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

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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