Methylation of the dopamine transporter gene in blood is associated with striatal dopamine transporter availability in ADHD: A preliminary study.

Dopamine transporters (DAT) are implicated in the pathogenesis and treatment of attention-deficit hyperactivity disorder (ADHD) and are upregulated by chronic treatment with methylphenidate, commonly prescribed for ADHD. Methylation of the DAT1 gene in brain and blood has been associated with DAT expression in rodents' brains. Here we tested the association between methylation of the DAT1 promoter derived from blood and DAT availability in the striatum of unmedicated ADHD adult participants and in that of healthy age-matched controls (HC) using Positron Emission Tomography (PET) and [11 C]cocaine. Results showed no between-group differences in DAT1 promoter methylation or striatal DAT availability. However, the degree of methylation in the promoter region of DAT1 correlated negatively with DAT availability in caudate in ADHD participants only. DAT availability in VS correlated with inattention scores in ADHD participants. We verified in a postmortem cohort with ADHD diagnosis and without, that DAT1 promoter methylation in peripheral blood correlated positively with DAT1 promoter methylation extracted from substantia nigra (SN) in both groups. In the cohort without ADHD diagnosis, DAT1 gene expression in SN further correlated positively with DAT protein expression in caudate; however, the sample size of the cohort with ADHD was insufficient to investigate DAT1 and DAT expression levels. Overall, these findings suggest that peripheral DAT1 promoter methylation may be predictive of striatal DAT availability in adults with ADHD. Due to the small sample size, more work is needed to validate whether DAT1 methylation in blood predicts DAT1 methylation in SN in ADHD and controls.

Methylomics of breast cancer: Seeking epimarkers in peripheral blood of young subjects.

Critical roles of epigenomic alterations in the pathogenesis of breast cancer have recently seized great attentions toward finding epimarkers in either non-invasive or semi-non-invasive samples as well as peripheral blood. In this way, methylated DNA immunoprecipitation microarray (MeDIP-chip) was performed on DNA samples isolated from white blood cells of 30 breast cancer patients compared to 30 healthy controls. A total of 1799 differentially methylated regions were identified including SLC6A3, Rab40C, ZNF584, and FOXD3 whose significant methylation differences were confirmed in breast cancer patients through quantitative real-time polymerase chain reaction. Hypermethylation of APC, HDAC1, and GSK1 genes has been previously reported in more than one study on tissue samples of breast cancer. Methylation of those aforementioned genes in white blood cells of our young patients not only relies on their importance in breast cancer pathogenesis but also may highlight their potential as early epimarkers that makes further assessments necessary in large cohort studies.

Identification of the dopamine transporter SLC6A3 as a biomarker for patients with renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is among the most common human malignancies. METHODS: In order to provide better understanding of the molecular biology of ccRCC and to identify potential diagnostic/prognostic biomarker and therapeutic targets, we utilized a microarray to profile mRNA expression of corresponding normal and malignant renal tissues. Real-time PCR, Western Blot and immunohistochemistry were applied to study the expression of candidate biomarkers. ccRCC cell lines were treated with sertraline to inhibit the dopamine transporter SLC6A3. RESULTS: Differential expression of fourteen mRNAs, yet not studied in ccRCC in depth, was confirmed using qPCR (upregulation: SLC6A3, NPTX2, TNFAIP6, NDUFA4L2, ENPP3, FABP6, SPINK13; downregulation: FXYD4, SLC12A1, KNG1, NPHS2, SLC13A3, GCGR, PLG). Up-/downregulation was also confirmed for FXYD4, KNG1, NPTX2 and SLC12A1 by Western Blot on the protein level. In contrast to the mRNA expression, protein expression of the dopamine transporter SLC6A3 was lower in ccRCC compared to normal renal tissue. Immunohistochemistry indicated that this decrease was due to higher concentrations of SLC6A3 in the proximal tubules. Immunohistochemical analyses further demonstrated that high SLC6A3 expression in ccRCC tissue was correlated with a shorter period of recurrence-free survival following surgery. Treatment of ccRCC cells with the SLC6A3 inhibitor sertraline induced dose-dependent cell-death. CONCLUSION: Our study identified several novel biomarkers with diagnostic potential and further investigations on sertraline as therapeutic agent in ccRCC patients are warranted.

Epigenetic dysregulation of dopaminergic genes in eating disorders.

OBJECTIVE: The pathophysiology of eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN) has been linked to an impaired dopaminergic neurotransmission, still the origin of this disturbance remains unknown. The aim of the present study was, therefore, to evaluate whether the expression of dopaminergic genes is altered in the blood of patients suffering from eating disorders and if these alterations can be explained by changes in the promoter specific DNA methylation of the genes. METHOD: We used quantitative real-time PCR to measure both the expression and the promoter specific DNA methylation of the dopamine transporter (DAT), and the D2 (DRD2) and D4 receptor (DRD4) gene in the blood of 46 patients (22 AN, 24 BN) and 30 healthy controls. RESULTS: Patients showed an elevated expression of DAT mRNA when compared with the controls and a downregulation of the DRD2 expression. The upregulation of the DAT gene was accompanied by a hypermethylation of the gene's promoter in the AN and BN group while a significant hypermethylation of the DRD2 promoter was only present in the AN group. No differences in expression or methylation were found for the other dopamine receptors investigated. DISCUSSION: Our study shows a disturbed expression of dopaminergic genes that is accompanied by a dysregulation of the epigenetic DNA methylation. Further studies are necessary to provide more insight into the epigenetic dysregulation of the dopaminergic neurotransmission in the pathophysiology of eating disorders.

Dopamine transporter immunoreactivity in peripheral blood lymphocytes discriminates Parkinson's disease from essential tremor.

Peripheral blood lymphocytes (PBL) provide a model to study the changes of neurotransmitter-receptor systems in neurodegenerative disorders, including Parkinson's disease (PD). In this study, densitometric analysis was applied to measure dopamine transporter (DAT) immunoreactivity in PBL from dopaminergic drug-free patients suffering PD or essential tremor (ET) with respect to healthy subjects. The results showed a significant reduction of DAT immunoreactivity in PBL in PD but not in ET. These finding suggests that DAT immunoreactivity in PBL may discriminate between PD and ET in the early clinical stages.

Serum Ceruloplasmin and Striatal Dopamine Transporter Density in Parkinson Disease: Comparison With 123I-FP-CIT SPECT.

PURPOSE: In patients with Parkinson disease (PD), decreased serum ceruloplasmin levels have been observed. This study investigated a correlation between serum ceruloplasmin-along with its related serum markers- and striatal presynaptic dopaminergic denervation measured with I-FP-CIT SPECT. METHODS: We analyzed a total of 141 de novo patients divided into 2 groups: the PD group (107 patients with PD) and the disease control group (34 patients with vascular pseudoparkinsonism, essential tremor, or drug-induced parkinsonism). Serum ceruloplasmin and related serum markers, such as copper, iron, total iron-binding capacity, and ferritin, were measured. Specific binding ratios of the striatum, caudate nucleus, putamen, and posterior putamen were obtained by I-FP-CIT SPECT. RESULTS: There was no difference in the serum markers, except for ceruloplasmin, between the 2 groups. Ceruloplasmin level was significantly lower in PD patients with longer symptom duration (>2 years) than in the disease control group (21.4 ± 3.4 vs 24.0 ± 3.8, P = 0.03). Serum ceruloplasmin had a significant correlation with specific binding ratios of the striatum, caudate nucleus, and putamen in a subgroup with longer symptom duration (P = 0.01, P = 0.02, P = 0.02, respectively, for the subgroup with symptom duration >1 year, and P < 0.01, P < 0.01, P = 0.04, respectively, for the subgroup with symptom duration >2 years). CONCLUSIONS: Decrease in serum ceruloplasmin had a positive correlation with a decrease in dopamine transporter density in PD patients with symptom duration of more than 1 year.

Characterization of the neuronal dopamine transporter DAT in human blood platelets.

Compelling evidence suggests a monoaminergic dysfunction in the aetiology of various neuro-psychiatric diseases such as depression, attention deficit hyperactivity disorder (ADHD), schizophrenia, addiction and Parkinson's disease. The efficiency of monoaminergic neurotransmission is controlled by rapid and efficient reuptake of dopamine out of the synaptic cleft by specific transporters for dopamine, serotonin and noradrenaline. In case of the serotonin transporter, many investigators have determined its function and expression also on peripheral cells such as blood platelets under the assumption that changes in protein expression in these cells might reflect neuronal changes. No comparable studies have so far been performed with respect to the dopamine transporter due to the lack of information about the existence of this protein in platelets. Here, we present pharmacological, immunological as well as microarray and PCR data that human blood platelets express the dopamine transporter protein (DAT), which is identical to that first identified in neurons. Because DAT expression is modulated also in non-neuronal cells independently of gene transcription, platelets may well serve as an easy accessible peripheral system to study DAT regulation in mental diseases or during drug treatment or drug abuse.

GABA and glutamate transporters are expressed in human platelets.

GABA and glutamate are the major neurotransmitters in the human central nervous system. Disturbances in these transmitter systems have been suggested to influence a variety of neurological and psychiatric diseases. Human platelets have been used as a model for neural amino acid transport, although it has not been known exactly which transporters participate in the transport process. In this study, we identify with reverse transcription-polymerase chain reaction (RT-PCR) BGT-1 and EAAT3 as transporters for GABA and glutamate, respectively. We also show that platelets contain transporters for dopamine, taurine and creatine. The cloning of these transporters confirms that blood platelets can be used as a model for neurotransmitter transport in the CNS.

Pilot study: potential transcription markers for adult attention-deficit hyperactivity disorder in whole blood.

Attention-deficit hyperactivity disorder (ADHD) is a common behavioural disorder that affects not only children and adolescents but also adults; however, diagnosis of adult ADHD is difficult because patients seem to have reduced externalized behaviour. ADHD is a multifactorial disorder in which many genes, all with small effects, are thought to cause the disorder in the presence of unfavourable environmental conditions. Therefore, in this pilot study, we explored the expression profile of a list of previously established candidate genes in peripheral blood samples from adult ADHD subjects (n = 108) and compared these results with those of healthy controls (n = 35). We demonstrate that combining the gene expression levels of dopamine transporter (SLC6A3), dopamine D5 receptor, tryptophan hydroxylase-1, and SNAP25 as predictors in a regression model resulted in sensitivity and specificity of over 80 % (ROC: max R(2) = 0.587, AUC = 0.917, P < 0.001, 95 % CI: 0.900-0.985). In conclusion, the combination of these four genes could represent a potential method for estimating risk and could be of diagnostic value for ADHD. Nevertheless, further investigation in a larger independent population including different subtypes of ADHD (inattentive, hyperactive, or combined type) patients is required to obtain more specific sets of biomarkers for each subtype as well as to differentiate between child, adolescent, and adulthood forms.