5-HTT Promoter Research Articles

Predicting anxiety-related personality traits by means of serotonergic VNTR variants STin2 and 5-HTTLPR

Polymorphisms of the serotonergic system are amongst the most commonly investigated genetic variants with respect to anxiety-related personality traits and affective disorders. Mostly the prominent 5-HTTLPR, a functional VNTR in the 5-HTT promoter region, is intensively analysed but effect sizes in meta-analyses are small and results are inconsistent. We reinvestigated the association of 5-HTTLPR with harm avoidance (HA) and neuroticism taking another functional 5-HTT-VNTR (STin2) into account, as both VNTRs have transcription regulating properties and research points to combinatorial effects on transcription efficacy. N = 2969 participants, among them 447 inpatients suffering from affective disorders, were genotyped and filled in the TCI, NEO-FFI personality inventories besides the CLEq measuring the extent of experienced stressful life events. Significant main effects for the 5-HTTLPR with inpatients carrying the L+ allele having lower HA scores as well as for the STin2 with healthy controls carrying at least one STin2.12R allele having lower neuroticism scores were observed. Besides no gene-interaction occurred. However, specific haplotype effects were observed in healthy participants as well as in the total sample. More specifically, the 12/L variant was associated with significant lower HA and neuroticism scores. Results highlight the multifactorial interplay of 5-HTT genetic variants and the use of haplotypes in association studies on anxiety-related personality traits with impact on affective disorders.

Cortico-limbic functional connectivity mediates the effect of early life stress on suicidality in bipolar depressed 5-HTTLPR*s carriers

BackgroundIn bipolar disorder (BD) the risk of suicide in adult life can be influenced by the interaction of adverse childhood experiences with the serotonin transporter polymorphism (5-HTTLPR). The cortico-limbic connectivity is a candidate endophenotype for the disorder, also related to suicidality and affected by the 5-HT system. MethodsIn 64 (*s carriers = 41; l/l = 23) depressed BD patients, we explored the effect of 5-HTTLPR on corticolimbic functional connectivity (FC) during emotional processing, and the role of FC in moderating/mediating the effect of early stressful events on suicidality among 5-HTTLPR groups, by implementing Generalized Structural Equation Model. Results5-HTTLPR affects FC between amygdala (Amy) and anterior cingulate cortex (ACC), temporal pole, putamen/thalamus, and precuneus. The short allele was associated to a more inefficient corticolimbic connectivity. In 5-HTTLPR*s carriers, but not in l/l, the Amy-ACC functional coupling mediated the relationship between stress load and current suicidality. LimitationsPatients were not drug-naive, and the recruitment took place in a single center, thus raising the possibility of population stratifications. The sample size is relatively small, but our findings can provide the background for replication study in independent and larger datasets. ConclusionsOur results confirm the link between the 5-HTT promoter polymorphism and susceptibility to stress in BD, and suggest that cortico-limbic functional connectivity mediates these effects. This pattern could identify a vulnerability factor for the exacerbation of mood episodes after stressful life events particularly relevant in *s carriers.

M85 - DNA METHYLATION AND 5-HTTLPR GENOTYPE OF THE SEROTONIN TRANSPORTER GENE (SLC6A4) IN ANTIDEPRESSANT TREATMENT RESPONSE OF MAJOR DEPRESSIVE DISORDER

Background The current process used to prescribe antidepressant medication is markedly inefficient, as more than 50% of treated patients fail to reach remission. Antidepressant treatment success has been associated with genetic variation, but studies are not well replicated and epigenetic mechanisms remain under investigated. DNA methylation may provide more information regarding antidepressant response and guide physicians in choosing the most effective medication for each patient. We investigated the influence of SLC6A4 methylation and 5-HTTLPR genotypes on antidepressant response in our sample. Methods A subset of depression patients with complete clinical data (European Caucasian, n=166), were selected from our discovery sample (IMPACT; N=8,000). Saliva samples were collected at enrollment and genetic testing was done of cytochrome P450 liver enzyme, 5HTTLPR and 5HT2A variants to guide clinical choice and dosage of psychotropic medications. As such, a significant component of our sample includes treatment refractory, as well as medication intolerant patients. DNA was bisulfite converted and methylation profiles were interrogated using the Infinium HumanMethylation450 Beadchip array. Nine CpG probes located across the SLC6A4 promoter region were selected, based on previous studies, to quantify methylation levels. Change in Beck Depression Inventory II (BDI-II) score was used to measure antidepressant response over eight weeks. The relationship among genotype, methylation levels, and antidepressant response was analysed using linear regression modeling. Results Increased methylation in the promoter region of the SLC6A4 gene was nominally associated with impaired response to antidepressants in our sample. The effect was driven by methylation site cg05016953, located in exon 1 A. Increased methylation level as measured by CpG cg05016953 was associated with less improvement in BDI-II score over eight weeks of antidepressant treatment (F(3,163)=6.14, puncorr=0.012). 5-HTTLPR genotype was not associated with SLC6A4 DNA methylation or antidepressant treatment response. Discussion An increase in SLC6A4 DNA methylation at cg05016953 may be associated impaired response to antidepressant medications in our sample. The SLC6A4 transcriptional control region contains a CpG island, previously shown to functionally influence mRNA levels depending on 5-HTTLPR genotypes. Typically, hypermethylation in promoter regions is associated with decreased gene expression. Theoretically, hypermethylation of the 5-HTT promoter would reduce the number of serotonin transporters, and reduce serotonin re-uptake, which in simple terms should improve response to SSRI treatment. Further pharmaco-epigenetic studies are required to elucidate the effect of DNA methylation changes on antidepressant response.

The influence of the serotonergic system on the personality and quality of life of postmenopausal women.

The aim of this study was to establish the relationship between personality traits of postmenopausal women and the presence of the 44-bp VNTR polymorphism in the serotonin transporter (5-HTT) (SLC6A4) promoter region and the 30-bp VNTR polymorphism in the MAO-A promoter region. The study’s aim was also to determine the influence of personality traits on the quality of postmenopausal women’s lives. The study involved 214 postmenopausal women from northwest Poland, with an average age of 56.8±4.08 years. It was performed using the Temperament and Character Inventory-Revised and the Short Form Health Survey. DNA polymorphisms were identified by means of polymerase chain reaction. Analysis demonstrated that the s/s genotype was significantly more common than the l/l genotype in women with higher fear of uncertainty. In a group with higher enlightened second nature and empathy, the l/s genotype was considerably more common than the l/l genotype. There were statistically significant associations between selected aspects of quality of life and personality traits such as enlightened second nature, transpersonal identification, purposefulness, and self-transcendence. The s/s genotype of the 44-bp VNTR polymorphism in the 5-HTT (SLC6A4) promoter region may increase the tendency to avoid harm within the fear of uncertainty dimension. Carriers of this genotype may have predisposition to anxiety and depressive disorders. The l/s genotype of the 44-bp VNTR polymorphism in the 5-HTT (SLC6A4) promoter region contributes to increased expression of enlightened second nature and empathy. Some personality traits may influence the quality of women’s lives.

The analysis of anxiety and mood in healthy late-reproductive-stage women with regard to hormonal and genetic factors.

The purpose of this study was to determine whether anxiety and mood disorders in late-reproductive-stage women are related to the serotonin transporter and monoamine oxidase A gene polymorphisms. Research instrument used in this study were the State-Trait Anxiety Inventory and the UWIST Mood Adjective Checklist. The 44-bp VNTR polymorphism in the 5-HTT (SLC 6A4) promoter region and the 30-bp VNTR polymorphism in the MAO-A promoter region were analyzed. The study included 345 healthy Polish women in the late reproductive stage. The mean age of the participants was 42.3 ± 4.5 years. State anxiety was observed in 16.8 % of the women and trait anxiety in 14.5 %. There were no statistically significant differences in the mood and the mean levels of anxiety depending on the presence of the polymorphisms analyzed in this study. Depressed mood is frequent among healthy women in the late reproductive stage. Anxiety is definitely less common. The study did not demonstrate the relationship between the 5-HTT and MAO-A gene polymorphisms, and the severity of anxiety and mood disorders in healthy late-reproductive-stage women.

Analysis of personality traits and their influence on the quality of life of postmenopausal women with regard to genetic factors.

BackgroundQuality of life can be perceived as a subjective assessment of different aspects of human functioning. Personality is a factor which determines actions taken by individuals and their tendency to perceive reality in a particular way. Therefore, the assumption that personality may influence the QoL assessment seems reasonable. Our purpose was to assess the relationships between personality traits and the presence of the 44-bp VNTR polymorphism in the 5-HTT (SLC 6A4) promoter region and the 30-bp VNTR polymorphism in the MAO-A promoter region. We also wanted to determine the influence of personality on the quality of life of postmenopausal women.MethodsThe study involved 214 postmenopausal women from northwest Poland. It was conducted using the NEO-FFI and the SF-36 questionnaires. DNA polymorphisms were identified using the polymerase chain reaction (PCR).ResultsThe average age of the women was 56.8 ± 4.08 years. Half of the respondents had completed second-level education, 69.2 % had life partners, and 53.3 % were professionally active. Women with the 3/3 genotype were characterized by significantly lower openness to experience than respondents with other MAO-A genotypes (p < 0.05). There was a significant correlation between the quality of life and the levels of neuroticism and extroversion, as well as between selected quality of life domains and the levels of agreeableness and conscientiousness.Conclusions(1)Women with the 3/3 genotype of the 30-bp VNTR polymorphism in the MAO-A promoter region are characterized by lower levels of openness to experience than women with other MAO-A genotypes in our study (2) Personality traits may contribute to the assessment of the quality of life.

Impact of genetic variation in the 5-HT transporter and receptor on platelet function in patients with stable CAD taking aspirin

BackgroundSerotonin (5-HT) induces platelet aggregation by activating its 5-HT2A receptor. Platelet uptake is mediated by the 5-HT transporter (5-HTT). A common 5-HTT promoter (5-HTTLPR) splice variant results in long (L) and short (S) alleles. 5-HTTLPR genotype has been associated with increased platelet activation and risk of MI. Variation within HTR2A gene (C1354T) that encodes the 5-HT2A receptor has also been associated with enhanced platelet aggregation. We hypothesised that 5-HTT and/or HTR2A variation may influence platelet response to aspirin in patients with stable CAD. MethodsPatients (n=144) with stable cardiovascular disease taking aspirin were genotyped for the 5-HTTLPR and HTR2A variants. Platelet inhibition was assessed by serum thromboxane and arachidonic acid-induced platelet aggregation assay. Results5-HTT genotype (LL vs *S) was a significant determinant of serum TX level (8.9±2.6ng/ml vs 6.0±1.6ng/ml respectively; p<0.02) and 5-HTT LL genotype predicted an incomplete aspirin response (serum TXB2>2.2ng/ml) (p=0.04; OR=2.22, CI=1.03–4.79). Odds ratio of the effect of LL genotype on TX elevation was 3.8 (95% CI 1.2–11.6) in younger patients (under 64) compared to 1.0 (95% CI=0.3–3.8) in older subjects. LL genotype did not influence AA aggregation (p=0.83, OR=1.2, CI=0.3–4.1). The HTR2A variant had no effect on TX generation (p=0.70; OR=1.22, CI=0.45–3.26) nor AA aggregation (p=0.99; OR=1.0, CI=0.2–4.9). ConclusionsIn younger patients with stable CAD 5HTT LL genotype carried by almost one third of our cohort is associated with a diminished response to aspirin that may increase cardiovascular risk. Genotypic variation in platelet activation may be a contributing mechanism.

The mathematical formula of the intravaginal ejaculation latency time (IELT) distribution of lifelong premature ejaculation differs from the IELT distribution formula of men in the general male population.

PurposeTo find the most accurate mathematical description of the intravaginal ejaculation latency time (IELT) distribution in the general male population.Materials and MethodsWe compared the fitness of various well-known mathematical distributions with the IELT distribution of two previously published stopwatch studies of the Caucasian general male population and a stopwatch study of Dutch Caucasian men with lifelong premature ejaculation (PE). The accuracy of fitness is expressed by the Goodness of Fit (GOF). The smaller the GOF, the more accurate is the fitness.ResultsThe 3 IELT distributions are gamma distributions, but the IELT distribution of lifelong PE is another gamma distribution than the IELT distribution of men in the general male population. The Lognormal distribution of the gamma distributions most accurately fits the IELT distribution of 965 men in the general population, with a GOF of 0.057. The Gumbel Max distribution most accurately fits the IELT distribution of 110 men with lifelong PE with a GOF of 0.179. There are more men with lifelong PE ejaculating within 30 and 60 seconds than can be extrapolated from the probability density curve of the Lognormal IELT distribution of men in the general population.ConclusionsMen with lifelong PE have a distinct IELT distribution, e.g., a Gumbel Max IELT distribution, that can only be retrieved from the general male population Lognormal IELT distribution when thousands of men would participate in a IELT stopwatch study. The mathematical formula of the Lognormal IELT distribution is useful for epidemiological research of the IELT.

Link-Polymorphism of 5-HTT Promoter Region Is Associated with Autoantibodies in Patients with Systemic Lupus Erythematosus.

Serotonin transporter linked polymorphic region (5-HTTLPR) was reported to associate with depression in systemic lupus erythematosus (SLE) patients by our team. To explore whether 5-HTTLPR plays a role in the pathogenesis of SLE, we tested 138 SLE patients and 138 age and sex matched health controls (HCs) for 5-HTTLPR by polymerase chain reaction (PCR) and agarose gel electrophoresis. Interestingly, the results suggest that the frequencies of SS genotype and S allele in SLE patients with positive anti-Sm antibody and anti-U1RNP antibody were both significantly higher than the other genotypes and alleles. However, the frequencies of 5-HTTLPR genotypes and alleles were of no significant difference between SLE patients and HCs. This suggested that 5-HTTLPR was not a high-risk susceptible gene in SLE but might relate to SLE by affecting production of some autoantibodies, especially anti-Sm and anti-U1RNP antibody.

Meta-analysis of the Association between Serotonin Transporter Polymorphisms and Sudden Infant Death Syndrome

The serotonin transporter (5-HTT) gene has been considered one of the risk factors for sudden infant death syndrome (SIDS), but the association remains unconfirmed. This meta-analysis was performed to quantitatively summarize the evidence for such a relationship. PubMed, EMBASE, and China National Knowledge Infrastructure databases were searched for eligible studies within a range of published years from 1990 to December 2015. The odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the different associations. A total of 8 studies with 624 cases and 796 controls were included for 5-HTT promoter polymorphism, 5 studies with 418 cases and 542 controls for intron 2, and 3 studies with 253 cases and 334 controls for haplotype. The pooled examinations showed an overall increased SIDS risk for the 5-HTT promoter polymorphism (OR = 1.65, 95% CI = 1.03–2.63, P = 0.035 for LL vs. LS and SS; OR = 1.46, 95% CI = 1.04–2.04, P = 0.028 for L vs. S), but no association (OR = 1.00, 95% CI = 0.75–1.33, P = 0.994 for 10 + 9 carriers vs. 12/12; OR = 0.97, 95% CI = 0.79–1.19, P = 0.753 for 10 + 9 vs. 12) for intron 2 polymorphism, and an unreliable association (OR = 0.52, 95% CI = 0.31–0.87, P = 0.013) for S-9 and S-10 haplotypes. This meta-analysis suggests that the L allele or LL homozygote of 5-HTT promoter polymorphism has an increased risk for SIDS, while intron 2 polymorphism has no association with SIDS.

Histone deacetylase HDAC1 downregulates transcription of the serotonin transporter (5-HTT) gene in tumor cells

Serotonin (5-HT) has been reported to be involved in cancer progression by stimulating angiogenesis and cell growth. In this study, we examined the expression of the serotonin transporter (5-HTT) and the role of histone deacetylases (HDACs) in regulating the 5-HTT gene in tumor cells. The 5-HTT gene expression was almost silenced in chicken lymphoma DT40, myelomonocytic tumor HD11 and hepatoma DU249 cells, compared to their physiological counterpart. In contrast, HDAC1 mRNA expression was increased in these cell lines. Indeed, the pan-HDAC inhibitor trichostatin A (TSA) enhanced the 5-HTT mRNA expression in several tumor cell lines including the human cell lines HepG2 and THP-1 and increased the 5-HT uptake in HD11 cells. In addition, treatment with parthenolide, which is capable of depleting HDAC1, and knockdown of HDAC1 using siRNA resulted in increased 5-HTT mRNA expression, confirming the role of HDAC1 in the down-regulation of 5-HTT in the tumor cells. Deletion analysis of the 5-HTT promoter and site-directed mutagenesis revealed that the transcription factor CCAAT/enhancer binding protein beta (C/EBPß), in interacting with the 5-HTT promoter, mediated both the inhibition of the 5-HTT expression by HDAC1 and the activation by CREB-binding protein (CBP). Using a chromatin immunoprecipitation assay, we found increased acetylation of histone H4 associated with the 5-HTT promoter in cells treated with TSA. Our results suggest that the 5-HTT gene is epigenetically downregulated by HDAC1 in several types of cancer.

Involvement of serotonin transporter gene polymorphisms (5-HTT) in impulsive behavior in the japanese population.

The serotonergic pathway has been implicated in the pathogenesis of impulsivity, and sensitivity to aversive outcomes may be linked to serotonin (5-HT) levels. Polymorphisms in the gene that encodes the serotonin transporter (5-HTT), which have differential effects on the level of serotonin transmission, display alternate responses to aversive stimuli. However, recent studies have shown that 5-HT does not affect motor function, which suggests that the functioning of the serotonin-transporter-linked polymorphic region (5-HTTLPR) does not directly affect the behavioral regulatory process itself, but instead exerts an effect via the evaluation of the potential risk associated with particular behavioral outputs. The aim of the present study was to examine the effect of specific 5-HTTLPR genotypes on the motor regulatory process, as observed during a Go/Nogo punishment feedback task. 5-HTT gene-linked promoter polymorphisms were analyzed by polymerase chain reaction, using lymphocytes from 61 healthy Japanese volunteers. Impulsivity was defined as the number of commission errors (responding when one should not) made during a Go/Nogo task. We found that the s/s genotype group made fewer impulsive responses, specifically under aversive conditions for committing such errors, compared to those in the s/l group, without affecting overall motor inhibition. These results suggest that 5-HTTLPRs do not directly affect the behavioral regulatory process itself, but may instead exert an effect on the evaluation of potential risk. The results also indicate that under such aversive conditions, decreased expression of 5-HTT may promote motor inhibitory control.

Evaluation of the relationship between 5-HTT and MAO gene polymorphisms, mood and level of anxiety among postmenopausal women.

Objective: The aim of this study was to analyze how mood and anxiety level are related to the functional genetic polymorphism in the promoter region of SLC6A4 (5-HTTLPR) and the 30-bp VNTR polymorphism in the MAO A promoter region. Methods: The study involved 272 postmenopausal women from Poland. The authors employed the State-Trait Anxiety Inventory for measuring levels of anxiety, the Mood Adjective Check List for measuring mood, and genetic tests. Results: Analysis did not show any statistically significant differences in the mean levels of anxiety, and mood disorders in women in relation to genotypes of the 5-HTTLPR (SLC6A4) polymorphism and the 30-bp VNTR polymorphism in the MAO A promoter region. However, these problems were more severe among women with s/s genotype. In the case of MAO A gene polymorphism, the level of anxiety was higher in women with a 4/4 genotype. Conclusions: The study did not prove the possibility of the identification of homogeneous groups of women with an elevated risk of developing anxiety and mood disorders during the post-menopausal period. Nevertheless, it showed that respondents with s/s genotype of the 44-bp polymorphism in the 5-HTT (SLC6A4) promoter region had the highest average anxiety levels both as a state and as a trait. Furthermore, the analysis of the 30-bp VNTR polymorphism in the MAO A promoter region demonstrated slight differences in anxiety levels between the women, indicating that those with a 4/4 genotype had higher severity of anxiety symptoms.

Inhibition of serotonin transporter expression by C/EBPβ in LPS-activated macrophage cells (HD11)

Serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) is involved in inflammation and the stress response. In this study, we examined the regulation of 5-HTT expression in macrophage HD11 cells in response to bacterial LPS. Long-term exposure of cells to LPS (6-18 h) produced a decrease in 5-HTT mRNA expression. Accordingly, reduced 5-HTT activity measured by 5-HT uptake was also observed in LPS-treated HD11 cells. Moreover, LPS treatment, as well as co-transfection with an expression vector encoding the chicken CCAAT/enhancer binding protein beta (C/EBPβ), resulted in inhibition of 5-HTT promoter activity. Indeed, sequence analysis revealed several C/EBPβ binding motifs in the upstream region of the 5-HTT gene, which specifically interacted with C/EBPβ both in an in vitro band shift assay and in living HD11 cells. The C/EBPβ binding was activated in cells treated with LPS. The role of C/EBPβ in LPS inhibition of 5-HTT expression was further confirmed by small interfering RNA interference, which demonstrated that knockdown of endogenous C/EBPβ attenuated the inhibition of 5-HTT expression in LPS-treated cells. Taken together, the results suggest that C/EBPβ plays a critical role in regulating the 5-HTT gene in macrophages in response to pro-inflammatory stimuli.

A functional variant in the 5'-flanking region of the chicken serotonin transporter gene is associated with increased body weight and locomotor activity.

In this study, we identified a polymorphism in the 5'-flanking region of the chicken serotonin transporter (5-HTT) gene. Sequencing analysis revealed that in comparison with the wild-type variant (W), a deleted variant (D) is generated by deletion of four nucleotides (5'-AATT-3') and a single nucleotide change (A→T). Using a polyacrylamide gel electrophoresis system, we found that the 360-bp DNA fragment containing the W variant with the wild-type sequence 5'-AATTAATT-3' shows intrinsic DNA curvature while the 356-bp fragment containing the D variant lacking the four base pairs AATT is not curved. Quantitative real-time RT-PCR and ELISA demonstrated that the expression of 5-HTT in D/D chickens was higher than that in W/W and W/D chickens. In addition, transient transfection experiments with chloramphenicol acetyltransferase reporter gene constructs revealed increased 5-HTT promoter activity mediated by the D variant and a silencer activity of the W variant. Interestingly, females and males with D/D genotype showed significant greater increase in body weight from 6 weeks and 16 weeks of age, respectively, and higher body mass index. Moreover, we found that D/D chickens of both genders were physically more active than W/W and W/D chickens.

Impaired Fear Extinction as Displayed by Serotonin Transporter Knockout Rats Housed in Open Cages Is Disrupted by IVC Cage Housing

Anxiety disorders are influenced by both environmental and genetic factors. A well-known example for gene x environment interactions in psychiatry is the low activity (s) allelic variant of the serotonin transporter (5-HTT) promoter polymorphism (5-HTTLPR) that in the context of stress increases risk for depression and post-traumatic stress disorder (PTSD). Previously, we observed robust anxiety-related phenotypes, such as an impairment in fear extinction, in 5-HTT knockout (5-HTT−/−) versus wild-type (5-HTT+/+) rats housed in open cages. Recently, housing conditions were changed from open cages to individually ventilated cages (IVC), which are associated with a high ventilation fold and noise. This switch in housing conditions prompted an unplanned 5-HTT gene x environment interaction study in our rats. The current study shows that lifetime stress by means of IVC cage housing abolished genotype differences in fear extinction between 5-HTT−/− and 5-HTT+/+ rats. Although this effect was not attributed specifically to either the 5-HTT+/+ or the 5-HTT−/− genotype, the findings are in agreement with the modulatory role of serotonin in the processing of environmental stimuli. Our findings also underline the possibility that housing conditions confound the interpretation of anxiety-related behaviours in rodents.

DNA hypermethylation of serotonin transporter gene promoter in drug naïve patients with schizophrenia

IntroductionDysfunctional serotonin signaling has been linked to the pathogenesis of autism, obsessive compulsive disorder, mood disorders and schizophrenia. While the hypo-activity of serotonin signaling is involved in the pathogenesis of depression, anxiety and obsessive compulsive disorder; LSD, an agonist of serotonin type 2 receptor (5-HTR2A) induces psychosis. Therefore, anxiety and depressive disorders are treated by SSRIs which inhibit serotonin transporter (5-HTT) while psychotic disorders are controlled by drugs that block serotonin and/or dopamine receptors. Since genetic polymorphisms and epigenetic dysregulation of 5-HTT are involved in the pathogenesis of mental diseases, we analyzed DNA methylation of 5-HTT promoter in post-mortem brains and saliva samples of patients with schizophrenia (SCZ) and bipolar disorder (BD) to evaluate its potential application as a diagnostic and/or therapeutic biomarker in SCZ and BD. MethodsWhole genome DNA methylation profiling was performed for a total of 24 samples (including two saliva samples) using the Illumina 27K (for 12 samples) and 450K DNA methylation array platform (for another 12 samples), followed by bisulfite sequencing to identify candidate CpGs for further analysis. Quantitative methylation specific PCR (qMSP) was used to assess the degree of CpG methylation of 5-HTT promoter in 105 post-mortem brains (35 controls, 35 SCZ and 35 BD) and 100 saliva samples (30 controls, 30 SCZ, 20 BD and 20 first degree relatives of SCZ or BD). The U133 2.0 Plus Human Transcriptome array for a total of 30 post-mortem brain samples (each group 10) followed by quantitative real-time PCR was used to study 5-HTT expression in 105 post-mortem brain samples. ResultsThe qMSP analysis for 5-HTT promoter region showed DNA hypermethylation in post-mortem brain samples of SCZ patients (~30%), particularly in drug free patients (~60%, p=0.04). Similarly, there was a trend for DNA hypermethylation in antipsychotic free BD patients (~50%, p=0.066). qMSP analysis of DNA extracted from the saliva samples also exhibited hypermethylation of 5-HTT promoter in patients with SCZ (~30%, p=0.039), which was more significant in drug naïve SCZ patients (>50%, p=0.0025). However, the difference was not significant between the controls and unaffected first degree relatives of patients with SCZ (p=0.37) and versus patients using antipsychotic drugs (p=0.2). The whole genome transcriptome analysis of post-mortem brain samples showed reduced expression of 5-HTT in SCZ compared to the control subjects (~50%, p=0.008), confirmed by quantitative real-time PCR analysis (~40%, p=0.035) which was more significant in drug free SCZ patients (~70%, p=0.022). ConclusionA correlation between reduction in 5-HTT expression and DNA hypermethylation of the 5-HTT promoter in drug naïve SCZ patients suggests that an epigenetically defined hypo-activity of 5-HTT may be linked to SCZ pathogenesis. Furthermore, this epigenetic mark in DNA extracted from saliva can be considered as one of the key determinants in a panel of diagnostic and/or therapeutic biomarkers for SCZ.

The attribution of incentive salience to an appetitive conditioned cue is not affected by knockout of the serotonin transporter in rats

Understanding the neurobiological basis underlying individual differences in conditioned stimulus (CS) sensitivity is pertinent, given that excessive conditioned responses to CSs is a key feature of anxiety-related disorders and drug addiction. We have previously shown that behaviour of serotonin transporter knockout (5-HTT−/−) rats-mimicking the common 5-HTT promoter polymorphism in humans–is strongly driven by Pavlovian CSs. To investigate whether the knockout rats attribute greater incentive salience to CSs, we tested the 5-HTT−/− rats and their wild-type counterparts in the sucrose-reinforced sign-versus goal-tracking task. We also assessed whether motivational properties of the unconditioned stimulus (sucrose pellet) are involved in the individual differences under investigation, by testing the animals in a sucrose-reinforced progressive ratio schedule of reinforcement. We found no genotype differences in sign-versus goal-tracking behavior, despite that progressive ratio responding was increased in 5-HTT−/− rats. In conclusion, the high CS sensitivity in 5-HTT−/− rats cannot be explained by enhanced incentive salience attribution to the CS as measured by the sign- versus goal-tracking paradigm. Rather, 5-HTT−/− rats may be more sensitive to the motivational properties of the unconditioned stimulus.

How the cerebral serotonin homeostasis predicts environmental changes: a model to explain seasonal changes of brain 5-HTT as intermediate phenotype of the 5-HTTLPR

Molecular imaging studies with positron emission tomography have revealed that the availability of serotonin transporter (5-HTT) in the human brain fluctuates over the course of the year. This effect is most pronounced in carriers of the short allele of the 5-HTT promoter region (5-HTTLPR), which has in several previous studies been linked to an increased risk to develop mood disorders. We argue that long-lasting fluctuations in the cerebral serotonin transmission, which is regulated via the 5-HTT, are responsible for mediating responses to environmental changes based on an assessment of the expected "safety" of the environment; this response is obtained in part through serotonergic modulation of the hypothalamic-pituitary-adrenal (HPA) axis. We posit that the intermediate phenotype of the s-allele may properly be understood as mediating a trade-off, wherein increased responsiveness of cerebral serotonin transmission to seasonal and other forms of environmental change imparts greater behavioral flexibility, at the expense of increased vulnerability to stress. This model may explain the somewhat higher prevalence of the s-allele in some human populations dwelling at geographic latitudes with pronounced seasonal climatic changes, while this hypothesis does not rule out that genetic drift plays an additional or even exclusive role. We argue that s-allele manifests as an intermediate phenotype in terms of an increased responsiveness of the 5-HTT expression to number of daylight hours, which may serve as a stable surrogate marker of other environmental factors, such as availability of food and safety of the environment in populations that live closer to the geographic poles.

Depression in systemic lupus erythematosus patients is associated with link-polymorphism but not methylation status of the 5HTT promoter region

A higher prevalence of depression in systemic lupus erythematosus (SLE) patients has been reported, though the mechanism underlying this phenomenon remains unclear. The present study was conducted to explore whether the polymorphism and methylation status of the serotonin transporter gene (5HTT) promoter region (PR-5HTT) contribute to depression in SLE patients from both genetic and epigenetic perspectives. In this study, 96 SLE patients and 96 healthy controls (HCs) were recruited. Depression levels of all subjects were evaluated using the Hamilton Depression Rating Scale (HDRS). The serotonin transporter-linked polymorphism (5HTTLPR) and the DNA methylation status of PR-5HTT were detected in peripheral lymphocytes of SLE patients and HCs. The differences in 5HTTLPR and DNA methylation of PR-5HTT between SLEs and HCs were compared. In SLE patients, the frequencies of short allele (S) and SS genotype of 5HTTLPR were higher in depressive SLE (SLE-D) patients than in non-depressive SLE (SLE-ND) patients. The mean HDRS score of SS homozygote patients was higher than that of patients with SL/LL genotypes. Conversely, PR-5HTT was hypomethylated in HCs as well as SLE patients. There was no difference in the methylation status between HCs and SLEs. Thus, the functional expression of PR-5HTT may be primarily regulated by gene polymorphism and not by DNA methylation. The risk allele of 5HTTLPR appears to be a major contributor to depression in SLE patients.