No association between DNA methylation in BDNF gene and schizophrenia patients in Turkish population

INTRODUCTION: Schizophrenia is a multifactorial disorder. Genetic and environmental factors are involved in the etiology of schizophrenia. Although genetic factors are risk factors for schizophrenia, it is assumed that some environmental factors are required for the manifestationof disease1 . Epigenetic mechanisms regulate gene functions without causing change in the nucleotide sequence of DNA. These regulations are reversible; the most commonly studied epigenetic mechanisms are DNA methylation and histone modification2 . DNA methylation and epigenetic mechanisms are associated with psychiatric disorders such as depression, psychotic disorders, post-traumatic stress disorder, autism, eating disorders, and substance dependence3 . Epigenetics has an important role in gene and environment interactions. This means environmental factors influence the genomic expressions through epigenetic mechanisms. DNA methylation is caused by coupling of a methyl group to CpG sites with the DNA methyltransferase enzyme. A normal level of DNA methylation is required for controlling genomic expressions. Brain Derived Neurotropic Factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity, and it has a role in proliferation, differentiation, survival and death of neuronal and non-neuronal cells. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. Genetic studies show an association between BDNF and schizophrenia. In this study we aimed to investigate the DNA methylation status of the BDNF gene in patients with schizophrenia.

METHODS: The study included 49 patients (aged 35.31±10.35 years, 33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (aged 35.18±9.05 years, 46 male and 19 female). Volunteers in the control group had no personal or familial history of schizophrenia. Individuals with known major health problems, diabetes and malignancies were excluded from the study. Patients were diagnosed with schizophrenia according to the 4th edition of the Diagnostic and Statistical Manual for Mental Disorders (DSM-IV). The severity of schizophrenia symptoms in the patients was evaluated using the Positive and Negative Syndrome Scale (PANSS) and the Clinical Global Impression severity scale (CGI-S). DNA was extracted from blood samples by using salt-chloroform method. Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA would result in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues would remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA. The primers were designed for 2 different CpG islands in the BDNF promoter. 100 ng DNA samples were treated with EpiMark Bisulfite Conversion Kit (Catalog No: E3318, New England Biolabs), in accordance with the manufacturer’s standard instructions. The collected data were analyzed using the SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). Both descriptive and analytical statistics were used. Chi-square/Fisher’s Exact test was used to compare categorical variables. To compare the continuous variables, t test was used.

RESULTS: The mean ages and the gender distribution of the study groups were similar. There were no significant differences in the methylated or un-methylated status for each area between schizophrenia patients and controls (Table 1). When patients were compared with clinical parameters such as duration of the illness, CGI-S, PANSS scores, BMI, and methylated or un-methylated status for each areas, we did not find any significant difference (p>0.05).

DISCUSSION: This study found no difference in methylation status of two regions of the BNDF gene between schizophrenia patients and healthy controls. Although the methylation status of some other genes has been studied in schizophrenia patients, there are limited studies about the methylation status of the BNDF gene in schizophrenia patients. However, in one study BDNF gene methylation status and BDNF gene expression were investigated in schizophrenia patients; BDNF gene methylation was found lower, BDNF gene expression higher in schizophrenia patients compared to controls. The methylation status of some genes other than BDNF has been studied in schizophrenia patients, e.g., MB-COMT and RELN. One study found hypomethylation in the MB-COMT gene and increased transcript levels of MB-COMT in schizophrenia patients. In that study, it is suggested that MB-COMT hypomethylation is a major risk factor for schizophrenia. Another study shows hypermethylation of the RELN promoter. But in contrast to these other studies, it shows that there is no difference in methylation status of COMT and RELN genes in schizophrenia patients compared to healthy controls4 . As far as we can understand from these studies, data on DNA methylation status in patients with schizophrenia are conflicting. However, there must be an association with DNA methylation and schizophrenia according to our hypothesis, and again the methylation status must be different in schizophrenia patients compared to controls. Bu our findings do not support our hypothesis. As mentioned above, there are inconsistent results in methylation studies. Several other factors such as patients’ received antipsychotics or analyzing methylation in blood may affect the methylation status. In a study which examined the effect of antipsychotics on DNA methylation, it is found that clozapine and quetiapine reduced DNA methylation, but there are no similar effects of haloperidol and risperidone on the DNA methylation status. Another study in animals shows that clozapine reduced BDNF methylation and increased social interaction5 . The authors are aware of some limitations of this study. First, we analyzed DNA methylation in peripheral blood, which does not reflect directly the central nervous system. Second, this is a naturalistic and cross-sectional study and patients were continuing their medication. Therefore, antipsychotics received may have affected the methylation status. DNA methylation is a dynamic and reversible process, and many other environmental factors also affect methylation. For these reasons, examining the methylation status and protein levels of the BDNF gene in certain intervals throughout the treatment period in the same patients may be more worthwhile. In conclusion, there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls. Further studies are necessary with more patients, and the limitations referred in this paper should be considered.