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 norepinephrine; 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 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. 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. 10.1371/journal.pgen.1000318.g001 Figure 1 Ordered subset analysis (OSA) using age-of-onset in GENECARD, chromosome 7. We constructed a CAD linkage map using the GENECARD microsatellite genome screen on chromosome 7. Results are depicted for all 420 families (grey), and for the subset generated by OSA (black), demonstrating increased LOD at the peak microsatellite from 1.04 (420 families) to 4.22 (97 lowest age-of-onset families, mean 37.8 years), p = 0.004 for increase in LOD. 10.1371/journal.pgen.1000318.t001 Table 1 Baseline characteristics. Variable GENECARD Affected (N = 946) GENECARD Probands (N = 221) CATHGEN Cases (N = 556) CATHGEN Very-Young Cases (N = 74) CATHGEN Controls (N = 256) Age 51.5 (7.1) 50.8 (6.6) 51.1 (7.9) 43.4 (8.1) 69.3 (6.5) Age-of-onset 43.7 (5.8) 43.8 (5.7) 46.2 (6.4) 34.7 (3.3) N/A Sex (% male) 68.0% 68.0% 79.7% 83.8% 38.7% Race % Caucasian 91.9% 83.1% 71.9% 68.9% 77.4% % African- American 2.3% 7.4% 20.5% 21.9% 16.7% Dyslipidemia 82.4% 83.7% 72.5% 81.2% 41.8% Lipids TC 205.6 (57.4) 216.3 (59.0) 194.2 (55.8) 201.1 (71.6) 192.5 (50.3) TG 221.6 (166.5) 224.7 (149.5) 225.7 (263.2) 281.2 (391.8) 163.4 (140.2) HDL 38.2 (10.7) 35.5 (9.6) 39.8 (11.8) 38.5 (9.6) 51.7 (17.7) LDL 117.9 (45.9) 131.6 (53.0) 113.5 (43.0) 115.2 (46.3) 106.3 (34.7) Hypertension 55.2% 60.4% 68.5% 66.2% 65.2% Diabetes 20.9% 25.6% 31.7% 27.0% 16.4% BMI (SD) 29.7 (5.7) 30.6 (6.3) 30.8 (6.5) 31.9 (6.5) 28.8 (7.4) Smoking 33.2% 26.5% 68.7% 75.7% 38.3% History of MI 63.1% 61.7% 52.2% 52.7% 0% Family history CAD 100% 100% 54.1% 70.3% 22.3% * Continuous variables presented as mean (SD). NPY Variants Are Associated with Early-Onset CAD Twenty-four SNPs were genotyped and were in Hardy-Weinberg equilibrium. Rs5571 was monomorphic and not analyzed further. Consistent with microsatellite results, nine NPY SNPs had LOD>1.0 in GENECARD, with higher LODs for several SNPs in the subset of 97 very-young-age-of-onset OSA families. Six of these linked SNPs also showed family-based association with CAD by PDT in GENECARD (Table 2). These SNPs are in varying degrees of LD (Figure 2). Association with CAD was validated in the non-familial dataset CATHGEN for five SNPs within the 6-SNP block; the sixth SNP (rs16147) demonstrated borderline significance (Table 3). CATHGEN results also validated our GENECARD findings for age-of-onset effects, with stronger association in the youngest age-of-onset CAD cases ( 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 µm. All images are representative of ≥4 independent specimens stained with each modality. 10.1371/journal.pgen.1000318.g005 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 µm. B, Computerized morphometry was used to quantitate the indicated carotid artery dimension, as described in Methods; the means±S.E. of 5 independent carotid arteries from each treatment group are shown. Compared with control: p 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 laboratories 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 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 discrimination 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 −80°C, 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 (apoe −/−) C57BL/6 mice were used, at the age of 10±2 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 µl 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 µmol/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-µm 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 IgG1κ anti-mouse Mac3 or negative control FITC-conjugated rat IgG1κ (Pharmingen, Inc.)); smooth muscle cells (SMCs) (Cy3-conjugated 1A4 IgG targeting SMC α-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 immunofluorescence 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 [40], and has been successfully used for gene mapping in complex diseases [41]–[44]. Similar methods were used to identify the BRCA1 breast cancer susceptibility gene, where evidence for linkage was only present in earlier-onset families [22],[45]. Subsets of families with earlier ages-of-onset may help evaluate genetic heterogeneity and define subgroups with a stronger genetic effect. A priori specification of the age-of-onset threshold is unnecessary, as OSA uses maximal linkage evidence to define subsets. OSA was conducted using nonparametric multipoint family-specific LODs from microsatellite genotypes from the original screen as input [7], with age-of-onset as a covariate. A permutation procedure provides empirical p-values for the significance of the increase in the maximum-subset-LOD from the overall LOD. To assess SNP linkage, two-point LODs were calculated using Merlin [46]. The Pedigree Disequilibrium Test (PDT) and Genotype-PDT were used for family-based association. PDT, an extension of the transmission-disequilibrium-test (TDT), allows incorporation of extended pedigrees and is valid even with population substructure [47]. Power calculations (QUANTO:http://hydra.usc.edu/) showed power ≥0.80 to detect effect sizes of ≥2.45 for the lowest allele frequency SNP (0.02, rs16139), and effect sizes ≥1.35 for the highest allele frequency SNP (0.49, rs16147). In CATHGEN, association was assessed using logistic regression models adjusting for race and sex; and for race, sex, hypertension, diabetes, dyslipidemia, smoking and body-mass-index (BMI) (multivariable model). Measured genotype analysis using generalized linear models was performed to compare differences in means of quantitative traits (NPY levels, age-of-onset) by NPY genotype. Baseline differences were assessed using a chi-square or t-test. The Graphical Overview of Linkage Disequilibrium (GOLD) program [48] was used to assess LD. Haplotype analysis used HaploStats 1.1.0 (Mayo Clinic, Rochester, MN). We report p-values uncorrected for multiple comparisons, but also present results in the context of correction for LD between SNPs [35]. The extent of atherosclerosis was compared between control and NPY1 receptor antagonist groups in mice with one-way ANOVA and Tukey's post-hoc test for multiple comparisons. SAS 9.1 (SAS Institute, Cary, NC) was used for statistical analysis. Supporting Information Table S1 Race-stratified analyses: association of NPY SNPs with early-onset CAD in CATHGEN Caucasians. (0.04 MB DOC) Click here for additional data file. Table S2 Minor allele frequencies and replication of associated allele for six NPY SNPs associated with CAD. (0.03 MB DOC) Click here for additional data file. Table S3 CAD risk factors in multivariable regression model for rs16120, CATHGEN Cases vs. Controls. (0.03 MB DOC) Click here for additional data file. Text S1 Generalizability of NPY Genetic Variant Association with Cardiovascular Phenotypes: Results from Framingham SHARe database. (0.03 MB DOC) Click here for additional data file.
