POLYMORPHISM OF ANGIOTENSIN-CONVERTING ENZYME (rs4340) AND DIABETIC NEPHROPATHY IN CAUCASIANS WITH TYPE 2 DIABETES MELLITUS
Šeruga M, Makuc J,, Završnik M, Cilenšek I, Ekart R, Petrovič D M. Šeruga and J. Makuc contributed equally to this study.
*Corresponding Author: Professor Dr. Daniel Petrovič, M.D., Ph.D., Institute of Histology and Embryology, Faculty of Medicine, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia. Tel: +386-1-543-7360. Fax: +386-1-543-7361. E-mail: daniel.petrovic@mf.uni-lj.si
page: 29

MATERIALS AND METHODS

We enrolled 651 unrelated Caucasians with T2DM from outpatient clinics of the University Medical Centre Maribor and the General Hospitals in Murska Sobota and Slovenj Gradec, Slovenia. Two hundred and seventy-six patients also had DN (cases) and 375 subjects with T2DM of more than 10 years duration but no clinical signs of DN (controls) were enrolled in the study. Diagnosis of DN was made according to the World Health Organization 1999 diagnostic criteria [16]. We excluded patients with overt nephropathy, poor glycemic control, significant heart failure New York Heart Association classification II-IV (NYHA II-IV), alcoholism, infection and other causes of renal disease. The study was approved by the National Medical Ethics Committee and was performed in compliance with the Helsinki declaration. After informed consent for participation in the study was obtained from all patients, a detailed interview was made. Biochemical Analyses. Total serum cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL), triglycerides (TGs), serum cystatin C, fastingserum glucose, serum glycemic Hb (Hb A1c), serum urea, serum creatinine were determined by standard biochemical methods. Albumin-to-creatinine ratio was also determined for each patient in three urine samples, according to the diagnostic criteria. To assess the kidney function, we used the modification diet in renal disease (MDRD) equation and serum-cystatin C [21,22]. Genotyping. Genomic DNA was extracted from 100 μL of whole blood using a Qiagen isolation kit (Qiagen GmbH, Hilden, Germany) following the blood and body fluid spin V3 protocol. The protocol was supported by five different reagents (QIAgene DNA Blood Mini Kit; Qiagen GmbH): buffer AL, 96.0% ethanol, buffer AW1, buffer AW2, buffer AE and appropriate amount of proteases (285 μL of proteases/200 μL of blood). From 200 μL of blood, 3-12 mg of genomic DNA, i.e., 30-40 ng/ mL were extracted according to the instructions of the manufacturer. The protocol for rs4340 polymorphism of the ACE gene was as follows: one cycle at 94 C for 5 min. followed by 30 cycles (94 C for 60 seconds, 58 C for 60 seconds and 72 C for 60 seconds) and finishing with one cycle at 72 C for 5 min. Statistical Analyses. Statistical analyses were conducted with the use of the Statistical Package for the Social Sciences program for Windows, version 20 (SPSS Inc., Chicago, IL, USA). Continuous clinical data were compared by unpaired Students t-test, while the χ2 test was used to compare discrete variables. Data were expressed as mean SD (standard deviation) (continuous variables) or as the number and percent of patients (categorical variables). All variables that showed significant differences by univariate analysis (with a p values of <0.05 were considered significant) were analyzed together using a logistic regression method. A p value of <0.05 was considered statistically significant. Deviation from Hardy-Weinberg equilibrium (HWE) was assessed by the exact test (http://ihg. gsf.de/).



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