| Background and purposesArtery calcification is a dysregulation of matrix mineral metabolism involved in atherosclerotic lesions and is generally regarded as a quantitative index for the presence and progress of atherosclerosis. The most frequently involved artery segments of atherosclerotic calcification include aortic, coronary, carotid and femoral arteries. Concomitant calcifications in different vessel beds are common due to shared risk factors (hypertension, diabetic mellitus, dyslipidemia and smoking) and the systematical distribution of atherosclerosis. However, the severity of atherosclerotic calcifications usually varies across vessel beds, and the calcifications often initiate sequentially, although sometimes overlap, in different arteries. These observed phenomena indicate similar pathogenesis of, but dissimilar predisposition to atherosclerotic calcification in different vascular beds. In recent years, genetic factors are increasingly regarded as a pathological basis for artery calcification. Genome-wide association studies (GWASs) have identified a number of single-nucleotide polymorphisms (SNPs) responsible for coronary artery calcification (CAC). The present GWASs and other association studies on calcification mainly focused on coronary artery, and rarely involved in other vascular beds. Considering the universality of genetic effects as well as the varied structures of different arteries, we hypothesis that the same SNPs related to CAC may also influence calcifications in other arteries, but to different extends. To test this hypothesis, we evaluated the impacts of SNPs associated with CAC on calcifications in carotid artery and aortic arch in a cohort of Chinese patients with ischemic stroke.Materials and methodsConsecutive patients with acute ischemic stroke and aged 18 years or older were enrolled from Nanjing Stroke Registry Program (NSRP) during December 2009 and June 2013. All eligible patients underwent a neck computed tomography angiography (CTA) within 7 days after stroke onset. Exclusion criteria included:concurrent malignant neoplasm, severe liver or kidney diseases, parathyroid gland disease, or calcium-phosphorus metabolism disorders. Considering that implanted stent may influence the accuracy of calcification assessment, patients with history of carotid artery or aortic arch stenting were also excluded. Finally, 878 patients were enrolled. CTA was performed using a dual-source 64 slice CT system (Siemens, Forchheim, Germany) to quantify carotid artery calcification (CarAC) and aortic arch calcification (AorAC). Calcification scores in aortic arch and carotid artery were measured with commercially available software, Syngo Calcium Scoring (Siemens, Forchheim, Germany) according to the Agatston method. MEDLINE database was searched for all GWAS studies on CAC-related SNPs published before December 31, 2013.Four SNPs from 2 GWA studies were identified based on the P values less than the significant threshold of 5 × 10-8 and a minor allele frequency (MAF)> 0.05 for Chinese Han population indexed in Hapmap Data Phase III (http://www.hapmap.org). Two of the four CAC-related SNPs, rs1333049 and rs1537370 are near the two cyclin-dependent kinase inhibitors, CDKN2A and CDKN2B. The other two SNPs, rs2026458 and rs9349379, are located in phosphatase and actin regulator 1 gene (PHACTR1). The 4 SNPs were genotyped using improved multiplex ligase detection reaction (iMLDR). Hardy-Weinberg equilibrium was calculated with Chi-square goodness-of-fit test or Fisher exact test to examine the frequency distribution of each SNP between patients with and without artery calcification.Linkage disequilibrium (LD) was analyzed with Haploview 4.2. IBM SPSS Statistics Version 22.0 (Armonk, NY:IBM Corp.) was applied for statistical analyses. Linear regression was used to explore the association between each SNP and transformed calcification quantity. We further constructed a genetic risk score (GRS) to test the accumulative effects of CAC-SNPs on artery calcification. In addition, we applied generalized multifactor dimensionality reduction (GMDR, http://sourceforge.net/projects/gmdr/) to identify the potential interactions of SNPs on degree of artery calcification. All analyzes were performed with an additive model of inheritance and adjusted for age, gender, DM, hypertension, triglyceride, total cholesterol and smoking.ResultsOf the 860 patients included for final analysis, the average age at baseline was 62.4±11.4 years old,599 (69.7%) of the patients were male,629 (73.1%) with hypertension and 209 (24.3%) with diabetes. Based on the CTA results,541 (62.9%) patients have carotid artery calcification (CarAC) and 692 (80.5%) have aortic arch calcification (AorAC). The genotype distributions of all SNPs were in accordance with Hardy-Weinberg equilibrium (P> 0.05). According to the linear regression adjusted for main cardiovascular risk factors including age, gender, diabetes, hypertension, triglyceride level, total cholesterol level and smoking, rs2026458 was significantly associated with CarAC (β= 0.31,95% confidence interval [CI] 0.10 to 0.52, P=0.003) and AorAC (β=0.32,95% CI 0.10 to 0.54, P=0.004), while rs1333049 associated with CarAC (β=0.28,95% CI 0.06 to 0.50, P=0.011), but not with AorAC (P=0.163). For rs1537370 and rs9349379, no significant effects were detected on CarAC or AorAC. When patients were stratified by gender, different effects of SNPs on calcifications were detected between male and female patients. Rs1537370 was associated with CarAC (β=-0.51,95% CI-0.90 to-0.11, P=0.013) in female, but not in male patients. By contrast, rs2026458 was significantly associated with both CarAC (β= 0.38,95% CI 0.13 to 0.64, corrected P=0.012) and AorAC (β=0.35,95% CI0.09 to 0.61, corrected P=0.008) in male, but not in female patients. The GRS of the joint CAC-SNPs was significantly associated with calcification in both carotid artery (p=0.77,95% CI 0.34 to 1.09, P< 0.001) and aortic arch (β=0.88,95% CI 0.36 to 1.40, P=0.001), and more prominent in male patients (CarAC:β=0.79,95% CI 0.37 to 1.20, P< 0.001;AorAC:β= 1.05,95% CI 0.47 to 1.62, P< 0.001).ConclusionIn a cohort of Chinese patients with ischemic stroke, we found that SNPs associated with calcification in coronary artery may also increase the risk of calcification in other arteries such as carotid artery and aortic arch. This finding implicates that different arteries might have shared genetic factors but different susceptibility to undergo calcification, which is helpful for understanding the mechanism of atherosclerosis at a molecular level. |
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