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
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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 Student’s 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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