THE IMPACT OF THE D727E POLYMORPHISM HAS
NO SIGNIFICANT ROLE IN MULTI NODULAR GOITER Tug E1,*, Sengül N2, Aydin H3, Yilmaz EE2 *Corresponding Author: Esra Tug, M.D., Ph.D., Gazi University, Faculty of Medicine, Department of Medical
Genetics, 06500 Ankara, Turkey; Tel.: +90-312-202-69-44; Fax: +90-312-202-46-35; E-mail: esratug@hotmail.com page: 67
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MATERIALS AND METHODS
We studied 31 consecutive Turkish patients with
MNG who underwent total thyroidectomy at the
Department of General Surgery, Abant Izzet Baysal
Medical School, Bolu, Turkey. The Abant Izzet Baysal
University Ethics Committee had approved the
study (#2009/100-78) and written informed consent
was obtained from all subjects after full description
of the objectives and procedures of the study.
Every patient underwent radioimmunology analysis
of serum TSH, free thyroxine (FT4), free triiodothyronine
(FT3) and thyroid autoantibodies (TPOAb,
TGAb) before surgery and medical therapy for hyperthyroidy.
According to the results of thyroid function
tests, thyroid ultrasonography and a thyroid technetium
99 m (99mTc) scintigraphy, the patients were divided
into toxic (subclinical hyperthyroidism and hypertyroidism)
MNG, and non toxic MNG. None of the
patients had mental illnesses, systemic diseases (neurological
or other endocrine), acute or chronic inflammatory
or autoimmune diseases. Seventy-five percent
of the patients have a positive family history of MNG.
Thirty healthy subjects attending the outpatient
laboratory of the hospital with no history or evidence
of MNG and no first-degree relatives with hyperthyroidism
were recruited as control group. The patients
and controls were unrelated Turkish subjects residing
in the Bolu region, in northwest Turkey. The demographic
characteristics and laboratory criterion of the
patients and controls are shown in Table 1.
Venous blood (3 mL) was collected from subjects
into EDTA-coated tubes, and genomic DNA
was extracted using the QIAamp DNA Blood Mini
Kit (Qiagen, Hilden, Germany). A pair of primers
was designed to flank the region in exon 10 of the
TSHR gene that contains the p.D727E polymorphism
(rs1991517), as previously described [11]. To amplify
the 196 bp DNA product containing the polymorphic
site, primers 5’-CTC AGC AAG TTT GGC
ATC TG-3’’ (forward) and 5’-CTT CTG AGA TTT
GGC CTT GC-3’ (reverse) were used [http://innere.
uniklinikum-leipzig.de/tsh/frame.html].
Polymerase chain reaction (PCR) was used to
amplify genomic DNA in a 25 μL reaction volume
containing 2.5 μL genomic DNA (20-50 ng), 1.5 μL
MgCl2 (25 mM), 1 μL dNTP (2.5 mM) 1 μL forward
primer (20 ng/μL) and 1 μL reverse primer (20 ng/μL)
and 0.1 μL Taq DNA Polymerase (5U/μL) (Vivantis
Technologies, Selangor Malaysia), in the PCR buffer
provided by the manufacturer (10mmol/L Tris-HCl,
pH 8.0, and 100 mmol/L KCl). The PCR involved an
initial denaturation at 95°C for 3 min., followed by 35
cycles of denaturation at 95°C for 30 seconds, annealing
at 56°C for 30 seconds, and extension at 72°C for
30 seconds, with a final extension at 72°C for 4 min.,
using a thermocycler (Takara, Shiga, Japan). The PCR
amplification product was 196 bp long. To detect the
C>G substitution, we used the NlaIII (Hin1II) (Fermentas,
Vilnius, Lithuania) restriction enzyme. The G
allele fragments were 129 and 67 bp long, while those
for the C allele were 108, 67 and 21 bp long (Figure 1). Statistical analysis was done by using SPSS software
package. Mann-Whitney U tests and Chi-square
tests were used whenever appropriate and a p value
of <0.05 was considered significant.
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