Table 4. Association of NPY SNPs with early-onset CAD, GENECARD probands compared with CATHGEN controls, adjusted for race and sex. SNP RS16147 RS9785023 RS5574 RS16474 RS16120 RS16119 GENECARD Probands Genotype OR (95% CI) 1.55 (1.15–2.09) 1.54 (1.15–2.06) 1.57 (1.17–2.11) 1.63 (1.21–2.19) 1.58 (1.18–2.12) 1.56 (1.15–2.11) 0.004 0.004 0.003 0.001 0.002 0.004 p-value OR (95%CI) p-value Allele 1.79 (1.12–2.88) 1.88 (1.18–3.00) 1.89 (1.21–2.96) 2.06 (1.28–3.31) 2.02 (1.25–3.24) 1.98 (1.21–3.24) 0.016 0.008 0.005 0.003 0.004 0.007 OR: Odds Ratio; 95% CI: 95% Confidence Interval. doi:10.1371/journal.pgen.1000318.t004 allele leads to increased plasma NPY levels, which, acting on NPY1 (and perhaps other) receptors, promotes arterial smooth muscle cell proliferation [30], thereby promoting atherogenesis. To our knowledge, ours is the first study to take a gene-wide approach to NPY in cardiovascular disease. Furthermore, the NPY variant rs16147 has never been reported for any cardiovascular phenotypes. Because rs16147 is not in LD with upstream SNPs, it appears that the LD block tagged by rs16147 does not extend beyond the gene (www.hapmap.org). This inference is supported by our own data: the upstream rs7800861 is not in LD with rs16147 or other key SNPs. The allele-specific effects of the NPY promoter SNP rs16147 (A/ G) on NPY expression have been documented in three previous Neuropeptide Y and Early-Onset Atherosclerosis Figure 3. Rs16147 is associated with higher plasma NPY levels. NPY levels were measured in fasting plasma (N = 220). Box-and-whisker plots are presented displaying mean (+), median, and 25–75th interquartile range (shaded boxes), according to number of risk alleles at rs16147. *P = 0.04 for comparison of 1 or 2 vs. 0 risk alleles. doi:10.1371/journal.pgen.1000318.g003 studies [23–25]. In accord with our work, two of these studies have observed that the rs16147 G allele (reverse strand C allele) increases NPY expression [23,24]. Furthermore, a putative SP1 transcription factor binding site within the rs16147 stretch of NPY sequence is lost with the rs16147 A allele [24]; consequently, we Figure 4. Mouse carotid atherogenesis: cellular effects of a NPY Y1 receptor antagonist. Immediately after wire-mediated endothelial denudation, carotid arteries of apoe2/2 mice were encased in Pluronic gel lacking (‘‘None,’’ ‘‘control’’) or containing the NPY1 receptor-selective antagonist BIBP 3226 (‘‘BIBP’’). Carotid arteries were harvested 6 wk (A–C) or 2 wk (D–G) postoperatively, and processed as described in Methods. A, B, Typical atherosclerosis in a representative control-treated carotid artery. Frozen sections were stained for DNA and (immunofluorescently) with IgG specific for SMC a-actin and either the macrophage marker Mac3 (A), or an isotype negative control rat IgG (B). Green autofluorescence of the elastic laminae can be seen in both A and B. Alternatively, frozen sections were stained for cholesteryl ester with Sudan IV and hematoxylin counterstain (C). Scale bar = 100 mm. D, E, Carotid sections were stained with IgG for cleaved caspase-3 (apoptosis marker) and counterstained for DNA; specimens stained with non-immune rabbit IgG yielded no red immunofluorescence (not shown). Scale bar = 50 mm. F, G, Carotid sections were stained with IgG for proliferating cell nuclear antigen (PCNA), counterstained for DNA, and oriented with the lumen side upward. The closed arrow designates a single PCNA-positive nucleus in the tunica media, representative of .20 such nuclei in the field shown; specimens stained with non-immune rabbit IgG (not shown) yielded only elastic lamina autofluorescence, like that observed in panels F and G. IEL, internal elastic lamina, indicated by the open arrows. Scale bar = 50 mm. All images are representative of $4 independent specimens stained with each modality. doi:10.1371/journal.pgen.1000318.g004 PLoS Genetics | www.plosgenetics.org 6 January 2009 | Volume 5 | Issue 1 | e1000318