complete understanding of the relationship between NPY, CAD risk factors, and CAD will require additional studies. In our study, the correlation between SNPs in the form of LD makes a Bonferroni correction for multiple comparisons too conservative. Using a method that corrects for LD [35] resulted in 11 minimally redundant SNPs (corrected p,0.0047). Thus, one SNP for our CATHGEN very-young-age-of-onset group (rs16119, p = 0.0004) and most SNPs for the GENECARD proband comparison group would remain significant. More importantly, we have used additional statistical and functional studies to demonstrate that association of NPY SNPs with CAD does not result from chance alone: the convergence of evidence from linkage and association, as well as replication of the same SNP/ CAD association in independent cohorts. The possibility that NPY contributes to atherosclerosis is supported by the correlation of plasma NPY levels with NPY SNP risk alleles, and the inhibition of murine atherosclerosis with an antagonist of the NPY1 receptor, which mediates most cardiovascular effects of NPY [8]. In summary, NPY is a strong candidate gene for early-onset CAD. NPY SNPs may help refine CAD risk estimates and target therapies for young members of families with CAD. Methods Study Sample and Phenotype Description The GENECARD study enrolled 920 families with early-onset CAD (age-of-onset before 51 years for men, 56 years for women) to perform affected-sibling-pair linkage [7]. GENECARD families were recruited based on the presence of at least two siblings with early-onset CAD defined by: stress testing with ischemia; myocardial infarction (MI); coronary revascularization; or angi- ography with $50% stenosis in one major vessel. Unaffected family members had no clinical evidence of CAD and age .55 years for men (.60 years for women). The 420 families from the initial linkage scan [7] are included in this report, including a limited number of unaffected family members (N = 37). An independent non-familial validation cohort was selected from the CATHGEN biorepository, consisting of subjects recruited sequentially through the cardiac catheterization labora- tories at Duke University Medical Center (Durham, NC). Fasting whole blood and plasma samples were obtained from the femoral artery during cardiac catheterization and frozen prior to use. CAD cases were defined as CAD-index$32 (at least one vessel with $95% stenosis) with age-of-onset ,56 years. CADi is a numerical summary of angiographic data directly related to outcome [36]. Age-of-onset was defined as age at first MI, coronary revascular- ization, or catheterization meeting CADi threshold. Controls were defined as age-at-catheterization .60 years; CAD-index#23, no history of cardiovascular disease, and no clinically significant CAD. Dyslipidemia was defined as a previous diagnosis and/or treatment of hypercholesterolemia (yes/no), confirmed by review of medical records. We also constructed a third case-control group comprising GENECARD probands included in the original linkage scan from United States sites (N = 221) and CATHGEN controls. Institutional Review Boards approved all study protocols. Informed consent was obtained. Genotyping and Plasma NPY Levels Tagged NPY SNPs were identified using the SNPSelector program, which employs a linkage disequilibrium (LD) tagging algorithm that prioritizes functional SNPs [37], and were then genotyped using either Taqman or Illumina BeadArray (www. illumina.com). The 7900HT Taqman SNP genotyping system incorporates a standard PCR-based, dual fluor, allelic discrimina- Neuropeptide Y and Early-Onset Atherosclerosis tion assay with a dual laser scanner. Assays were purchased through Applied Biosystems (www.appliedbiosystems.com). QC samples, composed of 12 reference controls, were included in each quadrant of the plate. Illumina BeadStation genotyping was performed using the 500G system. All SNPs examined were successfully genotyped for $95% of the individuals in the study. Error rate estimates for SNPs meeting QC benchmarks were ,0.2%. Plasma NPY levels were measured by an NPY-specific radioimmunoassay (Alpco Diagnostics, Salem NH) on 220 CATHGEN subjects, randomly selected (regardless of case/ control status) from all subjects included in the genotyping studies. NPY levels were measured in fasting unextracted plasma collected at time of cardiac catheterization (prior to administration of supplementary anticoagulants if given), which was subsequently frozen and stored at 280uC, and analyzed in a single RIA run. Cross-reaction of the NPY RIA assay as reported by the company is: human NPY 100%; human PYY ,2.0%; human pancreatic polypeptide (PP) ,1.0%; NPY 1–21 ,0.1%; NPY 20–36 ,0.4%. The assay was characterized as a mean recovery of 82% (range 75–88%) for NPY-spiked plasma. Precision was characterized by an intra-assay CV of 2.6–3.9% in our samples, which were run in one batch. NPY levels were approximately normally distributed. Mouse Atherosclerosis Studies All animal experiments complied with institutional guidelines. Gender-matched apolipoprotein E-deficient (apoe2/2) C57BL/6 mice were used, at the age of 1062 weeks. To accelerate atherosclerosis, we performed endothelial denudation of the left common carotid artery with a 0.36-mm flexible angioplasty guidewire (Johnson and Johnson), inserted via the external carotid artery as described [28,38]. After ligating the external carotid artery, we encased the common carotid in 150 ml of 22.5% (w/v) Pluronic gel, containing either 1% (v/v) water that lacked (control) or contained the NPY1 receptor antagonist BIBP 3226 [27] (21 mmol/ L, [final]; Sigma-RBI, Inc.). The skin was closed after the Pluronic gelled (virtually instantaneously). Postoperatively, mice were fed a Western diet (Harlan Teklad #88137) ad libitum for two-six weeks (until arterial harvest), and there were no differences in weight between mice treated with vehicle or BIBP 3226. BIBP 3226 does not affect blood pressure in rodents [26]. As we previously described [39], arteries were perfusion/fixed and embedded in paraffin, or embedded in OCT and frozen. Five-mm sections were stained with a modified connective tissue stain (paraffin sections), Sudan IV (frozen sections, for cholesteryl ester), or immunostained (frozen sections) as previously described [38,39]: for macrophages (FITC- conjugated rat IgG1k anti-mouse Mac3 or negative control FITC- conjugated rat IgG1k (Pharmingen, Inc.)); smooth muscle cells (SMCs) (Cy3-conjugated 1A4 IgG targeting SMC a-actin (Sigma)); for apoptotic cells (monoclonal rabbit IgG targeting cleaved (activated) caspase-3 (Cell Signaling, Inc.)); or for proliferating cell nuclear antigen (Santa Cruz Biotechnology, Inc). All sections were digitally photographed with fixed light settings for all immunoflu- orescence specimens within a single staining batch, to facilitate quantitation [39]. Atherosclerosis, cellular (or protein antigen) prevalence, and PCNA-positive nuclear prevalence [38] were quantified with Scion Image (www.scioncorp.com) or by manual counting by an observer blinded to specimen identity [39], from three cross sections obtained from the distal, middle, and proximal portions of each specimen, as described [39]. Data from these locations were averaged for each carotid specimen. Statistical Analysis Ordered subset analysis (OSA) examines the impact of covariates by defining homogeneous subsets of families for linkage PLoS Genetics | www.plosgenetics.org 8 January 2009 | Volume 5 | Issue 1 | e1000318