Balkan Journal of Medical Genetics

DUAL EFFECT OF THE GHRL GENE VARIANT IN THE MOLECULAR PATHOGENESIS OF OBESITY
Becer E1,2, Ergoren MC2,3,*
*Corresponding Author: Associate Professor Mahmut C. Ergoren, Ph.D., Department of Medical Biology, Faculty of Medicine, Near East University, Near East Boulevard, 99138 Nicosia, Cyprus. Tel.: +90-392-675-1000, Ext: 3035. Fax: +90-392-223-6461. Mobile: +90-0548-865-8889. E-mail: mahmutcerkez.ergoren@ neu.edu.tr
page: 27

MATERIALS AND METHODS

Study Design and Studied Subjects. The current investigation involved 106 adult obese patients and 95 non obese subjects from the Turkish-Cypriot population. Turkish-Cypriots are defined as residing in North Cyprus as well as being born to parents who were born before 1974. Considering the Turkish-Cypriot population in the Island, the sample size is convenient. Every participant was provided with a questionnaire that included ethnicity, age, socioeconomic status and general health conditions. Subjects with systemic and metabolic disorders such as diabetes mellitus T2DM, hypertension, dyslipidemias, cirrhosis, cancer, kidney lithiasis, thyroid, cardiovascular disorders, or any active inflammatory disease, were excluded. The participants did not receive any medications or conduct any dietary or exercise program during the sample collection. Written informed consent was obtained from all the subjects. The study was approved by the Research Ethics Committee of the University [ethics committee application number: YDU/2017/43-354; project no: SAG-2016-2-036]. Anthropometric Measurements and Biochemical Parameters. Anthropometric measurements [height (m), weight (kg), waist circumference (cm) and hip circumference (cm)] were performed at the fasting state from each subject. Hip circumference was measured by placing a measuring tape around fullest portion of the patient’s hips. Waist circumference was measured using soft tape. Waist was defined midway between the lowest rib (laterally) and the iliocristale landmark. Body mass index (BMI) was calculated by dividing body weight (kg) by the square of height (m2). A BMI of >30 kg/m2 was considered to be obese [26]. Peripheral blood samples were collected after overnight fasting. Serum levels of triglycerides (TG), glucose, high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were measured using an automated analyzer (Abbott Architect C8000; Abbott Laboratories, Abbott Park, IL, USA). Insulin concentrations were measured using an electrochemiluminescence assay (Ref. 12017547; Elecsys Corporation, Lenexa, KS, USA). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated according to the formula: fasting insulin (μU/ mL) × fasting glucose (mmol/L) divided by 22 [27]. Genotyping. Each participant’s genomic DNA was isolated from EDTA-containing whole blood using PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To minimize the risk of DNA contamination, all procedures were conducted in a class II laminar flow. The genotyping of the GHRL rs34911341 (C>T) (Arg51Gln) and rs696217 (G>T) (Leu72Met) was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique. The detail of PCR primers, restriction endonucleases and digestion patterns of DNA fragments are shown in Table 1 [28]. The PCR reaction was performed on a conventional thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) in a total volume of 25 μL containing PCR Master Mix (2×) (Thermo Fisher Scientific, Waltham, CA, USA), 10 nM of each primer (Hibrigen Biyoteknoloji Ar-Ge San Tic Ltd. Sti, Gebze, Koceli, Turkey), and ~10 ng of the genomic DNA template. Digested products were segregated on 2.0% agarose gels and visualized by ethidium bromide staining and subsequent UV transillumination. Genotypes were determined on the basis of the presence or absence of restriction sites (Table 1). Statistical Analyses. Continuous variables were expressed as means ± SD. Comparison between groups were analyzed using Student’s t-test for continuous variables. Comparison of anthropometric and biochemical parameters between genotypes was performed by the one-way analysis of variance (ANOVA) test. Allele frequencies were calculated and the Hardy-Weinberg equilibrium (HWE) was evaluated by the goodness-of-fit χ2 test. The odds ratio (OR) and the 95% confidence interval (95% CI) were determined to calculate the strength of the association between genotypic alleles and the obese and non obese subjects. All statistical analyses were carried out using the GraphPad Prism 7 software (GraphPad Software, Inc, San Diego, CA, USA).

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