Abstract:
To investigate the possible association between V249I and T280M polymorphisms and carotid atherosclerosis (CAS) in a Han population in northern China we studied 328 patients with increased carotid artery intima-media thickness (C-IMT) and 292 healthy controls with normal C-IMT. T280M and V249I polymorphic genotypes of CX3CR1 were determined by polymerase chain reaction restriction fragment length polymorphism. Results showed that the M280 allele frequency in CAS group was significantly lower than that in the controls. Logistic regression analysis revealed that the decreased frequency of the M280 allele was an independent risk factor for CAS.
INTRODUCTION
Studies have shown that atherosclerosis is a chronic inflammatory process. Many factors can lead to progressive vascular lesions, which then induce inflammatory reactions at the vessel wall [Citation1]. The main characteristics of inflammatory reactions at vessel wall stem from interactions between white blood cells (WBC), endothelial cells, smooth muscle cells, and fibroblasts. At the molecular level, these characteristic inflammatory reactions involve interactions between cytokines, adhesion molecules, and chemokines [Citation2–4].
Fractalkine (FKN), a newly identified, unique chemokine, is present in both soluble and membrane bound forms. Soluble FNK displays chemotaxis activity towards WBC. CX3CR1, a novel seven-transmembrane receptor for fractalkine, is expressed mainly on CD4-T cells, CD8+ T cells, NK cells, and monocytes, providing a new pathway for WBC activation [Citation5,Citation6]. The Fractalkine/ CX3CR1 system functions in the pathological mechanisms of vascular and tissue lesions through enhancement of cell adhesion and promotion of the transmembrane migration of CX3CR1+ monocytes and lymphocytes [Citation7].
Recent studies have identified two single nucleotide polymorphisms (SNPs) in the gene encoding CX3CR1: 839 C→T(rs3732378; T280M) and 745 G→A(rs3732379; V249I). I249 and M280 have been shown to be in linkage disequilibrium and have been associated with interindividual differences in susceptibility to atherosclerotic diseases [Citation8–12]. Currently, the function of V249I and T280M genetic polymorphisms in atherosclerosis remains controversial. In the present study, we investigated the possible association between V249I and T280M polymorphisms and carotid atherosclerosis (CAS) in a Han population in northern China.
SUBJECTS
Before study, the research plan was approved by the clinical research ethics committee in Tiantan Hospital. All subjects provided signed informed consent prior to participation in this study.
The CAS group was comprised of 318 cases with increased carotid artery intima-media thickness (C-IMT), including 118 cases with TIA and 200 cases with asymptomatic carotid atherosclerosis. TIA patients were recruited from inpatients and outpatients of the neurology department in our hospital between June 2005 and May 2007. TIA was defined as a documented neurological deficit lasting <24 hours without definite radiographic evidence of acute ischemia. Among TIA patients, only those confirmed with increased C-IMT were enrolled in this study and those without increased C-IMT were excluded. Cases with asymptomatic carotid atherosclerosis were recruited from regular healthy check-ups. The control group consisted of 292 cases with normal C-IMT who had regular check-ups; those with a clinical history of cerebrovascular disease, myocardial infarction, or peripheral vascular disease were excluded.
All the subjects were of the Han population in China without relationship. Hypercholesterolaemia was defined as fasting total cholesterol ≥ 5.18mmol/L (200 mg/dl) or LDL cholesterol ≥ 3.37 mmol/L (130 mg/dl) or current treatment with cholesterol-lowering medication. DM was defined as a fasting blood glucose level >7 mmol/L (125 mg/dl), or current treatment with insulin or oral hypoglycaemic agents. Hypertension was defined as a seated systolic blood pressure ≥ 140 mmHg (1 mmHg = 0.133kPa) and/or diastolic pressure ≥ 90 mmHg or a history of hypertension leading to antihypertensive treatment. Current smoking was defined as smoking tobacco within the last month.
METHODS
Carotid Ultrasonography
C-IMT was detected in all subjects using cervical color ultrasonic Doppler. We measured C-IMT in 3 different points of the carotid artery on both sides, the common carotid artery (CCA), internal carotid artery bulb (ICA) and carotid bifurcation (BIF). Increased IMT was defined as 0.9mm≤C-IMT≤ 1.3mm in this study.
Blood Sample Collection and DNA Preparation
Genomic DNA from patients or controls was isolated from venous blood using a commercially available genomic DNA purification kit [Promega Corporation, Madison, WI, USA].
Determination of V249I and T280M Polymorphisms
According to the gene sequence in Genbank, primers were designed using Premier Primer 5 software. The upstream primer was 5′-CCTGCAAGAACC-ACAAGAAAGC-3′, the downstream primer was 5′-TGCTCAGAACACT-TC-CATGCC-3′. Both primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The product length obtained after PCR was 360 bp. PCR-RFLP-based genotyping of V249I and T280M polymor- phisms were performed in 25μl containing 5μl of extracted Genomic DNA, 100μmol/L of dNTPs, 1.5 mmol/L of MgCl2, 1U of Taq polymerase (TaKaRa BIOINC, Shiga Japan), and the 2 primers. After an initial denaturation step at 94°C for 3min, PCR was carried out for 30 cycles at 94°C for 1 minute, 57°C for 45 s, and 72°C for 1 minute, followed by a final extension at 72°C for 10 min. A volume of 5 μl PCR products was digested with Acl I restriction enzyme or HpyCH4 III restriction enzyme (New England Biolabs, MA, USA), then visualized with ethidium bromide after electrophoresis in 1.5% agarose gel. Results are shown in and . The PCR result was confirmed by direct sequencing in randomly selected samples.
Figure 1. PCR-RELP analysis of V249I CX3CR1 gene polymorphisms. Amplified PCR products were digested with Acl I enzyme.
Lane M, molecular marke, Lane 1, genotype VV, Lane 2, genotype II, Lane 3, genotype VI
Figure 2. PCR-RELP analysis of T280M CX3CR1 gene polymorphisms. Amplified PCR products were digested with HpyCH4 III enzyme.
Lane M, molecular marker, Lane 1,genotype MM, Lane 2, genotype TM, Lane 3,genotype TT
Statistical Analysis
Quantitative variables are presented as (mean ± standard deviation) and qualitative variables as percentages. Genotype distributions and allele frequencies between patients and controls were compared by chi-square (χ2) test. Multiple regression analyses were performed to assess the independent contribution of risk factors to CAS. Hardy– Weinberg equilibrium was tested using a χ2 test. Statistical analysis was performed with SPSS for Windows, version 13.0 (SPSS Inc.). SHEsis online software was used to calculate the linkage disequilibrium coefficient D’.
RESULTS
The 318 patients and 292 controls were well matched in terms of age [(61.8± 10.6) VS (60.3±9.1) years] and gender. Smoking, hypercholesterolemia, hypertension and DM were significantly more frequent among patients ().
Table 1. Baseline characteristics in patients and controls
The distributions of V249I and T280M genotypes were under Hardy-Weinberg equilibrium in patients and controls (χ2 =0.365-1.430, P=0.833-0.489). There were no significant differences in genotypic distribution between patients and controls or in allelic distribution regarding the V249I variant (P =0.940, P=0.121, respectively). However, the variant T280M revealed significant differences in the genotypes and allele frequencies. The genotype frequencies of T280M in the two groups were significantly different (OR =0.646, 95%CI 0.451 ∼ 0.928, P=0.017). The M280 allele frequency in CAS group was significantly lower than that in the control (OR =0.677, 95% CI0.499 ∼ 0.918; P=0.010) (). The V249I and T280M polymorphisms were in complete linkage disequilibrium (D’ = 1.000, r2 =0.445) , forming six combined genotypes of the nine theoretically possible ().
Table 2. CX3CR1 genotype and allele frequencies in patients and controls
Table 3. Combined genotype frequencies of V249I and T280M polymorphisms in two groups.
In logistic regression analysis, sex, age, hypertension, hypercholesterolemia, DM, smoking, family history of stroke and CX3CR1 genotypes (VV versus VI + II or TT versus TM+MM) were included as independent variables. The result revealed that decreased frequency of the M280 allele was an independent risk factor for CAS ().
Table 4. Multiple logistic regression analysis
DISCUSSIONS
Atherosclerosis is a systemic disease that is responsible for most cardiovascular events and stroke. The measurement of carotid intima-media thickness (C-IMT) is an accepted surrogate marker of atherosclerosis [Citation13–15] .
In the present study, we investigated the correlation of CAS and V249I and T280M polymorphisms of CX3CR1 for the first time in a Han population in northern China. Results showed that MM and TM genotype frequencies for the T280M polymorphism and M280 allele frequency were significantly lower in CAS group, implying that the M280 allele may be related to the decreased incidence of CAS[Citation16]. To further investigate the possible functions of CX3CR1 I249 and M280 alleles on CAS in the Han population in northern China, we performed a multi-factorial logistic regression analysis. The results showed that with the exception of individuals who smoked, had hypertension, and had hyperlipidemia, decreased M280 allele expression was an independent risk factor for CAS.
Animal experiments have shown that in CX3CRl knockouts, monocytes have decreased ability to bind to FKN and exhibit decreased chemotaxis activity. Comparing CX3CRl and apoE double knockout mice with mice with only apoE knockout, macrophage infiltration in the artery wall was significantly decreased in the former. Therefore, the CX3CR1/FKN pathway showed direct and major effects on atherosclerosis foci with abundant macrophages [Citation17]. Current reports on the function of M280 and I249 alleles in atherosclerotic disease are inconsistent [Citation8–12]. Possible reasons for these discrepancies may be as follows. Firstly, CX3CR1-T280M variation does not influence ligand binding, but rather negatively influences receptor-mediated reactions, including chemotaxis and adhesion ability [Citation11]. Secondly, in the monocytes of VI genotype carriers, the number of binding sites for FKN is significantly lower, enhancing the diastolic functions of the endothelium and stabilizing atherosclerosis plaques [Citation8,Citation9,Citation18]. Finally, peripheral CX3CR1 is abnormally expressed, significantly decreasing the chemotaxis and adhesion abilities of membrane-bound FKN on endothelial cells and reducing damage to endothelial cells [Citation18].
We confirmed that the V249I and T280M polymorphisms were in complete linkage disequilibrium. As reported in the white population[Citation10], only six (VV-TT, VI-TT, II-TT, VI-TM, II-TM,II-MM) of the nine possible combined genotypes were observed, and only three haplotyes (V249-T280, I249-T280, and I249-M280) of the four possible haplotypes were detected in this study. However, in the report of Norata et al., additional combined genotype VI-MM was observed, and with which subjects showed a reduced C-IMT) [Citation16]. It is worth noting that, in the above studies including ours, the combined genotype VV-TT is the dominating type, the frequencies of which are 58%–61% and 51%–56% in patients and in controls, respectively [Citation10,Citation11,Citation16]. But somewhat different from these studies, Hattori H. et al. [Citation12] showed that the frequency of the combined genotype VV-TT in the Japanese population was much higher and suggested that the V249-T280 haplotype has a high proportion of the nine possible combined genotypes in Japanese. In addition, they identified eight combined genotypes including VV-TM and VV-MM, demonstrating the presence of a new haplotype, V249-M280, in Japanese. Therefore, whether V249I and T280M polymorphisms of CX3CR1 can contribute to the development of atherosclerostic diseases remains controversial.
We limited our analysis to a Han population in nothern China. As is usually the case in genetic association studies, these discrepancies may arise from the variability of these linkage relationships among different ethnic groups and population substrata. Furthermore, it is difficult to completely ensure that the result is not secondary to another closely linked site. The disease association with M280 reported here may be influenced by I249 or other as-yet-unknown variants in the CX3CR1 gene promoter. Finally, the observed associations might result from linkage disequilibrium with unmeasured functional variants in closely linked genes,such as CCR8 [Citation19] .
The presence of the M280 polymorphism of CX3CR1 is associated with a decreased C-IMT, whereas the presence of the I249 polymorphism does not play a major role on the progression of carotid atherosclerosis. Because of the complex mechanism underlying CAS, further studies are needed to confirm these findings.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
This paper was first published online as an Early Online article on 5 January 2010.
Related Research Data
REFERENCES
- Ross, R. (1999). Atherosclerosis: an inflammatory disease. N Engl J Med 340 (2): 115–126.
- Libby, P., Ridker, P.M., Maseri, A. (2002). Inflammation and atherosclerosis. Circulation 105 (9): 1135–1143.
- McDermott, D.H.(2000). Chemokines and their receptors in infectious disease. Springer Semin Immunopathol 22 (4): 393–415.
- Murphy, P.M., Baggiolini, M., Charo I,F. . (2000). International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 52 (1): 145–176.
- Fong, A.M., Robinson, L.A., Steeber, D.A., Tedder, T.F., Yoshie, O., Imai, T., Patel, D.D.(1998). Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow. J Exp Med 188 (8): 1413–1419.
- Imai, T., Hieshima, K., Haskell, C., . (1997). Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91 (4): 521–530.
- Umehara, H., Bollm, E.T., Okazaki, T., . (2001). Fractalkine and vascular injury. Trends Immunol 22 (11): 602–607.
- McDermott, D.H., Halcox, J.P., Schenke, W.H., . (2001). Association between polymorphism in the chemokine receptor CX3CR1 and coronary vascular endothelialdysfunction and atherosclerosis. Circ Res 89 (5): 401–407.
- Moatti, D., Faure, S., Fumeron, F, . (2001). Polymorphism in the fractalkine receptor CX3CR1 as a genetic risk factor for coronary artery disease. Blood 97 (7): 1925–1928.
- Ghilardi, G., Biondi, M.L., Turri, O. . (2004). internal rotid artery occlusive disease and polymorphisms of fractalkine receptor CX3CR1: a genetic risk factor. Stroke; 35 (6): 1276–1279.
- Lavergne, E., Labreuche, J., Daoudi, M., . (2005) adverse associations between CX3CR1 polymorphisms and risk of cardiovascular or cerebrovascular disease. Arterioscler Thromb Vasc Biol 25 (4): 847–853.
- Hattori, H., Ito, D., Tanahashi, N., . (2005). T280M and V249I polymorphisms of fractalkine receptor CX3CR1 and ischemic cerebrovascular disease. Neurosci Lett 374 (2): 132–135.
- Coll, B., Feinstein, S.B.(2008). Carotid intima-media thickness measurements: techniques and clinical relevance. Current Atherosclerosis Reports 10 (5): 444–450.
- Lorenz, M.W., Markus, H.S., Bots, M.L., . (2007). Prediction of clinical cardiovascular events with carotid intima-media thickness: a systematic review and meta-analysis. Circulation 115 (4): 459–467.
- Prati, P., Tosetto, A., Vanuzzo, D., . (2008). Carotid intima media thickness and plaques can predict the occurrence of ischemic cerebrovascular events. Stroke 39 (9): 2470–2476.
- Norata, G.D., Garlaschelli, K., Ongari, M., . (2006). Effects of fractalkine receptor variants on common carotid artery intima-media thickness. Stroke 37 (6): 1558–1561.
- Combadiere, C., Potteaux, S., Gao, J.L., . (2003). Decreased atherosclerotic lesion formation in CX3CR1/ apolipoprotein E double knockout mice. Circulation, 107 (7): 1009–1013.
- Olliviera, V.,Faure, S., Tarantino, N., . (2003). Fractalkine/CX3CR1 production by human aortic smooth muscle cells impairs monocyte procoagulant and inflammatory responses. Cytokine 21 (6): 303–311.
- Myron, I., Cybulsky, Robert A., Hegele. The fractalkine receptor CX3CR1 is a key mediator of atherogenesis. J Clin. Invest 111 (8): 1118–1120.