Neuropeptide Y Gene Polymorphisms Confer Risk of Early-Onset Atherosclerosis Svati H. Shah1,2*, Neil J. Freedman1, Lisheng Zhang 1 , David R. Crosslin2, David H. Stone3, Carol Haynes2, Jessica Johnson2, Sarah Nelson2, Liyong Wang4, Jessica J. Connelly2, Michael Muehlbauer5, Geoffrey S. Ginsburg1,6, David C. Crossman 7 , Christopher J. H. Jones8, Jeffery Vance4, Michael H. Sketch, Jr.1, Christopher B. Granger1, Christopher B. Newgard5, Simon G. Gregory2, Pascal J. Goldschmidt-Clermont4, William E. Kraus1, Elizabeth R. Hauser2 1 Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America, 2 Center for Human Genetics, Duke University Medical Center, Durham, North Carolina, United States of America, 3 Department of Surgery, Duke University, Durham, North Carolina, United States of America, 4 Miller School of Medicine, University of Miami, Miami, Florida, United States of America, 5 Sarah W. Stedman Center for Nutrition and Metabolism at Duke, Durham, North Carolina, United States of America, 6 Duke Institute for Genome Sciences and Policy, Durham, North Carolina, United States of America, 7 University of Sheffield School of Medicine, Sheffield, United Kingdom, 8 The Wales Heart Research Institute, Cardiff, United Kingdom Abstract Neuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58–2.72), family-based association of this block with CAD (p = 0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46–1.65, p = 0.01–0.05), showing stronger association in youngest cases (OR 1.84–2.20, p = 0.0004–0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79–2.06, p = 0.003–0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor–antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis. Citation: Shah SH, Freedman NJ, Zhang L, Crosslin DR, Stone DH, et al. (2009) Neuropeptide Y Gene Polymorphisms Confer Risk of Early-Onset Atherosclerosis. PLoS Genet 5(1): e1000318. doi:10.1371/journal.pgen.1000318 Editor: Gregory A. Cox, The Jackson Laboratory, United States of America Received July 7, 2008; Accepted November 25, 2008; Published January 2, 2009 Copyright: 2009 Shah et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the American Heart Association FTF Award (SHS); NIH grants HL73042 (PJGC, WEK), HL073389 (ERH), and HL3036648 (NJF); the Joseph C. Greenfield Scholars Award (SHS), and a MEDUSA educational grant to Duke University from Medtronic, Inc. (SHS). Competing Interests: The authors have declared that no competing interests exist. * E-mail: svati.shah@duke.edu Introduction The prevalence of early-onset cardiovascular disease in Americans (under 40 years of age) is approximately 10–15% [1] and the hereditary nature of coronary artery disease (CAD) is well- established [2]. The relative risk of developing CAD in a first degree relative is 3.8 to 12.1, with higher risk correlating with earlier age-of-onset [3]. Recent successes suggest that CAD genes can be identified through comprehensive genetic and functional studies [4–6]. However, the genetic architecture of CAD remains complex and poorly understood. To identify genetic risk factors in early-onset CAD, we implemented a strategy combining the strengths of family-based studies with validation by case-control association, in conjunction with careful consideration of phenotype and functional data. In our own GENECARD linkage study of early-onset CAD, we have found five genomic regions of linkage with multipoint linkage odds (LOD) scores .1.0 [7]. The neuropeptide Y gene (NPY) is located adjacent to the peak microsatellite marker in the 7p14 peak. Because of its proximity to the peak marker, and because NPY has been implicated in disorders of vascular smooth muscle cell proliferation [8,9], we sought to examine NPY further as a candidate gene for early-onset CAD. NPY is the most abundant peptide in the heart and brain, and is produced by sympathetic neurons, endothelial cells [10], and platelets [11]. NPY has diverse functions including roles in sympathetic nerve stimulation through co-release with norepi- nephrine; immune function [12]; regulation of food consumption [9]; and modulation of heart rate, vasoconstriction, coronary blood flow and ventricular function [13]. These cardiovascular functions are primarily mediated through the NPY1 receptor [12,14,15]. In injured rat carotid arteries, non-atherosclerotic neointimal hyperplasia is aggravated by local delivery of NPY, and ameliorated by treatment with NPY1 receptor antagonist [8,9]. In PLoS Genetics | www.plosgenetics.org 1 January 2009 | Volume 5 | Issue 1 | e1000318