Association of the dopamine transporter gene (DAT1) with poor methylphenidate response

OBJECTIVE: This study attempted to relate the alleles of the D2 (DRD2), D4 (DRD4), and dopamine transporter (DAT1) genes to the behavioral outcome of methylphenidate therapy. METHOD: African-American children with attention-deficit hyperactivity disorder were treated with methylphenidate in doses not in excess of 60 mg/day. The dosage was increased until behavioral change was achieved, using a decrement in scores of less than or equal to 1 on a commonly used rating scale or until the maximum tolerated dose was achieved. Blood samples were obtained at that point, and genotypes for polymorphism at the respective genes were identified. RESULTS: Genotypes were then tested by chi 2 to assess the significance of any association with drug response. Only the dopamine transporter gene was found to be significant. Homozygosity of the 10-repeat allele was found to characterize nonresponse to methylphenidate therapy (p = .008). CONCLUSIONS: While the results suggest that alleles of the dopamine transporter gene play a role in methylphenidate response, replication in additional studies is needed.     

Identification of a novel polymorphism of the human dopamine transporter (DAT1) gene and the significant association with alcoholism

Human dopamine transporter gene (DAT1) has a variable number of tandem repeats (VNTR) in its 3'-untranslated region (UTR). The association between the VNTR polymorphism and neuropsychiatric disorders has been studied, but their relationship is still unclear. Here we identified a novel polymorphism in the 3'-UTR of the DAT1 gene, G2319A, and a significant association between the polymorphism and alcoholism was observed in both genotypic and allelic frequencies (P = 0.040 and 0.019, extended Fisher's exact test, respectively). There was a significant gene dose effect on the risk for alcoholism associated with the 2319-A allele (chi2 = 6.16, df = 2, P = 0.046, linearity tendency test: Cochranq-Armitage analysis). Moreover, in the haplotype analysis with G2319A- and VNTR-polymorphisms, a positive gene dose efffect on the risk with the A10 allele (P = 0.044, linearity tendency test) and a negative gene dose effect with the G10 allele (P = 0.010, linearity tendency test) for alcoholism were significantly detected. Odds ratio for alcoholism with the A10 and G10 alleles were 1.76 (1.12-2.76) and 0.53 (0.32-0.79), respectively. These results indicate that the DAT1 gene may confer vulnerability to alcoholism.     

The effect of manganese on dopamine toxicity and dopamine transporter (DAT) in control and DAT transfected HEK cells

Chronic exposure to Mn results in the development of a neurological disorder known as manganism characterized by neurological deficits resembling that seen in Parkinsonism. Although dopaminergic neurons within the nigrostriatal pathway appear intact, Mn-induced irregularities in DA transmission have been observed including decreased amphetamine-induced DA release and loss of the dopamine transporter (DAT). Results of studies to evaluate the effect of Mn and DA on cell viability in control and DAT-transfected HEK cells reveal that Mn is equally toxic to both cell lines whereas DA was only toxic to cells containing DAT. DA toxicity was saturable suggesting that transport may be rate limiting. When Mn and DA were added simultaneously to the media, cell toxicity was similar to that produced by Mn alone suggesting that Mn may suppress DA uptake in the DAT containing cells. Preincubation of DA prior to the addition of Mn resulted in cell death which was essentially additive with that produced independently by the two agents. Mn was also shown to decrease DA uptake and amphetamine-induced DA efflux in DAT containing cells. Time-lapsed confocal microscopy indicates that Mn can promote trafficking of cell surface DAT into intracellular compartments which may account for the decrease in DA uptake and DA efflux in these cells. Mn-induced internalization of DAT may provide an explanation for disruption in DA transmission previously reported in the striatum.     

Interaction between serotonin 5-HT2A receptor gene and dopamine transporter (DAT1) gene polymorphisms influences personality trait of persistence in Austrian Caucasians

We examined 89 normal volunteers using Cloninger's Temperament and Character Inventory (TCI). Genotyping the 102T/C polymorphism of the serotonin 5HT2A receptor gene and the ser9gly polymorphism in exon 1 of the dopamine D3 receptor (DRD3) gene was performed using PCR-RFLP, whereas the dopamine transporter (DAT1) gene variable number of tandem repeats (VNTR) polymorphism was investigated using PCR amplification followed by electrophoresis in an 8% acrylamide gel with a set of size markers. We found a nominally significant association between gender and harm avoidance (P=0.017; women showing higher scores). There was no association of either DAT1, DRD3 or 5HT2A alleles or genotypes with any dimension of the TCI applying Kruskal–Wallis rank-sum tests. Comparing homozygote and heterozygote DAT1 genotypes, we found higher novelty seeking scores in homozygotes (P=0.054). We further found a nominally significant interaction between DAT1 and 5HT2A homo-/heterozygous gene variants (P=0.0071; DAT1 and 5HT2A genotypes P value of 0.05), performing multivariate analysis of variance (MANOVA). Examining the temperamental TCI subscales, this interaction was associated with persistence (genotypes: P=0.004; homo-/heterozygous gene variants: P=0.0004). We conclude that an interaction between DAT1 and 5HT2A genes might influence the temperamental personality trait persistence.     

Dopamine transporter (DAT1) VNTR polymorphism in 12 Indian populations

The dopamine transporter (DAT1) is a membrane spanning protein that binds the neurotransmitter dopamine and performs re-uptake of dopamine from the synapse into a neuron. The gene encoding DAT1 consists of 15 exons spanning 60 kb on chromosome 5p15.32. Several studies have investigated the possible associations between variants in DAT1 gene and psychiatric disorders. The present study aimed to determine the distribution of the variable number of tandem repeat (VNTR) polymorphism in the 3′ untranslated region of DAT1 in 12 Indian populations. A total of 471 healthy unrelated individuals in 12 Indian populations from 3 linguistic groups were included in the present study. The analysis was carried out using PCR and electrophoresis. Overall, 4 alleles of the DAT1 40-bp VNTR, ranging from 7 to 11 repeats were detected. Heterozygosity indices were low and varied from 0.114 to 0.406. The results demonstrate the variability of the DAT1 40-bp VNTR polymorphism in Indian populations and revealed a high similarity with East Asian populations.     

A 4-year dopamine transporter (DAT) imaging study in neuroleptic-naive first episode schizophrenia patients

Alterations in the dopaminergic system have long been implicated in schizophrenia. A key component in dopaminergic neurotransmission is the striatal dopamine transporter (DAT). To date, there have been no longitudinal studies evaluating the course of DAT in schizophrenia. A 4-year follow-up study was therefore conducted in which single photon emission computed tomography was used to measure DAT binding in 14 patients and 7 controls. We compared the difference over time in [¹²³I] FP-CIT striatal/occipital uptake ratios (SOUR) between patients and controls and the relationship between this difference and both symptomatology and functional outcome at follow-up. We also calculated the relationship between baseline SOUR, symptoms and functional outcome at follow-up. There were no statistically significant differences between patients' SOUR changes over time and those of controls. A significant negative correlation was observed between patients' SOUR changes over time and negative symptomatology at follow-up. A significant negative correlation was also found between baseline SOUR in patients and negative symptomatology, and there was a significant association between lower SOUR at baseline and poor outcome. Although the study found no overall differences in DAT binding during follow-up between schizophrenia patients and controls, it demonstrated that differences in DAT binding relate to patients' characteristics at follow-up.     

Postnatal rearing conditions influence ontogeny of adult dopamine transporter (DAT) immunoreactivity of the striatum in gerbils

Summary. In the present study, the influence of postnatal rearing conditions on the structural maturation of the striatum of adult male gerbils (Meriones unguiculatus) was investigated. For that purpose, animals were bred and reared either grouped in an object-filled environment (EC) or isolated under restricted environmental conditions (IC). At the age of postnatal day 90, dopamine fibers were stained immunocytochemically using an antibody against the dopamine transporter (DAT). Innervation density was determined along the entire rostrocaudal axis of the ventromedial and dorsolateral part of the striatum. As a result, restricted rearing produced a significant restraint of the maturation of striatal dopamine (DA) innervation, leading to adult fiber densities which were approximately 9% below those in semi-naturally reared gerbils. Results are discussed with structural and functional alterations observed in the brain of IC animals. Received May 9, 2001; accepted June 3, 2002 Published online July 26, 2002     

Oestrogens prevent loss of dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) in substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice

Previous results from our laboratory have shown that 17beta-oestradiol prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) striatal dopamine depletion. 17beta-oestradiol, oestriol and oestrone are the naturally occurring oestogens in humans. Using various dopamine markers, the present study investigated whether oestrone and oestriol such as 17beta-oestradiol have neuroprotective activity in MPTP-treated mice. Male mice were treated with 17beta-oestradiol, oestriol or oestrone for 5 days before and after MPTP administration, and were compared with nonlesioned mice receiving the same treatment. Striatal concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were assayed by high-performance liquid chromatography. Dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) specific binding were measured by autoradiography. DAT, VMAT2 and tyrosine hydroxylase mRNA levels were measured by in situ hybridisation. MPTP induced a loss of DAT and VMAT2 specific binding in the striatum and substantia nigra, as well as a decrease of VMAT2 mRNA in the substantia nigra. 17beta-oestradiol treatment prevented the loss of these dopaminergic markers, as well as striatal concentrations of dopamine, DOPAC and HVA. Mice receiving oestriol and oestrone showed catecholamine concentrations comparable to MPTP mice. Oestriol treatment had no effect on dopaminergic markers in MPTP mice whereas oestrone prevented striatal DAT loss and the decrease of VMAT2 mRNA in the substantia nigra. In nonlesioned mice, 17beta-oestradiol, oestriol or oestrone had no effect on all the dopaminergic markers investigated. In conclusion, a weak or a lack of effect of oestriol and oestrone was observed compared to 17beta-oestradiol in MPTP mice and none of these steroids had an effect in nonlesioned mice. A DAT and VMAT2 specific binding decrease after MPTP in the striatum and substantia nigra, as well as a decrease of substantia nigra VMAT2 mRNA, was observed and could be prevented by oestradiol.     

Rapid and differential losses of in vivo dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) radioligand binding in MPTP-treated mice

The dose- and time-dependent changes of in vivo radioligand binding to the neuronal membrane dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were examined in mouse brain after MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) administrations. Regional brain distribution studies were done in male C57BL/6 mice using simultaneous injections of d-threo-[(3)H]methylphenidate (DAT) and (+)-alpha-[(11)C]dihydrotetrabenazine (VMAT2). Single (55 mg/kg i.p. ) or multiple (4 x 10 mg/kg i.p., 1-hour intervals) administration of MPTP caused significant reductions in [(3)H]methylphenidate and [(11)C]dihydrotetrabenazine specific striatal binding, measured 14 days later. The single high dose of MPTP produced greater losses of [(11)C]dihydrotetrabenazine binding than did the multiple MPTP dosing regimen. Using the single high dose of MPTP, changes of in vivo binding of the two radioligands were determined at 1, 3, and 14 days after neurotoxin injection. At 1 day, there are large losses of [(3)H]methylphenidate binding (DAT) but no changes in [(11)C]dihydrotetrabenazine binding to the VMAT2 site in the striatum. At 3 and 14 days, there were >50% losses of binding of both bot radioligands, but significantly (P < 0.001) greater losses of VMAT2 binding of [(11)C]dihydrotetrabenazine. These studies indicate that the losses of the neuronal membrane and vesicular transporters are not always equal, and do not occur in the same time frame, after administration of the neurotoxin MPTP.     

The role of the dopamine transporter DAT1 genotype on the neural correlates of cognitive flexibility

Cognitive flexibility, the ability to adapt goal‐oriented behaviour in response to changing environmental demands, varies widely amongst individuals, yet its underlying neural mechanisms are not fully understood. Neuropharmacological and human clinical studies have suggested a critical role for striatal dopaminergic function mediated by the dopamine transporter (DAT). The present study aimed at revealing the role of the DAT in the individual brain response stereotypy underlying cognitive flexibility. A task‐switching protocol was administered to a sample divided according to the presence or absence of the 9‐repeat (9R) allele of the DAT1 polymorphism, while registering behavioural and electrophysiological novelty‐P3 responses. The absence of the 9R (higher gene expression) is related to less striatal DA availability. Individuals lacking the 9R (9R?) showed specific response time (RT) increases for sensory change and task‐set reconfiguration, as well as brain modulations not observed in participants with the 9R allele (9R+), suggesting that task performance of the former group depended on immediate local context. In contrast, individuals displaying high striatal DA showed larger RT costs than 9R? individuals to any sensory change, with no further increase for task‐set reconfiguration, and a larger early positive brain response irrespective of the task condition, probably reflecting larger inhibition of any previous interference as well as stronger activation of the current task set. However, the polymorphic groups did not differ in their mean RTs in trials requiring task‐set reconfiguration. This distinct stereotypy of cerebral responses reveals different patterns of cognitive control according to the DAT1 gene polymorphism.     

Multimodal dopamine transporter (DAT) imaging and magnetic resonance imaging (MRI) to characterise early Parkinson's disease

Idiopathic Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterised by the progressive loss of dopaminergic nigrostriatal terminals. Currently, in early idiopathic PD, dopamine transporter (DAT)-specific imaging assesses the extent of striatal dopaminergic deficits, and conventional magnetic resonance imaging (MRI) of the brain excludes the presence of significant ischaemic load in the basal ganglia as well as signs indicative of other forms of Parkinsonism.In this article, we discuss the use of multimodal DAT-specific and MRI protocols for insight into the early pathological features of idiopathic PD, including: structural MRI, diffusion tensor imaging, nigrosomal iron imaging and neuromelanin-sensitive MRI sequences. These measures may be acquired serially or simultaneously in a hybrid scanner.From current evidence, it appears that both nigrosomal iron imaging and neuromelanin-sensitive MRI combined with DAT-specific imaging are useful to assist clinicians in diagnosing PD, while conventional structural MRI and diffusion tensor imaging protocols are better suited to a research context focused on characterising early PD pathology. We believe that in the future multimodal imaging will be able to characterise prodromal PD and stratify the clinical stages of PD progression.     

Global variation of a 40-bp VNTR in the 3'-untranslated region of the dopamine transporter gene (SLC6A3).

BACKGROUND: The dopamine transporter (DAT) is the primary mechanism for dopamine clearance from the synapse in midbrain dopaminergic neurons, and the target of psychostimulant and neurotoxic drugs such as cocaine, amphetamine, and MPTP. Consequently, the gene for DAT (SLC6A3) has been the focus of many population-based case-control association studies using a 40-bp VNTR in the 3'-untranslated region. Results have differed depending on the population studied, suggesting allele frequency effects are involved. For this reason, a global survey of allele frequencies for this VNTR polymorphism was performed. METHODS: Individuals (n = 1528) from 30 populations around the world were typed for this VNTR using PCR and agarose gel electrophoresis. RESULTS: As with previous studies, the ten-repeat allele is most common, except for a Middle Eastern population in which the nine-repeat allele is most frequent. Frequencies of the nine- and ten-repeat alleles vary widely even among European populations. CONCLUSIONS: Many previous association studies have used "white" or "black" U.S. populations. However, many different ethnic groups have contributed to these populations. The large variation in allele frequencies observed in this study emphasizes the inadequacy of most past studies using the case-control design and the importance of matching patient and control populations in future association studies.     

Dopamine transporter gene (DAT1) VNTR polymorphism in major psychiatric disorders: family-based association study in the Bulgarian population

OBJECTIVE: A 40-bp variable number tandem repeat in the 3'-UTR of dopamine transporter gene (DAT1) has been examined for association with major psychiatric disorders in several case-control studies. No significant results have been found. We used a new collection of parent-offspring trios to test for association with schizophrenia (SZ), bipolar 1 disorder (BPI) and schizoaffective (SA) disorder. METHOD: We genotyped trios from Bulgarian origin where the proband had SZ (178 trios), BPI (77 trios) and SA (29 trios). Alleles ranging from 5 to 11 repeats were observed. The results were analysed with the extended TDT (ETDT). RESULTS: No preferential transmission of alleles was observed for any diagnostic group. The presence of allele DAT*10 was associated with the severity and frequency of auditory hallucinations, however, this result is not significant if corrected for multiple testing. CONCLUSION: Our results are in agreement with previous reports of a lack of association between this polymorphism and major psychiatric disorders.     

Polymorphisms in the 3'-untranslated region of human and monkey dopamine transporter genes affect reporter gene expression.

Dopamine transporter (DAT) levels vary in normal subjects and deviate from the normal range in pathological states. We investigated mechanisms by which the DAT gene may influence DAT protein expression. As the 3'-untranslated region (3'-UTR) of the DAT gene varies with regard to length and single nucleotide polymorphisms (SNPs), we addressed whether the 3'-UTR of sequence-defined DAT alleles can differentially affect the level of reporter gene expression in vitro. We first established that within individual rhesus monkeys, two alleles of the DAT gene were expressed in the substantia nigra. We then transfected HEK-293 cells with HSV-TK- and SV40-driven luciferase expression vectors harboring downstream DAT 3'-UTR segments of alleles containing polymorphisms of length (human: 9 or 10 repeat units) or SNPs within alleles of fixed length (human: DraI-sensitive (DraI+) vs. DraI-insensitive (DraI-) 10-repeat alleles; rhesus monkey: Bst1107I-sensitive (Bst+) vs. Bst1107I-insensitive (Bst-) 12-repeat alleles). Vectors containing the 3'-UTR segment of a human DAT allele containing nine tandem repeat units resulted in significantly higher levels of luciferase production than analogous vectors containing 10 tandem repeat units. Depending on the promoter used, vectors containing the human or monkey 3'-UTR segments that differed on the basis of an SNP resulted in increases or decreases in luciferase gene expression. This report provides experimental evidence that variability in the length or the sequence of the 3'-UTR of the DAT gene may influence levels of DAT protein in the brain.     

Association of the dopamine transporter (DAT1) 10/10-repeat genotype with ADHD symptoms and response inhibition in a general population sample

Association between attention-deficit hyperactivity disorder (ADHD) and the 10-repeat allele of the dopamine transporter gene (DAT1) has been reported in independent clinical samples using a categorical clinical definition of ADHD. The present study adopts a quantitative trait loci (QTL) approach to examine the association between DAT1 and a continuous measure of ADHD behaviours in a general-population sample, as well as to explore whether there is an independent association between DAT1 and performance on neuropsychological tests of attention, response inhibition, and working memory. From an epidemiological sample of 872 boys aged 6-11 years, we recruited 58 boys scoring above the 90th percentile for teacher reported ADHD symptoms (SWAN ADHD scale) and 68 boys scoring below 10th percentile for genotyping and neuropsychological testing. A significant association was found between the DAT1 homozygous 10/10-repeat genotype and high-scoring boys (chi(2)square=4.6, P<0.03; odds ratio=2.4, 95% CI 1.1-5.0). Using hierarchical linear regression, a significant independent association was found between the DAT1 10/10-repeat genotype and measures of selective attention and response inhibition after adjusting for age, IQ, and ADHD symptoms. There was no association between DAT1 and any component of working memory. Furthermore, performance on tasks of selective attention although associated with DAT1 was not associated with SWAN ADHD high scores after controlling for age and IQ. In contrast, impairment on tasks that tapped sustained attention and the central executive component of working memory were found in high-scoring boys after adjusting for age and IQ. The results suggest that DAT1 is a QTL for continuously distributed ADHD behaviours in the general population and the cognitive endophenotype of response inhibition.     

Studies of the coding region of the neuronal glutamate transporter gene in amyotrophic lateral sclerosis

Studies of the coding region of the neuronal glutamate transporter of 6 amyotrophic lateral sclerosis (ALS) patients and 10 controls show an identical pattern of four reported amino acid variations. No mutations and polymorphisms were detected in 5 sporadic ALS patients and a single patient with the familial form of the disease.     

Attention deficit/hyperactivity disorder-derived coding variation in the dopamine transporter disrupts microdomain targeting and trafficking regulation

Attention deficit/hyperactivity disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain-imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine- and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at presynaptic and postsynaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a hDAT (human DAT) coding variant, R615C, located in the distal C terminus of the transporter, a region previously implicated in constitutive and regulated transporter trafficking. Here, we demonstrate that, whereas wild-type DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively and demonstrate insensitivity to the endocytic effects of AMPH and PKC (protein kinase C) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered CaMKII (calcium/calmodulin-dependent protein kinase II) and flotillin-1 interactions. Using C-terminal peptides derived from wild-type DAT and the R615C variant, we establish that the DAT 615C C terminus can act dominantly to preclude AMPH regulation of wild-type DAT. Mutagenesis of DAT C-terminal sequences suggests that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.