Figure 5. Reduction of atherosclerosis by antagonism of the NPY Y1 receptor. Mice treated identically to those in Figure 4 underwent carotid harvest 6 weeks after endothelial denudation, as described in Methods. A, Carotid sections from mice subjected to the indicated treatment were stained with a modified connective tissue stain (green = collagen, black = elastin, red = cytoplasm) [39]. Sections shown are representative of 3 obtained from each of 5 mice in each treatment group. Scale bar = 100 mm. B, Computerized morphometry was used to quantitate the indicated carotid artery dimension, as described in Methods; the means6S.E. of 5 independent carotid arteries from each treatment group are shown. Compared with control: p,0.03. doi:10.1371/journal.pgen.1000318.g005 would expect the A allele to demonstrate lower NPY expression. In contrast, Zhou et al recently studied NPY expression in post- mortem brain and virally transformed lymphoblastoid cells and found that the rs16147 A allele results in higher, rather than lower expression of NPY [25]. Zhou et al. also found that haplotypes reporting on the rs16147 A allele result in higher NPY plasma levels [25]. However, it is important to note that Zhou et al. collected plasma from patients under resting conditions, whereas we collected plasma from patients under arguably stressful conditions, immediately before they underwent coronary angiog- raphy. Furthermore, differences in cell and tissue types examined in these studies may underlie the discordant results obtained for NPY expression. Given previous studies showing that NPY levels are higher in cardiovascular disease [16,31] (findings that were corroborated in our study), and the strong concordance of our results showing a higher frequency of the rs16147 G allele in CAD cases, it follows that the G allele would result in increased NPY Neuropeptide Y and Early-Onset Atherosclerosis expression, as observed in the previous NPY studies [23,24] and in our study. Taken together, all of these studies nevertheless imply a functional role for rs16147. One prior linkage scan has implicated chromosome 7p in atherogenesis, showing modest linkage to carotid plaque (LOD 0.60–0.78, p = 0.03–0.05) [32]. A recently published study has also shown linkage on chromosome 7p15 (LOD 3.3) to pulse pressure, a measure of central arterial stiffness and a predictor of cardiovascular mortality [33]. None of the genomewide associa- tion studies (GWAS) of atherosclerosis have published association with variants on chromosome 7p. However, we reviewed individual SNPs through the Framingham SHARe GWAS database (dbGAP, http://www.ncbi.nlm.nih.gov/projects/gap) and found association of several flanking NPY SNPs with cardiovascular phenotypes (Text S1, p = 0.04–0.0005). These results provide further support for a role for NPY in human atherosclerosis. Although previously associated with atherosclerosis primarily in Scandinavian subjects [18–20], the NPY SNP rs16139 was not associated with CAD in our sample. Our inability to replicate association for rs16139 may have resulted from the low minor allele frequency of rs16139 in our cohorts (similar to other non- Scandinavian populations [34]), and hence the small power of our studies for rs16139. Alternatively, the relationship between rs16139 and atherosclerosis in Scandinavians, which is as yet mechanistically obscure, may not pertain to our genetically more diverse cohorts. Concomitantly, we note that rs16147 is very common, and despite a modest relative risk conferred, this variant may have a higher population attributable risk in genetically diverse cohorts. In fact, 21% of our CATHGEN subjects are homozygous for the rs16147 risk allele. This prevalence is similar in magnitude to that observed with the recently identified chromosome 9p21 CAD susceptibility variant discovered through GWAS [5]. We cannot exclude the possibility that our CATHGEN data are affected by population stratification; however we adjusted for race and CATHGEN subjects were recruited from only one site. Furthermore, race-stratified analyses showed consistent, and often more significant, association between NPY SNPs and CAD in Caucasians, though our analyses were underpowered to assess such relationships in non-Caucasians. Importantly, there is no evidence of population stratification in previous GENECARD analyses [7] and, unlike the non-familial case-control regression analyses which are sensitive to population stratification, the family- based association analyses are robust to population stratification. The allele frequencies of the NPY SNPs were remarkably similar in very-young-age-of-onset CATHGEN cases and GENECARD probands. Consequently, these independent cohorts appear to be genetically as well as clinically similar, in that they demonstrate a strong genetic component to their risk profiles. These very young individuals represent a prime target for prevention. In identifying the association between NPY variants and atherosclerosis, our results highlight the importance of pursuing a comprehensive gene-wide SNP survey. The replication of our GENECARD findings in our CATHGEN validation dataset is very consistent, not just for the NPY gene itself but also for the association of the individual associated alleles of each SNP, suggesting it is extremely unlikely that results are due to chance alone. It remains to be determined whether association between NPY and CAD is influenced by traditional CAD risk factors, particularly dyslipide- mia, and whether a particular constellation of risk factors along with NPY SNPs is related to the very early-onset CAD associations observed in both GENECARD and CATHGEN. A more PLoS Genetics | www.plosgenetics.org 7 January 2009 | Volume 5 | Issue 1 | e1000318