Human heart rate: heritability of resting and stress values in twin pairs, and influence of genetic variation in the adrenergic pathway at a microribonucleic acid (microrna) motif in the 3'-UTR of cytochrome b561 [corrected].

OBJECTIVES: The goal of this study was to understand the role of genetic variation in the catecholamine biosynthetic pathway for control of human heart rate (HR). BACKGROUND: Human HR is an integrated cardiovascular trait predictive of morbidity and survival. Because the autonomic pathway exerts rapid control over the heart, we probed the role of heredity in the control of HR, focusing on a component of the autonomic sympathetic pathway already predictive of outflow responses: cytochrome b561 (CYB561), the electron shuttle in catecholamine vesicle membranes for transmitter biosynthesis. METHODS: We studied hereditary control of HR with the twin pair design, at rest and during environmental (cold) stress. Single nucleotide polymorphism disruption of a microribonucleic acid (microRNA) recognition motif in the human CYB561 3'-UTR was identified computationally, and its differential effect on gene expression was demonstrated in a transfected luciferase reporter/3'-UTR variant. We exposed stem cell-derived human embryoid bodies to the microRNA mimic or antagomir oligonucleotides, and we observed the effects on contraction rate in proto-hearts. RESULTS: Substantial heritability (h(2)) was demonstrated by using twin pair variance components for both basal/resting HR (h(2) 50.9 ± 6.4% of trait variation, p = 2.47 × 10(-10)) and stress-augmented HR (h(2) 55.1 ± 5.9%, p = 8.79 × 10(-13)), and the 2 HR traits shared genetic determination (genetic covariance ρG 0.747 ± 0.058, p = 2.85 × 10(-9)). CYB561 displayed 1 common genetic variant in the transcript region: A+1485G (rs3087776), in the 3'-UTR, 1485 bp downstream of the termination codon, in a conserved region, with the A-allele ancestral in primates. In a twin/sibling sample (n = 576), A+1485G influenced HR, both at rest (p = 0.010) and after environmental stress (p = 0.002), with the minor (A) allele displaying a recessive effect with lower HR. The effect of A+1485G on HR was extended by meta-analysis into 2 additional population samples (total n = 2,579), and the influence remained directionally consistent and significant (p = 0.007). A+1485G disrupted a microRNA (human microribonucleic acid-1294 [hsa-miR-1294]) recognition motif in the 3'-UTR, as demonstrated by a transfected luciferase reporter/human 3'-UTR variant system in 2 different neuronal/neuroendocrine cell types. The microRNA effect was further documented by cotransfection of an hsa-miR-1294 mimic, yielding an exaggerated decline in expression of the A-allele (better match) reporter (p = 4.3 × 10(-5)). Similar findings of differential 3'-UTR allelic susceptibility to hsa-miR-1294 were noted during expression of the full-length human CYB561 messenger ribonucleic acid with its cognate 3'-UTR. Finally, exposure of stem cell-derived human embryoid bodies to hsa-miR-1294 mimic or antagomir oligonucleotides yielded directionally opposite effects on contraction rate in proto-hearts. CONCLUSIONS: HR is a substantially heritable trait, with genetic influence by variation in the adrenergic pathway, here shown for messenger ribonucleic acid translational control at the CYB561 step of transmitter formation. The results have implications for potentially modifiable autonomic pathways that influence this risk trait in the population.

Total sleep time and other sleep characteristics measured by actigraphy do not predict incident hypertension in a cohort of community-dwelling older men.

STUDY OBJECTIVE: To evaluate whether actigraphy-measured total sleep time and other sleep characteristics predict incident hypertension in older men. METHODS: Study subjects were community-dwelling participants in the ancillary sleep study of the Osteoporotic Fractures in Men Study (MrOS) who were normotensive at the time of actigraphy (based on self-report, lack of antihypertensive medication use, and with systolic blood pressure < 140 mm Hg and diastolic blood pressure < 90 mm Hg). In 853 community-dwelling men 67 years and older (mean 75.1 years), sleep measures (total sleep time [TST]), percent sleep [%-sleep], latency, and wake after sleep onset [WASO]) were obtained using validated wrist actigraphy with data collected over a mean duration of 5.2 consecutive 24-h periods. We evaluated incident hypertension (based on self-report, use of antihypertensive medication, or measured systolic blood pressure ≥ 140 mm Hg or diastolic blood pressure ≥ 90 mm Hg) at a follow-up visit an average of 3.4 years later. Baseline prehypertension was defined as a systolic blood pressure 120 to < 140 mm Hg or diastolic blood pressure 80 to < 90 mm Hg. RESULTS: At follow-up, 31% of initially normotensive men were hypertensive (264 of 853). Those with incident hypertension had higher baseline body mass index (BMI; kg/m(2)) and were more likely to have had prehypertension at the sleep visit than those men who remained normotensive. However, neither TST (reference 6 to < 8 h; < 6 h OR 0.96 [95% CI 0.7, 1.3] and ≥ 8 h OR 0.93 [0.5, 1.7]) nor the other actigraphic-measured sleep variables, including % -sleep (reference > 85%; < 70% OR 1.17 [0.66, 2.08]) and 70% to ≤ 85% OR 1.23 (0.9, 1.68), sleep latency (reference < 30 min; ≥ 30 min OR 1.29 [0.94, 1.76]), or WASO (reference < 30 min; 30 to < 60 min OR 0.7 [0.43, 1.14] and ≥ 60 min OR 0.92 [0.58, 1.47]) differed in those community-dwelling men who developed incident hypertension compared to those who remained normotensive. CONCLUSION: TST and other sleep parameters determined by wrist actigraphy were not associated with incident hypertension in community-dwelling older men.

Central hemodynamics in prehypertension: effect of the ß-adrenergic antagonist nebivolol.

The aim of the current study was to characterize the effects of the novel ß-adrenergic antagonist nebivolol on central aortic blood pressures, arterial properties, and nitroxidergic activity in individuals with prehypertension. Prehypertension is emerging as a major risk factor for several adverse cardiovascular consequences. Increased pulse wave velocity, aortic augmentation index, and aortic blood pressures have been linked with augmented risk of cardiovascular disease and mortality. While the effects of antihypertensive drugs on these parameters in hypertensive patients have been studied, there are limited data so far in prehypertension. Fifty individuals with prehypertension were randomized to either nebivolol (5 mg per day) or placebo in a double-blind clinical trial. Patients underwent measurement of pulse wave velocity as well as aortic blood pressure and aortic augmentation index via pulse wave analysis at baseline and 8 weeks. Patients also had blood and urine biochemistries done at each visit. Nebivolol achieved significant reductions in central aortic systolic (P=.011), diastolic (P=.009), and mean arterial blood pressure (P=.002). Pulse wave velocity trended toward improvement but did not achieve significance (P=.088). Nitric oxide production, measured as urinary nitrite/nitrite excretion, also rose substantially in the nebivolol group (by approximately 60%, P=.030). Central blood pressures can be effectively lowered by ß-blockade while patients are still in the prehypertension phase, and the effects may be coupled to improve nitric oxide release by the drug.

Heritable influence of DBH on adrenergic and renal function: twin and disease studies.

BACKGROUND: Elevated sympathetic activity is associated with kidney dysfunction. Here we used twin pairs to probe heritability of GFR and its genetic covariance with other traits. METHODS: We evaluated renal and adrenergic phenotypes in twins. GFR was estimated by CKD-EPI algorithm. Heritability and genetic covariance of eGFR and associated risk traits were estimated by variance-components. Meta-analysis probed reproducibility of DBH genetic effects. Effect of DBH genetic variation on renal disease was tested in the NIDDK-AASK cohort. RESULTS: Norepinephrine secretion rose across eGFR tertiles while eGFR fell (p<0.0001). eGFR was heritable, at h(2) = 67.3±4.7% (p = 3.0E-18), as were secretion of norepinephrine (h(2) = 66.5±5.0%, p = 3.2E-16) and dopamine (h(2) = 56.5±5.6%, p = 1.8E-13), and eGFR displayed genetic co-determination (covariance) with norepinephrine (ρG = -0.557±0.088, p = 1.11E-08) as well as dopamine (ρG = -0.223±0.101, p = 2.3E-02). Since dopamine ß-hydroxylase (DBH) catalyzes conversion of dopamine to norepinephrine, we studied functional variation at DBH; DBH promoter haplotypes predicted transcriptional activity (p<0.001), plasma DBH (p<0.0001) and norepinephrine (p = 0.0297) secretion; transcriptional activity was inversely (p<0.0001) associated with basal eGFR. Meta-analysis validated DBH haplotype effects on eGFR across 3 samples. In NIDDK-AASK, we established a role for DBH promoter variation in long-term renal decline rate (GFR slope, p = 0.003). CONCLUSIONS: The heritable GFR trait shares genetic determination with catecholamines, suggesting new pathophysiologic, diagnostic and therapeutic approaches towards disorders of GFR as well as CKD. Adrenergic activity may play a role in progressive renal decline, and genetic variation at DBH may assist in profiling subjects for rational preventive treatment.

Neuropeptide Y (NPY): genetic variation in the human promoter alters glucocorticoid signaling, yielding increased NPY secretion and stress responses.

OBJECTIVES: This study sought to understand whether genetic variation at the Neuropeptide Y (NPY) locus governs secretion and stress responses in vivo as well as NPY gene expression in sympathochromaffin cells. BACKGROUND: The NPY is a potent pressor peptide co-released with catecholamines during stress by sympathetic axons. Genome-wide linkage on NPY secretion identified a LOD (logarithm of the odds ratio) peak spanning the NPY locus on chromosome 7p15. METHODS: Our approach began with genomics (linkage and polymorphism determination), extended into NPY genetic control of heritable stress traits in twin pairs, established transcriptional mechanisms in transfected chromaffin cells, and concluded with observations on blood pressure (BP) in the population. RESULTS: Systematic polymorphism tabulation at NPY (by re-sequencing across the locus: promoter, 4 exons, exon/intron borders, and untranslated regions; on 2n = 160 chromosomes of diverse biogeographic ancestries) identified 16 variants, of which 5 were common. We then studied healthy twin/sibling pairs (n = 399 individuals), typing 6 polymorphisms spanning the locus. Haplotype and single nucleotide polymorphism analyses indicated that proximal promoter variant ∇-880Δ (2-bp TG/-, Ins/Del, rs3037354) minor/Δ allele was associated with several heritable (h(2)) stress traits: higher NPY secretion (h(2) = 73 ± 4%) as well as greater BP response to environmental (cold) stress, and higher basal systemic vascular resistance. Association of ∇-880Δ and plasma NPY was replicated in an independent sample of 361 healthy young men, with consistent allelic effects; genetic variation at NPY also associated with plasma NPY in another independent series of 2,212 individuals derived from Australia twin pairs. Effects of allele -880Δ to increase NPY expression were directionally coordinate in vivo (on human traits) and in cells (transfected NPY promoter/luciferase reporter activity). Promoter -880Δ interrupts a novel glucocorticoid response element motif, an effect confirmed in chromaffin cells by site-directed mutagenesis on the transfected promoter, with differential glucocorticoid stimulation of the motif as well as alterations in electrophoretic mobility shifts. The same -880Δ allele also conferred risk for hypertension and accounted for approximately 4.5/approximately 2.1 mm Hg systolic BP/diastolic BP in a population sample from BP extremes. CONCLUSIONS: We conclude that common genetic variation at the NPY locus, especially in proximal promoter ∇-880Δ, disrupts glucocorticoid signaling to influence NPY transcription and secretion, raising systemic vascular resistance and early heritable responses to environmental stress, eventuating in elevated resting BP in the population. The results point to new molecular strategies for probing autonomic control of the human circulation and ultimately susceptibility to and pathogenesis of cardiovascular and neuropsychiatric disease states.

Autonomic and hemodynamic origins of pre-hypertension: central role of heredity.

OBJECTIVES: The purpose of this study is to better understand the origins and progression of pre-hypertension. BACKGROUND: Pre-hypertension is a risk factor for progression to hypertension, cardiovascular disease, and increased mortality. We used a cross-sectional twin study design to examine the role of heredity in likely pathophysiological events (autonomic or hemodynamic) in pre-hypertension. METHODS: Eight hundred twelve individuals (337 normotensive, 340 pre-hypertensive, 135 hypertensive) were evaluated in a sample of twin pairs, their siblings, and other family members. They underwent noninvasive hemodynamic, autonomic, and biochemical testing, as well as estimates of trait heritability (the percentage of trait variance accounted for by heredity) and pleiotropy (the genetic covariance or shared genetic determination of traits) by variance components. RESULTS: In the hemodynamic realm, an elevation of cardiac contractility prompted increased stroke volume, in turn increasing cardiac output, which elevated blood pressure into the pre-hypertension range. Autonomic monitoring detected an elevation of norepinephrine secretion plus a decline in cardiac parasympathetic tone. Twin pair variance components documented substantial heritability as well as joint genetic determination for blood pressure and the contributory autonomic and hemodynamic traits. Genetic variation at a pathway locus also indicated pleiotropic effects on contractility and blood pressure. CONCLUSIONS: Elevated blood pressure in pre-hypertension results from increased cardiac output, driven by contractility as well as heart rate, which may reflect both diminished parasympathetic and increased sympathetic tone. In the face of increased cardiac output, systemic vascular resistance fails to decline homeostatically. Such traits display substantial heritability and shared genetic determination, although by loci not yet elucidated. These findings clarify the role of heredity in the origin of pre-hypertension and its autonomic and hemodynamic pathogenesis. The results also establish pathways that suggest new therapeutic targets for pre-hypertension, or approaches to its prevention.

Genetic variation within a metabolic motif in the chromogranin a promoter: pleiotropic influence on cardiometabolic risk traits in twins.

BACKGROUND: The cardiometabolic syndrome comprised of multiple correlated traits, but its origin is incompletely understood. Chromogranin A (CHGA) is required for formation of the catecholamine secretory pathway in sympathochromaffin cells. In twin pair studies, we found that CHGA traits aggregated with body mass index (BMI), as well as its biochemical determinant leptin. METHODS Here we used the twin method to probe the role of heredity in generating such risk traits, and then investigated the role of risk-trait-associated CHGA promoter genetic variation in transfected chromaffin cells. Trait heritability (h(2)) and shared genetic determination among traits (pleiotropy, genetic covariance, ρ(G)) were estimated by variance components in twin pairs. RESULTS: CHGA, BMI, and leptin each displayed substantial h(2), and the traits also aggregated with several features of the metabolic syndrome (e.g., insulin resistance, blood pressure (BP), hypertension, catecholamines, and C-reactive protein (CRP)). Twin studies demonstrated genetic covariance (pleiotropy, ρ(G)) for CHGA, BMI, and leptin with other metabolic traits (insulin resistance, BP, and CRP). We therefore investigated the CHGA locus for mechanisms of codetermination with such metabolic traits. A common functional variant in the human CHGA promoter (G-462A, rs9658634, minor allele frequency ~21%) was associated with leptin and CRP secretion, as well as BMI, especially in women; marker-on-trait effects on BMI were replicated across twin populations on two continents. In CHGA promoter/luciferase reporter plasmids transfected into chromaffin cells, G-462A alleles differed markedly in reporter expression. The G-462A variant disrupted predicted transcriptional control by a PPARγ/RXRα motif and costimulation by PPARγ/RXRα and their cognate ligands, differentially activated the two alleles. During chromatin immunoprecipitation, endogenous PPARγ bound the motif. CONCLUSIONS: Multiple features of the metabolic syndrome are thus under joint (pleiotropic) genetic determination, with CHGA as one such contributory locus: a common polymorphism in the promoter (G-462A) of CHGA predicts such heritable metabolic traits as BMI and leptin. CHGA promoter variant G-462A was not only associated with such metabolic traits but also disrupted a PPARγ/RXRα motif and responded differentially to characteristic trans-activators of that motif. The results suggest novel links between the catecholaminergic system and risk for the metabolic syndrome as well as systemic hypertension.

Methylenetetrahydrofolate reductase (MTHFR) polymorphism A1298C (Glu429Ala) predicts decline in renal function over time in the African-American Study of Kidney Disease and Hypertension (AASK) Trial and Veterans Affairs Hypertension Cohort (VAHC).

BACKGROUND: Hyperhomocysteinemia is associated with increased venous thrombosis and cardiovascular disease (CVD). Mutations in the human methylenetetrahydrofolate reductase (MTHFR) gene have been associated with increased homocysteine levels and risks of CVD in various populations including those with kidney disease. Here, we evaluated the influence of MTHFR variants on progressive loss of kidney function. METHODS: We analyzed 821 subjects with hypertensive nephrosclerosis from the longitudinal National Institute of Diabetes and Digestive and Kidney Diseases African-American Study of Kidney Disease and Hypertension (AASK) Trial to determine whether decline in glomerular filtration rate (GFR) over ∼4.2 years was predicted by common genetic variation within MTHFR at non-synonymous positions C677T (Ala222Val) and A1298C (Glu429Ala) or by MTHFR haplotypes. The effect on GFR decline was then supported by a study of 1333 subjects from the San Diego Veterans Affairs Hypertension Cohort (VAHC), followed over ∼4.5 years. Linear effect models were utilized to determine both genotype [single-nucleotide polymorphism (SNP)] and genotype (SNP)-by-time interactions. RESULTS: In AASK, the polymorphism at A1298C predicted the rate of GFR decline: A1298/A1298 major allele homozygosity resulted in a less pronounced decline of GFR, with a significant SNP-by-time interaction. An independent follow-up study in the San Diego VAHC subjects supports that A1298/A1298 homozygotes have the greatest estimated GFR throughout the study. Haplotype analysis with C677T yielded concurring results. CONCLUSION: We conclude that the MTHFR-coding polymorphism at A1298C is associated with renal decline in African-Americans with hypertensive nephrosclerosis and is supported by a veteran cohort with a primary care diagnosis of hypertension. Further investigation is needed to confirm such findings and to determine what molecular mechanism may contribute to this association.

Decreased slow wave sleep increases risk of developing hypertension in elderly men.

The importance of sleep to health and cardiovascular disease has become increasingly apparent. Sleep-disordered breathing, sleep duration, and sleep architecture may all influence metabolism and neurohormonal systems, yet no previous study has evaluated these sleep characteristics concurrently in relation to incident hypertension. Our objective was to determine whether incident hypertension is associated with polysomnography measures of sleep-disordered breathing, sleep duration, and sleep architecture in older men. Participants were 784 community-dwelling, ambulatory men ≥65 years of age (mean age: 75.1±4.9 years) from the Outcomes of Sleep Disorders in Older Men Study who did not have hypertension at the time of their in-home polysomnography sleep studies (2003-2005) and who returned for follow-up (2007-2009). Of 784 older men included in this report, 243 met criteria for incident hypertension after a mean follow-up of 3.4 years. In unadjusted analyses, incident hypertension was associated with increased hypoxemia, increased sleep stages N1 and N2, and decreased stage N3 (slow wave sleep [SWS]). After adjustment for age, nonwhite race, study site, and body mass index, the only sleep index to remain significantly associated with incident hypertension was SWS percentage (odds ratio for lowest to highest quartile of SWS: 1.83 [95% CI: 1.18 to 2.85]). No attenuation of this association was seen after accounting for sleep duration, sleep fragmentation, and indices of sleep-disordered breathing. Percentage time in SWS was inversely associated with incident hypertension, independent of sleep duration and fragmentation, and sleep-disordered breathing. Selective deprivation of SWS may contribute to adverse blood pressure in older men.

A cross-sectional and 10-year prospective study of postmenopausal estrogen therapy and blood pressure, renal function, and albuminuria: the Rancho Bernardo Study.

OBJECTIVE: Blood pressure, urine albumin-to-creatinine ratio, and estimated glomerular filtration rate (GFR) are highly correlated conditions. The longitudinal effect of exposure to postmenopausal estrogen therapy on these traits studied together has not been reported. METHODS: This was a cross-sectional study of 1,044 older postmenopausal community-dwelling women from the Rancho Bernardo Study (1992-1996); 443 women were reevaluated ∼ 10 years later (2002-2005). We determined the cross-sectional and prospective association of baseline postmenopausal estrogen therapy and blood pressure, urine albumin-to-creatinine ratio, GFR, and the odds of categorical hypertension (physician diagnosis, medication, or blood pressure ≥ 140/≥ 90 mm Hg), chronic kidney disease (GFR ≤ 60 mL/min per 1.73 m2), and albuminuria (urine albumin-to-creatinine ratio ≥ 25 mg/g). RESULTS: At baseline, the mean age was 68.3 years for current estrogen users, 75.4 years for past users, and 74.3 years for never users. In the cross-sectional analyses, current users had lower diastolic blood pressure and lower odds of having chronic kidney disease, independent of covariates. In the ∼ 10-year follow-up, comparisons between never, past, and current estrogen use (91% continuous use since baseline), the mean diastolic blood pressure declined over time in current users, whereas systolic blood pressure increased among never users. Urine albumin-to-creatinine ratio also increased in never users and decreased in current users; GFR did not differ by estrogen use. CONCLUSIONS: In cross-sectional analyses, estrogen users had better GFR and blood pressure than nonusers did, but the 10-year follow-up showed improved blood pressure and decreased urine albumin-to-creatinine ratio among mostly long-term current users, without differences in GFR by estrogen use. This study suggests no association of GFR with 10 years of continuous estrogen use and an inverse association with albuminuria.

Catecholamine storage vesicles: role of core protein genetic polymorphisms in hypertension.

Hypertension is a complex trait with deranged autonomic control of the circulation. The sympathoadrenal system exerts minute-to-minute control over cardiac output and vascular tone. Catecholamine storage vesicles (or chromaffin granules) of the adrenal medulla contain remarkably high concentrations of chromogranins/secretogranins (or "granins"), catecholamines, neuropeptide Y, adenosine triphosphate (ATP), and Ca(2+). Within secretory granules, granins are co-stored with catecholamine neurotransmitters and co-released upon stimulation of the regulated secretory pathway. The principal granin family members, chromogranin A (CHGA), chromogranin B (CHGB), and secretogranin II (SCG2), may have evolved from shared ancestral exons by gene duplication. This article reviews human genetic variation at loci encoding the major granins and probes the effects of such polymorphisms on blood pressure, using twin pairs to probe heritability and individuals with the most extreme blood pressure values in the population to study hypertension.

Early inflammatory and metabolic changes in association with AGTR1 polymorphisms in prehypertensive subjects.

BACKGROUND: The Seventh Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure in 2003 created a prehypertension category for persons with blood pressures ranging from systolic blood pressure (SBP) of 120-139 mm Hg or diastolic blood pressure (DBP) from 80 to 89 mm Hg, due to increased risk of cardiovascular disease. METHODS: Our study utilized the University of California-San Diego (UCSD) Twin Hypertension Cohort. We measured comprehensive plasma cholesterol levels and metabolic (glucose, insulin, leptin) and inflammatory markers (interleukin-6 (IL-6), C-reactive protein (CRP), free fatty acids) to determine the differences between normotensive and prehypertensive subjects. Additionally, we determined whether angiotensin II receptor type-1 (AGTR1) polymorphisms, previously associated with hypertension, could predict prehypertension. RESULTS: A total of 455 white subjects were included in the study (mean age 37.1 years). Prehypertensive subjects were older with greater body mass index (BMI) than the normotensives, and after adjusting for sex and age, had greater plasma glucose, insulin, and IL-6. The common AGTR1 A1166C (rs5186) polymorphism in the 3'-UTR region, particularly the presence of the 1166C allele, which fails to downregulate gene expression, predicted greater likelihood of being in the prehypertension group and higher SBP. A lesser-studied polymorphism in intron-2 of AGTR1 (A/G; rs2276736) was associated with plasma high-density lipoprotein (HDL) and apolipoprotein A-1. In a subgroup analysis of nonobese subjects (N = 405), similar associations were noted. CONCLUSION: Prehypertensive subjects already exhibit early pathophysiologic changes putting them at risk of future cardiovascular disease, and AGTR1 may also contribute to this increased risk. Further investigation is needed to confirm these findings and the precise molecular mechanisms of action.

Contemporary approaches to genetic influences on hypertension.

PURPOSE OF REVIEW: Essential hypertension has long been considered to be primarily 'genetic,' though recent studies have only revealed minor contributions to blood pressure. Technology has advanced tremendously in the recent years, with much focus on DNA studies utilizing both candidate gene and genome-wide association studies. However, many new areas that need continued investigation have arisen. RECENT FINDINGS: In addition to DNA studies, genetic studies are actively pursuing previously unexplored areas of potential variation, such as that which occurs posttranscriptionally in RNA and posttranslationally in protein structure. Advances have also been made in animal models and systems biology for large-scale integrative approaches. However, many other areas need continued investigation in the genetics of hypertension, including improved phenotyping and trait definition, gene-by-gene interactions (epistasis), and gene-by-environment interactions. 'Next generation' sequencing will assist researchers in performing more extensive genetic studies even more quickly, especially on unusual (rare) genetic variants. SUMMARY: Hypertension appears to have many genetic contributions from each regulatory area ranging from DNA to RNA to protein to postprotein to interactive influences of the environment on genes. New technologies have enabled such research to advance in the recent years. However, for this complex trait of hypertension, continued efforts must progress in all of these areas as well as in increased modeling and sequencing, so that the knowledge may be united for a comprehensive understanding of this common disease, such that diagnosis and treatment options in hypertensive patients and those at risk are facilitated.

Naturally occurring genetic variants in human chromogranin A (CHGA) associated with hypertension as well as hypertensive renal disease.

Chromogranin A (CHGA) plays a fundamental role in the biogenesis of catecholamine secretory granules. Changes in storage and release of CHGA in clinical and experimental hypertension prompted us to study whether genetic variation at the CHGA locus might contribute to alterations in autonomic function, and hence hypertension and its target organ consequences such as hypertensive renal disease (nephrosclerosis). Systematic polymorphism discovery across the human CHGA locus revealed both common and unusual variants in both the open reading frame and such regulatory regions as the proximal promoter and 30-UTR. In chromaffin cell-transfected CHGA 30-UTR and promoter/luciferase reporter plasmids, the functional consequences of the regulatory/non-coding allelic variants were documented. Variants in both the proximal promoter and the 30-UTR displayed statistical associations with hypertension. Genetic variation in the proximal CHGA promoter predicted glomerular filtration rate in healthy twins. However, for hypertensive renal damage, both end-stage renal disease and rate of progression of earlier disease were best predicted by variants in the 30-UTR. Finally, mechanistic studies were undertaken initiated by the clue that CHGA promoter variation predicted circulating endothelin-1. In cultured endothelial cells, CHGA triggered co-release of not only the vasoconstrictor and pro-fibrotic endothelin-1, but also the pro-coagulant von Willebrand Factor and the pro-angiogenic angiopoietin-2. These findings, coupled with stimulation of endothelin-1 release from glomerular capillary endothelial cells by CHGA, suggest a plausible mechanism whereby genetic variation at the CHGA locus eventuates in alterations in human renal function. These results document the consequences of genetic variation at the CHGA locus for cardiorenal disease and suggest mechanisms whereby such variation achieves functional effects.

Reprint of: Catestatin: a multifunctional peptide from chromogranin A.

In 1997, we identified a novel peptide, catestatin (CST: bovine chromogranin A [CHGA]344₋364: RSMRLSFRARGYGFRGPGLQL; human CHGA352₋372: SSMKLSFRARGYGFRGPGPQL), which is a potent inhibitor of nicotinic-cholinergic-stimulated catecholamine secretion. CST shows characteristic inhibitory effects on nicotinic cationic (Na+, Ca²+) signal transduction, which are specific to the neuronal nicotinic receptor. Utilizing systematic polymorphism discovery at the human CHGA locus we discovered three human variants of CST: G³64S, P³7°L, and R³74Q that showed differential potencies towards the inhibition of catecholamine secretion. In humans, CHGA is elevated and its processing to CST is diminished in hypertension. Diminished CST is observed not only in hypertensive individuals but also in the early-normotensive offspring of patients with hypertension, suggesting that an early deficiency of CST might play a pathogenic role in the subsequent development of the disease. Consistent with human findings, prevention of endogenous CST expression by targeted ablation (knockout) of the mouse Chga locus (Chga-KO) resulted in severe hypertension that can be "rescued" specifically by replacement of the CST peptide. CST acts directly on the heart to inhibit the inotropic and lusitropic properties of the rodent heart and also acts as a potent vasodilator in rats and humans. While the G³64S CST variant caused profound changes in human autonomic activity and seemed to reduce the risk of developing hypertension, CST replacement rescued Chga-KO mice from dampened baroreflex sensitivity. In addition, CST has been shown to induce chemotaxis and acts as an antimicrobial as well as an antimalarial peptide. The present review summarizes these multiple actions of CST.

Isoprostane, an "intermediate phenotype" for oxidative stress heritability, risk trait associations, and the influence of chromogranin B polymorphism.

OBJECTIVES: The purpose of this study is to understand whether isoprostane, a biomarker of oxidative stress, is subject to heritable control; whether it shares heritability with other cardiometabolic risk traits; and finally whether genetic variation at a specific candidate locus contributes to isoprostane variability. BACKGROUND: Isoprostane marks oxidative stress, and elevated isoprostane excretion might be involved in cardiovascular target organ damage. Here we used the classical twin pair method to probe the role of heredity in generating the isoprostane trait. METHODS: Trait heritability (h(2)) and shared genetic determination among traits (pleiotropy, genetic covariance, ρ(G)) were estimated by variance components in twin pairs. Because the isoprostane and Chromogranin B (CHGB) traits shared ρ(G), we examined the CHGB locus for effects on the traits. RESULTS: Urinary isoprostane excretion was substantially heritable (h(2) = 65.8 ± 4.3%), and the isoprostane trait aggregated with multiple traits (CHGB, catecholamines, autonomic/baroreceptor, and renal function), including several features of the metabolic syndrome (body mass index, insulin resistance, dyslipidemia). Isoprostane excretion also aggregated with systemic hypertension. Twin studies demonstrated genetic covariance (pleiotropy) for the isoprostane and CHGB traits (ρ(G) = 0.27), and therefore we investigated the CHGB locus for trait effects. A common variant in the 3'-UTR of CHGB (C+84A) associated with plasma CHGB as well as isoprostane excretion. The C+84A disrupted an A/U-rich messenger ribonucleic acid stability element, and in transfected luciferase/3'-UTR plasmids, the C+84 and +84A alleles differed markedly in reporter expression in chromaffin and neuroblastoma cells, whereas site-directed mutagenesis confirmed the importance of this variant within the context of the A/U-rich motif. CONCLUSIONS: Isoprostane excretion is substantially heritable and shares joint genetic determination with CHGB as well as multiple features of the metabolic syndrome. A common polymorphism in the 3'-UTR (C+84A) of CHGB, which disrupts an A/U-rich messenger ribonucleic acid stability element, associates with not only CHGB secretion but also excretion of isoprostane. We propose a chain of events whereby CHGB genetic variation results in oxidative stress, with isoprostane formation. The results suggest novel links among the catecholaminergic system, oxidative pathways, and systemic hypertension.

Common charge-shift mutation Glu65Lys in K+ channel ß1-Subunit KCNMB1: pleiotropic consequences for glomerular filtration rate and progressive renal disease.

BACKGROUND: Glomerular filtration rate (GFR) is a heritable trait, and hyperfiltration (GFR increment in remnant nephrons) may accelerate renal functional decline in chronic kidney disease (CKD). Mesangial and vascular smooth myocytes control GFR by contraction, dependent on voltage-gated Ca(2+) influx, which is controlled by the regulatory ß1-subunit (KCNMB1) of large-conductance heteromeric K+ ('BK') channels. KCNMB1 gain-of-function variant Glu65Lys results in generalized vasorelaxation and thus protection against systemic hypertension. Here we asked whether the Glu65Lys variant influences GFR, in the basal state or during progressive renal decline. METHODS: We explored Glu65Lys effects on GFR in three populations spanning two ethnicities and two diseases (hypertension and nephrosclerosis). GFR was either estimated (eGFR from serum creatinine) or directly measured (iothalamate clearance). RESULTS: The 65Lys variant was relatively common, occurring on ∼5-10% of chromosomes in different biogeographic ancestry groups, and 65Lys carriers exhibited higher eGFR in two primary care populations: extreme BP values in Kaiser clinics (p = 0.029, accounting for ∼0.2% of trait variance), or treated hypertensives in VA clinics (p = 0.017, accounting for ∼0.9% of trait variance). In blacks with progressive renal disease (NIDDK AASK), 65Lys carriers displayed a steeper slope in GFR chronic decline (p = 0.030, accounting for ∼0.4% of trait variance), and Glu65Lys genotype also predicted time of onset of renal failure (log rank p = 0.019). CONCLUSIONS: Common KCNMB1 gain-of-function variant Glu65Lys influences GFR, and 65Lys carriers exhibit not only elevated baseline GFR, but also more rapid GFR decline (and consequent development of renal failure) in CKD. The results suggest that profiling patients at Glu65Lys can assist in gauging renal prognosis as well as selection of rational therapy in hypertension with progressive renal disease.

Direct vasoactive effects of the chromogranin A (CHGA) peptide catestatin in humans in vivo.

Catestatin is a bioactive peptide of chromogranin A (CHGA) that is co-released with catecholamines from secretory vesicles. Catestatin may function as a vasodilator and is diminished in hypertension. To evaluate this potential vasodilator in vivo without systemic counterregulation, we infused catestatin to target concentrations of approximately 50, approximately 500, approximately 5000 nM into dorsal hand veins of 18 normotensive men and women, after pharmacologic venoconstriction with phenylephrine. Pancreastatin, another CHGA peptide, was infused as a negative control. After preconstriction to approximately 69%, increasing concentrations of catestatin resulted in dose-dependent vasodilation (P = 0.019), in female subjects (to approximately 44%) predominantly. The EC(50) (approximately 30 nM) for vasodilation induced by catestatin was the same order of magnitude to circulating endogenous catestatin (4.4 nM). No vasodilation occurred during the control infusion with pancreastatin. Plasma CHGA, catestatin, and CHGA-to-catestatin processing were then determined in 622 healthy subjects without hypertension. Female subjects had higher plasma catestatin levels than males (P = 0.001), yet lower CHGA precursor concentrations (P = 0.006), reflecting increased processing of CHGA-to-catestatin (P < 0.001). Our results demonstrate that catestatin dilates human blood vessels in vivo, especially in females. Catestatin may contribute to sex differences in endogenous vascular tone, thereby influencing the complex predisposition to hypertension.

Human tyrosine hydroxylase natural allelic variation: influence on autonomic function and hypertension.

The catecholamine biosynthetic pathway consists of several enzymatic steps in series, beginning with the amino acids phenylalanine and tyrosine, and eventuating in the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Since the enzyme tyrosine hydroxylase (TH; tyrosine 3-mono-oxygenase; EC 1.14.16.2; chromosome 11p15.5) is generally considered to be rate-limiting in this pathway, probed as to whether common genetic variation at the TH gene occurred, and whether such variants contributed to inter-individual alterations in autonomic function, either biochemical or physiological. We began with sequencing a tetranucleotide (TCAT) repeat in the first intron, and found that the two most common versions, (TCAT)(6) and (TCAT)(10i), predicted heritable autonomic traits in twin pairs. We then conducted systematic polymorphism discovery across the ~8 kbp locus, and discovered numerous variants, principally non-coding. The proximal promoter block contained four common variants, and its haplotypes and SNPs (especially C-824T, rs10770141) predicted catecholamine secretion, environmental stress-induced BP increments, and hypertension. Finally, we found that two of the common promoter variants, C-824T (rs10770141) and A-581G (rs10770140), were functional in that they differentially affected transcriptional activity of the isolated promoter, disrupted recognition motifs for specific transcription factor binding, altered the promoter responses to the co-transfected (exogenous) factors, and bound the endogenous factors in the chromatin fraction of the nucleus. We concluded that common variation in the proximal TH promoter is functional, giving rise to changes in autonomic function and consequently cardiovascular risk.