THE LRP1 GENE POLYMORPHISM IS ASSOCIATED WITH INCREASED RISK OF METABOLIC SYNDROME PREVALENCE IN THE SERBIAN POPULATION
Vučinić N1,*, Stokić E1,2, Djan I3,4, Obreht D5, Veličković N5, Stankov K6, Djan M5
*Corresponding Author: Nataša Vučinić, Ph.D., University of Novi Sad, Faculty of Medicine, Department of Pharmacy, Hajduk Veljkova 3, 21000 Novi Sad, Serbia. Tel: +38121422760. Mobile: +381652452456. Fax: +38121450620. E-mail: natasa.vucinic@mf.uns.ac.rs
page: 51

MATERIALS AND METHODS

Study Subjects. The study included 93 individuals (males and females), aged from 19 to 65 [X standard deviation (SD)], attending the Clinical Center of Vojvodina, Novi Sad, Serbia. All subjects were divided in two groups. The MetS group consisted of 63 unrelated men and women who were diagnosed as MetS patients. The control group consisted of 30 unrelated healthy men and women, sex- and age-matched with the test group. The International Diabetes Federation (IDF) definition was applied to define the MetS group of patients [24]. To be diagnosed as MetS, participants needed to fulfill the following criteria: to have central obesity defined by waist circumference (WC) at least 94 cm in men and 80 cm in women, plus two of the following, hyperglycemia defined as fasting plasma glucose of at least 5.6 mmol/L, high blood pressure (BP) defined as resting BP of at least 130/85 mmHg or known treatment of hypertension hypertriglyceridemia defined as triglycerides (TG) at least 1.7 mmol/L low high-density lipoprotein (HDL) defined as fasting HDL cholesterol less than 1.0 mmol/L in men and less than 1.3 mmol/L in women [24,25]. Signed informed consent was obtained from all participants and the study was approved by the Institutional Ethics Review Committee and was performed according to the Declaration of Helsinki. Anthropometric Measurements and Biochemical Parameters Determination. Anthropometric measurements [body mass (BM), body height (BH) and WC), and cardiovascular risk factors assessment [systolic and diastolic BP, fasting serum lipids levels, glucose levels, Creactive protein (CRP)] were determined. With the subjects wearing light indoor clothes and no shoes, BM and BH were measured using a calibrated beam-type balance to the nearest 0.1 kg and a Harpenden anthropometer to the nearest 0.1 cm, respectively, and body mass index (BMI) was calculated [BMI = (body weight) BW/BH2 (kg/m2)]. Waist circumference was measured using flexible tape to the nearest 0.1 cm at the level midway between the lowest point on the rib margin and the highest point on the iliac crest. Systolic and diastolic BP were measured in a fasting state, early in the morning, using sphygmomanometer by Scipione Riva-Rocci (Italian inventor of cuff-based version of the mercury sphygmomanometer for the measurement of blood pressure; 1863-1937) in sitting position after a 10-15 min. rest period. The mean of three measurements was taken as the most valid value. Total cholesterol (TC) and TG were determined using a commercial kit (Boehringer Manheim GmbH, Mannheim, Germany). Highdensity lipoprotein cholesterol was estimated using the method of precipitation with sodium phospho-wolframate, while LDL-cholesterol was calculated using the formula by Friedewald et al. [26]. Fasting plasma glucose was measured using the Dialab glucose GOD-PAP (Dialab GmbH, Wiener Neudorf, Austria), method. The CRP levels were done by Latex (Dialab GmbH) immunoturbidimetric method. All blood samples were drawn after an overnight 12-hour fast. Genotyping of the LRP1 Gene. Total genomic DNA was isolated from EDTA-anticoagulated blood using phenol chloroform isoamylalcohol extraction [27]. Exon 3 of the LRP1 gene was amplified with standard primer set F (5-CCA TAG CCA GCT TGT TCA TG-3) and R 5-ACG GGA GAG TAG AGA GTG G-3) [19]. Polymerase chain reaction (PCR) amplification was done according to the modified method of Kang et al. [20]. The PCR reaction was performed in a 25 L final volume containing 100 ng of genomic DNA as a template with 0.4 M of each primer, 200 M dNTPs (dATP, dTTP, dCTP, dGTP), 1 × Buffer, 1.5U Taq polymerase, 0.5 × Q solution and 4 mM. The LRP1 gene was amplified for 30 cycles, consisting of 30 seconds at 94 C, 30 seconds at 55 C and 30 seconds at 72 C. Final extension at 72 C for 10 min. was applied. The PCR products were digested with FokI over a minimum period of 5 hours at 55 C. The LRP1 fragments obtained were separated by electrophoresis on a 4.0% MetaPhor agarose gel in 1 × TAE buffer and visualized by ethidium bromide fluorescence. The genotype of each person was determined from the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) profile. Data Analyses. Statistical analysis of data was performed by software package STATISTICA, version 10.0 (http://www.statsoft.com/Products/STATISTICA-Features/Version-10) [28]. Odds ratio (OR) as estimate of relative risk for disease were calculated and 95% confidence intervals (CI) obtained by logistic regression, were used to test the relationship between MetS and LRP1 alleles, as well as their associations with selected anthropometric and biochemical parameters.



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