ASSOCIATION BETWEEN OBSESSIVE COMPULSIVE DISORDER AND TUMOR NECROSIS FACTOR-α GENE –308 (G>A) AND –850 (C>T) POLYMORPHISMS IN TURKISH CHILDREN
Lüleyap HU1, Onatoğlu D1, Tahiroğlu AY2, Alptekin D1,*, Yılmaz MB1, Çetiner S3, Pazarbaşı A1, Ünal İ4, Avcı A2
*Corresponding Author: Professor Dr. Davut Alptekin, Department of Medical Biology and Genetics, Medical Faculty, Çukurova University, 01330 Adana, Turkey; Tel.: +90-322-3386060/3498; Fax: +90-322-3386572; E-mail: alptekin@cu.edu.tr
page: 61

MATERIALS AND METHODS

This study included 49 children with OCD attending the Department of Child and Adolescent Psychiatry, Medical Faculty, Çukurova University, Adana, Turkey, and 58 healthy children attending the Department of Social Pediatrics, Medical Faculty, Çukurova University, Adana, Turkey. Ages of the patients and those of the control group ranged between 4 and 12 years. Psychiatric diagnoses were determined by the specialist doctors from the Department of Child and Adolescent Psychiatry, Medical Faculty, Çukurova University, Adana, Turkey in accordance with the Diagnostic and Statistical Manual of Mental Disorder (DSM-IV) criteria and with Kiddie Schedule for Affective Disorders and Schizophrenia-Present and Lifetime (KSAD-S-PL) version. The Children’s Yale-Brown Obsessive Compulsive Scale (CY-BOCS) was used to evaluate the severity of OCD symptoms [20,21]. Demographic data and previous infection histories of patients and their families included a positive account of infection: tonsillitis (more than six tonsillitis attacks/year), prophylactic antibiotic/penicillin use, tonsillectomy, ASO titers, as well as symptom aggravation associated with infections. This study was approved by the Ethics Committee of the Medical Faculty of Çukurova University, Adana, Turkey. The patient and control groups were informed and we obtained written parental consent; all consented to be a part of this study before peripheral blood samples were drawn. In order to identify the TNF-α gene polymorphisms, we isolated the DNA from blood employing the salting out method of Miller et al. [22] and it was stored at 4°C until use. Areas with the –308 and –850 polymorphisms were amplified with polymerase chain reaction (PCR). Poly-morphisms were genotyped using available restriction enzymes with restriction fragment length polymorphism (RFLP) and polyacrylamide gel electrophoresis (PAGE) methods [23]. For the –308 polymorphism, the 142 bp product was amplified with the following primers: F-5’-GGG ACA CAC AAG CAT CAA GG-3’ and R-5’-AAT AGG TTT TGA GGG CCA TG-3’; the 126 bp restricted product was named as the mutant AA genotype; the non restricted 142 bp product was named as the normal GG genotype. This polymorphism includes only one base variation (G>A), is differentiated by the NcoI restriction enzyme on an 8.0% polyacrylamide gel. For the –850 polymorphism, the 131 bp product was amplified with primers F-5’-AAG TCG AGT ATG GGG ACC CCC CGT TAA-3’ and R-5’-CCC CAG TGT GTG GCC ATA TCT TCT T- 3’; the 106 bp restricted product was named as the normal CC genotype; the non restricted 131 bp product was named as the mutant TT genotype. This polymorphism, which includes only one base variation (C>T) was differentiated by the HindII enzyme on an 8.0% polyacrylamide gel [24,25]. The normal value of ASO is in fact 200, but in our study the cut-off value was taken as 400, considering these children were already in the active infection period. Antristreptolysin O was considered by the TODD (Strepto-lysin O enzyme) unit. Values above 200 were accepted as significant and referred to a new infection as an indicator [26]. Serum ASO titers of patients were determined using the Beckman Delta Nephelometor at the Department of Central Laboratory Biochemistry, Balcalı Hospital, Medical Faculty, Çukurova University, Adana, Turkey. This laboratory is accredited by the International Joint Commission, (IL, USA). Statistical analyses for comparing the prevalence of mutant genotypes between OCD patients and the control group was carried out using the Chi-square (χ2) test. To measure the effect of ASO titer values on the mutant genotypes in OCD patients, the Kruskal- Wallis test was used for overall comparison and Bonferroni adjustments (dividing the type I error by three, the number of subgroups, or equivalently, multiplying the p values by three, i.e., α = α/3 = 0.017) were used for multiple comparisons. The Mann Whitney U test was applied. Throughout the analysis, the SPSS 15.0 package program was used with the level of statistical significance accepted as p <0.05.



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