DO WE USE METHYLATION OF NFATC1 AND FOS GENES AS
A BIOMARKER FOR POSTMENOPAUSAL OSTEOPOROSIS?
Kalkan R, Tosun O
*Corresponding Author: Associate Professor Rasime Kalkan, Department of Medical Genetics, Faculty
of Medicine, Near East University, Near East Boulevard, Nicosia, Cyprus, 99138. Tel: +903-92-
223-6464. Fax: +903-92-223-6461. E-mail: rasime.kalkan@neu.edu.tr, kalkanr@yahoo.com
page: 35
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MATERIALS AND METHODS
The study protocol was approved by the Near East
University Local Ethics Committee [YDU/2016/42-352],
Nicosia, Cyprus. Written informed consent was obtained
from all participants. Genomic DNA was extracted from
blood samples according to the AllPrep DNA/RNA/Protein
(Qiagen GmbH, Hilden, Germany) isolation kit, and
its quantity was measured using a NanoDrop ND-1000
Spec-trophotometer (Thermo Fisher Scientific, Waltham,
MA USA) [19].
Subjects. In this study, the subjects were divided
into two groups. One group consisted of 35 postmenopausal
women with a mean age of 56.7 ± 4.9, and the
other group consisted of 30 premenopausal women with
a mean age of 33.5 ± 6.9. None of the participants had
hypertension, liver, kidney, diabetes, thyroid or cardiovascular
disease. Medical therapy history was obtained
for all subjects. They did not receive any medications or
participate in any dietary or exercise program during the
study. All participants were genetically unrelated postmenopausal
females. Subjects with abnormal menopause,
took medications such as anxiolytics, anti-depressants and/
or exogenous hormone. Women who had serious disease
or mental retardation, smoking, alcohol usage and had
weight loss therapy, food allergies, heart disease history,
insulin-dependent diabetes mellitus type 2 (T2DM), kidney
disease or liver disease, were excluded. Individuals
who had been in postmenopause for a minimum of 1 year
were recruited.
Determination of NFATC1 and FOS Methylation
Status. Bisulfite modification was applied according to
the manufacturer’s protocol (EpiTect Bisulfite Kit; Qiagen
GmbH) and 1.3 μg DNA was used for bisulfite treatment
reaction. The Rotor Gene Q (Qiagen GmbH) was used for
methylation sensitive-high resolution melting (MS-HRM)
analysis to detect the methylation status of NFATC1 and
FOS genes, and universal methylated and unmethylated
DNA (EpiTect Control DNA Set, Cat No. 59568; Qiagen
GmbH) was also used as a control in each reaction. The
primers for each were designed according to the EpiTect®
HRM™ PCR Handbook (Qiagen GmbH) [19].
Statistical analysis. The statistical analyses and their
associations with patient characteristics were performed by
Pearson’s χ2 test and two-tailed Fisher’s exact test. Calculations
were performed using the Statistical Package for
Social Science (SPSS) version 16.0 software (SPSS Inc.,
Chicago, IL, USA), and a p level of 0.05 was considered
to be statistically significant.
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