human lineage. Why such ancient variants should persist in the population is a matter of speculation; perhaps such nonneutral variants might confer selective advantage under particular environmental circumstances. We propose that heightened baroreflex control of the circulation (Figure 2) may benefit the longevity of 364Ser carriers.

	Acknowledgments

 
We appreciate the assistance of the General Clinical Research Center (RR00827) and its core laboratory.

Sources of Funding

This work was supported by the Department of Veterans Affairs and the National Institutes of Health, Bethesda, Md.

Disclosures

None.

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