We studied Slovene patients of Slavic origin from independent families who had DM2 lasting more than 10 years: 142 with MI (MI group) and 310 DM2 patients with no history of CAD, and no signs of ischemic changes on electrocardiogram and no ischemic changes during sub­maximal stress testing [17]. The diagnosis of MI was based on the World Health Organization criteria [18]. Patients with MI were included in the study 1-9 months after the acute event. After informed consent was obtained from the patients and control subjects, we conducted a detailed interview. Arterial hypertension and cigarette smoking were defined as binary variables. Patients were classified as having DM2 according to the American Diabetes Association criteria for the diagnosis and classification of diabetes [7]. Body mass index (BMI) was calculated as weight in kilograms divided by the height in square meters. Total serum cholesterol, low density lipoprotein (LDL) cholestetol, high density lipoprotein (HDL) cholesterol and triglycerides serum levels were determined by standard biochemical methods. We analyzed serum TNFa levels in a subpopulation of 70 consecutive DM2 patients: 10 with MI and 60 without MI; in this group there were eight smokers and 62 non smokers, and the mean age was 62.5 ± 10.9 years.

      The G(–308)A polymorphism was evaluated by polymerase chain reaction (PCR) using the following primers: sense (5'-AGG CAA TAG GTT TTG AGG GCC AT-3'), antisense (5'-TCC TCC CTG CTC CGA TTC CG-3'). The cycling conditions were: two cycles at 94°C for 3 min., 60°C for 1 min., 72°C for 1 min.; 35 cycles at 94°C for 1 min., 60°C for 1 min., 72°C for 1 min.; two cycles at 94°C for 1 min., 60°C for 1 min. and 72°C for 5 min. The PCR product of 107 bp was digested with the NcoI restriction enzyme (Fermentas, St. Leon-Rot, Austria) into two fragments of 87 and 20 bp, respectively. The products of 87 and 20 bp represented the G allele, whereas undigested PCR products represented the A allele. The products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining [19]. Two of us (KS, DP), who were blinded for the origin of the DNA sample, performed the genotype classification.         

     Differences in mean values were analyzed by the Student t-test. The chi-square test was used to compare discrete variables and genotype distributions. Genotypic odds ratios (ORs) for MI with 95% confidence intervals (CIs) with two-tailed p values were calculated by the chi-square test. Statistical analysis was performed using the SPSS program 15 for Windows (SPSS Inc., Chicago, IL, USA).