CXCL16 Gene Tagging Snps And The Risk Of Coronary Artery Disease In Chinese Han Population

Objective Coronary artery disease (CAD) is one of the leading causes of death and disability worldwide. CXC ligand 16 is a newly discovered chemokine of the CXC family that is expressed in soluble and transmembrane forms. A series of studies conducted described CXCL16/SR-PSOX combining three important functions, chemokines, scavenger receptors and adhesion molecules, and suggested that CXCL16/SR-PSOX can be a potential player in atherogenesis. Initial ex vivo studies showed that CXCL16/SR-PSOX is abundant in human and murine atherosclerotic lesions. Following in vitro studies suggested that CXCL16/SR-PSOX might mediate T-cell adhesion to the endothelium, drive T-cell migration, stimulate cell proliferation and elicit inflammatory phenotype in smooth muscle cells and mediate uptake of atherogenic lipoproteins by macrophages. All these effects are known to be pro-atherogenic. Surprisingly, in vivo studies performed in murine models of atherosclerosis suggested that CXCL16/SR-PSOX is atheroprotective, while its receptor CXCR6 is harmful. In addition, studies investigating the association of circulating CXCL16/SR-PSOX plasma concentrations with the presence and extent of CAD in humans are controversial suggesting both positive, negative and no association. Furthermore, in patients with an acute coronary syndrome, CXCL16 levels obtained within 24 hours of admission are associated with long-term mortality after adjustment for other risk factors. Whether CXCL16/SR-PSOX can serve as a causative factor, biomarker or even a therapeutic target in atherosclerosis is still under debate. Genetic variation of CXCL16 could modulate CXCL16 production and function. A preliminary study found that a single nucleotide polymorphism (SNP) in the fourth exon of the CXCL16 gene was significantly associated with the severity of coronary artery stenosis in CAD patients. However, the association study is limited to a SNP, which fails to include all the potential risk-conferring variations in the CXCL16 gene and their potential effects on CAD risk. Besides, the relatively small size can affect the results, and there are no study performed in Chinese Han population. To address these issues, we first conducted an analysis of HapMap-tagged SNPs in the CXCL16 gene, including 181Ala >Val (rs2277680), and its relation to the risk of CAD in a Chinese Han population. Next, we investigated the association of CXCL16 genetic variation with its plasma levels in a subgroup of patients.Methods: (1) Study Populations. A total of 1176 patients with CAD and 850 control individuals, aged between 29 and 75 years, were enrolled in the study from June 2006 to April 2009. Patients and controls were unrelated individuals of Chinese Han from the northeast region of China. All the subjects had undergone coronary angiography for the evaluation of suspected or established CAD at Northern Hospital, Shenyang, China. Subset population was selected from our original cohort between March 2008 and April 2009. They consisted of 160 subjects with first episode of acute MI and 160 subjects with completely normal coronary arteries. The subset cases and controls were individually matched in terms of age (≤5 years), gender, smoking status, presence of hypertension, and diabetes mellitus. (2) Tag SNPs selection and genotyping. Five tag SNPs were retrieved from Hapmap database for Han population sample using the r2-based Tagger program with pairwise r 2≥0.80 and minor allele frequency (MAF)≥5%. The polymorphism rs2277680 was genotyped by polymerase chain reaction (PCR)-based RFLP analysis. The polymorphisms rs1051009, rs3744700, rs2304973, and rs2250333 were genotyped by PCR direct sequencing. (3) Determination of plasma CXCL16 levels. AMI patients had blood withdrawal at admission to the hospital before any invasive procedure and therapy. The median symptom onset-entry time was 5.3 hours (range 1 to 12 hours). Fasting blood samples were collected from CAD-free subjects after angiography. CXCL16 concentrations in plasma were measured with the human CXCL16 Quantikine ELISA Kit according to the manufacturer's instructions.Results (1) Baseline characteristics of the study population. There were significant differences in age, gender, smoker, hypertension, diabetes, BMI, LDL-C, HDL-C triglyceride and fasting glucose levels between the case and control groups. However, they were no significant differences in the these variables between the cases and controls in subgroup. (2) Association of CXCL16 SNPs with CAD. All five SNPs investigated in both case and control samples followed Hardy-Weinberg equilibrium (HWE). Both the genotypes distribution and allele frequencies of rs3744700 were significantly different between the cases and control subjects (P = 0.001 and P <0.001, respectively). To control Type I error the Bonferroni correction was used, with the adjusted alpha value of 0.01. The association between rs3744700 and CAD was still significant. On the basis of multivariable logistic regression analysis with adjustment for cardiovascular risk factors, subjects bearing the GG homozygotes had significantly increased susceptibility to CAD compared with the T allele carriers (GT + TT) (adjusted OR, 1.77; 95% CI, 1.28-2.43; P < 0.001). There was no significant difference in allele frequencies or genotypes distribution of the other four SNPs between CAD patients and controls. Further analysis didn't observed any significant association between the five SNPs of CXCL16 gene and the angiographic severity of CAD. Also, no significant differences were seen between the genotypes of the five SNPs of the CXCL16 gene and plasma lipid levels (TG, TC, LDL-C, and HDL-C) in control subjects without lipid-lowering threapy (n = 771). (3) Identification of regulatory elements in intron 4. Using a online splice site prediction program from Berkeley Drosophila Genome Project, one alternative splice acceptor site demonstrating a confidence score 1.00 was identified within intron 4 of CXCL16, and rs3744700 locates just 61 bp upstream form it. We also found that intron 4 harbors several putative regulatory elements, including the consensus sequence of GATA, which is affected by the rs3744700 polymorphism. (4) Plasma CXCL16 levels in subset subjects. Plasma CXCL16 levels were significantly higher in AMI patients than CAD-free controls (2.36±0.40 ng/ml versus 1.88±0.36 ng/ml, P < 0.01). However, there was no association between CXCL16 levels and the severity of coronary atherosclerosis. We found that CXCL16 level was significantly higher in subjects with GG genotype than those with T allele (GT and TT genotypes) in the control group (P=0.015). Similarly, AMI patients with GG homozygote had higher plasma CXCL16 concentration than that those with T allele (P = 0.027).Conclusions SNP rs3744700 of CXCL16 gene is independently associated with the development of CAD in Chinese Han population, and GG homozygote which is associated with increased expression of CXCL16 may have a promoting effect on CAD. However, we found no evidence for an association of the other gene variations with CAD in Chinese Han population.