Balkan Journal of Medical Genetics

DETERMINATION OF THE RELATIONSHIP BETWEEN DNA METHYLATION STATUS OF KLOTHO AND ARNTL GENES WITH HYPERTENSION
Osum M, Tosun O, Birtan H, Kalkan R
*Corresponding Author: Prof. Dr.Rasime Kalkan, PhD, Faculty of Medicine, European University of Lefke, Mersin 10, Lefke 99728, Northern Cyprus, Turkey, kalkanr@yahoo.com, rkalkan@eul.edu.tr
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MATERIAL AND METHODS

Study subjects 78 hypertensive patients (31 female and 47 male) and 49 control subjects (36 female and 13 male) were included in this study, and peripheral blood specimens of the participants were collected. Participants were recruited from Burhan Nalbantoğlu State Hospital, Nicosia (from November 2022 to April 2023). The diagnostic criteria of hypertension were defined as systolic blood pressure (SBP)/diastolic blood pressure (DBP) >140/90 mmHg or antihypertensive medication use for decreasing high blood pressure [23]. The participants with cancer, respira- tory diseases, cerebral infarction, congenital heart disease, diabetes mellitus or chronic kidney diseases, and autoim- mune diseases were excluded, moreover, subjects who were discontent to participate in the study were excluded as well. The medical history of all participants was ques- tioned. All clinical investigations performed for this study were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Scientific Research Ethics Committee of the Near East University (YDU/2020/80-1066). All subjects signed the written consent form before participating in the study. Measurements of the Biochemical Parameters Peripheral blood samples were collected from par- ticipants after overnight fasting. All participants’ serum was obtained by centrifugation at 2000 rpm for 20 min at 4 °C. The fasting blood sugar (FBS), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), so- dium (Na), potassium (K), creatinine (Cr), and urea levels were determined with an automated clinical biochemistry analyzer (Abbott Architect C8000). Methylation Analysis Peripheral blood samples of hypertensive patients and normal controls were transferred to 2 ml vacuum tubes with K2EDTA. Genomic DNA was isolated from whole blood samples by using the AllPrep DNA/RNA/Protein isolation kit (Qiagen in Manchester, United Kingdom). A NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA) was used to analyze the amount and purification of DNA samples. Sodium bisulfite modification of DNA samples was done by using the Epi- Tect Bisulfite Modification Kit (Qiagen, Manchester, UK). According to the EpiTect® MS-HRM PCR Handbook (Qiagen, Manchester, UK), primers were designed. The MS-HRM analysis was performed to analyze the promotor methylation status of KLOTHO and ARNTL genes accord- ing to the EpiTect® HRM™ PCR Handbook protocol (Rotor-Gene Q, Qiagen). As methylated and unmethylated controls, universal methylated and unmethylated DNA (EpiTect Control DNA Set, Cat No./ID: 59568) were pre- ferred [24]. Statistical Analysis Descriptive statistics for qualitative (frequency and percentage) and quantitative varaibles (arithmetic mean, standard deviation) were calculated. Pearson’s Chi-Square test or Fisher’s exact test was used to investigate the as- sociations between gene methylation status and several patient characteristics, where appropriate. The McNemar test was used to investigate the association of methylation in both genes. A two-way Analysis of Variance test was performed to analyze the effects of gene methylation and hypertension on several biochemical parameters. Sidak’s posthoc test was applied to investigate the pairwise differ- ences, in the case of statistical significance. The Statistical Package for Social Science (SPSS) (Demo Version 26.0 for Mac) and GraphPad Prism (Demo Version 9.51 for Mac) software were used for all statistical calculations. The level of significance was accepted to be 0.05.

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