Balkan Journal of Medical Genetics

RLIP76 GENE VARIANTS ARE NOT ASSOCIATED WITH DRUG RESPONSE IN TURKISH EPILEPSY PATIENTS
Manguoğlu E1,*, Akdeniz S1, Dündar NO2, Duman Ö2, Aktekin B3, Haspolat Ş2, Bilge U4, Özel D4, Lüleci G1
*Corresponding Author: Esra Manguoğlu, Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University, Antalya, Turkey; Tel.: +90-242-249-6977; Fax: +90-242-249-6906; E-mail: emanguoglu@akdeniz.edu.tr
page: 25

MATERIALS AND METHOD

Patients. A total of 146 (81 male and 65 female) refractory and 155 (79 male and 76 female) non refractory patients were recruited for the study. The mean age for refractory and non refractory groups were 34.4 ± 17.8 (ranging from 3 to 83 years) and 25.4 ± 17.5 (ranging from 2 to 78 years), respectively. One-hundred and forty-three the patients were above the age of 18, while 158 patients were below the age of 18. Patients who were seizure free for at least 1 year were regarded as non refractory epilepsy patients. Patients, who had seizures, in spite of taking at least three different AEDs with maximum tolerable doses, were considered to be refractory epilepsy patients. This study was approved by the Akdeniz University Ethics Committee (Antalya, Turkey). Information about the study was given to all patients. A signed, informed consent was obtained from all patients or from adults responsible for them. Patients were recruited from Akdeniz University Hospital, Departments of Pediatrics and Neurology (Antalya, Turkey). All patients were Turkish. Genotyping. DNA isolation from peripheral leukocytes was performed by a standard salting-out method [13]. All coding exons (exons 2-10) and exon-intron boundaries of the RLIP76 (RALBP1, NM_006788.3) gene were screened for sequence changes by denaturing high performance liquid chromatography (HPLC). The polymerase chain reaction (PCR) primer sequences are given in Table 1. The PCR reactions were set up in 20 ML volume with 10-20 ng DNA, 0.5 U Optimase Taq DNA polymerase, using a PE9700 thermal cycler [Applied Biosystems Inc. (ABI), Foster City, CA, USA]. Amplifications were done by touch-down PCR with annealing temperature with decreasing increments of 0.5°C in each cycle starting from 61°C to 54°C. Denaturing HPLC analyses were performed on a Transgenomic (Omaha, NE, USA) Wave HS nucleic acid fragment analysis system with DNASep cartridges, WAVE Optimized buffers and standards. To increase the sensitivity of these analyses, they were performed on at least two different oven temperatures for each PCR fragment. All regions that have denaturing HPLC patterns suggesting a sequence change were sequenced successfully to define the sequence change. The PCR products were purified using a Roche High Pure PCR product purification kit (catalog no. 11 732 676 001; Roche Applied Science, Indianapolis, IN, USA). A direct sequencing protocol was applied and BigDye Terminator v.1.1 cycle sequencing system was used (Applied Biosystems). Sequencing samples were separated using an ABI 3130™ system (Applied Biosystems). Statistical Analysis. When minimum expected values were less than 5, the Fisher’s Exact test was used. When the minimum expected value was between 5 and 25, the Yate’s Continuity Correction test was used. In all other circumstances, the Pearson χ2 (Chi-square) test was used. Statistical analysis was performed with SPSS version 15 (SPSS Inc., Chicago, IL, USA); p <0.05 was considered to be statistically significant.

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