Balkan Journal of Medical Genetics

EPIGENETIC ALTERATIONS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS
Karachanak-Yankova S1,a, Dimova R2,a, Nikolova D1, Nesheva D1, Koprinarova M3, Maslyankov S4, Tafradjiska R5, Gateva P6, Velizarova M7, Hammoudeh Z1, Stoynev N2, Toncheva D1, Tankova T2, Dimova I1,*
*Corresponding Author: Ivanka Dimova, Associate Professor, Department of Medical Genetics, Medical University Sofia, Zdrave str. 2, 1431 Sofia, Bulgaria. Tel: +359-2-91-72-735. E-mail: ivanka.i.dimova@ gmail.com
page: 15

MATERIALS AND METHODS

Subjects. We collected blood samples from healthy controls (n = 12) and T2DM patients (n = 27). The patients were subdivided according to diabetes duration: newly-diagnosed T2DM (n = 9); T2DM duration of <5 years (n = 9); and T2DM duration of >5 years (n = 9). The participants were recruited at the Department of Diabetology, Clinical Centre of Endocrinology at the Medical University, Sofia, Bulgaria. All subjects provided written informed consent and were familiar with the aims, methods and risks of participating in the study in accordance with the Helsinki Declaration and rules of Good Clinical Practice. The study was approved by the Ethics Committee of the Medical University, Sofia, Bulgaria. Methods. The patients were interviewed for T2DM duration, presence of arterial hypertension and other chronic diseases. Body mass index (BMI) was calculated. Arterial blood pressure was measured in standard conditions. Laboratory parameters were measured in all participants at fasting: Hb A1c in whole blood by immuneturbidimetric method (Roche Diagnostics Deutschland GmbH, Mannheim, Germany); total cholesterol, high density lipoprotein (HDL) cholesterol, and triglycerides by enzymatic colorimetric method (Roche Diagnostics); low density lipoprotein (LDL) cholesterol was calculated using Fridewald’s formula. The 2005 International Diabetes Federation (IDF) definition of the metabolic syndrome (MetS) was used. The main characteristics of the subgroups are summarized in Table 1. A subset of the blood samples from the patients (n = 16) and controls (n = 12) were used for total RNA purification for the analysis of the MBD2 gene expression. The epigenetic changes in the 22 genes related to stress and cell cycle regulation were analyzed in DNA specimens extracted from all blood samples. MBD2 mRNA Expression Analysis. RNA was purified using QIAamp® RNA Blood Kit (Qiagen GmbH, Hilden, Germany). An amount of 250 ng of each RNA sample was reverse transcribed to cDNA (QuantiTect Reverse Transcription kit; Qiagen). The cDNAs were further examined using the quantitative polymerase chain reaction (qPCR) Primer Assay for Human MBD2 (Cat. #PPH08621A-200; Qiagen). The real-time PCR (RT-PCR) amplification was performed on an ABI PRISM® 7500 instrument and the results were analyzed with ABI PRISM® Sequence Detection Software, ver.1.4.0 (Applied Biosystems, Foster City, CA, USA). The total volume of each reaction was 50 μL with less than 100 ng cDNA per reaction. The amplification conditions of each RTPCR cycle were as follows: denaturation at 94 °C for 15 seconds, primer annealing at 55 °C for 30 seconds, followed by primer extension at 72 °C for 30 seconds. The expression levels of MBD2 were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the ΔΔCT method. In order to avoid contamination, we included an internal non template control in each run. DNA Methylation Analysis of 22 Stress and Toxicity Genes. Whole genome DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen). The DNA samples were combined into four DNA pools each of nine healthy controls, newly-diagnosed T2DM patients, patients with T2DM duration of less and more than 5 years. DNA in each pool was adjusted to a final concentration of 50 ng/μL. Each DNA pool was analyzed for the promoter methylation status of 22 genes included in the Human Stress & Toxicity PathwayFinder EpiTect Methyl II Signature PCR Array (Qiagen Sciences Inc., Germantown, MD, USA) (Table 2). The method is based on detection of remaining input DNA after cleavage with a methylation-sensitive and/or a methylationdependent restriction enzyme. These enzymes will digest unmethylated and methylated DNA, respectively. Following digestion, the remaining DNA in each individual enzyme reaction is quantified by RT-PCR using primers that flank a promoter (gene) region of interest. The relative fractions of methylated and unmethylated DNA are subsequently determined by comparing the amount in each digest with that of a mock (no enzymes added) digest using a ΔCT method. Unmethylated represents the fraction of input genomic DNA containing no methylated CpG sites in the amplified region of a gene. Methylated represents fraction of input genomic DNA containing two or more methylated CpG sites in the targeted region of a gene. Data quality control parameters were strictly accomplished according to the protocol of the manufacturer.

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