GENE-39876; No. of pages: 5; 4C: Gene xxx (2014) xxx–xxx
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Article history: Received 25 March 2014 Received in revised form 1 August 2014 Accepted 5 August 2014 Available online xxxx
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Keywords: Coronary artery disease Heat shock protein70-2 HSP70-2 gene +1267ANG polymorphism PCR-RFLP
Cardiovascular Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Division of Applied Medicine, Medical School, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK c Department of Modern Sciences & Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran d Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran e Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran f Department of Biochemistry, Golestan University, Gorgan, Iran g Health Sciences Research Center, Department of Health and Management, School of Health, Mashhad University of Medical Sciences, Mashhad, Iran h Biochemistry of Nutrition Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran i Brighton & Sussex Medical School, Division of Medical Education, Falmer, Brighton, Sussex BN1 9PH, UK
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Background: Coronary artery disease (CAD) is an inflammatory process and a major cause of mortality and morbidity. The (heat shock protein70-2) HSP70-2 gene is reported to be associated with coronary artery disease possibly by affecting the regulation of pro-inflammatory cytokines such as TNF-α. The association between CAD and the HSP70-2 gene +1267ANG polymorphism has been studied in some population but there are no data about this association in the Iranian population. Aim: We have investigated the association between the HSP70-2 gene +1267ANG polymorphism and angiographically defined CAD within an Iranian population. Methods: We determined the presence of the HSP70-2 gene +1267ANG polymorphism in 628 patients with CAD and 307 healthy individuals using PCR-RFLP. Of the patients, 433 (68%) had N50% stenosis (CAD+) and the remaining 195 patients had b50% stenosis (CAD−), based on coronary angiography. Angiogram positive patients were subdivided into three groups: those with single (n = 113), double (n = 134), and triple vessels (n = 186) disease. Results: A significant higher frequency of AG + GG genotypes (G allele carriers) was observed in angiogram positive and angiogram negative groups compared to controls in a dominant analysis model of the HSP70-2 gene +1267ANG position (51.2 vs. 43.2, P = 0.002, OR = 1.37) (51.0 vs. 43.2, P = 0.01, OR = 1.37). The allele frequency of the HSP70-2 G was also significantly higher in angiogram positive and angiogram negative groups compared to the control group (51.2 vs. 43.2, P = 0.002, OR = 1.37) (51.0 vs. 43.2, P = 0.01, OR = 1.37). Conclusion: These results suggest that HSP70-2 +1267 polymorphism may influence the risk of CAD in Iranian population, however further studies are needed to clarify the role of other HSP70-2 gene polymorphisms in the pathogenesis of the CAD. © 2014 Published by Elsevier B.V.
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Maryam Mardan-Nik a,1, Alireza Pasdar b,c,1, Khadijeh Jamialahmadi d,e,1, Atefeh Biabangard-Zak f, Seyed Reza Mirhafez a, Marzieh Ghalandari a, Mohammad Tajfard g, Mohsen Mohebati a, Habibollah Esmaeili h, Gordon A. Ferns i, Majid Ghayour-Mobarhan a,h,⁎
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Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population
Abbreviations: CAD, coronary artery disease; HSP70-2, heat shock protein70-2; TNF-α, tumor necrosis factor-α; RFLP, restriction fragment length polymorphism; OR, odd ratio; P, p value; HSP, heat shock proteins; MHC, major histocompatibility class; BMI, body mass index; FBG, fasting blood glucose; dNTPs, deoxynucleotide triphosphate; SPSS, statistical package for social science; WC, waist circumference; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; HC, hip circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; MI, myocardial infarction; MUMS, Mashhad University of Medical Sciences; SVD, single vascular disease; 2VD, two vascular disease; 3VD, three vascular disease; CI, confidence interval; MetS, metabolic syndrome. ⁎ Corresponding author at: Biochemistry of Nutrition Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad 99199-91766, Iran. E-mail address: [email protected] (M. Ghayour-Mobarhan). 1 These authors contribute equally.
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1. Introduction Coronary artery disease (CAD) is the most common form of cardiovascular disease and one of the major causes of mortality and morbidity (Go et al., 2013; Lozano, 2013). CAD seems to remain as the most important cause of mortality in the world until 2020 (Hatmi et al., 2007; Murray and Lopez, 1997). The prevalence of CAD and risk factors for CAD is high in Iran (Ebrahimi et al., 2011). Therefore identifying those individuals at high risk for coronary diseases is important. The risk of CAD can be partially evaluated through the ascertainment of the risk factor profile, that includes family history, hyperlipidemia, excessive weight, hypertension and smoking (Ebrahimi et al., 2011). Among
http://dx.doi.org/10.1016/j.gene.2014.08.012 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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t1:3
Characteristics
Control (n = 307)
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22
Age, year Gender, no. (%) Male BMI (kg/m2) Weight (kg) WC (cm) Height (cm) TC (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) FBG (mg/dl) HC (cm) Waist/hip ratio SBP (mm Hg) DBP (mm Hg) MI, no. (%) Hypertension, no. (%) Diabetes, no. (%)
t1:23 t1:24 t1:25 t1:26 t1:27
Angio− (n = 195)
Comparison between the groups P1
P2
P3
52.2 ± 8.6 232 (77.9)
58.7 ± 10 271 (63.0)
53.2 ± 12 66 (34.2)
b0.001 b0.001
0.50 b0.001
b0.001 b0.001
28.8 ± 4.6 73.8 ± 12.1 97.9 ± 11.8 160 (14) 193 (64) 122 (71) 43 (11) 119 (38) 82 (20) 104 (12) 0.9 (0.1) 122 (28) 80 (9) – 52 (19.0) 20 (9.2)
26.6 ± 4.7 69.7 ± 14.4 92.2 ± 13.2 162 (15) 165 (85) 127 (92) 39 (15) 96 (50) 103 (53) 95 (12) 0.9 (0.09) 140 (40) 80 (17) 121 (28.9) 208 (49.3) 125 (29.6)
26.7 ± 5.5 67.9 ± 14.5 90.4 ± 13.9 158 (12) 159 (61) 114 (75) 43 (17) 86 (40) 96 (24) 97 (15) 0.9 (0.1) 130 (40) 70 (10) 18 (9.5) 80 (41.7) 33 (17.2)
b0.001 b0.001 b0.001 0.08 b0.001 0.05 0.002 b0.001 b0.001 b0.001 b0.001 b0.001 0.001 – 0.001 b0.001
b0.001 b0.001 b0.001 0.50 b0.001 0.20 0.70 b0.001 b0.001 b0.001 0.40 b0.001 b0.001 – b0.001 0.01
0.90 0.20 0.20 0.01 0.30 0.006 0.006 0.10 b0.001 0.20 b0.001 0.20 0.03 – 0.07 b0.001
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Angio+ (n = 433)
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Table 1 Characteristics of Angio+, Angio− and control subjects.
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expression in human endothelial and smooth muscle cells has been documented in response to the oxidized LDL in vitro (Zhu et al., 1994, 1995). Overexpression of HSP70 in advanced atherosclerotic lesions occurs in several cell types including macrophages, dendritic cells and smooth muscle cells, which leads to expression of pro-inflammatory cytokines from macrophages (Bobryshev and Lord, 2002). Expression of cytokines such as TNF-α from atheromas may stimulate the innate immune response. Induction of HSP70 prevents NF- B activation and leads to reduction of inflammatory cytokine activity. This pathway partly reflects the anti-inflammatory activity of HSP70 (Shimizu et al., 2002). High levels of HSP70 expression lead to cardiac cell protection from stressful damage by binding to denatured or inappropriately folded proteins (Snoeckx et al., 2001; Xu, 2002). Increased serum levels of HSP70 are associated with reduced atherosclerotic intima thickness and risk of coronary artery disease (Neschis et al., 1998; Zhu et al., 2003). Previous studies suggested that the HSPA1B + 1267 allele G was associated with coronary artery disease (Giacconi et al., 2006). Another study suggests that HSP70-2 gene + 1267ANG polymorphism is associated with diabetic nephropathy (Buraczynska et al., 2009).
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these, family history is an important factor because genetics may play a substantial role in the development of CAD. Study of genetic variants in human and animal models has so far shown the role of more than 100 genes involved in the development of atherosclerotic plaques (Lusis et al., 2004). HSP70-2 is one of the strong candidate genes for the evaluation of coronary artery disease risk. HSP70-2 gene is a member of multigene HSP70 family. Heat shock proteins (HSPs) are expressed in almost all cells and organisms from bacteria to humans, in response to a variety of different stress stimuli including heat, cold, heavy metals, inflammatory cytokines, oxidized LDL and hypoxia. The expression of these proteins might be constitutive or inducible (Kiang and Tsokos, 1998; Whitley et al., 1999). In humans, three genes encode members of the HSP70 class including HSP70-1 (HSPA1A), HSP70-2 (HSPA1B) and HSP70-hom (HSPA1L) (Milner and Campbell, 1990; Sargent et al., 1989). All three heat shock protein70 (HSP70) genes are located within the MHC class III region (6p21.3) (Milner and Campbell, 1992). Antibody titers related to HSP70 is shown to be associated with coronary risk factors, increased risk and severity of cardiovascular disease (Ghayour-Mobarhan et al., 2008). HSP70
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Fig. 1. PCR-RFLP and sequence of the amplified segment in HSP70-2 gene +1267ANG. The genotype was labeled on corresponding sequences, and the sites which were marked with black arrows were the SNP of HSP70-2 gene electrophoresed on 1.5% agarose, stained with ethidium bromide. Lane 2, 100 bp DNA ladder. Lane 3, heterozygote, Lane 4 homozygote GG and Lane 1 homozygote AA for HSP70-2 genotype.
Values are expressed as mean ± SD, median and interquartile range for normally and non-normally distributed variables, respectively. Comparisons were performed by one-way ANOVA and Kruskal–Wallis test. Also the post hoc test and Mann–Whitney U test were used for comparison between groups. Χ2 of test results for categorical data. BMI: body mass index; WC: waist circumference, TC: total cholesterol; TG: triglycerides; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; FBG: fasting blood glucose; HC: hip circumference, SBP: systolic blood pressure; DBP: diastolic blood pressure. MI: myocardial infarction. P1: comparison between groups of Angio+ and control, P2: comparison between groups of Angio− and control, P3: comparison between groups of Angio+ and Angio−.
Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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Table 2 Genotype and allele frequencies of the HSP70-2 gene + 1267ANG polymorphism in Angio+, Angio− and control subjects.
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Genotypes
Angio+ (n = 433) N (%) Angio− (n = 195) N (%) Control (n = 307) N (%) P-value
Alleles
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AG
GG
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45(10.4)
333(76.9)
55(12.7)
423(48.8)
443(51.2)
20(10.3)
151(77.4)
24(12.3)
191(49.0)
199(51.0)
91(29.6)
167(54.4)
49(16.0)
349(56.8)
265(43.2)
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0.005
5. Genetic analysis
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Peripheral blood was obtained from the subjects, and the genomic DNA was extracted using commercial kit (Genet bio, Korea). Quality DNA concentration (ng/μl) was measured by spectrophotometry (Nano Drop 1000, Thermo Scientific). Genotyping of HSP70-2 +1267 polymorphism was performed by PCR-FFLP technique. The sequence of forward and reverse primers used for amplification of the A1267G region was 5′CATCGACTTCTACACGTCC-3′ and 5′-CGGAGTAGGTGGTGAAGATC-3′ respectively. The PCR was performed in 25 μl final volume, using 100 ng of genomic DNA, 0.2 mM dNTPs, 2 mM MgCl2, 1 × Taq DNA polymerase buffer, 0.32 pmol of each primer and 1 unit of Taq DNA polymerase (Genet bio, Korea). The amplification conditions for PCR were 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 61 °C for 30 s, DNA extension at 72 °C for 1 min and the final extension at 72 °C for 7 min. All amplification cycles were performed in PCR system Verity 96 well thermocycler (Applied Biosystems, USA). 10 μl of each PCR product was then digested for 16 h at 37 °C with 1 μl of PstI (Fermentas, Lithuania). The DNA lacking polymorphic PstI site within the HSP70-2 gene produced a fragment of 428 bp (A allele), whereas the presence of 1267 G allele generated two fragments of 247 and 181 bp after digestion. Digested products were identified by 1.5% agarose gel electrophoresis with ethidium bromide staining and visualized by UV light. Finally, a direct sequencing approach (Company of Sequetech, USA) was used to confirm the genotypes obtained by PCR-RFLP for some of the subjects in different groups (Fig. 1).
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Comparisons are performed using χ2 test.
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In addition, a high frequency of GG-HSP70-2 genotype has been found in obese Tunisians which suggests that HSP70-2 gene polymorphism might have susceptibility implications in both obesity and diabetes (ZouariBouassida et al., 2004). For this current study, we evaluated the genotypic frequency of HSP70-2 gene + 1267ANG polymorphism in patients with CAD in an Iranian population.
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2. Study population
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Patients were selected from those subjects who underwent coronary angiography in the Ghaem Hospital Medical Center, Mashhad, Iran. Prior to recruitment, all participants were asked to complete the consent form which was approved by the Mashhad University of Medical Sciences (MUMS) Ethics Committee. The angiograms were interpreted by a cardiologist. The study group consisted of 935 subjects (61.8% males and 38.2% females), of whom 628 were patients and 307 were healthy subjects without evidence of CAD. 433 patients had more than 50% stenosis (68%) and 195 patients had less than 50% stenosis (31.1%), (age ranged from 19 to 89). Individuals with artery stenosis ≥50% (left main, right coronary artery, left anterior descending, circumflex) were selected as the significant CAD (CAD+) and artery stenosis less than 50% were considered as angiogram negative (CAD−). In addition to this classification, CAD patients were further classified into 3 groups according to the number of vessels with stenosis ≥50% as 1-vessel (n = 113, 26.1%), 2-vessel (n = 134, 30.9%) and 3-vessel (n = 186, 43.0%). Pregnant and lactating women, patients who had SLE (Systemic Lupus Erythematosus), renal disease, and those who had a history of past angiography were excluded from the study.
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3. Anthropometric measurements
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Anthropometric parameters of individuals including weight, height, BMI, waist circumference, hip circumference and waist/hip ratio as well as systolic and diastolic blood pressures were measured as previously described (Ghayour-Mobarhan et al., 2008).
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4. Biochemical analysis
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A full fasted lipid profile was determined for each subject. Serum lipids and fasting blood glucose (FBG) concentrations were measured by enzymatic methods as previously described (Ghayour-Mobarhan et al., 2008).
t3:1 t3:2 Q3
Table 3 Dominant analysis model of HSP70-2 gene +1267ANG polymorphism in angiogram positive and control groups.
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AA, no. (%) AG − GG, no. (%)
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6. Statistical analyses
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Statistical analyses were conducted using the SPSS for Windows™, version 16 software package (SPSS Inc., Chicago, Illinois). Normal distribution was tested using Kolmogorov–Smirnov test. Continuous variables were expressed as mean ± standard deviation (SD) for parameters with normal distribution and median and interquartile range (IQ3–IQ1) for data with non-normal distribution. Categorical variables were assessed by the χ2 test. One-way ANOVA (Tukey as post hoc test) was applied to compare the differences in the mean across groups. Kruskal–Wallis analysis of median test was used followed by the Mann–Whitney U test for pairwise comparisons. Differences in the alleles and genotype frequencies of the HSP70-2 gene + 1267ANG polymorphism between the patients and controls were calculated using a Chi-square test. Hardy–Weinberg equilibrium at each polymorphism was examined by χ2 test. Binary logistic regression was also used to adjust for confounders. A 2-sided P b 0.05 was considered significant.
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7. Results
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7.1. Demographics
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Comparisons of anthropometric and metabolic characteristics of the subjects are shown in Table 1. 54.1% of subjects who underwent angiography were males. Among those who had angiography, 37% of females and 63% of males, had N 50% artery stenosis. There was a significant difference in the mean age of angiogram positive compared to the controls (58.7 ± 10 vs. 52.2 ± 8.6). The mean BMI (body mass index), LDL (lowdensity lipoprotein cholesterol), TC (total cholesterol) and weight were
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Angio+ (n = 433)
Control (n = 307)
P-value
OR (95% CI)
Adj.a P-value
Adj. OR (95% CI)
45(10.4) 388(89.6)
91(29.6) 216(70.4)
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3.61(2.450–5.387)
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3.64 (2.026–5.924)
CI: confidence interval, OR: odds ratio. a Adjusted for age, sex, BMI (body mass index), LDL (low-density lipoprotein cholesterol), smoking and MetS-IDF (metabolic syndrome).
Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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Table 4 Dominant analysis model of HSP70-2 gene +1267ANG polymorphism in angiogram negative and control groups.
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AA, no. (%) AG − GG, no. (%)
Angio− (n = 195)
Control (n = 307)
P-value
OR (95% CI)
Adj.a P-value
Adj. OR (95% CI)
20(10.3) 175(89.7)
91(29.6) 216(70.4)
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3.68(2.184–6.22)
0.010
2.62(1.236–5.563)
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CI: Confidence interval, OR: odds ratio. a Adjusted for age, sex, BMI (body mass index), LDL (low-density lipoprotein cholesterol), smoking and MetS-IDF (metabolic syndrome).
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significantly different in angiogram positive and in angiogram negative compared to controls. Significant difference in FBG (fasting blood glucose) and diastolic blood pressure (DBP) was observed between the groups. Significant difference in systolic blood pressure (SBP) was also observed in angiogram positive and in angiogram negative compared to the controls. The HDL (high-density lipoprotein cholesterol) and TG (triglycerides) were significantly different in the angiogram positive group compared to angiogram negative. In addition, hypertension and diabetes mellitus were significantly higher in the patient group compared to the controls.
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7.2. Genotypes
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7.2.1. HSP70-2 gene +1267ANG polymorphism and baseline characteristics The analyses of anthropometric and metabolic characteristics in relation to HSP70-2 genotypes demonstrated that the mean BMI in controls with the GG genotype was slightly higher than individual with AA genotype (30 ± 4 vs. 27 ± 4, p = 0.01). Individuals with GG genotype had also a higher systolic blood pressure (SBP) compared to AA genotype in negative-angiograph group (p = 0.04). However, the results indicated no significant difference between the other characteristics and genotypes in both control and patient groups.
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8. Discussion
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Chronic inflammation plays a major role in the initiation and progression of coronary artery disease (Hansson, 2005). Coronary artery disease is the major cause of morbidity and mortality in the world
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7.2.2. Association between HSP70-2 gene +1267ANG polymorphism and CAD Table 2 shows the distribution of genotypes and alleles for HSP70-2 gene +1267ANG polymorphism in the angiogram positive patient, angiogram negative and control groups. The genotype distribution was not consistent with the Hardy–Weinberg equilibrium in the patients (P-value b 0.05). To ensure that samples were correctly genotyped, 10% of samples were regenotyped and no technical issues were found. The distribution of HSP70-2 gene +1267A/G genotypes showed a significant difference between all groups (X2 = 62.9; df = 4; P b 0.01). An increased frequency of the G allele was observed in angiogram positive (51.2 vs. 43.2) (P = 0. 002, OR = 1.37; 95% CI 1.120–1.698) and angiogram negative (51.0 vs. 43.2) (P = 0. 01, OR = 1.37; 95% CI 1.064– 1.770) compared to the control group. In a dominant analysis model of the HSP70-2 gene +1267A/G position (AA vs. AG + GG), the percentage of subjects who were either homo or heterozygous for the G allele (1267AG and 1267GG) was significantly higher in angiogram positive than controls (89.6 vs. 70.4) (Adj. P b 0.001, Adj. OR = 3.64; 95% CI 2.026–5.924). This was also observed in angiogram negative (89.7 vs. 70.4) (Adj. P = 0. 01, Adj. OR = 2.62; 95% CI 1.236–5.563) (Tables 3 and 4). In addition, there was a significant difference in the frequency of the HSP70-2 +1267 genotypes, when a dominant analysis model was applied (AA vs. AG + GG) between SVD (89.4 vs. 70.4) (Adj. P = 0.001, Adj. OR = 4.47; 95% CI 1.882– 10.649), 2VD (94.0 vs. 70.4) (Adj. P b 0.001, Adj. OR = 7.63; 95% CI 2.839–20.503) and 3VD (86.6 vs. 70.4) (Adj. P = 0.007, Adj. OR = 2.46; 95% CI 1.283–4.722) compared to controls.
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including Iran (Hatmi et al., 2007). In this study, our patients were cases who were referred to the hospital due to CAD symptoms and underwent coronary angiography where 63% males and 37% females had artery stenosis above 50%. This figure is consistent with other data indicating that males are more prone to cardiovascular disease and may develop it earlier in their life compared to females. In our study there was no significant relationship between lipid profile and this polymorphism, although it is acknowledged that this could be due to antilipid treatments for some of the patients. Gombos et al. suggested that HSP70 level in chronic heart failure patients is associated with HSP70-2 gene +1267ANG polymorphism and the disease severity (Gombos et al., 2008). A recent report by Bouassida et al. also found that HSP70-2 polymorphism is associated with an increased risk of type 2 diabetes in Tunisians (ZouariBouassida et al., 2004). Similar study in stroke patients by Wie et al. showed that there were no significant differences in +1267A/G allele or genotype frequencies between patients with stroke and healthy controls (Wei et al., 2012). Our results showed that G allele frequency of HSP70-2 was significantly higher in angiogram positive and angiogram negative compared to controls. In addition, dominant model analysis revealed that the AG + GG genotype was also significantly higher in individuals with angiogram positive and angiogram negative than controls. Other studies indicated a significant increase of GG genotype in the patients with CAD compared to controls (Giacconi et al., 2006; Hrira et al., 2012). Possible reasons for such differences may be related to different environmental risk factors, patient selection criteria and other possible genetic factors. In conclusion, our present results may suggest an association between HSP70-2 gene +1267ANG polymorphism and CAD in an Iranian population, however further studies are needed to clarify the role of other HSP70-2 polymorphisms in the pathogenesis of the CAD. Functional studies are also needed to elucidate the involvement of HSP70-2 gene in coronary artery disease.
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The authors declare that there is no conflict of interests.
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Acknowledgments
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The authors frankly thank all of the patients and their family members who volunteered to participate in this study. We are also particularly grateful to the Research Council of the Mashhad University of Medical Sciences (MUMS) for the financial support of the study.
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Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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Go, A.S., et al., 2013. Heart disease and stroke statistics—2013 update: a report from the American Heart Association. Circulation 127 (1), e6. Gombos, T., et al., 2008. Interaction of serum 70-kDa heat shock protein levels and HspA1B (+1267) gene polymorphism with disease severity in patients with chronic heart failure. Cell Stress Chaperones 13 (2), 199–206. Hansson, G.K., 2005. Inflammation, atherosclerosis, and coronary artery disease. N. Engl. J. Med. 352 (16), 1685–1695. Hatmi, Z., et al., 2007. Prevalence of coronary artery disease risk factors in Iran: a population based survey. BMC Cardiovasc. Disord. 7 (1), 32. Hrira, M.Y., et al., 2012. Hsp70-2 gene polymorphism: susceptibility implication in Tunisian patients with coronary artery disease. Diagn. Pathol. 7, 88. Kiang, J.G.,Tsokos, G.C., 1998. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol. Ther. 80 (2), 183–201. Lozano, R., 2013. The world Health Report 2013: a path for research on universal health coverage. Salud Publica Mex. 55 (5), 536–537. Lusis, A.J.,Fogelman, A.M.,Fonarow, G.C., 2004. Genetic basis of atherosclerosis: part I new genes and pathways. Circulation 110 (13), 1868–1873. Milner, C.M., Campbell, R.D., 1990. Structure and expression of the three MHC-linked HSP70 genes. Immunogenetics 32 (4), 242–251. Milner, C.M., Campbell, R.D., 1992. Polymorphic analysis of the three MHC-linked HSP70 genes. Immunogenetics 36 (6), 357–362. Murray, C.J., Lopez, A.D., 1997. Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study. Lancet 349 (9064), 1498–1504. Neschis, D.G., et al., 1998. Thermal preconditioning before rat arterial balloon injury limitation of injury and sustained reduction of intimal thickening. Arterioscler. Thromb. Vasc. Biol. 18 (1), 120–126.
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Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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Article history: Received 25 March 2014 Received in revised form 1 August 2014 Accepted 5 August 2014 Available online xxxx
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Keywords: Coronary artery disease Heat shock protein70-2 HSP70-2 gene +1267ANG polymorphism PCR-RFLP
Cardiovascular Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Division of Applied Medicine, Medical School, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK c Department of Modern Sciences & Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran d Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran e Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran f Department of Biochemistry, Golestan University, Gorgan, Iran g Health Sciences Research Center, Department of Health and Management, School of Health, Mashhad University of Medical Sciences, Mashhad, Iran h Biochemistry of Nutrition Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran i Brighton & Sussex Medical School, Division of Medical Education, Falmer, Brighton, Sussex BN1 9PH, UK
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Background: Coronary artery disease (CAD) is an inflammatory process and a major cause of mortality and morbidity. The (heat shock protein70-2) HSP70-2 gene is reported to be associated with coronary artery disease possibly by affecting the regulation of pro-inflammatory cytokines such as TNF-α. The association between CAD and the HSP70-2 gene +1267ANG polymorphism has been studied in some population but there are no data about this association in the Iranian population. Aim: We have investigated the association between the HSP70-2 gene +1267ANG polymorphism and angiographically defined CAD within an Iranian population. Methods: We determined the presence of the HSP70-2 gene +1267ANG polymorphism in 628 patients with CAD and 307 healthy individuals using PCR-RFLP. Of the patients, 433 (68%) had N50% stenosis (CAD+) and the remaining 195 patients had b50% stenosis (CAD−), based on coronary angiography. Angiogram positive patients were subdivided into three groups: those with single (n = 113), double (n = 134), and triple vessels (n = 186) disease. Results: A significant higher frequency of AG + GG genotypes (G allele carriers) was observed in angiogram positive and angiogram negative groups compared to controls in a dominant analysis model of the HSP70-2 gene +1267ANG position (51.2 vs. 43.2, P = 0.002, OR = 1.37) (51.0 vs. 43.2, P = 0.01, OR = 1.37). The allele frequency of the HSP70-2 G was also significantly higher in angiogram positive and angiogram negative groups compared to the control group (51.2 vs. 43.2, P = 0.002, OR = 1.37) (51.0 vs. 43.2, P = 0.01, OR = 1.37). Conclusion: These results suggest that HSP70-2 +1267 polymorphism may influence the risk of CAD in Iranian population, however further studies are needed to clarify the role of other HSP70-2 gene polymorphisms in the pathogenesis of the CAD. © 2014 Published by Elsevier B.V.
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Maryam Mardan-Nik a,1, Alireza Pasdar b,c,1, Khadijeh Jamialahmadi d,e,1, Atefeh Biabangard-Zak f, Seyed Reza Mirhafez a, Marzieh Ghalandari a, Mohammad Tajfard g, Mohsen Mohebati a, Habibollah Esmaeili h, Gordon A. Ferns i, Majid Ghayour-Mobarhan a,h,⁎
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Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population
Abbreviations: CAD, coronary artery disease; HSP70-2, heat shock protein70-2; TNF-α, tumor necrosis factor-α; RFLP, restriction fragment length polymorphism; OR, odd ratio; P, p value; HSP, heat shock proteins; MHC, major histocompatibility class; BMI, body mass index; FBG, fasting blood glucose; dNTPs, deoxynucleotide triphosphate; SPSS, statistical package for social science; WC, waist circumference; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; HC, hip circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; MI, myocardial infarction; MUMS, Mashhad University of Medical Sciences; SVD, single vascular disease; 2VD, two vascular disease; 3VD, three vascular disease; CI, confidence interval; MetS, metabolic syndrome. ⁎ Corresponding author at: Biochemistry of Nutrition Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad 99199-91766, Iran. E-mail address: [email protected] (M. Ghayour-Mobarhan). 1 These authors contribute equally.
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1. Introduction Coronary artery disease (CAD) is the most common form of cardiovascular disease and one of the major causes of mortality and morbidity (Go et al., 2013; Lozano, 2013). CAD seems to remain as the most important cause of mortality in the world until 2020 (Hatmi et al., 2007; Murray and Lopez, 1997). The prevalence of CAD and risk factors for CAD is high in Iran (Ebrahimi et al., 2011). Therefore identifying those individuals at high risk for coronary diseases is important. The risk of CAD can be partially evaluated through the ascertainment of the risk factor profile, that includes family history, hyperlipidemia, excessive weight, hypertension and smoking (Ebrahimi et al., 2011). Among
http://dx.doi.org/10.1016/j.gene.2014.08.012 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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t1:3
Characteristics
Control (n = 307)
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22
Age, year Gender, no. (%) Male BMI (kg/m2) Weight (kg) WC (cm) Height (cm) TC (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) FBG (mg/dl) HC (cm) Waist/hip ratio SBP (mm Hg) DBP (mm Hg) MI, no. (%) Hypertension, no. (%) Diabetes, no. (%)
t1:23 t1:24 t1:25 t1:26 t1:27
Angio− (n = 195)
Comparison between the groups P1
P2
P3
52.2 ± 8.6 232 (77.9)
58.7 ± 10 271 (63.0)
53.2 ± 12 66 (34.2)
b0.001 b0.001
0.50 b0.001
b0.001 b0.001
28.8 ± 4.6 73.8 ± 12.1 97.9 ± 11.8 160 (14) 193 (64) 122 (71) 43 (11) 119 (38) 82 (20) 104 (12) 0.9 (0.1) 122 (28) 80 (9) – 52 (19.0) 20 (9.2)
26.6 ± 4.7 69.7 ± 14.4 92.2 ± 13.2 162 (15) 165 (85) 127 (92) 39 (15) 96 (50) 103 (53) 95 (12) 0.9 (0.09) 140 (40) 80 (17) 121 (28.9) 208 (49.3) 125 (29.6)
26.7 ± 5.5 67.9 ± 14.5 90.4 ± 13.9 158 (12) 159 (61) 114 (75) 43 (17) 86 (40) 96 (24) 97 (15) 0.9 (0.1) 130 (40) 70 (10) 18 (9.5) 80 (41.7) 33 (17.2)
b0.001 b0.001 b0.001 0.08 b0.001 0.05 0.002 b0.001 b0.001 b0.001 b0.001 b0.001 0.001 – 0.001 b0.001
b0.001 b0.001 b0.001 0.50 b0.001 0.20 0.70 b0.001 b0.001 b0.001 0.40 b0.001 b0.001 – b0.001 0.01
0.90 0.20 0.20 0.01 0.30 0.006 0.006 0.10 b0.001 0.20 b0.001 0.20 0.03 – 0.07 b0.001
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Angio+ (n = 433)
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70 71
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Table 1 Characteristics of Angio+, Angio− and control subjects.
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expression in human endothelial and smooth muscle cells has been documented in response to the oxidized LDL in vitro (Zhu et al., 1994, 1995). Overexpression of HSP70 in advanced atherosclerotic lesions occurs in several cell types including macrophages, dendritic cells and smooth muscle cells, which leads to expression of pro-inflammatory cytokines from macrophages (Bobryshev and Lord, 2002). Expression of cytokines such as TNF-α from atheromas may stimulate the innate immune response. Induction of HSP70 prevents NF- B activation and leads to reduction of inflammatory cytokine activity. This pathway partly reflects the anti-inflammatory activity of HSP70 (Shimizu et al., 2002). High levels of HSP70 expression lead to cardiac cell protection from stressful damage by binding to denatured or inappropriately folded proteins (Snoeckx et al., 2001; Xu, 2002). Increased serum levels of HSP70 are associated with reduced atherosclerotic intima thickness and risk of coronary artery disease (Neschis et al., 1998; Zhu et al., 2003). Previous studies suggested that the HSPA1B + 1267 allele G was associated with coronary artery disease (Giacconi et al., 2006). Another study suggests that HSP70-2 gene + 1267ANG polymorphism is associated with diabetic nephropathy (Buraczynska et al., 2009).
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these, family history is an important factor because genetics may play a substantial role in the development of CAD. Study of genetic variants in human and animal models has so far shown the role of more than 100 genes involved in the development of atherosclerotic plaques (Lusis et al., 2004). HSP70-2 is one of the strong candidate genes for the evaluation of coronary artery disease risk. HSP70-2 gene is a member of multigene HSP70 family. Heat shock proteins (HSPs) are expressed in almost all cells and organisms from bacteria to humans, in response to a variety of different stress stimuli including heat, cold, heavy metals, inflammatory cytokines, oxidized LDL and hypoxia. The expression of these proteins might be constitutive or inducible (Kiang and Tsokos, 1998; Whitley et al., 1999). In humans, three genes encode members of the HSP70 class including HSP70-1 (HSPA1A), HSP70-2 (HSPA1B) and HSP70-hom (HSPA1L) (Milner and Campbell, 1990; Sargent et al., 1989). All three heat shock protein70 (HSP70) genes are located within the MHC class III region (6p21.3) (Milner and Campbell, 1992). Antibody titers related to HSP70 is shown to be associated with coronary risk factors, increased risk and severity of cardiovascular disease (Ghayour-Mobarhan et al., 2008). HSP70
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Fig. 1. PCR-RFLP and sequence of the amplified segment in HSP70-2 gene +1267ANG. The genotype was labeled on corresponding sequences, and the sites which were marked with black arrows were the SNP of HSP70-2 gene electrophoresed on 1.5% agarose, stained with ethidium bromide. Lane 2, 100 bp DNA ladder. Lane 3, heterozygote, Lane 4 homozygote GG and Lane 1 homozygote AA for HSP70-2 genotype.
Values are expressed as mean ± SD, median and interquartile range for normally and non-normally distributed variables, respectively. Comparisons were performed by one-way ANOVA and Kruskal–Wallis test. Also the post hoc test and Mann–Whitney U test were used for comparison between groups. Χ2 of test results for categorical data. BMI: body mass index; WC: waist circumference, TC: total cholesterol; TG: triglycerides; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; FBG: fasting blood glucose; HC: hip circumference, SBP: systolic blood pressure; DBP: diastolic blood pressure. MI: myocardial infarction. P1: comparison between groups of Angio+ and control, P2: comparison between groups of Angio− and control, P3: comparison between groups of Angio+ and Angio−.
Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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Table 2 Genotype and allele frequencies of the HSP70-2 gene + 1267ANG polymorphism in Angio+, Angio− and control subjects.
t2:4
t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11
Genotypes
Angio+ (n = 433) N (%) Angio− (n = 195) N (%) Control (n = 307) N (%) P-value
Alleles
AA
AG
GG
A
G
45(10.4)
333(76.9)
55(12.7)
423(48.8)
443(51.2)
20(10.3)
151(77.4)
24(12.3)
191(49.0)
199(51.0)
91(29.6)
167(54.4)
49(16.0)
349(56.8)
265(43.2)
b0.001
0.005
5. Genetic analysis
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Peripheral blood was obtained from the subjects, and the genomic DNA was extracted using commercial kit (Genet bio, Korea). Quality DNA concentration (ng/μl) was measured by spectrophotometry (Nano Drop 1000, Thermo Scientific). Genotyping of HSP70-2 +1267 polymorphism was performed by PCR-FFLP technique. The sequence of forward and reverse primers used for amplification of the A1267G region was 5′CATCGACTTCTACACGTCC-3′ and 5′-CGGAGTAGGTGGTGAAGATC-3′ respectively. The PCR was performed in 25 μl final volume, using 100 ng of genomic DNA, 0.2 mM dNTPs, 2 mM MgCl2, 1 × Taq DNA polymerase buffer, 0.32 pmol of each primer and 1 unit of Taq DNA polymerase (Genet bio, Korea). The amplification conditions for PCR were 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 61 °C for 30 s, DNA extension at 72 °C for 1 min and the final extension at 72 °C for 7 min. All amplification cycles were performed in PCR system Verity 96 well thermocycler (Applied Biosystems, USA). 10 μl of each PCR product was then digested for 16 h at 37 °C with 1 μl of PstI (Fermentas, Lithuania). The DNA lacking polymorphic PstI site within the HSP70-2 gene produced a fragment of 428 bp (A allele), whereas the presence of 1267 G allele generated two fragments of 247 and 181 bp after digestion. Digested products were identified by 1.5% agarose gel electrophoresis with ethidium bromide staining and visualized by UV light. Finally, a direct sequencing approach (Company of Sequetech, USA) was used to confirm the genotypes obtained by PCR-RFLP for some of the subjects in different groups (Fig. 1).
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Comparisons are performed using χ2 test.
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In addition, a high frequency of GG-HSP70-2 genotype has been found in obese Tunisians which suggests that HSP70-2 gene polymorphism might have susceptibility implications in both obesity and diabetes (ZouariBouassida et al., 2004). For this current study, we evaluated the genotypic frequency of HSP70-2 gene + 1267ANG polymorphism in patients with CAD in an Iranian population.
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2. Study population
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Patients were selected from those subjects who underwent coronary angiography in the Ghaem Hospital Medical Center, Mashhad, Iran. Prior to recruitment, all participants were asked to complete the consent form which was approved by the Mashhad University of Medical Sciences (MUMS) Ethics Committee. The angiograms were interpreted by a cardiologist. The study group consisted of 935 subjects (61.8% males and 38.2% females), of whom 628 were patients and 307 were healthy subjects without evidence of CAD. 433 patients had more than 50% stenosis (68%) and 195 patients had less than 50% stenosis (31.1%), (age ranged from 19 to 89). Individuals with artery stenosis ≥50% (left main, right coronary artery, left anterior descending, circumflex) were selected as the significant CAD (CAD+) and artery stenosis less than 50% were considered as angiogram negative (CAD−). In addition to this classification, CAD patients were further classified into 3 groups according to the number of vessels with stenosis ≥50% as 1-vessel (n = 113, 26.1%), 2-vessel (n = 134, 30.9%) and 3-vessel (n = 186, 43.0%). Pregnant and lactating women, patients who had SLE (Systemic Lupus Erythematosus), renal disease, and those who had a history of past angiography were excluded from the study.
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3. Anthropometric measurements
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Anthropometric parameters of individuals including weight, height, BMI, waist circumference, hip circumference and waist/hip ratio as well as systolic and diastolic blood pressures were measured as previously described (Ghayour-Mobarhan et al., 2008).
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4. Biochemical analysis
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A full fasted lipid profile was determined for each subject. Serum lipids and fasting blood glucose (FBG) concentrations were measured by enzymatic methods as previously described (Ghayour-Mobarhan et al., 2008).
t3:1 t3:2 Q3
Table 3 Dominant analysis model of HSP70-2 gene +1267ANG polymorphism in angiogram positive and control groups.
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AA, no. (%) AG − GG, no. (%)
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6. Statistical analyses
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Statistical analyses were conducted using the SPSS for Windows™, version 16 software package (SPSS Inc., Chicago, Illinois). Normal distribution was tested using Kolmogorov–Smirnov test. Continuous variables were expressed as mean ± standard deviation (SD) for parameters with normal distribution and median and interquartile range (IQ3–IQ1) for data with non-normal distribution. Categorical variables were assessed by the χ2 test. One-way ANOVA (Tukey as post hoc test) was applied to compare the differences in the mean across groups. Kruskal–Wallis analysis of median test was used followed by the Mann–Whitney U test for pairwise comparisons. Differences in the alleles and genotype frequencies of the HSP70-2 gene + 1267ANG polymorphism between the patients and controls were calculated using a Chi-square test. Hardy–Weinberg equilibrium at each polymorphism was examined by χ2 test. Binary logistic regression was also used to adjust for confounders. A 2-sided P b 0.05 was considered significant.
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7. Results
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7.1. Demographics
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Comparisons of anthropometric and metabolic characteristics of the subjects are shown in Table 1. 54.1% of subjects who underwent angiography were males. Among those who had angiography, 37% of females and 63% of males, had N 50% artery stenosis. There was a significant difference in the mean age of angiogram positive compared to the controls (58.7 ± 10 vs. 52.2 ± 8.6). The mean BMI (body mass index), LDL (lowdensity lipoprotein cholesterol), TC (total cholesterol) and weight were
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Angio+ (n = 433)
Control (n = 307)
P-value
OR (95% CI)
Adj.a P-value
Adj. OR (95% CI)
45(10.4) 388(89.6)
91(29.6) 216(70.4)
1 b0.001
3.61(2.450–5.387)
b0.001
3.64 (2.026–5.924)
CI: confidence interval, OR: odds ratio. a Adjusted for age, sex, BMI (body mass index), LDL (low-density lipoprotein cholesterol), smoking and MetS-IDF (metabolic syndrome).
Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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Table 4 Dominant analysis model of HSP70-2 gene +1267ANG polymorphism in angiogram negative and control groups.
t4:3 t4:4 t4:5
AA, no. (%) AG − GG, no. (%)
Angio− (n = 195)
Control (n = 307)
P-value
OR (95% CI)
Adj.a P-value
Adj. OR (95% CI)
20(10.3) 175(89.7)
91(29.6) 216(70.4)
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3.68(2.184–6.22)
0.010
2.62(1.236–5.563)
t4:6 t4:7
CI: Confidence interval, OR: odds ratio. a Adjusted for age, sex, BMI (body mass index), LDL (low-density lipoprotein cholesterol), smoking and MetS-IDF (metabolic syndrome).
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significantly different in angiogram positive and in angiogram negative compared to controls. Significant difference in FBG (fasting blood glucose) and diastolic blood pressure (DBP) was observed between the groups. Significant difference in systolic blood pressure (SBP) was also observed in angiogram positive and in angiogram negative compared to the controls. The HDL (high-density lipoprotein cholesterol) and TG (triglycerides) were significantly different in the angiogram positive group compared to angiogram negative. In addition, hypertension and diabetes mellitus were significantly higher in the patient group compared to the controls.
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7.2. Genotypes
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7.2.1. HSP70-2 gene +1267ANG polymorphism and baseline characteristics The analyses of anthropometric and metabolic characteristics in relation to HSP70-2 genotypes demonstrated that the mean BMI in controls with the GG genotype was slightly higher than individual with AA genotype (30 ± 4 vs. 27 ± 4, p = 0.01). Individuals with GG genotype had also a higher systolic blood pressure (SBP) compared to AA genotype in negative-angiograph group (p = 0.04). However, the results indicated no significant difference between the other characteristics and genotypes in both control and patient groups.
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8. Discussion
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Chronic inflammation plays a major role in the initiation and progression of coronary artery disease (Hansson, 2005). Coronary artery disease is the major cause of morbidity and mortality in the world
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7.2.2. Association between HSP70-2 gene +1267ANG polymorphism and CAD Table 2 shows the distribution of genotypes and alleles for HSP70-2 gene +1267ANG polymorphism in the angiogram positive patient, angiogram negative and control groups. The genotype distribution was not consistent with the Hardy–Weinberg equilibrium in the patients (P-value b 0.05). To ensure that samples were correctly genotyped, 10% of samples were regenotyped and no technical issues were found. The distribution of HSP70-2 gene +1267A/G genotypes showed a significant difference between all groups (X2 = 62.9; df = 4; P b 0.01). An increased frequency of the G allele was observed in angiogram positive (51.2 vs. 43.2) (P = 0. 002, OR = 1.37; 95% CI 1.120–1.698) and angiogram negative (51.0 vs. 43.2) (P = 0. 01, OR = 1.37; 95% CI 1.064– 1.770) compared to the control group. In a dominant analysis model of the HSP70-2 gene +1267A/G position (AA vs. AG + GG), the percentage of subjects who were either homo or heterozygous for the G allele (1267AG and 1267GG) was significantly higher in angiogram positive than controls (89.6 vs. 70.4) (Adj. P b 0.001, Adj. OR = 3.64; 95% CI 2.026–5.924). This was also observed in angiogram negative (89.7 vs. 70.4) (Adj. P = 0. 01, Adj. OR = 2.62; 95% CI 1.236–5.563) (Tables 3 and 4). In addition, there was a significant difference in the frequency of the HSP70-2 +1267 genotypes, when a dominant analysis model was applied (AA vs. AG + GG) between SVD (89.4 vs. 70.4) (Adj. P = 0.001, Adj. OR = 4.47; 95% CI 1.882– 10.649), 2VD (94.0 vs. 70.4) (Adj. P b 0.001, Adj. OR = 7.63; 95% CI 2.839–20.503) and 3VD (86.6 vs. 70.4) (Adj. P = 0.007, Adj. OR = 2.46; 95% CI 1.283–4.722) compared to controls.
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including Iran (Hatmi et al., 2007). In this study, our patients were cases who were referred to the hospital due to CAD symptoms and underwent coronary angiography where 63% males and 37% females had artery stenosis above 50%. This figure is consistent with other data indicating that males are more prone to cardiovascular disease and may develop it earlier in their life compared to females. In our study there was no significant relationship between lipid profile and this polymorphism, although it is acknowledged that this could be due to antilipid treatments for some of the patients. Gombos et al. suggested that HSP70 level in chronic heart failure patients is associated with HSP70-2 gene +1267ANG polymorphism and the disease severity (Gombos et al., 2008). A recent report by Bouassida et al. also found that HSP70-2 polymorphism is associated with an increased risk of type 2 diabetes in Tunisians (ZouariBouassida et al., 2004). Similar study in stroke patients by Wie et al. showed that there were no significant differences in +1267A/G allele or genotype frequencies between patients with stroke and healthy controls (Wei et al., 2012). Our results showed that G allele frequency of HSP70-2 was significantly higher in angiogram positive and angiogram negative compared to controls. In addition, dominant model analysis revealed that the AG + GG genotype was also significantly higher in individuals with angiogram positive and angiogram negative than controls. Other studies indicated a significant increase of GG genotype in the patients with CAD compared to controls (Giacconi et al., 2006; Hrira et al., 2012). Possible reasons for such differences may be related to different environmental risk factors, patient selection criteria and other possible genetic factors. In conclusion, our present results may suggest an association between HSP70-2 gene +1267ANG polymorphism and CAD in an Iranian population, however further studies are needed to clarify the role of other HSP70-2 polymorphisms in the pathogenesis of the CAD. Functional studies are also needed to elucidate the involvement of HSP70-2 gene in coronary artery disease.
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The authors declare that there is no conflict of interests.
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Acknowledgments
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The authors frankly thank all of the patients and their family members who volunteered to participate in this study. We are also particularly grateful to the Research Council of the Mashhad University of Medical Sciences (MUMS) for the financial support of the study.
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Please cite this article as: Mardan-Nik, M., et al., Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.012
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