Objectives The purpose of this study is to understand whether naturally occurring genetic variation in the promoter of chromogranin B (CHGB), a major constituent of catecholamine storage vesicles, is functional and confers risk for cardiovascular disease. Background CHGB plays a necessary (catalytic) role in catecholamine storage vesicle biogenesis. Previously, we found that genetic variation at CHGB influenced autonomic function, with association maximal toward the 5' region. Methods Here we explored transcriptional mechanisms of such effects, characterizing 2 common variants in the proximal promoter, A-296C and A-261T, using transfection/cotransfection, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP). We then tested the effects of promoter variation on cardiovascular traits. Results The A-296C disrupted a c-FOS motif, exhibiting differential mobility shifting to chromaffin cell nuclear proteins during EMSA, binding of endogenous c-FOS on ChIP, and differential response to exogenous c-FOS. The A-261T disrupted motifs for SRY and YY1, with similar consequences for EMSA, endogenous factor binding, and responses to exogenous factors. The 2-SNP CHGB promoter haplotypes had a profound (p = 3.16E-20) effect on blood pressure (BP) in the European ancestry population, with a rank order of CT <

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 del-880 Delta (2-bp TG/-, Ins/Del, rs3037354) minor/Delta 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 del-880 Delta 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 -880 Delta to increase NPY expression were directionally coordinate in vivo (on human traits) and in cells (transfected NPY promoter/luciferase reporter activity). Promoter -880 Delta 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 -880 Delta 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 del-880 Delta, 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. (J Am Coll Cardiol 2012;60:1678-89) (c) 2012 by the American College of Cardiology Foundation

Objectives We asked whether naturally occurring genetic variation at the human NPY1R locus alters autonomic traits that might predispose individuals to cardiovascular disease. Background Neuropeptide Y (NPY) interacts with the Y-1 receptor, NPY1R, to control adrenergic activity and blood pressure (BP). Methods We searched for polymorphism at NPY1R by systematic resequencing in ethnically diverse people. There were 376 twins/siblings who were evaluated for heritable autonomic traits: baroreflex function and pressor response to environmental stress. Results The common NPY1R variant A+1050G in the 3'-untranslated region (3'-UTR) predicted baroreceptor slope (p = 0.014-0.047) and BP change to cold stress (p = 0.0091-0.016), with minor allele homozygotes displaying blunted slope and exaggerated pressor response. In 936 individuals with the most extreme BPs in the population, not only 3'-UTR A + 1050G (p = 1.2 x 10(-4)) but also promoter A-585T (p = 0.001) affected both systolic BP and diastolic BP, in interactive fashion (p = 0.007), with combined homozygotes showing the highest diastolic BP (> 20 mm Hg). The 3'-UTR variant + 1050G decreased expression of a transfected luciferase reporter/NPY1R 3'-UTR plasmid; promoter variant A-585 also decreased expression of an NPY1R promoter/luciferase reporter. Thus, alleles that increased BP in vivo (3'-UTR + 1050G, promoter A-585) also decreased NPY1R expression in cella. Computational alignment showed that A + 1050G disrupted a microRNA motif. Conclusions Our results indicate that naturally occurring genetic variation at the NPY1R locus has implications for heritable autonomic control of the circulation, and ultimately, for systemic hypertension. The findings suggest novel pathophysiological links between the NPY1R locus, autonomic activity, and blood pressure, and suggest new strategies to approach the mechanism, diagnosis, and treatment of systemic hypertension. (J Am Coll Cardiol 2009; 54: 944-54) (C) 2009 by the American College of Cardiology Foundation