PECAM-1 GENE POLYMORPHISM (rs668) AND SUBCLINICAL MARKERS OF CAROTID ATHEROSCLEROSIS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS
Popović D, Nikolajević Starčević J, Šantl Letonja M, Makuc J, Cokan Vujkovac A, Reschner H, Bregar D, Petrovič D
*Corresponding Author: Professor Daniel Petrovič, M.D., Ph.D., Institute of Histology and Embryology, Faculty of Medicine University Ljubljana, Korytkova 2, SI-1000 Ljubljana, Slovenia. Tel: +386-1-543-7367. Fax: +386-1-543-7361. E-mail: daniel.petrovic@mf.uni-lj.si
page: 63

PATIENTS AND METHODS

This study included 595 consecutive subjects with T2DM, admitted to the diabetes outpatient clinics of the general hospitals at Murska Sobota and Slovenj Gradec, Slovenia, and from the outpatient department at the Medical Center Medicor, Ljubljana, Slovenia. The inclusion criteria for the control group was the absence of T2DM, and consisted of employees of the General Hospital Murska Sobota, Slovenia. Another inclusion criteria for the subjects with T2DM and for the subjects in the control group was the age from 40 to 70. The exclusion criteria for subjects with T2DM and for the subjects in the control group was a history of either myocardial infarction (MI) or ischemic stroke. The study protocol was approved by the Slovene Medical Ethics Committee (98/08/10). The patients and control subjects were enrolled and followed in the period from 2008 to 2014. Patients were classified as having T2DM according to the current report of the American Diabetes Association [26]. After informed consent was obtained from the patients, a detailed interview was conducted concerning smoking habits, the duration and treatment of diabetes, arterial hypertension, and hyperlipidemia. Patients were asked whether they were smokers at the time of recruitment (current smoker). Subjects with T2DM with systolic blood pressure ≥140.0 mm Hg or diastolic blood pressure ≥85.0 mm Hg and/or subjects who were taking anti hypertensive drugs were considered to be hypertensive. All ultrasound examinations were performed by two experienced doctors blinded to the participants’ diabetes status. The carotid intima-media thickness (CIMT), defined as the distance from the leading edge of the lumen-intima interface to the leading edge of the media-adventitia interface, was measured as previously described [27]. Plaques were defined as a focal intima-media thickening, and divided into five types according to their echogenic/ echolucent characteristics, as previously described [27]. The interobserver reliability for carotid plaque characterization was found to be substantial (κ = 0.64, p <0.001). After the patients with T2DM and subjects without T2DM (control group) were enrolled, they were prospectively followed-up for a few years. From the group with T2DM, 426 responded and participated in the control ultrasound examination of the neck artery, whereas, 132 re-sponded from the group of subjects without T2DM; 3.8 ± 0.5 years passed between the first and the control ultrasound examination. Biochemical Analyses. Fasting blood samples for biochemical analysis were collected twice patients: upon enrollment and upon follow-up after a few years. Analyses were made at the hospital ac credited laboratory. The following parameters were determined: total cholesterol, triglycerides, highdensity lipoprotein (HDL), low-density lipoprotein (LDL), blood sugar and high-sensitive C-reactive protein (hsCRP). Genotyping. Genomic DNA was extracted from 100 μL of whole blood using a FlexiGene DNA isolation kit (Qiagen GmbH, Hilden, Germany), in accordance with the recommended protocol. The rs668 polymorphism of the PECAM-1 gene was determined with the KASPar assay (LGC Genomics Ltd., Hoddesdon, Hertfordshire, UK) system. Statistical Analyses. Continuous variables that were normally distributed, were reported in the form of mean ± standard deviation (SD). Variables that were not normally distributed, were presented in the form of median (inter-quartile range). The normality of distribution of continuous variables was examined using the Kolmogorov-Smirnov test. We used the Student’s t test or the analysis of variance (ANOVA) to compare the numerical values of the continuous variables, to identify the distribution of variables. If the variables were asymmetrically distributed, we used the Mann-Whitney U test or the Kruskal-Wallis H test. The χ2 test was used to compare the frequency’s categorical variables, statistical evaluation of differences in the frequencies of different alleles and genotypes between the two groups, as well as in the case of determining the Hardy-Weinberg equilibrium. The Pearson analyses was performed to examine the correlation between the independent variables. The results showed a high degree of correlation between the serum levels of total and LDL cholesterol (r = 0.86; p <0.001), as well as systolic and diastolic blood pressure (r = 0.65; p <0.001). In the case of a high degree correlation between two variables, only one variable from each pair was included in the multivariate statistical models. The change in the value of ultrasound markers of carotid artery atherosclerosis was calculated by deducting the values measured at two ultrasound examinations. The criteria for a statistically significant difference was a p value of less than 0.05. To reduce the possibility of error due to the small number of subjects, we used the Bonferroni correction. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) computer program for Windows, version 20 (SPSS Inc., Chicago, IL, USA).



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