schizophrenia is a multifactorial disorder , and genetic and environmental factors are involved in its etiology . although genetic factors are risk factors for schizophrenia , it is believed that some environmental factors are required for the manifestation of disease . epigenetic mechanisms regulate gene functions without changing the nucleotide sequence of dna . these regulations are reversible , and the most commonly studied epigenetic mechanisms are dna methylation and histone modification . dna methylation and epigenetic mechanisms are associated with psychiatric disorders , such as depression , psychotic disorders , post - traumatic stress disorder , autism , eating disorders , and substance dependence [ 57 ] . epigenetic factors have an important role in gene and environment interactions , and environmental factors influence the genomic expressions through epigenetic mechanisms . epigenetic mechanisms are associated with many environmental factors , including nutrition , maternal care and behavior , hormones and drugs , early life experiences , and environmental agents in early development stages . dna methylation is caused by coupling of a methyl group to cpg sites with the dna methyltransferase enzyme disease - related hypermethylation is thought to be associated with decreased rna levels and hypomethylation is associated with increased rna levels . an animal study reported that animals showed schizophrenia - like behaviors and molecular changes , such as significant decrease in bdnf mrna variants , increase in dna methyltransferase1 ( dnmt1 ) , and ten - eleven translocase methylcytosine dioxygenase1 ( tet1 ) , no changes in histone deacetylases , histone methyltransferases and demethylases , or methyl cpg binding protein 2 in mice exposed to prenatal stress . this and several other studies show that environmental factors , such as prenatal stress , influence brain development and cause schizophrenia by epigenetic mechanisms , especially in early stage of brain development [ 1416 ] . brain - derived neurotrophic 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 plays a role in the pathophysiology of schizophrenia . a schizophrenia animal model induced by prenatal stress showed increased dna methylation of the bdnf gene and reduced bdnf transcription . reduced bdnf levels in the prefrontal cortex , serum and csf samples of patients with schizophrenia have been reported [ 2023 ] . bdnf protein level and mrna expression are lower in the prefrontal cortex of schizophrenia patients . however , other studies showed higher bdnf levels in serum of schizophrenia patients   and there are conflicting results regarding bdnf levels in the hippocampus . postmortem studies have shown increased bdnf levels in the parietal cortex and frontal cortex , but decreased levels in the temporal cortex and occipital cortex . another post - mortem study showed abnormalities of dna methylation in brains of patients with schizophrenia . methylation status of the bdnf gene is associated with fear learning , memory , and stressful social interactions . dna methylation has a role in regulation of the bdnf gene in schizophrenia [ 3133 ] and dna methylation and demethylation may be used as targets for antipsychotic therapy . in this study we aimed to investigate the dna methylation status of the bdnf gene in patients with schizophrenia . we report the methylation status of 2 cpg islands of the bdnf gene that are located on the promoter region and intron 3-exon 4 boundary , as shown in figure 1 . the study included 49 patients ( aged 35.3110.35 years , 33 male and 16 female ) with schizophrenia and 65 unrelated healthy controls ( aged 35.189.05 years , 46 male and 19 female ) . mean duration of illness was 11.68.7 ( min : 1 , max : 36 ) years , mean panss total score was 66.330.2 ( min : 30 , max : 129 ) , and mean cgi - s score was 3.92.16 ( min : 1 , max : 7 ) . thirty - two patients received atypical antipsychotic , 1 patient received typical and atypical antipsychotic combination , 11 patients received rlai and atypical antipsychotic combination , and 7 patients received zuclopenthixol or flupenthixol depot and atypical antipsychotic combination . volunteers in the control group had no personal or familial history of schizophrenia . individuals with known major health problems , including diabetes and malignancies , were excluded from the study . patients were diagnosed with schizophrenia according to the 4 edition of the diagnostic and statistical manual for mental disorders ( dsm - iv ) . patients and controls were of the same ethnic origin and from the same geographical area ( south - eastern turkey ) . 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 ) . the study was approved by the local ethics committee , informed consent was obtained from all participants , and the study was conducted in accordance with the declaration of helsinki . determination of methylation pattern of cpg islands was based on the principle that bisulfite treatment of dna results in conversion of unmethylated cytokine residues into uracil , whereas methylated cytokine residues 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 ( figure 1 ) . e3318 , new england biolabs ) , in accordance with the manufacturer s standard instructions . primer sequences were : bdnf_1_mfw : 5-gtagttttcgtaggatgaggaagc-3 and bdnf_1_mrv : 5-aatataaattaacaaccccgatacg-3 for the methylated ( m ) reaction and bdnf_1_ufw : 5-gtagtttttgtaggatgaggaagtg-3 and bdnf_1_urv 5-tataaattaacaaccccaatacaca-3 for the unmethylated ( u ) reaction . bdnf_2_mfw : 5-atgatagcgtacgttaaggtatcgt-3 , bdnf_2_mrv : 5-aacgaaaaactccatttaatctcg-3 and bdnf_2_ufw : 5-atatgatagtgtatgttaaggtattgt-3 , bdnf_2_urv : 5-acaaaaaactccatttaatctcaac-3. pcr mixture contained 10 ng of modified dna , 2.0 mmol each of datp , dgtp , dctp , and dttp , 1.0 pmol each of primer , 2.0 mmol mgcl2 , 1 reaction buffer , and 1.0 u taq polymerase . the pcr conditions were : 1 cycle at 95c for 1 min , 12 cycles at 95c for 10 s , at 54c to 48c ( touch - down ) for 40 s , at 72c for 60 s , then 25 cycles at 95c for 10 s , at 52c for 40 s , at 72c for 60 s , and 1 cycle at 72c for 5 min . amplified pcr products were electrophoresed on 2% agarose gels , visualized by staining with ethidium bromide , and illuminated under uv light . positive control sample ( fermentas , cat . the mean ages ( f=1.306 , p=0.947 ) and the sex distribution ( x=0.154 , df=1 , p=0.695 ) of the study groups were similar . for bdnf gene cpg island-1 , 3.9% ( n=2 ) was methylated , 68.6% ( n=35 ) was hemi - methylated , and 27.4% ( n=14 ) was unmethylated in he patient group . there were no methylated subjects , 67.7% ( n=44 ) were hemi - methylated , and 32.3% ( n=21 ) were unmethylated in the control group ( figure 2 ) . there were no significant difference in methylated or unmethylated status for each area between schizophrenia patients and controls ( p>0.05 ) . the comparisons of the methylated or unmethylated status for each area according to the study groups are presented in table 1 . when patients were compared with clinical parameters ( duration of the illness , cgi - s , panss scores , bmi , and methylated or unmethylated status for each area ) we did not find any significant difference ( p>0.05 ) . there were no differences in dna methylation status of bdnf gene cpg island-1 and cpg island-2 regions with respect to sex or age between the schizophrenia patients and the control group ( p>0.05 ) . the mean duration of illness was significantly lower in the hemi - methylated group compared to non - methylated group for bdnf gene cpg island-1 in schizophrenia patients ( p=0.017 ) . however , there was no difference in mean duration of illness between the hemi - methylated group compared to the non - methylated group for bdnf gene cpg island-2 in schizophrenia patients ( p>0.05 ) . there were no differences in median cgi - s panss total and subscale scores between hemi - methylated group compared to non - methylated group for bdnf gene cpg island-1 and cpg island-2 regions in schizophrenia patients ( p>0.05 ) . we found that there was no difference in methylation status of 2 regions of the bndf gene between schizophrenia patients and healthy controls , suggesting that the genetic component of schizophrenia is very complex . the most likely candidate genes are : gabaergic genes ( reln and gad1 ) , dopaminergic genes ( dat and drd2 , mb - comt ) , 5-ht2a receptor ( htr2a ) gene , and bdnf . it is suggested that down - regulation of gabaergic genes such as reln and gad1 are related to hypermethylation of their gene promoters , causing gabaergic dysfunction ; moreover , it plays a role in schizophrenia [ 3840 ] . however , in another study , decreased methylation of the gad1promoter and lower levels ofgad1mrna in schizophrenia patients were found . a postmortem study performed in patients with schizophrenia showed increased methylation of reln gene and decreased reln gene expression . another study showed no or less methylation in reln gene until puberty , while increased levels of methylation were found after puberty . 5-ht2a receptor ( htr2a ) gene is a serotonergic system gene and in a study it was found that methylation of the htr2a gene promoter correlated with htr2a expression levels . however , another study found no difference between schizophrenia patients compared to controls in htr2a methylation . epigenetic regulations of genes influencing the dopaminergic system , such as comt , drd2 , and dat genes , have been investigated in schizophrenia . it was shown that there was a hypomethylation in mb - comt gene and it was suggested that this methylation is a risk factor for schizophrenia . however , another study was found no difference in comt promoter methylation between schizophrenia patients and controls . it was found that there were no differences in drd2 and dat gene methylation between schizophrenia patients and controls . a postmortem study found that there were no differences in bdnf gene exon i , iii , vi , and ix regions methylation status between schizophrenia patients and controls , but the same study fond reduced dna methylation in the val66met snp in exon ix . there are 2 other studies on bdnf gene methylation with peripheral blood samples of schizophrenia patients  one study showed reduced bdnf promoter iv methylation , and the other one showed increased bdnf promoter i methylation in schizophrenia patients compared to controls . another study with peripheral blood samples of japanese schizophrenia patients found that there was a small but significant increase in methylation in the bdnf 1 promoter , and the same study suggested that epigenetic differences in peripheral blood might be a potential biomarker and reflect the pathophysiology of schizophrenia . a review by mitchelmore and gede suggested that bdnf had a central role in neuronal development and neuroplasticity , and dysregulation of the bdnf gene was associated with several psychiatric and neurologic disorders . in addition , epigenetic mechanisms that regulate bdnf gene expression could be used in diagnosis , prognoses , and as biomarkers of several psychiatric disorders . in the present study we found that there were no associations between dna methylation status of bdnf gene cpg island-1 and cpg island-2 regions with respect to sex or age . however , another study showed that the methylation status of bdnf promoter was associated with sex  there was more prominent methylation in male patients with schizophrenia . the mean duration of illness was significantly shorter in the hemi - methylated group compared to the non - methylated group for bdnf gene cpg island-1 in schizophrenia patients . to the best of our knowledge there is no published data about the association between duration of illness and dna methylation in schizophrenia . a review of dna hypomethylation and human diseases showed that dna methylation plays a role in neuronal cell survival and maturation ; the balance of the methylation level is important for neuronal survival and hypomethylation causes abnormalities in neuronal function . although the mechanism is not clear , a study mentioned that dna hypomethylation may cause cell death by apoptosis . considering these data , it appears that reduced methylation ratios with duration of disease may cause the neuronal dysfunction by altering neuronal survival , suggesting that there is an association between duration of illness and dna methylation . we hypothesized that there was a defect in the dna methylation mechanisms that affect the bdnf gene , and that there is an association between dna methylation and schizophrenia . several other factors , such as patients receiving antipsychotics and analyzing methylation in blood , may affect the methylation status . a study that examined the effect of antipsychotics on dna methylation found that clozapine and quetiapine reduced the dna methylation , but there are no similar effects of haloperidol and risperidone on dna methylation status . another study in animals showed that clozapine reduces bdnf methylation and that this increases social interaction . first , we analyzed dna methylation in peripheral blood , which does not directly reflect the central nervous system . second , this is a naturalistic and cross - sectional study and patients were continuing their medication . dna methylation is a dynamic and reversible process , and many other environmental factors also affect methylation . , examining the methylation status and protein levels of bdnf gene at certain intervals throughout the treatment period in the same patients may be more valuable . although there were no differences in bdnf gene methylation status between schizophrenia patients and healthy controls , there was an association between duration of illness and dna methylation . further studies with more patients are necessary , and the limitations referred to in this paper should be considered .
backgroundalthough genetic factors are risk factors for schizophrenia , some environmental factors are thought to be required for the manifestation of disease . 
 epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of dna . 
 brain - derived neurotrophic factor ( bdnf ) is a neurotrophin that regulates synaptic transmission and plasticity . 
 it has been suggested that bdnf may play a role in the pathophysiology of schizophrenia . 
 it is established that methylation status of the bdnf gene is associated with fear learning , memory , and stressful social interactions . in this study , we aimed to investigate the dna methylation status of bdnf gene in patients with schizophrenia.material/methodsthe study included 49 patients ( 33 male and 16 female ) with schizophrenia and 65 unrelated healthy controls ( 46 male and 19 female ) . 
 determination of methylation pattern of cpg islands was based on the principle that bisulfite treatment of dna results in conversion of unmethylated cytosine residues into uracil , whereas methylated cytosine residues remain unmodified . 
 methylation - specific pcr was performed with primers specific for either methylated or unmethylated dna.resultsthere was no significant difference in methylated or un - methylated status for bdnf promoters between schizophrenia patients and controls . 
 the mean duration of illness was significantly lower in the hemi - methylated group compared to the non - methylated group for bdnf gene cpg island-1 in schizophrenia patients.conclusionsalthough there were no differences in bdnf gene methylation status between schizophrenia patients and healthy controls , there was an association between duration of illness and dna methylation .