Author Summary Early-onset coronary artery disease (CAD) has a very strong genetic component as evidenced by the heritable nature of this disease. However, little is known about the actual genes underlying disease risk. Neuropeptide Y (NPY) is an abundant protein in humans that has been implicated in cardiovascular disease pathophysiology, but comprehen- sive evaluation of the gene coding for this protein has never been pursued in cardiovascular disease. Therefore, using gene-wide evaluation of variants within the NPY gene in a family-based as well as a non-familial study, we have shown that a cluster of six related NPY genetic variants is associated with early-onset CAD risk. We then show that one of these variants, which resides within the promoter region of this gene, is associated with higher NPY levels. Finally, to further support the functional role of this gene in CAD, we find that antagonism of the primary receptor of this gene results in marked attenuation of atherosclerosis in a mouse model. In conclusion, these findings demonstrate the role of the NPY gene in cardiovascular disease risk and add important additional information about the genetic architecture of this complex disease. humans, NPY levels predict cardiovascular complications in end- stage renal disease [16], and NPY is implicated in congestive heart failure [17]. An NPY variant rare in most populations has been associated in Scandinavian populations with hyperlipidemia and carotid atherosclerosis [18,19], CAD in type 1 diabetics [20], and MI in hypertensive patients [21]; however, the effects of this variant on NPY expression remain obscure. Table 1. Baseline characteristics. Neuropeptide Y and Early-Onset Atherosclerosis To date there have been no systematic studies of the role of the NPY gene, or of the functional consequences of genetic variation at this locus, in cardiovascular disease pathogenesis. In response to the results of the genome-wide linkage analyses, the phenotypic correlations, and the strong but limited prior published work, we proposed to test the hypothesis that NPY variants affect atherosclerosis through effects on NPY plasma levels. We pursued a comprehensive gene-wide approach to correlating NPY variants with CAD and plasma NPY levels in humans, and tested the effects of NPY1 receptor blockade on atherosclerosis in mice. Results Table 1 presents baseline characteristics of the three datasets: GENECARD (N = 946 affected, 37 unaffected individuals); CATHGEN (N = 556 cases, 256 controls); and GENECARD probands versus CATHGEN controls (N = 221 cases, 256 controls). Despite GENECARD families being selected on early age-of-onset, genetic heterogeneity manifest as differences in age- of-onset could still be present, as observed in the discovery of the BRCA1 breast cancer gene [22]. Hence, we performed ordered subset analysis (OSA) to understand the effect of age-of-onset on linkage to CAD. We found increased linkage on the chromosome 7p14 peak in a subset of 97 families with the youngest age-of-onset (overall LOD = 1.04; subset LOD = 4.22; p = 0.004 for increase, Figure 1). The mean age-of-onset in these families was 37.8 years, and they had significantly higher mean total- and LDL-cholesterol and were more often male, compared with affected members of the remaining 323 families. No other genomic regions showed a correlation between linkage and age-of-onset. The NPY gene resides within this linkage peak and is a strong biological candidate. As a result we aimed to evaluate NPY polymorphisms, NPY levels, and age-of-onset of CAD, along with evaluating the role of NPY in a mouse model system. % Caucasian % African- American Variable Age Age-of-onset Sex (% male) Race Dyslipidemia Lipids TC TG HDL LDL Hypertension Diabetes BMI (SD) Smoking History of MI Family history CAD *Continuous variables presented as mean (SD). doi:10.1371/journal.pgen.1000318.t001 51.5 (7.1) 43.7 (5.8) 68.0% 91.9% 2.3% 82.4% 205.6 (57.4) 221.6 (166.5) 38.2 (10.7) 117.9 (45.9) 29.7 (5.7) 55.2% 20.9% 33.2% 63.1% 100% GENECARD Affected (N = 946) GENECARD Probands CATHGEN Cases (N = 221) (N = 556) CATHGEN Very-Young CATHGEN Controls Cases (N = 74) (N = 256) 50.8 (6.6) 43.8 (5.7) 68.0% 83.1% 7.4% 83.7% 216.3 (59.0) 224.7 (149.5) 35.5 (9.6) 131.6 (53.0) 60.4% 25.6% 30.6 (6.3) 26.5% 61.7% 100% 51.1 (7.9) 46.2 (6.4) 79.7% 71.9% 20.5% 72.5% 194.2 (55.8) 225.7 (263.2) 39.8 (11.8) 113.5 (43.0) 30.8 (6.5) 68.5% 31.7% 68.7% 52.2% 54.1% 43.4 (8.1) 34.7 (3.3) 83.8% 68.9% 21.9% 81.2% 201.1 (71.6) 281.2 (391.8) 38.5 (9.6) 115.2 (46.3) 31.9 (6.5) 66.2% 27.0% 75.7% 52.7% 70.3% 69.3 (6.5) 38.7% 77.4% 16.7% 41.8% 192.5 (50.3) 163.4 (140.2) 51.7 (17.7) 106.3 (34.7) 28.8 (7.4) 65.2% 16.4% 38.3% 0% 22.3% PLoS Genetics | www.plosgenetics.org 2 January 2009 | Volume 5 | Issue 1 | e1000318