Balkan Journal of Medical Genetics

SUPEROXIDE DISMUTASE 1 AND 2 GENE POLYMORPHISM IN TURKISH VITILIGO PATIENTS
Tuna A1, Ozturk G1, Gerceker TB1, Karaca E2,*, Onay H2, Guvenc SM2, Cogulu O2
*Corresponding Author: Emin Karaca, M.D., Associacte Professor, Department of Dermatology and Venerology, Faculty of Medicine, Ege University, Kazım Dirik mah, Izmir, Turkey. Tel: +90-232-3903961-+90-532-2579285. Fax: +90-232- 3903971. E-mail: karacaemin@gmail.com
page: 67

MATERIALS AND METHODS

Patients. The study included 101 vitiligo patients, with a control group of 99. The diagnosis of vitiligo was based on clinical findings and the Wood lamp examination, while a vitiligo-free family history, and adequate general health, qualified probands for the control group. Types and volumes of vitiligo in relation to the study group are summarized in Table 1 (focal, segmental, acrofacial, generalized universal and mixed). Other factors, such as disease duration, family history and comorbidities including DM or Hashimoto’s thyroiditis were also noted. Written informed consent was obtained from all subjects. The study was approved by the Ethics Committee of Ege University Medical Faculty, Izmir, Turkey. DNA Isolation and Genetic Analyses. Two milliters of peripheral blood were extracted from each participant, collected into EDTA tubes and stored at –20 °C. Genomic DNA was then isolated from the peripheral blood using standard techniques (DNA Isolation Blood Mini Kit; Invitek, Berlin, Germany). PCR-RFLP Analyses. For the polymorphism analysis of SOD1 35 A/C and SOD2 A16V (C/T), polymerase chain reaction-restriction fragment length polymorphim (PCR-RFLP) was performed on both occasions. For SOD1 gene 35 A/C polymorphism, PCR was initiated using forward (5’-CTA TCC AGA AAA CAC GGT GGG CC-3’) and reverse (5’-TCT ATA TTC AAT CAA ATG CTA CAA AAC-3’) primers. For SOD2 gene A16V (C/T) polymorphism, forward (5’-AGC AGG CAG CTG GCG CCG G-3’) and reverse (5’-GCG TTG ATG TGA GGT TCC AG- 3’) primers were used. A total volume of 25 μL, containing 50 ng DNA, 2 mM dNTPs, 2 nmol of each related primer, 1.5 mM MgCl2 and 3U Taq polymerase, was used in each polymorphism PCR set up. Each PCR cycle consisted of denaturation at 94 °C for 30 seconds, extension at 72 °C for 30 seconds, and a final extension step of incubation at 72 °C for 7 min. Annealing temperatures were 55 °C and 59.6 °C for 30 seconds for SOD1 35 A/C and SOD2 A16V (C/T) polymorphisms, respectively. Genotyping of the SOD1 Gene 35 A/C Polymorphism. The 278 bp PCR products for the SOD1 gene 35 A/C polymorphism were digested using the restriction enzyme HhaI (Fermentas, The Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 3 hours with 5U enzymes. The presence of the 35C mutant allele manifested through the appearance of sites of restriction. Following enzyme digestion, products were then cut into two fragments of 207 and 71 bp (Figure 1). Genotyping of the SOD2 Gene A16V (C/T) Polymorphism. A 267 bp amplicon of the SOD2 gene A16V (C/T) polymorphism was digested using NgoMIV (Fermentas) restriction endonuclease, left overnight at 37 °C. Due to the presence of a mutant allele, the 267 bp PCR amplification product digested into fragments of 183 and 84 bp, respectively (Figure 1). All fragments were visualized via electrophoresis on 4.0% agarose gel after restriction. Positive and negative controls were used in each genotyping assay. Sequencing of the PCR Amplified Product. As a safeguard, Sanger sequencing of the PCR product was used to confirm the presence of the SOD1 and SOD2 gene sequences before continuing (Figure 2). Statistical Analyses. The SOD1 35 A/C and SOD2 A16V (C/T) polymorphism allele frequencies of Vitiligo patients were compared to those of a control group using the χ2 test; the application of statistical program SPSS 9.0. p Values of <0.05 (one-way) were considered to be significant.

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