INTERLEUKIN-18 PROMOTER GENE POLYMORPHISMS ARE NOT ASSOCIATED WITH MYOCARDIAL INFARCTION IN TYPE 2 DIABETES IN SLOVENIA
Kariž S1*, Petrovič D2
*Corresponding Author: Stojan Kariž, Department of Internal Medicine, General Hospital Izola, Polje 35, Izola 6310, Slovenia; Tel.: +386-5-660-6480; Fax: +386-660-6305; E-mail: stojan.kariz@siol.net
page: 3

PATIENTS AND METHODS

This cross-sectional analysis examined 495 subjects (263 males, 232 females; age range 46-78 years) with type 2 diabetes of more than 10 years duration: 169 subjects with MI (MI group) and 326 with no history of CAD, no signs of ischemic changes on the electrocardiogram and no ischemic changes during sub-maximal stress testing (control group). Subjects were classified as having type 2 diabetes according to the current criteria of the American Diabetes Association (ADA) [19]. The diagnosis of MI was made according to the criteria in [20], patients being studied 1 to 9 months after the acute event. All subjects were Slovenes of Slavic origin (Caucasians) and came from independent families. The body mass index (BMI) was calculated as weight in kilograms divided by the height in square meters. High sensitivity C-reactive protein (CRP), glycosylated hemoglobin (Hb A1c), total cholesterol, low density lipoproteins (LDL), high density lipoproteins (HDL) and triglycerides were determined by standard biochemical methods. We also measured serum IL-18 levels in 70 subjects with type 2 diabetes (20 patients with MI and 50 patients without CAD) and 22 subjects without diabetes. Plasma IL-18 was determined with enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The National Medical Ethics Committee approved the research protocol. All patients participating in the study gave written informed consent. Genomic DNA was isolated from peripheral blood leukocytes by standard methods and stored at –20°C. Genotyping was carried out by polymerase chain reaction- restriction fragment length polymorphism (PCRRFLP) analysis. The –137 and –607 polymorphisms in the promoter of the IL-18 gene were determined as described in [13]. For the –137 G>C genotyping, a common reverse primer (5’-AGG AGG GCA AAA TGC ACT GG-3’) and two sequence-specific forward primers [5’-CCC CAA CTT TTA CGG AAG AAA AG-3’ (for alelle G) and 5’-CCC CAA CTT TTA CGG AAG AAA AC-3’ (for alelle C)] were used to amplify a 261 bp product. A control forward primer was used to amplify a 446 bp fragment that contained the polymorphic site to serve as an amplification control. The PCR reaction was performed in a final volume of 5 μL containing 0.5 μL 10 mM dNTP, 1 μL 5 × PCR buffer, 0.2 μL 25 mM MgCl2, 0.15 μL 10 μM of each primer, 0.5 μL genomic DNA, 2.55 μL H2O and 0.1 μL (0.5 U) GoTaq DNA polymerase. Two PCR reactions were performed for each individual DNA sample (for both sequencespecific forward primers). The cycling conditions were: denaturation for 2 min. at 94°C, then five cycles each lasting 20 seconds at 94°C, 60 seconds at 68°C and 60 seconds at 68°C, respectively, followed by 25 cycles of 20 seconds at 94°C, 20 seconds at 62°C, 40 seconds at 72°C and a final elongation for 7 min. at 72°C. Polymerase chain reaction products were visualized by 2.0% agarose gel electrophoresis stained by »SYBR Green I« (Invitrogene, Carlbad, CA, USA). For the –607 C>A polymorphism, a common reverse primer (5’-TAA CCT CAT TCA GGA CTT CC-3’) and two sequence-specific forward primers [5’-GTT GCA GAA AGT GTA AAA ATT ATT AC-3’ (for alelle C) and 5’-GTT GCA GAA AGT GTA AAA ATT ATT AA-3’ (for alelle A)] were used to amplify a 196 bp product. A control forward primer (5’-CTT TGC TAT CAT TCC AGG AA-3’) was used to amplify a 301 bp fragment covering the polymorphic site as an internal positive amplification control. The PCR reaction was performed in a final volume of 5 μL consisting of 0.5 μL 10 mM dNTP, 1 μL 5 × PCR buffer, 0.2 μL 25 mM MgCl2, 0.15 μl 10 μM of each primer, 0.5 μl genomic DNA, 2.55 μL H2O and 0.1 μL (0.5 U) GoTaq DNA polymerase. The cycling conditions were: denaturation for 2 min. at 94°C, then seven cycles each lasting 20 seconds at 94 ºC, 40 seconds at 64 ºC and 40 seconds at 72 ºC, respectively, followed by 25 cycles of 20 seconds at 94 ºC, 40 seconds at 57 ºC, 40 seconds at 72 ºC and a final elongation for 7 min. at 72 °C. PCR products were separated by electrophoresis on a 2% agarose gel and visualized by »SYBR Green I« (Invitrogene). Two investigators (SK, DP), blinded for the case or control status of the DNA sample, performed the assignment of genotype. The χ2 test was used to compare discrete variables and to compare genotype distributions. Continuous clinical data were compared by unpaired Student’s t-test and presented as mean ± standard deviation (SD). The Hardy-Weinberg equilibrium was confirmed using the χ2 test. A p value of <0.05 was considered to be statistically significant. A statistical analysis was performed using the SPSS program for Windows 2000 version 17 (SPSS Inc., Chicago, IL, USA).



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