Ironments among female nurses. Individuals working in a high work stress environment have 21?5 lower levels of methylation compared to individuals in a low work stress environment. 
Methylation was not associated with 5-HTTLPR genotype. However, we observed 1.4-fold higher methylation levels among individuals carrying the S allele on both of their chromosomes than among L/L individuals, which is concurrent with previous studies showing higher SLC6A4 methylation in blood cells of S-allele carriers [28,29]. We found no evidence in our dataFigure 3. Methylation levels based on burnout scores in high and low work stress environments. 12 nurses in the high stress environment exhibit moderate burnout (MBI-GS.1.5). Nurses in the low stress environment report no signs of burnout (MBI-GS,1.5). doi:10.1371/journal.pone.0045813.gthat the 5-HTTLPR polymorphism would contribute to methylation, burnout or environmental stress, which 1326631 contradicts with earlier interaction studies [21,28], but our study was underpowered to detect such interaction as suggested by the calculated effect sizes and minimal detectable effect sizes. In a recent study, hundreds of methylation linkage disequilibrium (LD) blocks were identified in over 2000 CpG JI-101 site islands [30]. These methylation LD blocks not only cover imprinted genes and X-chromosome regions in females due to X-chromosome inactivation, but they are also found among other genes. Methylation levels in all five CpG residues of the present study, located at close vicinity to each other in a stretch of 30 bp, were highly correlated and shared a single latent factor according to structural equation model. Consequently, a summed methylation score (METsum) from the principal component analysis was used in subsequent analyses in order to represent general methylation level at the region of interest. In most cases methylation at gene promoters leads to silencing of the gene itself. An in vitro study shows that SLC6A4 promoter methylation results in decreased levels of mRNA, although this effect appears AKT inhibitor 2 site stronger when 5-HTTLPR polymorphism is taken into account [13]. The protein encoded by SLC6A4 is responsible for the reuptake of 5-HT from the synaptic clefts and higher levels of SLC6A4 expression will invariably lead to increased reuptake of 5-HT, which decreases the efficiency, magnitude and duration of 5-HT. In the context of this study, we hypothesize that hypomethylation of SLC6A4 presents an adaptational mechanism for stress. While this adaptation is physiological and initially serves to maintain the individual’s best possible functional capacity during stress, it might, eventually, increase risk for functional disturbances, such as decreased cognitive ability and depressed mood, simultaneously with failure of other coping mechanisms. Interestingly, relative lack of serotonin in brain is one of the major hypotheses of depressive disorder, and serotonin transporter is known to be one of the major targets of many antidepressants, including the selective serotonin reuptake inhibitors 
[31]. All nurses in the high stress environment had been working in the same ward for at least 3 consecutive years with relatively low absenteeism and they showed no signs of clinical depression, despite being exposed to a chronic stressful environment. Environmental stress resulted in decreased methylation of the SLC6A4 promoter in blood leucocytes. In theory, if this occurred also in neurons, it would lead to decreased amounts of extracellul.Ironments among female nurses. Individuals working in a high work stress environment have 21?5 lower levels of methylation compared to individuals in a low work stress environment. Methylation was not associated with 5-HTTLPR genotype. However, we observed 1.4-fold higher methylation levels among individuals carrying the S allele on both of their chromosomes than among L/L individuals, which is concurrent with previous studies showing higher SLC6A4 methylation in blood cells of S-allele carriers [28,29]. We found no evidence in our dataFigure 3. Methylation levels based on burnout scores in high and low work stress environments. 12 nurses in the high stress environment exhibit moderate burnout (MBI-GS.1.5). Nurses in the low stress environment report no signs of burnout (MBI-GS,1.5). doi:10.1371/journal.pone.0045813.gthat the 5-HTTLPR polymorphism would contribute to methylation, burnout or environmental stress, which 1326631 contradicts with earlier interaction studies [21,28], but our study was underpowered to detect such interaction as suggested by the calculated effect sizes and minimal detectable effect sizes. In a recent study, hundreds of methylation linkage disequilibrium (LD) blocks were identified in over 2000 CpG islands [30]. These methylation LD blocks not only cover imprinted genes and X-chromosome regions in females due to X-chromosome inactivation, but they are also found among other genes. Methylation levels in all five CpG residues of the present study, located at close vicinity to each other in a stretch of 30 bp, were highly correlated and shared a single latent factor according to structural equation model. Consequently, a summed methylation score (METsum) from the principal component analysis was used in subsequent analyses in order to represent general methylation level at the region of interest. In most cases methylation at gene promoters leads to silencing of the gene itself. An in vitro study shows that SLC6A4 promoter methylation results in decreased levels of mRNA, although this effect appears stronger when 5-HTTLPR polymorphism is taken into account [13]. The protein encoded by SLC6A4 is responsible for the reuptake of 5-HT from the synaptic clefts and higher levels of SLC6A4 expression will invariably lead to increased reuptake of 5-HT, which decreases the efficiency, magnitude and duration of 5-HT. In the context of this study, we hypothesize that hypomethylation of SLC6A4 presents an adaptational mechanism for stress. While this adaptation is physiological and initially serves to maintain the individual’s best possible functional capacity during stress, it might, eventually, increase risk for functional disturbances, such as decreased cognitive ability and depressed mood, simultaneously with failure of other coping mechanisms. Interestingly, relative lack of serotonin in brain is one of the major hypotheses of depressive disorder, and serotonin transporter is known to be one of the major targets of many antidepressants, including the selective serotonin reuptake inhibitors [31]. All nurses in the high stress environment had been working in the same ward for at least 3 consecutive years with relatively low absenteeism and they showed no signs of clinical depression, despite being exposed to a chronic stressful environment. Environmental stress resulted in decreased methylation of the SLC6A4 promoter in blood leucocytes. In theory, if this occurred also in neurons, it would lead to decreased amounts of extracellul.