FREQUENCIES OF THE COMMON MEFV GENE MUTATIONS IN ADIYAMAN, SOUTHEAST ANATOLIA, TURKEY
Korkmaz DT1,*, Atak PG2, Çelik Ç3
*Corresponding Author: Deniz Taştemir Korkmaz, Ph.D., Vocational School of Health Services, Adıyaman University, TR-02040 Adıyaman, Turkey. Tel: +90-416-223-3800/4154. Fax: +90-416-223-2071. E-mail: deniz-tbio@hotmail.com
page: 67

MATERIALS AND METHODS

Study Population. The study involved 767 healthy individuals who had applied for premarital tests at the Adıyaman Obstetrics and Gynecology & Child Diseases Hospital, Adıyaman, Turkey. It was granted ethical approval by the local health committee and written consent was obtained from all individuals. Collective data (gender, age, hometown) for each individual were obtained. The study group consisted of 395 females and 372 males, and their ages ranged from 18 to 64 years with a mean age of 26.45 5.54 years. Screening the Common MEFV Gene Mutations. Peripheral blood samples were collected from a total of 767 healthy individuals who had applied for premarital tests at the Adıyaman Obstetrics and Gynecology & Child Diseases Hospital, Adıyaman, Turkey. Genomic DNA was isolated from 0.2 mL whole blood using a precipitation method in which saturated saline solution was used [7]. Four common MEFV gene mutations (M694V, M680I, M694I, V726A) located in exon 10 were screened with polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) methods. Primers used in M694V, M680I, M694I and V726A mutations were selected from previous studies [8]. Internal control primers (forward: 5-TGT ATC ATT GTT CTG GGC TCT-3, reverse: 5-AGG GCT GAA GAT AGG TTG AA-3) were also used in the study. The PCR mixture (25 μL) included 1 × PCR buffer, 1.25 mM MgCl2, 0.02 mM dNTPs, 5 pmol normal/mutant primers, 2.5 pmol internal control primers, 50 ng DNA, and 2 U Taq Polymerase (Fermantas , Thermo Scientific, Waltham, MA, USA). The PCR conditions for M694V, M680I and V726A identification were as follows: one cycle of initial denaturation at 94 C for 9 min., followed by 35 cycles of denaturation at 94 C for 10 seconds, annealing at 61 C for 10 seconds and elongation at 72 C 30 seconds; and for identification for M694I was as follows: once cycle of initial denaturation at 94 C for 5 min., followed by 35 cycles of denaturation at 94 C for 1 min., annealing at 60 C for 1 min. and elongation at 72 C for 2 min. The PCR products were electrophoresed in 3.0% agarose gels in 0.5 × Tris-Borate- EDTA (TBE) and visualized with ethidiumbromide. The product sizes were 212 bp for M694V, 220 bp for M680I, 184 bp for M694I, 247 bp for V726A, and 360 bp for internal control (Figure 1).



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