Polymorphisms at the F12 and KLKB1 loci have significant trait association with activation of the renin-angiotensin system.

BACKGROUND: Plasma coagulation Factor XIIa (Hageman factor; encoded by F12) and kallikrein (KAL or Fletcher factor; encoded by KLKB1) are proteases of the kallikerin-kinin system involved in converting the inactive circulating prorenin to renin. Renin is a key enzyme in the formation of angiotensin II, which regulates blood pressure, fluid and electrolyte balance and is a biomarker for cardiovascular, metabolic and renal function. The renin-angiotensin system is implicated in extinction learning in posttraumatic stress disorder. METHODS & RESULTS: Active plasma renin was measured from two independent cohorts- civilian twins and siblings, as well as U.S. Marines, for a total of 1,180 subjects. Genotyping these subjects revealed that the carriers of the minor alleles at the two loci- F12 and KLKB1 had a significant association with reduced levels of active plasma renin. Meta-analyses confirmed the association across cohorts. In vitro studies verified digestion of human recombinant pro-renin by kallikrein (KAL) to generate active renin. Subsequently, the active renin was able to digest the synthetic substrate angiotensinogen to angiotensin-I. Examination of mouse juxtaglomerular cell line and mouse kidney sections showed co-localization of KAL with renin. Expression of either REN or KLKB1 was regulated in cell line and rodent models of hypertension in response to oxidative stress, interleukin or arterial blood pressure changes. CONCLUSIONS: The functional variants of KLKB1 (rs3733402) and F12 (rs1801020) disrupted the cascade of enzymatic events, resulting in diminished formation of active renin. Using genetic, cellular and molecular approaches we found that conversion of zymogen prorenin to renin was influenced by these polymorphisms. The study suggests that the variant version of protease factor XIIa due to the amino acid substitution had reduced ability to activate prekallikrein to KAL. As a result KAL has reduced efficacy in converting prorenin to renin and this step of the pathway leading to activation of renin affords a potential therapeutic target.

The influence of chronic stress on dementia-related diagnostic change in older adults.

Increased susceptibility of the aging brain to both chronic stress and incipient dementia-related neuropathology may accelerate cognitive decline. We investigated associations between chronic stress and diagnostic change in 62 individuals (mean age, 78.7 y) participating in an Alzheimer disease research center longitudinal study. The subjects, diagnosed at baseline as cognitively normal (CN) or with mild cognitive impairment (MCI), were followed for an average of 2.5 years. Senior neurologists, blind to detailed measures of stress and cognition, assigned diagnoses annually. Logistic regression analyses assessed the accuracy with which measures of stress (event-based ratings, cortisol levels) predicted the conversion to MCI and dementia. Eleven individuals with MCI at baseline received a dementia diagnosis during follow-up. Sixteen converted from cognitively normal to MCI. Prolonged, highly stressful experiences were associated with conversion from MCI to dementia. The cortisol awakening response, with age and education, was associated with a diagnostic change to MCI. Cortisol measures were not associated with the progression from MCI to dementia, and there was no association between stressful experiences and the change to MCI. Mechanisms associated with the transition from normal cognition to MCI may differ from those associated with a diagnostic change to dementia. These findings could facilitate the identification of interventional strategies to reduce the risk of decline at different stages of susceptibility.

Genetic covariance between gamma-glutamyl transpeptidase and fatty liver risk factors: role of beta2-adrenergic receptor genetic variation in twins.

BACKGROUND & AIMS: Plasma levels of gamma-glutamyl transpeptidase (GGT) are associated with risk factors for nonalcoholic fatty liver disease (NAFLD), such as dyslipidemia, insulin resistance (IR), and hypertension. Limited data exist on whether there is genetic covariance between plasma levels of GGT and NAFLD risk factors. Variants of beta2-adrenergic receptor gene (ADRB2) have been associated with dyslipidemia, IR, and hypertension, but its effect on GGT secretion is not known. We estimated the heritability of GGT using a twin-study design and examined the genetic covariance between GGT levels, IR, hypertension, levels of low-density lipoproteins and triglycerides, and ADRB2 variants. METHODS: We studied phenotypes of 362 twins; the heritabilities of increased GGT activity and genetic covariance with NAFLD risk factors were estimated by variance-component methodology. ADRB2 genotype associations with plasma GGT activity were examined using generalized estimating equations to account for intra-twinship correlations. RESULTS: GGT activity was heritable at 49% +/- 8% of the twin cohort and had significant covariance with IR; insulin, triglyceride, and uric acid levels; and diastolic blood pressure. In generalized estimating equation models, the most common haplotype of ADRB2 was significantly associated with plasma GGT activity. Five single nucleotide polymorphisms in ADRB2 were associated with levels of GGT; ADRB2 haplotypes displayed pleiotropic effects on GGT and triglyceride levels. CONCLUSIONS: In a twin study, GGT shared genetic codetermination with traits of metabolic syndrome. The ADRB2 gene had pleiotropic effects on plasma levels of GGT and triglycerides, indicating linked pathways (eg, adrenergic) between genetic susceptibility to NAFLD and metabolic syndrome.

Baseline values of candidate urine acute kidney injury biomarkers vary by gestational age in premature infants.

Acute kidney injury (AKI) is common in premature infants and is associated with poor outcomes. Novel biomarkers can detect AKI promptly. Because premature infants are born with underdeveloped kidneys, baseline biomarker values may differ. We describe baseline values of urinary neutrophil gelatinase-associated lipocalin (NGAL), IL-18, kidney injury molecule-1 (KIM-1), osteopontin (OPN), beta-2 microglobulin (B2mG), and Cystatin-C (Cys-C). Next, we test the hypothesis that these biomarkers are inversely related to GA. Candidate markers were compared according to GA categories in 123 infants. Mixed linear regression models were performed to determine the independent association between demographics/interventions and baseline biomarker values. We found that urine NGAL, KIM-1, Cys-C, and B2mG decreased with increasing GA. With correction for urine creatinine (cr), these markers and OPN/cr decreased with increasing GA. IL-18 (with or without correction for urine creatinine) did not differ across GA categories. Controlling for other potential clinical and demographic confounders with regression analysis shows that NGAL/cr, OPN/cr, and B2mG/cr are independently associated with GA. We conclude that urine values of candidate AKI biomarkers are higher in the most premature infants. These findings should be considered when designing and analyzing biomarker studies in newborn with AKI.

Renal kallikrein excretion and epigenetics in human acute kidney injury: expression, mechanisms and consequences.

BACKGROUND: Renal kallikrein (KLK1) synthesis and urinary excretion are reportedly diminished during AKI (acute kidney injury) in animal models, and provision of kallikrein abrogates renal injury in this setting, but data in human AKI is limited. Therefore we first examined KLK1 renal excretion in human AKI, and then probed potential endocrine and epigenetic mechanisms for its alterations. METHODS: KLK1 enzymatic activity excretion was evaluated in urine from patients with established or incipient AKI, versus healthy/non-hospital as well as ICU controls. Endocrine control of KLK1 excretion was then probed by catecholamine and aldosterone measurements in established AKI versus healthy controls. To examine epigenetic control of KLK1 synthesis, we tested blood and urine DNA for changes in promoter CpG methylation of the KLK1 gene, as well as LINE-1 elements, by bisulfite sequencing. RESULTS: Patients with early/incipient AKI displayed a modest reduction of KLK1 excretion, but unexpectedly, established AKI displayed substantially elevated urine KLK1 excretion, ~11-fold higher than healthy controls, and ~3-fold greater than ICU controls. We then probed potential mechanisms of the change. Established AKI patients had lower SBP, higher heart rate, and higher epinephrine excretion than healthy controls, though aldosterone excretion was not different. Promoter KLK1 CpG methylation was higher in blood than urine DNA, while KLK1 methylation in blood DNA was significantly higher in established AKI than healthy controls, though KLK1 methylation in urine tended to be higher in AKI, directionally consistent with earlier/incipient but not later/established changes in KLK1 excretion in AKI. On multivariate ANOVA, AKI displayed coordinate changes in KLK1 excretion and promoter methylation, though directionally opposite to expectation. Control (LINE-1 repetitive element) methylation in blood and urine DNA was similar between AKI and controls. CONCLUSIONS: Unexpectedly, increased KLK1 excretion in AKI patients was found; this increase is likely to be due in part to increments in adrenergic tone during BP depression. Epigenetic changes at KLK1 may also play a role in early changes of KLK1 expression and thus AKI susceptibility or recovery.

Discovery of common human genetic variants of GTP cyclohydrolase 1 (GCH1) governing nitric oxide, autonomic activity, and cardiovascular risk.

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants' contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3'-untranslated region (3'-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3'-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension.

Chromogranin A regulates renal function by triggering Weibel-Palade body exocytosis.

Chromogranin A (CHGA), a protein released from secretory granules of chromaffin cells and sympathetic nerves, triggers endothelin-1 release from endothelial cells. CHGA polymorphisms associate with an increased risk for ESRD, but whether altered CHGA-endothelium interactions may explain this association is unknown. Here, CHGA led to the release of endothelin-1 and Weibel-Palade body exocytosis in cultured human umbilical vein endothelial cells. In addition, CHGA triggered secretion of endothelin-1 from glomerular endothelial cells and TGF-beta1 from mesangial cells cocultured with glomerular endothelial cells. In humans, plasma CHGA correlated positively with endothelin-1 and negatively with GFR. GFR was highly heritable in twin pairs, and common promoter haplotypes of CHGA predicted GFR. In patients with progressive hypertensive renal disease, a CHGA haplotype predicted rate of GFR decline. In conclusion, these data suggest that CHGA acts through the glomerular endothelium to regulate renal function.

Nocturnal levels of ghrelin and leptin and sleep in chronic insomnia.

Experimental sleep deprivation in healthy humans affects levels of ghrelin and leptin, two primary hormones involved in energy balance that regulate appetite and body weight. No study to date has examined levels of these hormones in patients with chronic insomnia. In this study, men diagnosed with primary insomnia using DSM-IV criteria (n=14) and age and body weight comparable healthy control men (n=24) underwent polysomnography. Circulating levels of ghrelin and leptin were measured at 2300h, 0200h and 0600h. As compared to controls, insomnia patients showed less total sleep time, stage 2 and REM sleep and decreased sleep efficiency and more stage 1 sleep than controls (p's<.05). Ghrelin levels across the night were significantly lower in insomnia patients (p<.0001). Leptin was not significantly different between the groups. In conclusion, decreased nocturnal ghrelin in insomnia is consistent with findings for nighttime levels in sleep deprivation studies in healthy sleepers. These findings suggest that insomnia patients have a dysregulation in energy balance that may play a role in explaining prospective weight gain in this population.

Matrix metalloproteinases: discrete elevations in essential hypertension and hypertensive end-stage renal disease.

The contribution of inflammation to hypertension and target organ damage is under investigation. The matrix metalloproteinase (MMP) enzymes are inflammatory mediators that may contribute to hypertension and its target organ consequences. Here we probe MMPs as inflammatory mediators in hypertension, by studying all three MMP classes in uncomplicated hypertension as well hypertension with profound renal damage, such as hypertensive end-stage renal disease (ESRD). We assayed plasma levels of five MMPs: one collagenase (MMP-1), two gelatinases (MMP-2, MMP-9), and two stromelysins (MMP-3, MMP-10). In hypertension, MMP-9 was elevated versus normotensive controls. Systolic blood pressure (SBP) in all three subject groups positively correlated with MMP-9. In hypertensive-ESRD, MMP-2 and MMP-10 were elevated compared to both hypertensive and normotensive subjects. Several correlations occurred across MMPs, suggesting coordinate biosynthetic control. Our results suggest discrete patterns of MMP overexpression in hypertension, with MMP-9 elevated early, and MMP-2 and MMP-10 linked to target organ damage.

Heredity of endothelin secretion: human twin studies reveal the influence of polymorphism at the chromogranin A locus, a novel determinant of endothelial function.

BACKGROUND: Endothelial dysfunction predisposes to vascular injury in association with hypertension. Endothelin (ET-1) is a potent vasoactive peptide that is synthesized and released by the vascular endothelium and is a marker of endothelial function. Chromogranin A (CHGA) regulates the storage and release of catecholamines and may have direct actions on the microvasculature. CHGA, a candidate gene for intermediate phenotypes that contribute to hypertension, shows a pattern of single nucleotide polymorphism variations that alter the expression and function of this gene both in vivo and in vitro. METHODS AND RESULTS: In a study of twins (n=238 pairs), plasma ET-1 was 58+/-5% (P<0.0001) heritable. Plasma ET-1 was both correlated and associated with chromogranin fragment levels, and the 2 were influenced by shared genetic determination (pleiotropy [rhoG]; for the CHGA precursor, rhoG=0.318+/-0.105; P=0.0032). We therefore hypothesized that variation in the CHGA gene may influence ET-1 secretion. Carriers of the CHGA promoter -988G, -462A, and -89A minor alleles showed significantly higher mean plasma ET-1 than their major allele homozygote counterparts (P=0.02, P=0.006, P=0.03, respectively). Analysis of a linkage disequilibrium block that spans these 3 single nucleotide polymorphisms showed a significant association between the GATACA haplotype and plasma ET-1 (P=0.0075). In cultured human umbilical vein endothelial cells, CHGA caused dose-dependent secretion of ET-1 over a brief (<1 hour) time course at relatively low concentrations of CHGA (10 to 100 nmol/L) with a threshold concentration (10 nmol/L) in the range found circulating in humans in vivo. CONCLUSIONS: These results suggest that common, heritable variation in expression of the human CHGA gene influences endothelial ET-1 secretion in vivo, explained by a CHGA stimulus/ET-1 secretion coupling in endothelial cells in vitro. The findings document a previously unsuspected interaction between the sympathochromaffin system and the endothelium and suggest novel genetic and cell biological approaches to the prediction, diagnosis, and mechanism of endothelial dysfunction in human disease.

Biological mediators of effect of diet and stress reduction on prostate cancer.

BACKGROUND: A 6-month pilot intervention trial was conducted to determine whether adoption of a plant-based diet, reinforced by stress reduction, could reduce the rate of prostate-specific antigen (PSA) increase, a marker of disease progression, in asymptomatic, hormonally untreated patients experiencing consistently increasing PSA levels after surgery or radiation. METHODS: A pre-post design was used to examine (1) the effect of intervention on potential mediators of disease progression, including body composition and weight-related biomarkers (sex steroid hormones and cytokines), and (2) whether changes in these variables were associated with change in rate of PSA increase. The baseline rate of PSA increase (from the time of posttreatment recurrence to the start of intervention) was ascertained from medical records. Body composition and biomarkers were assessed at baseline (prior to intervention), during the intervention (3 months), and at the end of the intervention (6 months). Changes in body composition and biomarkers were determined and compared with rates of PSA increase over the corresponding time intervals. RESULTS: There was a significant reduction in waist-to-hip ratio (P=.03) and increase in circulating sex hormone binding globulin (P=.04). The rate of PSA increase decreased from the preintervention period (PSA slope=0.059) to the period from 0 to 3 months (PSA slope=0.002, P<.01) and increased slightly, although not significantly, from 0 to 3 months to the period from 3 to 6 months (0.029, P=.43). CONCLUSIONS: Adoption of a plant-based diet and stress reduction may reduce central adiposity and improve the hormonal milieu in patients with recurrent PC. Changes in the rate of increase in PSA were in the same direction as changes in waist-to-hip ratio and opposite those of sex hormone binding globulin, raising the possibility that the effect of the intervention may have been mediated, in part, by these variables.

The effects of prolonged stress and APOE genotype on memory and cortisol in older adults.

BACKGROUND: Chronic elevations in cortisol associated with prolonged stress have been associated with memory loss, as has the apolipoprotein E gene (APOE-epsilon4) genotype. Combined effects of stress and APOE status on memory and cortisol in humans have not been studied. METHODS: A semistructured interview with standardized scoring was used to measure stress level and univariate analysis of variance to assess effects of stress and APOE-epsilon4 status on memory and salivary cortisol in 91 nondemented subjects (mean age 78.8 years). RESULTS: Low-stress subjects performed better than high-stress subjects on delayed recall of stories (p = .04), word lists (p = .02), and visual designs (p = .04). APOE-epsilon4-negative subjects obtained better scores than epsilon4-positive subjects on immediate (p = < .01) and delayed (p < .01) recall of visual designs. Significant stress by APOE-epsilon4 interaction effects on memory (p = .03) and cortisol (p < .01) resulted from consistently worse memory and higher cortisol concentrations in the high stress, epsilon4-positive group. CONCLUSIONS: These findings are consistent with a model in which prolonged exposure of older, nondemented individuals to stress in the presence of an epsilon4 allele leads to memory decline. Further studies will assess whether stress and APOE-epsilon4 interact to increase the risk of developing Alzheimer's disease.

C-reactive protein, an 'intermediate phenotype' for inflammation: human twin studies reveal heritability, association with blood pressure and the metabolic syndrome, and the influence of common polymorphism at catecholaminergic/beta-adrenergic pathway loci.

BACKGROUND: C-reactive protein (CRP) both reflects and participates in inflammation, and its circulating concentration marks cardiovascular risk. Here we sought to understand the role of heredity in determining CRP secretion. METHODS: CRP, as well as multiple facets of the metabolic syndrome, were measured in a series of 229 twins, both monozygotic (MZ) and dizygotic (DZ), to estimate trait heritability (h2). Single nucleotide polymorphism (SNP) genotyping was done at adrenergic pathway loci. Haplotypes were inferred from genotypes by likelihood methods. Association of CRP with hypertension and the metabolic syndrome was studied in a larger series of 732 individuals, including 79 with hypertension. RESULTS: MZ and DZ twin variance components indicated substantial h2 for CRP, at approximately 56 +/- 7% (P < 0.001). CRP was significantly associated (P < 0.05) with multiple features of the metabolic syndrome in twins, including body mass index (BMI), blood pressure (BP), leptin and lipids. In established hypertension, elevated CRP was associated with increased BP, BMI, insulin, HOMA (index of insulin resistance), leptin, triglycerides and norepinephrine. Twin correlations indicated pleiotropy (shared genetic determination) for CRP with BMI (P = 0.0002), leptin (P < 0.001), triglycerides (P = 0.002) and systolic blood pressure (SBP) (P = 0.042). Approximately 9800 genotypes (43 genetic variants at 17 loci) were scored within catecholaminergic pathways: biosynthetic, receptor and signal transduction. Plasma CRP concentration in twins was predicted by polymorphisms at three loci in physiological series within the catecholamine biosynthetic/beta-adrenergic pathway: TH (tyrosine hydroxylase), ADRB1 (beta1-adrenergic receptor) and ADRB2 (beta2-adrenergic receptor). In the TH promoter, common allelic variation accounted for up to approximately 6.6% of CRP inter-individual variance. At ADRB1, variation at Gly389Arg predicted approximately 2.8% of CRP, while ADRB2 promoter variants T-47C and T-20C also contributed. Particular haplotypes and diplotypes at TH and ADRB1 also predicted CRP, though typically no better than single SNPs alone. Epistasis (gene-by-gene interaction) was demonstrated for particular combinations of TH and ADRB2 alleles, consistent with their actions in a pathway in series. In an illustration of pleiotropy, not only CRP but also plasma triglycerides were predicted by polymorphisms at TH (P = 0.0053) and ADRB2 (P = 0.027). CONCLUSIONS: CRP secretion is substantially heritable in humans, demonstrating pleiotropy (shared genetic determination) with other features of the metabolic syndrome, such as BMI, triglycerides or BP. Multiple, common genetic variants in the catecholaminergic/beta-adrenergic pathway contribute to CRP, and these variants (especially at TH and ADRB2) seem to interact (epistasis) to influence the trait. The results uncover novel pathophysiological links between the adrenergic system and inflammation, and suggest new strategies to probe the role and actions of inflammation within this setting.

Plasma Levels of Advanced Glycation End Products Are Related to the Clinical Presentation and Angiographic Severity of Symptomatic Lower Extremity Peripheral Arterial Disease.

Evidence implicates a role of advanced glycation end products (AGEs) in the development of atherosclerosis. The present study examined the relationship between plasma levels of AGEs and the clinical and angiographic characteristics of patients with symptomatic peripheral arterial disease (PAD). A total of 40 consecutive patients with symptomatic lower extremity PAD undergoing invasive evaluation were enrolled. Clinical history, angiographic data, and plasma levels of total AGE (tAGE), N'-carboxymethyllysine (CML), and high-sensitivity C-reactive protein were obtained. In multivariate analyses, there were independent relationships noted between tAGE levels and the presence of critical limb ischemia (CLI) (r (2) = 0.195, p = 0.003), Rutherford stage (r (2) = 0.351, p < 0.001), and the average below the knee (BTK) score (r (2) = 0.119, p = 0.006). Presence of CLI (r (2) = 0.154, p = 0.012) and the Rutherford stage (r (2) = 0.194, p = 0.003) were associated with CML levels. We demonstrate a relationship between tAGE and the symptom profile of patients with PAD and an association between tAGE and infrapopliteal angiographic disease severity. Both tAGE and CML levels were related to the presence of CLI. These data suggest that AGE levels may reflect the severity of PAD and may be of importance in CLI.

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.

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.

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.

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.

Use of clinical markers to identify metabolic syndrome in antipsychotic-treated patients.

OBJECTIVE: Metabolic syndrome (MetS) is prevalent among antipsychotic-treated patients; however, in psychiatric clinics, scarce resources often limit the feasibility of monitoring all 5 criteria that are necessary for diagnosing MetS. As one goal of the MetS definition is to facilitate the clinical identification of insulin-resistant individuals, other biomarkers of insulin resistance have been explored. However, there are relatively few data from antipsychotic-treated patients, especially on the association between these markers and the clinical MetS diagnosis. METHOD: We analyzed data from 196 psychiatric patients over age 40 years enrolled in an ongoing study of antipsychotic-related metabolic effects that began in August 2005. In addition to anthropometric measures and MetS criteria, levels of certain metabolism-related peptides (ghrelin, adiponectin, peptide YY, leptin, and insulin) were measured. The utility of these clinical and metabolic markers to identify individuals with MetS was evaluated by constructing receiver operating characteristic curves. Optimal cutoff values were calculated for markers with the greatest area under the curve on the basis of sensitivities and specificities for MetS diagnosis. RESULTS: Ninety-nine subjects (50.5%) met MetS criteria. The receiver operating characteristic analysis found that waist circumference, triglyceride to high-density lipoprotein (TG:HDL) ratio, and body mass index had the greatest area under the curve. The waist circumference cutoff value of 40 inches, TG:HDL ratio of 2.6, and body mass index of 28 kg/m² yielded sensitivities and specificities of 73% and 80%, 74% and 78%, and 75% and 74%, respectively, for MetS diagnosis. CONCLUSIONS: Waist circumference, TG:HDL cholesterol ratio, or body mass index could be used as screens for identifying possible MetS in antipsychotic-treated patients to prompt complete investigation into all MetS criteria. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00245206.

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.