ASSOCIATION OF THE ACE rs4646994 AND rs4341 POLYMORPHISMS WITH THE PROGRESSION OF CAROTID ATHEROSCLEROSIS IN SLOVENIAN PATIENTS WITH TYPE 2 DIABETES MELLITUS
Merlo S1, Novák J2,3,4, Tkáčová N2, Nikolajević Starčević J5, Šantl Letonja M6, Makuc J7, Cokan Vujkovac A7, Letonja J5, Bregar D5, Zorc M5, Rojko M5, Mankoč S5, Kruzliak P8, Petrovič D5
*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: 37

MATERIALS AND METHODS

In this cross-sectional study, 595 (338 males; 257 females) subjects with T2DM and 200 (92 males; 108 females) subjects without T2DM (control group) were enrolled as described previously [4]. The study protocol was approved by the Slovene Medical Ethics Committee (128/09/ 2010). After informed consent for participation in the study was obtained, a detailed interview was made. All ultrasound examinations were performed by two experienced doctors blinded to the participants’ diabetes status. The 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 described previously [4]. Plaques were defined as a focal intima-media thickening and divided according to their echogenic/ echolucent characteristics into five types as described previously [4]. The inter-observer reliability for carotid plaque characterization was found to be substantial (κ = 0.64, p <0.001). Blood samples for biochemical analyses were collected as described previously [4]. The genomic DNA was extracted from 100 μL of whole blood using a FlexiGene DNA isolation kit, in accordance with the recommended protocol (Qiagen GmbH, Hilden, Germany). The ACE polymorphisms (rs4646994 and rs4341) were determined by a novel fluorescencebased competitive allele-specific polymerase chain reaction (PCR) (KASPar; Kbioscience Ltd., Hoddesdon, Hertfordshire, UK), assay. Details of the method used can be found at http://www.kbioscience. co.uk/. Continuous variables were expressed as means ± standard deviations (SDs) if normally distributed, and as median (interquartile range) if asymmetrically distributed. Continuous clinical data were compared using an unpaired Student’s t-test or analysis of variance (ANOVA) when normally distributed and the Mann-Whitney U test or the Kruskal-Wallis H test when asymmetrically distributed. The Pearson χ2 test was used to compare discrete variables. To determine the association of the ACE gene polymorphisms (rs4646994 and rs4341) with CIMT, a multiple linear regression analysis was performed. We used an additive model in which common allele homozygotes were coded as 1, heterozygotes as 2, and rare allele homozygotes as 3. All the regression models were adjusted for the presence of well-established cardiovascular risk factors. The results are presented as standardized β coefficients and p values for the linear regression and by odds ratios (ORs) and 95% confidence intervals (CIs) for the logistic regression. A two-tailed p value of less than 0.05 was considered statistically significant. A statistical analysis was performed using the Statistical Package for the Social Science (SPSS) software for Windows, version 20 (SPSS Inc., Chicago, IL, USA).



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