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The RANTES gene promoter polymorphisms are associated with the risk of atherothrombotic cerebral infarction in Northern Han Chinese
Clinica Chimica Acta 412 (2011) 1112–1115
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
The RANTES gene promoter polymorphisms are associated with the risk of atherothrombotic cerebral infarction in Northern Han Chinese Xue Qin a, Zhiyi He a,⁎, Dongxue Zhao b, Lei Li a, Liying Yuan a a b
Department of Neurology, The First Hospital of China Medical University, Shenyang 110001, China Department of Neurology, Shengjing Hospital of China Medical University, Shenyang 110001, China
a r t i c l e
i n f o
Article history: Received 24 November 2010 Received in revised form 23 February 2011 Accepted 25 February 2011 Available online 3 March 2011 Keywords: Regulated upon activation Normal T-cell expressed and secreted (RANTES) Single nucleotide polymorphism Atherothrombotic cerebral infarction (ACI)
a b s t r a c t Background: Regulated upon activation, normal T-cell expressed and secreted (RANTES) plays an important role in the inflammatory process. This study is aimed at evaluating the potential association of the − 403G/A (rs2107538) and − 28C/G (rs2280788) polymorphisms of the RANTES gene promoter with the risk of atherothrombotic cerebral infarction (ACI) in Northern Han Chinese. Method: A total of 314 patients with ACI and 389 unrelated aged-matched healthy controls were recruited. Their genotypes of the RANTES gene promoter − 403G/A (rs2107538) and − 28C/G (rs2280788) were analyzed by multiplex polymerase chain reaction (multiplex PCR) and multiplex SNaPshot analysis. The potential association of genotyping and allele frequencies with ACI in this population was assessed statistically. Results: The frequencies of − 403AA genotype and A allele in ACI male patients were significantly higher than that in healthy controls (P = 0.007, P = 0.009, respectively). Female patients were not different. Multiple logistic regression analysis revealed that the − 403AA genotype in males was significantly associated with an increased risk of ACI, even after adjusting for confounding factors (OR = 4.344; 95% CI = 1.969–9.582; P b 0.001). Although there was no significant association of the − 28C/G polymorphism with ACI, the A-403C-28 haplotype was significantly associated with an increased risk of ACI in Han Chinese [OR = 1.56, 95% CI = 1.23– 1.98, P b 0.001]. Conclusions: Our data suggest that the − 403AA genotype and A allele of the RANTES promoter were associated with increased risk for the development of ACI in male Northern Han Chinese. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Stroke is one of the leading causes of morbidity and mortality in both developed and developing countries [1]. Although non-modifiable (age, race, and gender) and acquired (hypertension, cigarette smoking, diabetes, and obesity) risk factors are crucial for the pathogenesis of stroke [2,3], previous genome-wide association studies (GWASs) have suggested that several susceptible genes are associated with the development of stroke [4,5]. However, little is known about genetic components associated with atherothrombotic cerebral infarction (ACI), particularly in Chinese. During the pathogenic process of atherosclerosis, chemokines are critical factors that regulate the inflammatory response, progression of atherosclerosis, and plaque destabilization [6,7]. RANTES (CCL5) is a member of the CC-chemokine family and can be produced by a variety of cells, including T-lymphocytes, platelets, endothelial cells,
⁎ Corresponding author at: Department of Neurology, The First Hospital of China Medical University, 155 Nanjing North Street, Shenyang 110001, China. Tel.: + 86 24 83282515. E-mail address: [email protected] (Z. He). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.02.033
smooth muscle cells, and glial cells [8]. RANTES can interact with the chemokine receptors, CCR1, CCR3, and CCR5, and it plays an active role in recruiting leukocytes into inflammatory sites, promoting the transendothelial migration of leukocytes, and contributing to the pathogenic process of arterial injury and atherosclerosis [9–11]. The human RANTES gene spans 8.8 kb on the chromosome 17q11.2–q12 region, and has the characteristic three exon–two intron organization of the CC chemokine family. The single nucleotide polymorphisms (SNPs) in the RANTES gene promoter of −28C/G and −403G/A have been suggested to affect the expression of RANTES and modulate other pathogenic processes [12–14]. Notably, the −403G/A mutation in the RANTES promoter has also been associated with the development of coronary artery disease (CAD) [15,16], internal carotid artery occlusive disease [17], cerebral infarction [18], and hypercholesterolemia [19]. In contrast, the RANTES − 403A allele is associated with a reduced risk of CAD in Koreans [20]. Apparently, this association is variable in different ethnic populations. Furthermore, whether these SNPs could be associated with the development of ACI in Han Chinese has not been explored. Given that the incidence of atherosclerosis and ACI is increasing in China, the discovery of genetic factors contributing to the development of ACI will be of great significance.
X. Qin et al. / Clinica Chimica Acta 412 (2011) 1112–1115
The present case–control study aimed at assessing the potential association of these two SNPs in the RANTES gene promoter with the development of ACI in a Northern Han Chinese population. 2. Subjects and methods 2.1. Study population A total of 314 Han Chinese patients (181 males and 133 females, mean age 64.43 ± 8.48 years) with ACI were recruited from the First Affiliated Hospital of China Medical University from 1 September 2009 to 31 May 2010. Individual patients with ACI were diagnosed by neurologists, according to the criteria of clinical features and ancillary laboratory examinations, including a sudden onset of non-conclusive and focal neurological deficit for more than 24 h with corresponding infarction on brain imaging (computed tomography and magnetic resonance imaging), echocardiography, and carotid duplex imaging. Additional 389 age and gender-matched unrelated control subjects without a history of stroke or cerebrovascular diseases were also obtained from the same geographic areas and their clinical histories were obtained through an interview by physicians. Participants with transient ischemic stroke (TIA), lacunar infarction, cardiogenic embolic infarction, or hemorrhagic stroke were excluded from this study. Individuals who had any historical and current atrial fibrillation, autoimmune diseases, renal and liver diseases, hematopathy, or phlebothrombosis of limbs were also excluded. Their selected characteristics are summarized in Table 1. Blood pressure was measured at least twice in the supine position after 15 min of rest. Hypertension was defined as being on antihypertensive therapy or having systolic and/or diastolic blood pressure equal to or greater than 140 mm Hg (18.7 kPa) and/or 90 mm Hg (12.0 kPa), respectively. Type 2 diabetes was diagnosed, according to the standard of American Diabetes Association (http://www.diabetes.org/home.jsp). Smoking was defined as current or historical tobacco user. Alcohol consumption was classified as 5 standard drinks (approximately 12 g of alcohol) or more per day at least 12 times per year [21]. Written informed consents were obtained from individual participants and the experimental protocols of this study were approved by the Ethics Committee of the Medical Faculty of The First Affiliated Hospital of China Medical University. 2.2. Genotype and biochemical parameters determination Venous blood samples (10 ml) were collected from individual subjects and their genomic DNA were extracted using the Wizard genomic DNA purification kit (Promega, Sunnyvale, USA), according to the manufacturers' instruction. Individual DNA samples with a ratio
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value of 1.8–2.0 absorbance at 260/280 nm were used for the following experiments, and DNA samples were stored at − 20 °C. The genotypes of individual DNA samples at positions of −403G/ A and − 28C/G in the RANTES gene promoter were characterized using a multiplex polymerase chain reaction (multiplex PCR) and Multiplex SNaPshot analysis. The DNA fragments for the −403G/A and −28C/G of the RANTES gene promoter were amplified from individual genomic DNA samples by multiplex PCR using specific primers. The sequences of primers were forward: 5′-TCTTGGGGACAACAAGGAGTGG-3′ and reverse primer: 5′-CCAATGCCCAGCTCAGATCAA3′ for −403G/A (249 bp); forward: 5′-CGGCCAATGCTTGGTTGCTAT-3′ and reverse: 5′-CGAGGTCCACGTGCTGTCTTG-3′ for −28C/G (124 bp), respectively. The PCR reactions (10 μl/tube) contained 10 ng of genomic template, 1 μmol/l of each primer, 1 × GC buffer I 10 μl (TAKARA), 3.0 mmol/l Mg2+, 0.3 mmol/l dNTPs, and 1 U HotStarTaq polymerase (Qiagen, Hilden, Germany). The PCR reactions were performed in duplicate at 95 °C for 15 min and subjected to 11 cycles of 94 °C for 20 s, 67.5 °C for 40 s, and 72 °C for 1.5 min. Subsequently, the PCR reactions were further amplified by 24 cycles of 94 °C for 20 s, 63 °C for 30 s, and 72 °C for 110 s, followed by extension at 72 °C for 2 min. The PCR products were then characterized using SNaPshot Multiplex kit and GeneMapper 4.0 (Applied Biosystems, Princeton, USA). The genotypes of individual samples were determined, according to the criteria of the PCR products with green signal for the wild-type allele, with blue signal for the mutant allele, and with both green and blue signals for the heterozygous. Individual samples with consistent data from duplicate tubes were counted. In addition, we randomly selected 10% of the positive samples for genotyping again to control experimental quality, and we obtained the same results. The levels of fasting plasma glucose (FPG) were measured by routine glucose oxidase procedure. The levels of serum total cholesterol and triglycerides were measured using commercial enzymatic methods (CHOD-PAP, GPO-PAP, Wako, Japan), respectively. The concentrations of serum HDL- and LDL-cholesterol were determined with homogeneous assays (Genzyme Diagnostics, Framingham, USA). 2.3. Statistical analysis Continuous variables were presented as mean ± SD and categorical variables as percentages. Allele frequencies were calculated from the genotypes of all subjects. Genotype distribution was compared to values predicted by the Hardy–Weinberg equilibrium (HWE) through χ2 analysis. The difference in the allele and genotype frequencies of SNP in the RANTES promoter between the patients and controls was analyzed by a chi-square test or Fisher's exact test, and analyzed by odds ratios (ORs) and 95% confidence interval (95%CI) using the most common genotype as the reference group. Statistical analyses were
Table 1 The demographic and clinical characteristics of subjects. Total
Number Age BMI (kg/m2) Hypertension (%) T2DM (%) Smoking (%) Alcohol consumption (%) Triglycerides (mmol/l) Total cholesterol (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) FPG (mmol/l)
Male
Cases
Controls
314 64.43 ± 8.48 23.66 ± 2.32 64.6 26.8 35.0 20.1 1.67 ± 1.02 4.96 ± 1.31 1.35 ± 0.35 2.85 ± 0.93 6.69 ± 2.25
389 63.93 ± 6.86 23.38 ± 1.76 20.1 7.5 15.7 12.1 1.56 ± 0.50 4.77 ± 0.72 1.34 ± 0.24 2.72 ± 0.76 4.96 ± 1.03
Female
P value
Cases
Controls
0.402 0.074 b0.001 b0.001 b0.001 0.004 0.079 0.021 0.562 0.048 b0.001
181 63.73 ± 8.44 23.55 ± 2.30 63.0 27.6 54.1 32.6 1.58 ± 1.03 4.78 ± 1.19 1.29 ± 0.33 2.78 ± 0.93 6.76 ± 2.43
215 63.59 ± 7.17 23.53 ± 1.87 20.9 7.4 20 18.6 1.46 ± 0.37 4.82 ± 0.75 1.37 ± 0.22 2.78 ± 0.74 5.08 ± 1.05
P value
Cases
Controls
P value
0.856 0.929 b 0.001 b 0.001 b 0.001 0.001 0.158 0.722 0.009 0.960 b 0.001
133 65.37 ± 8.48 23.82 ± 2.34 66.9 25.6 9.0 3.0 1.80 ± 1.01 5.21 ± 1.44 1.44 ± 0.37 2.95 ± 0.94 6.60 ± 1.97
174 64.35 ± 6.45 23.19 ± 1.58 19 7.5 10.3 9.5 1.68 ± 0.61 4.71 ± 0.67 1.31 ± 0.27 2.65 ± 0.77 4.81 ± 0.99
0.250 0.009 b 0.001 b 0.001 0.699 0.635 0.227 b 0.001 0.001 0.002 b 0.001
N: number; BMI: body mass index; T2DM: type 2 diabetes; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; FPG: fasting plasma glucose. Continuous and categorical variables were tested by Student's t-test and χ2-analysis, respectively.
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performed with Statistical Product and Service Solutions (SPSS) v13.0 software. Furthermore, the linkage disequilibrium index (D-prime and r2) and infer haplotype of these two SNPs were determined using the SHEsis analysis platform, as described previously [22,23]. After adjusting for the confounding variables, the remaining variables with significant difference were analyzed by multivariable logistic regression for the evaluation of the relationship between the genotypes of RANTES polymorphism and ACI. A value of p b 0.05 was considered statistically significant. 3. Results 3.1. Clinical characteristics of study subjects To determine the potential association of the SNPs of the RANTES promoter with the development of ACI, a total of 314 patients with ACI and 389 control subjects were recruited for this study. There was no significant difference in mean age, BMI, and the levels of serum HDL-C and total triglycerides between the patients and controls (Table 1). However, the percentage of subjects with hypertension, T2DM, smokers, or alcohol consumers, the levels of serum total cholesterol, LDL-C, and FPG in the ACI patients were significantly higher than that of controls. Further gender stratification revealed that the percentage of subjects with hypertension, T2DM, smokers, alcohol consumers, and FPG in male patients were significantly higher than that of controls, but the levels of HDL-C in male patients were significantly lower than that of controls. In addition, the percentage of subjects with hypertension or T2DM and the concentrations of serum total cholesterol, LDL-C, HDL-C, and FPG in female patients were significantly higher than that of controls. These data suggest that there are common factors (hypertension and T2DM) and genderspecific factors associated with the development of ACI in Han Chinese. 3.2. Genotype analysis Genotype distributions were in Hardy–Weinberg equilibrium in the entire population, and there was no significant difference in the distribution of each SNP in either the patients or controls (−403G/A for patient group: χ2 = 3.66, p = 0.06; control group: χ2 = 0.87, p = 0.35; −28C/G for patient group: χ2 = 0.03, p = 0.85; control group: χ2 = 0.69, p = 0.41). The allele and genotype distributions of both the −403G/A and − 28C/G polymorphisms in 314 ACI patients and 389 controls were analyzed in Table 2. The frequency of the −403AA genotype and − 403A allele in the patients was significantly
Table 3 Multivariable logistic regression analysis in males. Male
Hypertension Smoking FPG Genotype(AA)
OR
95%CI
p
5.132 2.565 2.936 4.344
3.016–8.734 1.440–4.570 2.068–4.170 1.969–9.582
b 0.001 0.001 b 0.001 b 0.001
Multivariable logistic regression analysis of individual variables was performed after adjustment of age, type 2 diabetes, hypertension, smoking, alcohol consumption, HDLC, and FPG.
higher than that of the controls. More importantly, the −403AA genotype was significantly associated with an increased risk of ACI, even after adjustment for confounding variables, determined by multivariate logistic regression analysis (p = 0.001, OR = 2.545, 95% CI = 1.441–4.493). Further gender stratification revealed that the frequencies of the −403AA genotype and A allele in male, but not in female, patients were significantly higher that of controls (Table 2). After adjusting for other risk factors, the −403AA genotype, together with hypertension, smoking, and high FPG was associated significantly with increased risk for ACI in males (Table 3). However, there was no significant difference in the distribution of − 28C/G genotype and allele polymorphisms between the patients and controls in this population. Haplotypes were analyzed using SHEsis program platform. These two SNPs were in linkage disequilibrium in this study population (Dprime = 0.912, r2 = 0.178). Of four possible haplotypes, only three haplotypes with a frequency of N0.03 among both cases and controls were included in the haplotype analysis. There was a statistically significant difference in the overall haplotype distribution between cases and controls (global test P b 0.001). According to our prior hypothesis and the SNP-based analyses, we considered the individuals with G-403C-28 haplotype to be the reference group for OR estimations. The frequency of A-403C-28 haplotype in the patients was significantly higher than that in the controls (P b 0.001, OR = 1.56, 95% CI = 1.23–1.98). 4. Discussion In this study, we explored the potential association of the SNPs of the RANTES promoter with ACI in Northern Han Chinese. We found that the frequency of the −403A allele and AA genotype, but not the −28C/G SNP, in male ACI patients was significantly higher than that of the controls. More importantly, even after multivariate adjustment
Table 2 Genotype and allele distributions in the studied groups. Total Cases (314)/controls (389) (%)
Male p
OR (95%CI)
− 403G/A Genotype GG 34.1/41.9 GA 43.9/44.0 AA 22.0/14.1
Reference 0.222 0.003
Allele A
Female
Cases (181)/controls (215) (%)
p
OR (95%CI)
1.23(0.88–1.71) 1.91(1.24–2.94)
32.6/41.4 45.9/46.5 21.5/12.1
Reference 0.316 0.007
0.003
1.39(1.12–1.72)
44.5/35.3
-28 C/G Genotype CC 79.6/75.3 CG 19.1/22.4 GG 1.27/2.3
Reference 0.259 0.283
0.81(0.56–1.17) 0.52(0.16–1.71)
Allele G
0.130
0.78(0.56–1.08)
43.9/36.1
10.8/13.5
Cases (133)/controls (174) (%)
p
OR (95%CI)
1.252(0.81–1.94) 2.263(1.25–4.10)
36.1/42.5 41.4/40.8 22.6/16.7
Reference 0.492 0.14
1.194(0.72–1.98) 1.595(0.85–2.98)
0.009
1.465(1.100–1.951)
43.2/37.1
0.122
1.293(0.933–1.791)
79.6/74.4 19.3/24.2 1.1/1.4
Reference 0.239 0.744
0.748(0.46–1.21) 0.741(0.12–4.50)
79.7/76.4 18.8/20.1 1.5/3.4
Reference 0.708 0.292
0.896(0.51–1.59) 0.418(0.083–2.115)
10.8/13.5
0.246
0.774(0.503–1.193)
10.9/13.5
0.332
0.784(0.479–1.283)
The difference in the allele and genotype frequencies of SNP between the patients and controls was analyzed by a chi-square test or Fisher's exact test.
X. Qin et al. / Clinica Chimica Acta 412 (2011) 1112–1115
for conventional risk factors of stroke, the AA genotype remained significantly associated with an increased risk of ACI in male Han Chinese. Therefore, the − 403A allele and AA genotype were associated with the development of ACI in male Han Chinese. Previous studies have shown that the RANTES promoter polymorphisms (−403G/A, −28C/G) are associated with chronic inflammatory diseases in advanced countries [24–26]. However, the association of −403A allele with the development of cardiovascular diseases is controversial in different disease models and ethnic populations [15,16,18,20]. We found that the − 403AA genotype and A allele were significantly associated with an increased risk of the development of ACI in male Han Chinese, even after adjusting for the conventional risk factors of stroke. Our findings support the notion that the AA genotype and A allele of the −403G/A in the RANTES promoter may be the risk factors for ACI in males. Thus, polymorphisms associated with ACI are different between women and men [19]. However, our data were in disagreement with other findings [20]. The different results may be attributed to the variables in ethnic populations, the methodological approaches, sample size, and patient selection. RANTES has been implicated in the inflammatory process of atherosclerosis and other cardiovascular diseases. Evidentially, the expression of RANTES is detected in atherosclerotic coronary arteries, but not in normal vessels [27], and the transcription of RANTES gene is markedly up-regulated within symptomatic atherosclerotic carotid plaque [28]. Although the association of serum RANTES levels with CAD remains controversial [29,30], the − 403A variant in the RANTES promoter has been demonstrated to up-regulate the expression of RANTES [14]. Given that RANTES is a strong chemokine and can recruit inflammatory leukocytes into inflammatory sites, the significantly up-regulated production of RANTES in individuals with the − 403A allele may promote the inflammatory process and the development of atherosclerosis and ACI. Indeed, mice deficient in the RANTES gene display significantly fewer T lymphocyte and monocyte infiltrates in inflammatory sites [31]. Furthermore, treatment with the RANTES antagonist Met-RANTES inhibits the arterial injury-mediated monocyte migration to carotid endothelium, neointima formation, and macrophage accumulation as well as atherosclerosis progression in uninjured arteries in apolipoprotein E-deficient mice [10,11,32]. Therefore, RANTES plays an important role in the development of atherosclerosis, potentially explaining the association between RANTES −403A and cardiovascular diseases. In summary, our data indicated that the −403AA genotype and A allele were significantly associated with an increased risk for the development of ACI in male Han Chinese. To the best of our knowledge, this is the first report on the SNPs of the RANTES promoter associated with ACI in Northern male Han Chinese. However, our study has limitations, such as a relatively small sample size, the lack of RANTES and other inflammatory marker (such as C-Reactive Protein, IL-6) measurements, and difference of many confounders between these two groups as well as the lack of a group of perfectly matched health controls in the current study. Therefore, further exploration of the current observations with more detailed genotyping, expression, and translational studies in a bigger population including optimal control subjects is warranted. Conceivably, after confirmation of its significance, the SNP may be used for the predisposition to ACI in Han Chinese. Acknowledgement This study was supported by a grant from the National Natural Science Foundation of China (30971018). References [1] Johnston SC, Mendis S, Mathers CD. Global variation in stroke burden and mortality: estimates from monitoring, surveillance, and modelling. Lancet Neurol 2009;8:345–54.
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
The RANTES gene promoter polymorphisms are associated with the risk of atherothrombotic cerebral infarction in Northern Han Chinese Xue Qin a, Zhiyi He a,⁎, Dongxue Zhao b, Lei Li a, Liying Yuan a a b
Department of Neurology, The First Hospital of China Medical University, Shenyang 110001, China Department of Neurology, Shengjing Hospital of China Medical University, Shenyang 110001, China
a r t i c l e
i n f o
Article history: Received 24 November 2010 Received in revised form 23 February 2011 Accepted 25 February 2011 Available online 3 March 2011 Keywords: Regulated upon activation Normal T-cell expressed and secreted (RANTES) Single nucleotide polymorphism Atherothrombotic cerebral infarction (ACI)
a b s t r a c t Background: Regulated upon activation, normal T-cell expressed and secreted (RANTES) plays an important role in the inflammatory process. This study is aimed at evaluating the potential association of the − 403G/A (rs2107538) and − 28C/G (rs2280788) polymorphisms of the RANTES gene promoter with the risk of atherothrombotic cerebral infarction (ACI) in Northern Han Chinese. Method: A total of 314 patients with ACI and 389 unrelated aged-matched healthy controls were recruited. Their genotypes of the RANTES gene promoter − 403G/A (rs2107538) and − 28C/G (rs2280788) were analyzed by multiplex polymerase chain reaction (multiplex PCR) and multiplex SNaPshot analysis. The potential association of genotyping and allele frequencies with ACI in this population was assessed statistically. Results: The frequencies of − 403AA genotype and A allele in ACI male patients were significantly higher than that in healthy controls (P = 0.007, P = 0.009, respectively). Female patients were not different. Multiple logistic regression analysis revealed that the − 403AA genotype in males was significantly associated with an increased risk of ACI, even after adjusting for confounding factors (OR = 4.344; 95% CI = 1.969–9.582; P b 0.001). Although there was no significant association of the − 28C/G polymorphism with ACI, the A-403C-28 haplotype was significantly associated with an increased risk of ACI in Han Chinese [OR = 1.56, 95% CI = 1.23– 1.98, P b 0.001]. Conclusions: Our data suggest that the − 403AA genotype and A allele of the RANTES promoter were associated with increased risk for the development of ACI in male Northern Han Chinese. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Stroke is one of the leading causes of morbidity and mortality in both developed and developing countries [1]. Although non-modifiable (age, race, and gender) and acquired (hypertension, cigarette smoking, diabetes, and obesity) risk factors are crucial for the pathogenesis of stroke [2,3], previous genome-wide association studies (GWASs) have suggested that several susceptible genes are associated with the development of stroke [4,5]. However, little is known about genetic components associated with atherothrombotic cerebral infarction (ACI), particularly in Chinese. During the pathogenic process of atherosclerosis, chemokines are critical factors that regulate the inflammatory response, progression of atherosclerosis, and plaque destabilization [6,7]. RANTES (CCL5) is a member of the CC-chemokine family and can be produced by a variety of cells, including T-lymphocytes, platelets, endothelial cells,
⁎ Corresponding author at: Department of Neurology, The First Hospital of China Medical University, 155 Nanjing North Street, Shenyang 110001, China. Tel.: + 86 24 83282515. E-mail address: [email protected] (Z. He). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.02.033
smooth muscle cells, and glial cells [8]. RANTES can interact with the chemokine receptors, CCR1, CCR3, and CCR5, and it plays an active role in recruiting leukocytes into inflammatory sites, promoting the transendothelial migration of leukocytes, and contributing to the pathogenic process of arterial injury and atherosclerosis [9–11]. The human RANTES gene spans 8.8 kb on the chromosome 17q11.2–q12 region, and has the characteristic three exon–two intron organization of the CC chemokine family. The single nucleotide polymorphisms (SNPs) in the RANTES gene promoter of −28C/G and −403G/A have been suggested to affect the expression of RANTES and modulate other pathogenic processes [12–14]. Notably, the −403G/A mutation in the RANTES promoter has also been associated with the development of coronary artery disease (CAD) [15,16], internal carotid artery occlusive disease [17], cerebral infarction [18], and hypercholesterolemia [19]. In contrast, the RANTES − 403A allele is associated with a reduced risk of CAD in Koreans [20]. Apparently, this association is variable in different ethnic populations. Furthermore, whether these SNPs could be associated with the development of ACI in Han Chinese has not been explored. Given that the incidence of atherosclerosis and ACI is increasing in China, the discovery of genetic factors contributing to the development of ACI will be of great significance.
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The present case–control study aimed at assessing the potential association of these two SNPs in the RANTES gene promoter with the development of ACI in a Northern Han Chinese population. 2. Subjects and methods 2.1. Study population A total of 314 Han Chinese patients (181 males and 133 females, mean age 64.43 ± 8.48 years) with ACI were recruited from the First Affiliated Hospital of China Medical University from 1 September 2009 to 31 May 2010. Individual patients with ACI were diagnosed by neurologists, according to the criteria of clinical features and ancillary laboratory examinations, including a sudden onset of non-conclusive and focal neurological deficit for more than 24 h with corresponding infarction on brain imaging (computed tomography and magnetic resonance imaging), echocardiography, and carotid duplex imaging. Additional 389 age and gender-matched unrelated control subjects without a history of stroke or cerebrovascular diseases were also obtained from the same geographic areas and their clinical histories were obtained through an interview by physicians. Participants with transient ischemic stroke (TIA), lacunar infarction, cardiogenic embolic infarction, or hemorrhagic stroke were excluded from this study. Individuals who had any historical and current atrial fibrillation, autoimmune diseases, renal and liver diseases, hematopathy, or phlebothrombosis of limbs were also excluded. Their selected characteristics are summarized in Table 1. Blood pressure was measured at least twice in the supine position after 15 min of rest. Hypertension was defined as being on antihypertensive therapy or having systolic and/or diastolic blood pressure equal to or greater than 140 mm Hg (18.7 kPa) and/or 90 mm Hg (12.0 kPa), respectively. Type 2 diabetes was diagnosed, according to the standard of American Diabetes Association (http://www.diabetes.org/home.jsp). Smoking was defined as current or historical tobacco user. Alcohol consumption was classified as 5 standard drinks (approximately 12 g of alcohol) or more per day at least 12 times per year [21]. Written informed consents were obtained from individual participants and the experimental protocols of this study were approved by the Ethics Committee of the Medical Faculty of The First Affiliated Hospital of China Medical University. 2.2. Genotype and biochemical parameters determination Venous blood samples (10 ml) were collected from individual subjects and their genomic DNA were extracted using the Wizard genomic DNA purification kit (Promega, Sunnyvale, USA), according to the manufacturers' instruction. Individual DNA samples with a ratio
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value of 1.8–2.0 absorbance at 260/280 nm were used for the following experiments, and DNA samples were stored at − 20 °C. The genotypes of individual DNA samples at positions of −403G/ A and − 28C/G in the RANTES gene promoter were characterized using a multiplex polymerase chain reaction (multiplex PCR) and Multiplex SNaPshot analysis. The DNA fragments for the −403G/A and −28C/G of the RANTES gene promoter were amplified from individual genomic DNA samples by multiplex PCR using specific primers. The sequences of primers were forward: 5′-TCTTGGGGACAACAAGGAGTGG-3′ and reverse primer: 5′-CCAATGCCCAGCTCAGATCAA3′ for −403G/A (249 bp); forward: 5′-CGGCCAATGCTTGGTTGCTAT-3′ and reverse: 5′-CGAGGTCCACGTGCTGTCTTG-3′ for −28C/G (124 bp), respectively. The PCR reactions (10 μl/tube) contained 10 ng of genomic template, 1 μmol/l of each primer, 1 × GC buffer I 10 μl (TAKARA), 3.0 mmol/l Mg2+, 0.3 mmol/l dNTPs, and 1 U HotStarTaq polymerase (Qiagen, Hilden, Germany). The PCR reactions were performed in duplicate at 95 °C for 15 min and subjected to 11 cycles of 94 °C for 20 s, 67.5 °C for 40 s, and 72 °C for 1.5 min. Subsequently, the PCR reactions were further amplified by 24 cycles of 94 °C for 20 s, 63 °C for 30 s, and 72 °C for 110 s, followed by extension at 72 °C for 2 min. The PCR products were then characterized using SNaPshot Multiplex kit and GeneMapper 4.0 (Applied Biosystems, Princeton, USA). The genotypes of individual samples were determined, according to the criteria of the PCR products with green signal for the wild-type allele, with blue signal for the mutant allele, and with both green and blue signals for the heterozygous. Individual samples with consistent data from duplicate tubes were counted. In addition, we randomly selected 10% of the positive samples for genotyping again to control experimental quality, and we obtained the same results. The levels of fasting plasma glucose (FPG) were measured by routine glucose oxidase procedure. The levels of serum total cholesterol and triglycerides were measured using commercial enzymatic methods (CHOD-PAP, GPO-PAP, Wako, Japan), respectively. The concentrations of serum HDL- and LDL-cholesterol were determined with homogeneous assays (Genzyme Diagnostics, Framingham, USA). 2.3. Statistical analysis Continuous variables were presented as mean ± SD and categorical variables as percentages. Allele frequencies were calculated from the genotypes of all subjects. Genotype distribution was compared to values predicted by the Hardy–Weinberg equilibrium (HWE) through χ2 analysis. The difference in the allele and genotype frequencies of SNP in the RANTES promoter between the patients and controls was analyzed by a chi-square test or Fisher's exact test, and analyzed by odds ratios (ORs) and 95% confidence interval (95%CI) using the most common genotype as the reference group. Statistical analyses were
Table 1 The demographic and clinical characteristics of subjects. Total
Number Age BMI (kg/m2) Hypertension (%) T2DM (%) Smoking (%) Alcohol consumption (%) Triglycerides (mmol/l) Total cholesterol (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) FPG (mmol/l)
Male
Cases
Controls
314 64.43 ± 8.48 23.66 ± 2.32 64.6 26.8 35.0 20.1 1.67 ± 1.02 4.96 ± 1.31 1.35 ± 0.35 2.85 ± 0.93 6.69 ± 2.25
389 63.93 ± 6.86 23.38 ± 1.76 20.1 7.5 15.7 12.1 1.56 ± 0.50 4.77 ± 0.72 1.34 ± 0.24 2.72 ± 0.76 4.96 ± 1.03
Female
P value
Cases
Controls
0.402 0.074 b0.001 b0.001 b0.001 0.004 0.079 0.021 0.562 0.048 b0.001
181 63.73 ± 8.44 23.55 ± 2.30 63.0 27.6 54.1 32.6 1.58 ± 1.03 4.78 ± 1.19 1.29 ± 0.33 2.78 ± 0.93 6.76 ± 2.43
215 63.59 ± 7.17 23.53 ± 1.87 20.9 7.4 20 18.6 1.46 ± 0.37 4.82 ± 0.75 1.37 ± 0.22 2.78 ± 0.74 5.08 ± 1.05
P value
Cases
Controls
P value
0.856 0.929 b 0.001 b 0.001 b 0.001 0.001 0.158 0.722 0.009 0.960 b 0.001
133 65.37 ± 8.48 23.82 ± 2.34 66.9 25.6 9.0 3.0 1.80 ± 1.01 5.21 ± 1.44 1.44 ± 0.37 2.95 ± 0.94 6.60 ± 1.97
174 64.35 ± 6.45 23.19 ± 1.58 19 7.5 10.3 9.5 1.68 ± 0.61 4.71 ± 0.67 1.31 ± 0.27 2.65 ± 0.77 4.81 ± 0.99
0.250 0.009 b 0.001 b 0.001 0.699 0.635 0.227 b 0.001 0.001 0.002 b 0.001
N: number; BMI: body mass index; T2DM: type 2 diabetes; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; FPG: fasting plasma glucose. Continuous and categorical variables were tested by Student's t-test and χ2-analysis, respectively.
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performed with Statistical Product and Service Solutions (SPSS) v13.0 software. Furthermore, the linkage disequilibrium index (D-prime and r2) and infer haplotype of these two SNPs were determined using the SHEsis analysis platform, as described previously [22,23]. After adjusting for the confounding variables, the remaining variables with significant difference were analyzed by multivariable logistic regression for the evaluation of the relationship between the genotypes of RANTES polymorphism and ACI. A value of p b 0.05 was considered statistically significant. 3. Results 3.1. Clinical characteristics of study subjects To determine the potential association of the SNPs of the RANTES promoter with the development of ACI, a total of 314 patients with ACI and 389 control subjects were recruited for this study. There was no significant difference in mean age, BMI, and the levels of serum HDL-C and total triglycerides between the patients and controls (Table 1). However, the percentage of subjects with hypertension, T2DM, smokers, or alcohol consumers, the levels of serum total cholesterol, LDL-C, and FPG in the ACI patients were significantly higher than that of controls. Further gender stratification revealed that the percentage of subjects with hypertension, T2DM, smokers, alcohol consumers, and FPG in male patients were significantly higher than that of controls, but the levels of HDL-C in male patients were significantly lower than that of controls. In addition, the percentage of subjects with hypertension or T2DM and the concentrations of serum total cholesterol, LDL-C, HDL-C, and FPG in female patients were significantly higher than that of controls. These data suggest that there are common factors (hypertension and T2DM) and genderspecific factors associated with the development of ACI in Han Chinese. 3.2. Genotype analysis Genotype distributions were in Hardy–Weinberg equilibrium in the entire population, and there was no significant difference in the distribution of each SNP in either the patients or controls (−403G/A for patient group: χ2 = 3.66, p = 0.06; control group: χ2 = 0.87, p = 0.35; −28C/G for patient group: χ2 = 0.03, p = 0.85; control group: χ2 = 0.69, p = 0.41). The allele and genotype distributions of both the −403G/A and − 28C/G polymorphisms in 314 ACI patients and 389 controls were analyzed in Table 2. The frequency of the −403AA genotype and − 403A allele in the patients was significantly
Table 3 Multivariable logistic regression analysis in males. Male
Hypertension Smoking FPG Genotype(AA)
OR
95%CI
p
5.132 2.565 2.936 4.344
3.016–8.734 1.440–4.570 2.068–4.170 1.969–9.582
b 0.001 0.001 b 0.001 b 0.001
Multivariable logistic regression analysis of individual variables was performed after adjustment of age, type 2 diabetes, hypertension, smoking, alcohol consumption, HDLC, and FPG.
higher than that of the controls. More importantly, the −403AA genotype was significantly associated with an increased risk of ACI, even after adjustment for confounding variables, determined by multivariate logistic regression analysis (p = 0.001, OR = 2.545, 95% CI = 1.441–4.493). Further gender stratification revealed that the frequencies of the −403AA genotype and A allele in male, but not in female, patients were significantly higher that of controls (Table 2). After adjusting for other risk factors, the −403AA genotype, together with hypertension, smoking, and high FPG was associated significantly with increased risk for ACI in males (Table 3). However, there was no significant difference in the distribution of − 28C/G genotype and allele polymorphisms between the patients and controls in this population. Haplotypes were analyzed using SHEsis program platform. These two SNPs were in linkage disequilibrium in this study population (Dprime = 0.912, r2 = 0.178). Of four possible haplotypes, only three haplotypes with a frequency of N0.03 among both cases and controls were included in the haplotype analysis. There was a statistically significant difference in the overall haplotype distribution between cases and controls (global test P b 0.001). According to our prior hypothesis and the SNP-based analyses, we considered the individuals with G-403C-28 haplotype to be the reference group for OR estimations. The frequency of A-403C-28 haplotype in the patients was significantly higher than that in the controls (P b 0.001, OR = 1.56, 95% CI = 1.23–1.98). 4. Discussion In this study, we explored the potential association of the SNPs of the RANTES promoter with ACI in Northern Han Chinese. We found that the frequency of the −403A allele and AA genotype, but not the −28C/G SNP, in male ACI patients was significantly higher than that of the controls. More importantly, even after multivariate adjustment
Table 2 Genotype and allele distributions in the studied groups. Total Cases (314)/controls (389) (%)
Male p
OR (95%CI)
− 403G/A Genotype GG 34.1/41.9 GA 43.9/44.0 AA 22.0/14.1
Reference 0.222 0.003
Allele A
Female
Cases (181)/controls (215) (%)
p
OR (95%CI)
1.23(0.88–1.71) 1.91(1.24–2.94)
32.6/41.4 45.9/46.5 21.5/12.1
Reference 0.316 0.007
0.003
1.39(1.12–1.72)
44.5/35.3
-28 C/G Genotype CC 79.6/75.3 CG 19.1/22.4 GG 1.27/2.3
Reference 0.259 0.283
0.81(0.56–1.17) 0.52(0.16–1.71)
Allele G
0.130
0.78(0.56–1.08)
43.9/36.1
10.8/13.5
Cases (133)/controls (174) (%)
p
OR (95%CI)
1.252(0.81–1.94) 2.263(1.25–4.10)
36.1/42.5 41.4/40.8 22.6/16.7
Reference 0.492 0.14
1.194(0.72–1.98) 1.595(0.85–2.98)
0.009
1.465(1.100–1.951)
43.2/37.1
0.122
1.293(0.933–1.791)
79.6/74.4 19.3/24.2 1.1/1.4
Reference 0.239 0.744
0.748(0.46–1.21) 0.741(0.12–4.50)
79.7/76.4 18.8/20.1 1.5/3.4
Reference 0.708 0.292
0.896(0.51–1.59) 0.418(0.083–2.115)
10.8/13.5
0.246
0.774(0.503–1.193)
10.9/13.5
0.332
0.784(0.479–1.283)
The difference in the allele and genotype frequencies of SNP between the patients and controls was analyzed by a chi-square test or Fisher's exact test.
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for conventional risk factors of stroke, the AA genotype remained significantly associated with an increased risk of ACI in male Han Chinese. Therefore, the − 403A allele and AA genotype were associated with the development of ACI in male Han Chinese. Previous studies have shown that the RANTES promoter polymorphisms (−403G/A, −28C/G) are associated with chronic inflammatory diseases in advanced countries [24–26]. However, the association of −403A allele with the development of cardiovascular diseases is controversial in different disease models and ethnic populations [15,16,18,20]. We found that the − 403AA genotype and A allele were significantly associated with an increased risk of the development of ACI in male Han Chinese, even after adjusting for the conventional risk factors of stroke. Our findings support the notion that the AA genotype and A allele of the −403G/A in the RANTES promoter may be the risk factors for ACI in males. Thus, polymorphisms associated with ACI are different between women and men [19]. However, our data were in disagreement with other findings [20]. The different results may be attributed to the variables in ethnic populations, the methodological approaches, sample size, and patient selection. RANTES has been implicated in the inflammatory process of atherosclerosis and other cardiovascular diseases. Evidentially, the expression of RANTES is detected in atherosclerotic coronary arteries, but not in normal vessels [27], and the transcription of RANTES gene is markedly up-regulated within symptomatic atherosclerotic carotid plaque [28]. Although the association of serum RANTES levels with CAD remains controversial [29,30], the − 403A variant in the RANTES promoter has been demonstrated to up-regulate the expression of RANTES [14]. Given that RANTES is a strong chemokine and can recruit inflammatory leukocytes into inflammatory sites, the significantly up-regulated production of RANTES in individuals with the − 403A allele may promote the inflammatory process and the development of atherosclerosis and ACI. Indeed, mice deficient in the RANTES gene display significantly fewer T lymphocyte and monocyte infiltrates in inflammatory sites [31]. Furthermore, treatment with the RANTES antagonist Met-RANTES inhibits the arterial injury-mediated monocyte migration to carotid endothelium, neointima formation, and macrophage accumulation as well as atherosclerosis progression in uninjured arteries in apolipoprotein E-deficient mice [10,11,32]. Therefore, RANTES plays an important role in the development of atherosclerosis, potentially explaining the association between RANTES −403A and cardiovascular diseases. In summary, our data indicated that the −403AA genotype and A allele were significantly associated with an increased risk for the development of ACI in male Han Chinese. To the best of our knowledge, this is the first report on the SNPs of the RANTES promoter associated with ACI in Northern male Han Chinese. However, our study has limitations, such as a relatively small sample size, the lack of RANTES and other inflammatory marker (such as C-Reactive Protein, IL-6) measurements, and difference of many confounders between these two groups as well as the lack of a group of perfectly matched health controls in the current study. Therefore, further exploration of the current observations with more detailed genotyping, expression, and translational studies in a bigger population including optimal control subjects is warranted. Conceivably, after confirmation of its significance, the SNP may be used for the predisposition to ACI in Han Chinese. Acknowledgement This study was supported by a grant from the National Natural Science Foundation of China (30971018). References [1] Johnston SC, Mendis S, Mathers CD. Global variation in stroke burden and mortality: estimates from monitoring, surveillance, and modelling. Lancet Neurol 2009;8:345–54.
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