NPR-C gene polymorphism is associated with increased susceptibility to coronary artery disease in Chinese Han population: a multicenter study

To find a new locus that confers significant susceptibility to CAD in Chinese Han population, a genome-wide association study in 200 “extreme individuals” from a Shandong cohort and a pathway-based candidate gene study from a Shanghai cohort (293 CAD/293 controls) were simultaneously performed. Amongst them, 13 SNPs associated with CAD were selected to conduct validation and replication studies in additional 3363 CAD patients and 3148 controls. A novel locus rs700926 in natriuretic peptide receptor C (NPR-C) was identified in Shandong and Hubei cohorts. Then rs700926 and other nine tag SNPs were genotyped in four geographically different populations (Shandong, Shaanxi, Hubei and Sichuan cohorts), and 6 SNPs (rs700926, rs1833529, rs2270915, rs17541471, rs3792758 and rs696831) showed stronger association with CAD, regardless of single or combined analysis. We further genotyped rs2270915 and 10 additional tag SNPs in a central China cohort and identified rs12697273 and rs10066436 as the loci associated with CAD. All these positive associations remained significant after adjustment for traditional risk factors of CAD. NPR-C gene SNPs significantly contribute to CAD susceptibility in the Chinese Han population.

3 mass index (BMI) was calculated as body weight (kg)/height (m) 2 . Hypertension was diagnosed if the systolic blood pressure ≧ 140mmHg and/or diastolic blood pressure ≧ 90 mmHg or there was a history of hypertension, or the subject was taking antihypertensive drugs. Diabetes mellitus was defined as ongoing therapy for diabetes or a fasting plasma glucose level ≥7.0 mM/l. Individuals with either HDL cholesterol (HDL-C) <40 mg/dl (1.03mM/L) or LDL cholesterol (LDL-C) >160 mg/dl (4.14 mM/L) or triglycerides >200 mg/dl (2.26mM/L) were considered dyslipidemic according to Adult Treatment Panel III (ATP III) Guidelines.
All patients and control subjects underwent selective coronary angiography or computed tomography angiography (CTA) and subjects with >50% coronary stenosis in at least one main vessel identified by coronary angiography or CTA and those who had myocardial infarction and/or underwent percutaneous coronary intervention or coronary artery bypass graft, were classified as CAD cases. The diagnosis of myocardial infarction was in accordance with the international guideline published by the Joint ESC/ACCF/AHA/WHF Task Force for the Redefinition of Myocardial Infarction. 2 Subjects with coronary arterial spasm or myocardial bridge identified by coronary angiography were excluded. The control subjects were evaluated by history, physical examinations and coronary angiography or CTA. Only subjects without any history of cardiovascular diseases and without angiographically visible coronary stenosis were included as controls. Subjects with congenital heart disease, cardiomyopathy, valvular heart disease, and renal or hepatic disease were also excluded. 4 The study protocol conformed to the principles of the Declaration of Helsinki and was approved by the local Ethics Committee in each hospital involving in this project. A written informed consent was given by all participants before enrollment. DNA Extraction. Venous blood was drawn from all subjects after an overnight fast.
Blood, serum, and plasma were separated immediately and stored at -70°C. Genomic DNA was extracted from EDTA-anticoagulated peripheral whole blood using the To determine whether common variants of the NPR-C gene might be associated with CAD, the Shandong research group firstly genotyped nine tag SNPs of NPR-C (rs1833529, rs2270915, rs17541471, rs3792758, rs696831, rs7715279, rs6450922, rs10941022 and rs976576) selected from the HapMap database. The Shanghai research group then genotyped rs2270915 and additional ten tag SNPs (rs9716700, rs11750438, rs6889608, rs10036648, rs12697273, rs10066436, rs3828586, rs10061804, rs2062708 and rs7730564). Moreover, the linkage disequilibrium (LD) of these tag SNPs with other SNPs were assessed respectively using the data from HapMap by D' and r 2 , and the results showed that the common variations could be tagged almost by these 20 chosen tag SNPs with a D' > 0.9 and an r 2 > 0.7 as standard (Supplementary Figure S.4). Thus, these tag SNPs had relatively strong representativeness for NPR-C SNPs.
In the candidate gene association study and at the validation and replication stages, genotyping was carried out with TaqMan technology (Applied Biosystems, Foster 7 City, CA) as described previously. 5  where ΔCt =Ct Target gene -Ct GAPDH .
Because rs700926 is located near intron 1 of NPR-C, we hypothesized that it may affect the expression levels of the NPR-C mRNA. Thus, we randomly selected 5 individuals with a minor genotype of GG, 27 individuals with genotype GT and 63 individuals with genotype TT of rs700926 for RT-PCR analysis. To validate the finding of a quantitative expression trait locus from the initial analysis, we performed a replication study in an independent population of 380 randomly selected individuals from the replication populations (northern and southern Chinese) as described above.
Linkage Disequilibrium (LD) and Statistical Genetics. The statistical analysis methodology of the GWAS was described previously. 6     For Table S2, please see the attached Excel file