GENE PROMOTER 469 E/K POLYMORPHISM OF THE INTERCELLULAR ADHESION MOLECULE-1 GENE ASSOCIATION WITH AMI IN TYPE 2 DIABETIC PATIENTS

Transcription

1 GENE PROMOTER 469 E/K POLYMORPHISM OF THE INTERCELLULAR ADHESION MOLECULE-1 GENE ASSOCIATION WITH AMI IN TYPE 2 DIABETIC PATIENTS *#Seham A Khodeer, **#Azza M. Abdu-Allah, ***^Mohamed Al-Assal & ****^Hesham K Rashid, *Clinical Pathology, ** Medical Biochemistry & ***Internal Medicine &Cardiology**** Departments, Faculty of Medicine #Menoufiya&^ Benha University, Egypt Abstract Background: ICAM-1 has a vital responsibility in the adhesion of circulating leucocytes to the blood vessel wall and subsequent transendothelial migration to the vascular intima, one of the earliest stepladders in atherogenesis. Objective: To investigate the possible role of the K469E polymorphism of the ICAM- 1 in genetic susceptibility to MI amongst the patients with type 2 diabetes. Subjects &Methods: The study included 70 patients divided into two groups: Group I included 35 patients suffering from type 2 DM with MI, Group II included 35 patients with type 2 DM without CAD, 30 apparently healthy age and gender matched subjects were involved as a control group. For all subjects the followings were done: History taking & clinical examination, ECG, lipid profile, HbA1c, sicam-1 by ELISA. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method was used to determine the distribution of allele and genotype frequencies of the 469 E/K polymorphism of the ICAM-1. Results: There were high statistical significant between ICAM level of Group 1 (468±40.4 ng/ml) and both Group II (266.4±44.1 ng/ml) & the controls (150.6±29.7ng/ml p <0.001 for both). Also there was a higher statistical significant between ICAM-1 in Group II and the control (p<0.001). The distribution of the EE genotype and E allele frequency of Group 1 (42.9%, 64%) was higher than those of Group II (20%, 41.7%) & the healthy controls (16.7%, 41.2%). Conclusion: The K469E polymorphism of theicam-1 gene may be employed as a genetic indicator for MI in patients with T2DM. INTRODUCTION: Type 2 diabetes mellitus (T2DM) is one of the major risk factors for the development of CAD and subsequent (myocardial infarction) MI (12) Various inflammatory mediators such as tumor necrosis factor-α increase the expression of cell adhesion molecules including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1 and E-selectin on endothelial cells (3).Thus, upon inflammatory stimulation, the endothelial barrier function is rapidly lost and preformed P-selectin is translocated to the luminal surface of endothelial cells, followed by expression and release of E-selectin, ICAM-1, and VCAM-1 substances that regulate the attachment and trans-endothelial migration of leukocytes. Both macrophages and endothelial cells produce ICAM-1 in response to inflammatory cytokines (21). ICAM-1 is a transmembrane glycoprotein of 505 amino acids, with a molecular mass of kda (depending on the degree of glycosylation), and contains five immunoglobulin (Ig)-like domains (20). ICAM-1 has a vital responsibility in the adhesion of circulating leucocytes to the blood vessel wall and subsequent transendothelial migration to the vascular intima, one of the earliest stepladders in atherogenesis (17). It provokes adhesive interactions by binding 1

2 to two surface membrane β2 integrin molecules present on leucocytes, namely leucocyte function associated-antigen-1 and macrophage antigen-1 (4). The ICAM-1 gene is located on chromosome 19p13 and consists of seven exons (23). Several single-nucleotide polymorphisms of the human ICAM-1 gene have been reported, and functional analyses regarding the association between each polymorphism and certain diseases have been performed (24). Vora et al. (22) searched for polymorphisms in the exons of ICAM-1 and described single nucleotide polymorphism K469E, is an A G substitution at position 1548 in codon 469 in exon 6, causing a lysine (AAG) to glutamine (GAG) change in Ig-like domain 5.1 E469K is known to be common in all populations and has been analyzed for its association with several inflammatory diseases. The object of this study was to investigate the possible role of the K469E polymorphism of the ICAM-1 in genetic susceptibility to MI amongst the patients with T2DM. Subjects &Methods: The present study was carried out at Clinical Pathology Department in collaboration with Medical Biochemistery, Cardiology & Internal Medicine Departments, Faculty of Medicine, Menoufiya & Benha University Hospitals in the period between August 2009 & December The study included 70 patients (47 males, 23 females), their age ranged between years divided into two groups: Group I included 35 patients (24 males, 11 females) with T2DM patients with MI (diagnosis was based on the characteristic ECG findings and cardiac specific enzymes, using diagnostic criteria of the American College of Cardiologists), their ages ranged between years. Group II included 35 patients with T2DM with no history of coronary artery disease (23 males, 12 females) lasting more than 10 years, their ages ranged between years. In addition, 30 apparently healthy(group III ) with age a matched age & sex were involved as a control group (21 males, 9 females), their ages ranged between years. Patients with a history of other metabolic, autoimmune and malignancy diseases or with a family history of such illness were excluded from the study. For all the subjects the followings were done: History taking and clinical examination, ECG, lipid profile (total cholesterol, triglyceride, LDL-c& HDL-c) & The polymerase chain reaction restriction fragment length polymorphism (PCR- RFLP) method was used to determine the distribution of allele and genotype frequencies of the 469 E/K polymorphism of the intercellular adhesion molecule-1. Written informed-consents were provided by all participants. - Sampling: under complete aseptic conditions, 6 ml of venous blood were collected after 12 hour fasting & divided as follows: 2 ml of blood collected in citrate (to prevent clotting and DNA degradation) for DNA extraction and kept immediately at - 20 C. 3 ml were collected, left to clot; serum was divided into two aliquots, one kept immediately at -20 C for further determination of sicam and the other used for immediate assay of lipid profile. 1ml of blood collected in EDTA for determination HbA1c. - Laboratory Methods: - Total cholesterol, triglyceride, and HDL-c concentrations were determined by using an enzymatic colorimetric assay on Synchron *Cx9. LDL-c concentration was calculated according to the Friedewald formula (6). HbA1c was determined by column chromatography**. 2

3 -sicam-1 was measured by ELISA*** an anti-icam-1 monoclonal antibody is adsorbed onto microwells. Soluble ICAM-1 present in a sample or standard then binds to antibodies adsorbed to the microwells. A second, HRP-conjugated monoclonal anti-icam-1 antibody is added and binds to ICAM-1 captured by the first antibody. Unbound enzyme-conjugated anti-icam-1 is removed with a wash step and HRP substrate solution is added to the wells. An amount of colored product is formed, proportional to the amount of soluble ICAM-1 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from ICAM-1 standard dilutions and the ICAM-1 sample concentration is determined..-dna analysis: PCR-RFLP method was used to determine the distribution of allele and genotype frequencies of the 469 E/K polymorphism of the intercellular adhesion molecule-1. The DNA was extracted using commercially available Spin-column technique kit for DNA extraction from human whole blood (QIAamp DNA Blood Mini Kit) ****. The DNA concentration was determined by measuring the OD at 260 nm. The PCR was performed on 300 ng DNA in a total volume of 25 μl of the reaction mixture containing 2.5 μl of 10X PCR buffer, 0.75 μl of 10 μm dntps, 0.75 μl of 50 μm MgCl2, 1 μl of 20 ρm primers and 2 U of Taq DNA polymerase, forward and reverse primers: forward primers: 5'-GGTGAGATTGCATTAAGGTC-3' and the reverse primers 5'-GGAACCCATTGCCCGAGC3. PCR was carried out with an initial denaturation at 95 C for 7 min and 35 cycles of 95 C for 35 s, 57 C for 45 s, and 72 C for 45 s with a final extension of 5 min at 72 C. The PCR fragments (223 bp) *Beckman Instrument. Inc. Fullerton, California USA ** Stanbio laboratory, Inc East Houston Street. San Antonio, Texas Stanbio@netexpress.com. *** Invitrogen corporation, Carlsbad, California techsupport@invitrogen.com ****QIAGEN, Avenue Stanford, Valencia, CA 91355, USA were digested using the restriction enzyme FastDigest BstUI*****. The digested samples were separated by electrophoresis on 3% agarose gel stained with ethidium bromide and visualized on a UV trans-illuminator. This resulted in three genotypes identified: (i) EE was divided into two fragments (87 and 136 bp) (ii) KK not divided (223bp) (iii) heterozygous EK divided into three fragments (223, 87 and 136 bp) (fig. 1). Statistical analysis: The statistical analysis was undertaken using SPSS software (version 17; SPSS Inc., Chicago, IL, USA). Descriptive statistics in the form of mean and standard deviation for parametric data were used. Chi-square test (χ2) was used for two qualitative variables. ANOVA test for comparison between three or more groups having quantitative variables normally distributed followed by LSD (least significant difference). Kruskal-Wallis test for comparison between three or more groups not normally distributed having quantitative variables. Odd ratios (ORs) and 95% confidence intervals (CI) were calculated by logistic regression analysis. The significance level was set at 0.05 or less (18). Results: The results were presented in the following figures and tables: 3

4 Fig (1) : Agarose gel electrophorsis 3.0% stained with ethidium bromide showing the ICAM- 1 DNA product after digestion with BstUI. Lanes 1& 2: EE allelic polymorphism lane 3&5: KK allelic polymorphism Lanes 4&6: KK allelic polymorphism. *****Fermentas International Inc., Canada. Table (1): Sociodemographic criteria & clinical parameters in the studied groups parameter Group ا (DM with MI) N = 35 Studied groups Group II (DM without CAD) N = 35 Group III (control group) N = 30 Age (years) X ± SD 51.71± ± ±5.86 Gender Male /Female (%) 25/10(71.4/28.6%) 22/13 (62.9/37.1%) 20/10 (66.7/33.3%) Test of significance P value f- test X Hypertension Positive /Negative (%) 31/4 (88.6/11.4%) 24/11(68.6/31.4%) 0/30 (0.0/100%) Smoking Positive/Negative (%) 24/11(68.6/31.4%) 22/13 (62.9/37.1%) 0/30 (0.0/100%) X * 37.1** 2.36 *** X * 28.5 ** 0.25 *** * Comparison between group I and group III ** comparison between group II and group III *** Comparison between group I and group II P: Probability of error P < 0.05 significant P> 0.05 non significant P< highly significant F test=analysis of variance (ANOVA) X 2 =chi square 0.124*** <0.001 * *** 4

5 Table (2): Laboratory parameters in the studied groups parameter Group I (DM with MI) N = 35 Group II (DM without CAD) N = 35 Group III (control group) N = 30 HbA1c (%) X ± SD 10.94± ± ± f- test P value <0.001 Triglycerides (mg/dl) X ± SD 212.5± ± ± <0.001 HS Cholesterol (mg/dl) X ± SD ± ± ± HS HDL-c (mg/dl) X ± SD ± ± ± <0.001 HS LDL-c (mg/dl) X ± SD ± ± ± <0.001 HS sicam1 (ng/ml) X ± SD ± ± ± <0.001 HS Post Hoc test (LSD) * 0.442*** 0.002* 0.021** 0.338*** 0.260*** 0.115** * * Comparison between group I and group III ** comparison between group II and group III *** Comparison between group I and group II P: Probability of error P < 0.05 significant P> 0.05 non significant P< highly significant F test=analysis of variance (ANOVA) LSD least significant difference. Table (3): Differences in allele distribution and genotype frequency of ICAM-1 E469K gene polymorphism between the studied groups parameter Group 1 N =35 No % GroupII N =35 No % Group III N =30 No % X 2 P value OR (95% CI) genoype EE EK KK * 0.84 ** *** 5.57 ( ) allele E K * 0.97 ** *** 2.54 ( ) * comparison between group I and group III ** comparison between groupii and groupiii *** comparison between group I and group II P: Probability of error P < 0.05 significant P> 0.05 non significant P< highly significant CI confidence interval OR odds ratio 5

6 sicam mean value (ng/ml) meanvalue of sicam(ng/ml) sicam1mean value (ng/ml) in the studied group groupi groupii Studied groups group III sicam-1 value in groupi stratified by E496k gene polymorphism EE EK Group I KK Fig. (2): sicam-1 mean value in the studied groups & group I stratified by ICAM-1 E469K gene polymorphism. Table (4): Sociodemographic criteria, clinical and laboratory parameters in the group I estratified by ICAM-1 E469K gene polymorphism. parameter EE N =15 Studied groups EK N =15 KK N = 5 Test of significance P value Hypertension (Positive/Negative) 15/0 (100/0.0%) 11/4 (73.3/26.7%) 4/1 (80.0/20.0%) Smoking Positive/Negative (%) 11/4 (73.3/26.7%) 9/6 (60.0/40.0%) 1/4 (20.0/80.0%) X X HbA1c X ± SD (%) 10.86± ± ±1.76 Kruskal Wallis test 1.38 Triglyceride (mg/dl) X ± S ± ± ± Cholesterol (mg/dl) X ± S ± ± ± HDL-c (mg/dl) X ± SD ± ± ± LDL-c (mg/dl) X ± SD ± ± ± sicam-1 (ng/ml) X ± SD ± ± ± P: Probability of error P> 0.05 non significant X 2 =chi square

7 Discussion CAD is one of the major causes for morbidity and mortality in the developing countries (2). One of the main and independent risk factors for the development of CAD and consequent myocardial infarction is type 2 diabetes (15). ICAM-1 mediates the interaction of activated endothelial cells with leukocytes and plays a fundamental role in the pathogenesis of coronary atherosclerosis (13). In the current study (table1), no statistical significant differences were found between the studied groups regarding to age and gender (p= 0.691, respectively). Also, there were high statistical significant differences between both diabetic groups and control group as regard hypertension and smoking (p<0.001 for both).while, no statistical significant differences were found between diabetic patients with MI and diabetic patients without CAD as regard hypertension and smoking (p=0.124, respectively). The present study (table2) showed that diabetic patients with MI had a higher statistical significant difference regarding HbA1c (10.94± 1.49%) compared with diabetic patients without CAD (9.96±1.11%, p=0.001). While, no statistical significant differences were found regarding total cholesterol (200.31±32.6 mg/dl vs ±25.66 mg/dl, p= 0.338), LDL-c (150.62±26.1 mg/dl vs ± mg/dl, p=0.115) levels, HDL-c (33.97 ± 5.3 mg/dl vs ± 4.03 mg/dl, p= 0.260), and triglyceride levels (212.5±23.3 mg/dl vs ± mg/dl, p=0.442) between group I & group II. These results were partially in agreement with Milutinović & Petrovič (12) who stated that their studied patients had higher total cholesterol and LDL-c levels, and lower HDL-c levels than their diabetic controls. There were no significant differences in triglyceride levels between the cases and the diabetic controls. ICAM1 is a member of the large immunoglobulin superfamily widely expressed at a basal level and can be upregulated by pro-inflammatory cytokines. Furthermore, circulating sicam1 concentrations are increased in various inflammatory conditions, including CVD, and have been associated with future CVD risk (27). Regarding sicam1 level in the current study (table2, fig.2), there were statistical significant differences between diabetic patients with MI (468.11±40.42 ng/ml) and both diabetic patients without CAD (266.48±44.15 ng/ml) and the control group(150.63±29.7 ng/ml, p<0.001 for both). Also, a higher statistical significant difference was found between diabetic patients without CAD and the control group (p <0.001). In agreement to these results, sicam1 level increase amongst patients with coronary disease was informed by Ridker et al.,(16).& Malik et al., (10). The study done by El-Mesalamy et al., revealed that sicam1 concentrations were significantly higher in the ischemic heart disease group and the diabetic patient with coronary artery disease group in comparison to those levels obtained in the healthy controls group (p 0.05). The mechanism of elevation of ICAM may be contributed to hyperglycemia, oxidative stress, and inflammation or insulin resistance (5). On the other hand, Mohamed et al., stated that there were no significant differences in sicam1 levels between CHD patients and control subjects (13). This difference in 7

8 results may be due to the fact that the patients in the current study had an acute state, whilst the patients in some of the previous studies had chronic CHD. T2DM group with MI had a statistical significant higher distribution of the EE genotype and E allele frequency (42.9%, 64%) than those of T2DM without CAD group (20%,41.7%) & the healthy controls (16.7%,41.2%). Meanwhile, there was no statistical significance between T2DM without CAD group & the healthy control group regarding EE genotype and E allele frequency. These results were in accordance with a study done in Caucasians in Italy (14), demonstrated a positive association between the EE genotype of the K469E polymorphism of the ICAM-1 gene and ischemic stroke. Also, the results of Yokoyama and colleagues found that the distribution of K469E polymorphism in T2DM and control subjects was not significantly different between both groups (25). Furthermore, in a study done in Egypt by Shaker et al.,(19) diabetic patients with peripheral arterial occlusive disease including MI had a higher statistical difference in the frequency of the EE genotype (29%) compared to control group (13%). This could be explained by Yokoyama et al., (25) who stated that EE genotype of the K469E polymorphism of the ICAM-1 gene has been reported to be associated with the lower plasma fibrinogen levels in patients with T2DM. In contrast to the present study, Milutinović & Petrovič (12) failed to reveal a statistical differences between the EE genotype of the K469E polymorphism of the ICAM-1 gene and T2DM in patients with MI (EE= 21.7 %, EK=47.4 %, KK=30.9 %) & T2DM patients without MI (EE=19.1 %, EK=50.7 %, KK=30.2 %). Also, McGlinchey et al., (11) found no association between the ICAM-1 K469E polymorphism and CHD in a well-defined Irish population. Similarly, Aminian et al., (1) found no significant differences between CHD, MI patients and controls as regards KK genotype in the studied population from Fars province, Iran. In a study done in Egypt by Mohamed et al., (13) ICAM-1 EK and KK variants represented 57% of the CHD patients and were associated with increased risk of disease development. In Chinese population, Zhang et al.,(27) found that the presence of KK genotype of ICAM-1 codon 469 conferred an increased risk for CHD. Whereas, a report in Chinese population demonstrated a positive association between the KK genotype and restenosis after coronary stenting (8). Similarly, Lu et al.,(9) found that K allele frequency was higher in CHD patients than in controls, and K allele carriers develop myocardial infarction more easily. Atherosclerosis is a multifactorial disease and evidence indicates that certain synergistic risk factors accelerate atherogenesis. In the current study by using logistic regression analysis (table3) revealed that T2DM patients with the EE genotype as well as E allel were at increased risk for MI (OR % CI , OR = % CI , respectively) compared with those having the KK genotype and K allel. In the first study documenting a role of the ICAM-1 gene polymorphism in the pathogenesis of a cardiovascular disease done by Gaetani et al.,(7) revealed EE genotype significantly increases the risk of PAOD (odds ratio, 3.5; 95% CI, ). To the opposite of the present results, Milutinović & Petrovič (12) demonstrated that the EE genotype was not associated with MI in subjects with T2DM (OR=1.2; 95 % CI= ).In another study done by McGlinchey and co-workers, stated that the effects of K469E polymorphism in families with a history of coronary artery disease were investigated and no meaningful relation was found between K469E polymorphism and occurrence of coronary artery disease (11). Aminian et al., (1) concluded that there was no strong relation between K469E polymorphisms and occurrence of CHD and MI in 8

9 the studied population from Fars province, Iran. Meanwhile, Mohamed et al., (13) suggest that the KK and EK genotypes of the ICAM-1 gene polymorphism in codon 469 are associated with the risk for CHD development in Egyptians (OR=3.8, 95% CI: ). The results obtained by this study (table4) revealed that in T2DM with MI, stratified by ICAM-1 E469K gene polymorphism showed no statistical significant differences regarding all studied parameters including sicam-1 levels (fig.2) (p<0.05 for all). These results were in accordance, with those of Milutinovic and Petrovic (12) who found no statistical significant association between the genotypes and coronary risk factors (clinical history and laboratory data). While in a study done by Shaker et al.,(19) showed no statistical difference between the different polymorphisms and the clinical history and laboratory data among the patient groups except in those with IHD, where there was significant difference between EE and EK.Whereas, in the Han population of China, individuals with the K allele had higher plasma level of sicam- 1 than those without the K allele (8). These discrepancies in results may be explained by variable sample sizes; also, the different populations represent different gene pools. This suggesting that gene-disease relationships can be expected to be different between populations due to the differences in a compound genetic set (26). Moreover, in type 2 diabetic patients, ICAM-1 may have to a certain extent different import in its role in sequence of vascular harm from non-diabetic subjects. Conclusion: the EE genotype was associated with MI in the present set of patients. Also, the circulating sicam-1 was elevated in diabetic patients with MI but did not swayed by this polymorphism. Further studies enrolling larger numbers of patients from different populations are needed to confirm these findings. Hence, the K469E polymorphism of the ICAM-1 gene may be employed as a genetic indicator for MI in patients with T2DM in the Egyptian population. Economic studies are recommended for the cost benefit of using it as a screening genetic marker for diabetic patients and how far this could be used in early prophylaxis against MI. References: 1-Aminian B, Abdi Ardekani AR& Arandi N(2007): ICAM-1 polymorphisms (G241R, K469E), in coronary artery disease and myocardial infarction. Iran J Immunol.; 4 (4): Anderson RN (2000): Deaths: leading causes for for 2000, Natl. Vital. Stat. Rep., 50: Bartzeliotou AI, Margeli AB& Tsironi M (2007): Circulating levels of adhesion molecules and markers of endothelial activation in acute inflammation induced by prolonged brisk exercise. Clin Biochem; 40: Diamond MS, Staunton DE, de Fougerolles AR, et al., (1990): ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).Journal of Cell Biology, 111: El-Mesallamy H, Suwailem S &Hamdy N (2007): Evaluation of C-Reactive Protein, Endothelin-1, Adhesion Molecule(s), and Lipids as Inflammatory Markers in Type 2 Diabetes Mellitus Patients. Mediators of Inflammation, 2007:1. 6-Friedewald W T, Levy R I & Fredrickson DS (1972): Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.clinical Chemistry, 18( 6) :

10 7-Gaetani E, Flex A, Pola R, et al., (2002): The K469E polymorphism of the ICAM-1 gene is a risk factor for peripheral arterial occlusive disease. Blood Coagul Fibrinolysis. 13(6): Liu ZP, Huo Y, Li JP et al., ( 2004): Polymorphism K469E of intercellular adhesion molecule-1 gene and restenosis after coronary stenting in Chinese patients. Chin Med J, 117: Lu FH, Shang Q, Wen PE, et al., (2006): A study on K469E polymorphism of ICAM1 gene and ICAM1 plasma level in patients with coronary heart disease. Zhonghua Yi Xue Yi Chuan Xue Za Zhi.; 23: Malik I, Danesh J, Whincup P, et al. (2001): Soluble adhesion molecules and prediction of coronary heart disease: A prospective study and meta-analysis.lancet.; 358: McGlinchey PG, Spence MS, Patterson CC, et al., (2004): The intercellular adhesion molecule-1 (ICAM-1) gene K469E polymorphism is not associated with ischaemic heart disease: an investigation using family-based tests of association. Eur J Immunogenet.; 31: Milutinović A & Petrovič D (2006): The K469E Polymorphism of the Intracellular AdhesionMolecule 1 (ICAM-1) Gene Is Not Associated withmyocardial Infarction in Caucasians with Type 2 Diabetes Folia Biologica (Praha) 52: Mohamed A, Rashed L, Amin H et al., (2010): K469E polymorphism of the intercellular adhesion molecule-1 gene in Egyptians with coronary heart disease Ann Saudi Med. 30(6): Pola R, Flex A, Gaetani E, et al., (2003): Synergistic effect of -174 G/C polymorphism of the interleukin-6 gene promoter and 469 E/K polymorphism of the intercellular adhesion molecule-1 gene in Italian patients with history of ischemic stroke. Stroke, 34: Reschner H, Milutinovic A& Petrovič D (2009): The PECAM-1 gene polymorphism - a genetic marker of myocardial infarction.cent. Eur. J. Biol. 4(4): Ridker PM, Hennekens CH, Buring JE, et al.,(200): C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med; 342: Rothlein R, Dustin ML, Marlin SD et al., (1986): A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. Journal of Immunology, 137: SAS Institute (2005): Statistical analytical measures. Version 6.11, Gary North Carolina, USA 19-Shaker O, Zahra A, Sayed A, et al. (2010): Role of ICAM-1 and E-selectin gene polymorphisms in pathogenesis of PAOD in Egyptian patients. Vasc Health Risk Manag ; 6:9. 20-van der Stolpe A & van der Saag, P (1996): Intercellular adhesion molecule-1. Journal of Molecular Medicine, 74: Tzoulaki I, Murray GD &Lee AJ (2005): C-reactive protein, interleukin-6, and soluble adhesion molecules as predictors of progressive peripheral atherosclerosis in the general population. Circulation; 112: Vora DK, Rosenbloom CL, Beaudet AL, et al., (1994): 10

11 Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster. Genomics, 21: Voraberger G, Schafer R & Stratowa C (1991): Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5 - regulatory region. Induction by cytokines and phorbol ester. Journal of Immunology, 147: Yamashita M, Yoshida S& Kennedy S (2005): Association study of endometriosis and intercellular adhesion molecule-1 (ICAM-1) gene polymorphisms in a Japanese population. J Soc Gynecol Investig; 12(4): Yokoyama H, Tahara H& Emoto M (2005): The K469E polymorphism of the intercellular adhesion molecule-1 gene is associated with plasma fibrinogen level in type II diabetes. Metabolism; 54: Zee RL, Cheng SA, Lindpaintner EK, et al., (2007): Intercellular Adhesion Molecule1 (ICAM1) Lys56Met and Gly241Arg Gene Variants, Plasma-Soluble ICAM1Concentrations, and Risk of Incident Cardiovascular Events in Initially Healthy White. Stroke; 38: Zhang SR, Xu LX, Gao QQ, et al., (2006): The correlation between ICAM-1 gene K469E polymorphism and coronary heart disease. Zhonghua Yi Xue Yi Chuan Xue Za Zhi.; 23: