No direct effect of the -521 C/T polymorphism in the human dopamine D4 receptor gene promoter on transcriptional activity
© Kereszturi et al; licensee BioMed Central Ltd. 2006
Received: 15 November 2005
Accepted: 24 May 2006
Published: 24 May 2006
The human dopamine D4 receptor (DRD4) gene has been studied extensively as a candidate gene for certain psychological traits and several behavioural and psychiatric disorders. Both the 5' regulatory region and the coding sequence contain a number of polymorphisms. The promoter variants have received particular attention in the past few years due to their possible role in the regulation of gene transcription. Previously, the -521C/T SNP was shown to influence promoter activity. The aim of this study is to perform an in-depth analysis of this effect in the context of various neural cell lines.
Endogenous mRNA expression of the DRD4 gene was demonstrated in two neuroblastoma (SK-N-F1, IMR32) and one retinoblastoma cell line (Y79) by RT-PCR. In addition, very low DRD4 mRNA levels were also detected in HeLa cells. The transcriptional activity of a series of 5' promoter deletion mutants was determined by transient transfection of luciferase reporter constructs. The activity profile of these promoter fragments was similar in each of the cell lines tested. The highest luciferase reporter activity was obtained with a construct containing promoter sequences between nucleotides -668 to -389, while a putative silencer region was localised spanning from nucleotide -1571 to -800. Surprisingly, the -521 C/T polymorphism had no significant effect on transcriptional activity of the reporter construct with the highest activity (-668 to -389) in any of the three cell lines tested.
Our results do not confirm previous data assigning different transcriptional activities to the -521 C/T alleles of the human DRD4 promoter. Furthermore, these findings highlight the need for further characterization of the 5' regulatory region of the DRD4 gene and identification of additional functional promoter polymorphic sites, especially in the context of haplotype.
Dopamine, an important neurotransmitter in the brain, plays a major role in the control of motor functions and behavioral patterns via interacting with specific cell surface receptors. Dopamine receptors belong to the large family of G protein-coupled receptors and can be classified as D1-like (D1, D5) and D2-like (D2, D3, D4) subgroups based on their sequence homology and pharmacological characteristics . The human dopamine D4 receptor (DRD4) gene was originally cloned by Van Tol et al. . The 5' flanking region of the gene was characterized by Kamakura et al.  as a housekeeping gene-like promoter. The promoter lacks a TATA or CAAT box but several putative transcription factor binding sites and CpG islands have been identified. 5'-RACE analysis of DRD4 mRNA revealed multiple transcription initiation sites located between nucleotides -501 and -436, relative to the translational start site. The region between -591 and -123 was found to contain the minimal promoter and to confer tissue-specific expression on the DRD4 gene and a putative repressor element was identified between nucleotides -770 and -679. Although high DRD4 mRNA expression was detected in the limbic area and frontal cortex associated with emotional function, motivation and cognition, the highest mRNA level was found in the retina . The DRD4 plays an important role in cognitive functions and it is a target of numerous antipsychotic drugs widely used in psychiatric disorders .
The 5' regulatory region and coding sequence of the DRD4 gene harbour a number of polymorphisms which may arise due to its telomeric location on chromosome 11. Promoter polymorphisms of the DRD4 gene, including the 120 base pair (bp) duplication and several SNPs (-521 C/T, -616 G/C SNP), have been extensively studied as possible risk factors of psychological traits as well as behavioural and psychiatric disorders. Despite the numerous polymorphic allele variants, only one mutation has been described in this region . A 27 bp deleted sequence was identified only in heterozygotes and with an extremely low allele frequency. To assess the potential functional role of the 27 bp deletion, an in silico analysis was performed that revealed various transcription factor binding sites within this short region.
An association between attention deficit hyperactivity disorder (ADHD) and the long form of the 120 bp duplication polymorphism was confirmed by a number of groups [7–9], although contradictory results have been reported by others . Interestingly, the short form of this promoter length polymorphism has been associated with novelty seeking, a human personality trait sharing common characteristics with ADHD . Seaman et al.  identified several consensus transcription factor binding sites within the 120 bp repeat, and there is in vitro evidence for enhanced Sp1 binding capacity of the duplicated form . The functional role of these genetic variants was studied recently employing in vitro luciferase reporter assays, demonstrating a lower transcriptional activity for the duplicated allele compared to the shorter variant .
Another well studied polymorphism in the DRD4 regulatory region is the -616 C/G SNP, which is thought to interact with the recently described -615 A/G variant in the adjacent position . It has been suggested that the -616 C/G SNP results in the creation of an AP-2 transcription factor binding site . An association of the -616 C/G SNP with ADHD , schizophrenia  and personality traits  have been reported in the literature but have not been confirmed by others. Although there are no association studies concerning the -1217G insertion/deletion polymorphism, its putative functional effect was examined by fMRI and a correlation was found between the insertion allelic variant and a greater activation in the anterior cingulate cortex, a brain area mediating conflict .
One of the most studied polymorphisms of the DRD4 promoter region is the -521 C/T SNP. An association between novelty seeking and the -521 CC genotype was first identified in a Japanese population , followed by a replication in a Caucasian (Hungarian) population , although negative results have also been reported [23, 24]. A recent meta-analysis showed a weak but positive association between this polymorphism and novelty seeking . Lakatos et al.  revealed an association between the infant disorganized attachment and the T allele of the -521 C/T SNP but only in combination with the 7-repeat form of the DRD4 exon III polymorphism. A family-based association approach demonstrating a low-rate transmission of the DRD4 7-repeat – -521 T haplotype in a large secure group of infants  supports this finding, although results to the contrary have also been published . Furthermore, a positive association was detected between the -521 C allele and schizophrenia , a disorder with elevated DRD4 mRNA levels in the frontal cortex , although others failed to replicate this result . A weak relationship between the -521 C/T SNP and ADHD has also been reported . To date, only one study has been performed that reveals the functional effect of the -521 C/T SNP , using chloramphenicol acetyltransferase reporter assay with the minimal promoter previously defined in the Y79 retinoblastoma cell line. The results from these experiments showed that the T allele reduced promoter activity by 40% relative to the C allele.
In the present study, we attempted to replicate the possible transcriptional effect of the -521 C/T SNP using a similar in vitro reporter system in a number of neuroblastoma and retinoblastoma cell lines.
DRD4 gene expression in neural and non-neural cell lines
DRD4 promoter deletional analysis in transiently transfected neural and non-neural cell lines
Relative luciferase activity of the DRD4 promoter constructs. Luciferase activities of the crude cell extracts were normalized to their β-galactosidase activities. Data are shown as the mean ± SD. Results of a representative experiment are shown as measured in triplicates. Similar data were obtained from three independent transfection experiments.
3.52 ± 1.43
3.87 ± 0.09
3.70 ± 0.29
3.59 ± 0.13
10.35 ± 1.00
8.80 ± 1.29
6.77 ± 0.86
8.71 ± 0.78
31.63 ± 4.7
26.89 ± 4.51
10.56 ± 0.52
27.08 ± 1.54
34.09 ± 3.34
28.98 ± 4.61
9.45 ± 0.46
21.89 ± 0.99
0.42 ± 0.02
1.75 ± 0.15
1.97 ± 0.12
2.56 ± 0.14
1.66 ± 0.20
4.18 ± 0.38
1.76 ± 0.22
2.51 ± 0.31
1.75 ± 0.17
4.21 ± 0.23
1.77 ± 0.16
2.63 ± 0.30
84.21 ± 10.76
114.97 ± 10.50
28.73 ± 2.29
172.35 ± 2.05
Effect of the -521 C/T SNP on transcriptional activity of DRD4 gene expression
Regulation of DRD4 gene expression is a significant issue, not only due to the well-known importance of this neurotransmitter receptor in brain function, but also because its highly polymorphic promoter has been implicated in a number of psychogenetic association studies. The human Y79 and IMR32 neuroblastoma cell lines reportedly express DRD4 mRNA . In addition to these cells, another neuroblastoma cell line (SK-N-F1) was also shown to produce high level of DRD4 mRNA in our study (Figure 1). These results are in agreement with the expression pattern of the DRD4 mRNA described by Van Tol et al . A low but detectable level of DRD4 mRNA was also observed in HeLa cells, indicating that the DRD4 promoter was slightly active in this cell line, in agreement with recent results , but in contrast to earlier data .
To characterize the DRD4 promoter, Kamakura et al.  performed CAT reporter assays using IMR32, Y79 and HeLa cell lines, and identified a putative promoter region between nucleotides -591 and -123 conferring tissue-specific DRD4 expression. In addition, a short region located 1.2 kb upstream from the initiation codon that harbours the 120 bp duplication, has also been shown to exhibit promoter activity . Notably, we could not map any region with cell type-specific transcriptional activity in our experimental system, as the in vitro transcriptional activity of the six promoter constructs showed a similar expression pattern in all cell lines, including the non-neural HeLa cells (Figure 3). It is important to note, however, that a cell type-specific feature is often typical for members of the dopamine receptor family [31–36], indicating the need for additional studies that explore tissue-specific DRD4 expression in experimental systems that more closely mimic in vivo conditions.
In terms of transcriptional activity, our constructs could be classified into two main groups: the pDRD4-D, E and F group exhibited about 10 fold lower activity than the pDRD4-A, B and C group. In fact, plasmids pDRD4-D, E and F possessed similar or even lower activities than the promoterless pGL3-Basic vector (see Table 1). These results were quite surprising since the D, E and F fragments were very similar to those used in a former study . A possible explanation for the inactivity of these promoter fragments could be due to the existence of four ATG codons residing with in this region (nucleotides -389 and -127) at positions -361, -346, -261 and -163. Three of these start codons are out of reading frame and could lead to the formation of a frame-shifted (inactive) luciferase gene. Another possibility is that a strong negative modulator element resides in this region.
The comparative analysis of constructs pDRD4-B (-799 to -389) and pDRD4-C (-668 to -389) was particularly interesting in light of a silencer region mapped to positions -770 and -679 by Kamakura et al. . We observed no significant difference between the transcriptional activities of constructs with (pDRD4-B) or without (p-DRD4-C) the previously identified silencer region (see Figure 3 and Table 1). On the other hand, a significant difference was found between the in vitro transcriptional activity of pDRD4-B (-799 to -389) and pDRD4-A (-1571 to -389), suggesting the presence of a negative modulator in the region between positions -1571 and -800. Thus, the transcriptional activity data generated with constructs A, B and C has revealed yet another silencer region upstream from the one previously described . Together, these results suggest that the human DRD4 gene promoter may possess two negative modulator regions, one of which did not function in our system. This hypothesized structure of the gene is reminiscent of that of the rat D2-, the Drosophila Dmdop1-, as well as the rat D3 dopamine receptor gene structure, which all contain two inhibitory elements in their promoter [32, 37, 38]. Nevertheless, the dopamine receptor promoters also show remarkable heterogeneity in their 5'-untranslated region. For example, the Drosophila Dmdop1 and the rat D2 dopamine receptor promoter contain an additional activating region beside the silencing elements [35, 37]. Similarly, the human D1A and D5 dopamine receptor genes also comprise varying numbers of positive and negative modulator elements [31, 34]. Beside the regulatory elements, the precise boundaries of the DRD4 promoter remain poorly defined. One study suggested that the promoter region might be within an intronic region . This theory was based on previously reported discrepancies in the size of the DRD4 mRNA which was originally reported as 5.3 kb in the SK-N-MC human neuroblastoma cell line and in the striatum, medulla, frontal cortex and limbic area of the rat and monkey brain . By contrast, the DRD4 mRNA was later found to be much shorter (1.5 kb), and expressed at higher levels, in the corpus callosum, medulla, subthalamic nucleus, as well as in the mesolimbic system and related regions. Furthermore, the DRD4 mRNA was barely detected in the striatum and the cerebral cortex  in contrast to the previous findings . These observed differences in mRNA size suggest that the DRD4 gene might have alternative transcription start sites, as in the case of the catechol-O-methyltransferase (COMT) gene, resulting in the formation of two distinct transcripts with differing 5' ends . Additionally, Todd and O'Malley suggested the possibility that a large intron might separate the coding exons of the DRD4 gene from the untranslated exons , as described for the dopamine D2 [35, 36, 42] and D3  receptors. In conclusion, the 5' untranslated region of the dopamine D4 receptor shows a high degree of complexity, emphasizing the need for further characterization to better define the core promoter and transcription initiation site.
Another aim of our study was to corroborate the effect of the -521 C/T SNP on the transcriptional activity previously described by Okuyama et al. [21, 29] however, in our system we observed no significant difference between the activities of the -521 C and -521 T alleles, in either the neuroblastoma or retinoblastoma cell lines (Figure 4). There were several differences in experimental setup between the previous and the present study, such as applying different reporter vectors and some variation in the length of the promoter fragment. The length of the cloned promoter fragments especially might have a significant effect on transcriptional activity . An additional possibility for these conflicting results is the highly polymorphic nature of this gene. Other identified polymorphisms in the DRD4 promoter region, such as the SNPs in positions -768, -616, -615, -603 and -600, may also influence promoter activity. For this reason, a novel approach for elucidating the effect of these various polymorphisms on DRD4 gene expression would be to perform a comparative analysis of the SNPs in combination in the context of different genetic haplotypes.
The aim of this study was to evaluate the effect of the -521 C/T SNP on DRD4 promoter activity. Our results do not confirm previous data assigning different transcriptional activities to the -521 C/T alleles in any of the three neuronal cell lines tested. The transcriptional activity of a series of 5' promoter deletion mutants was determined by luciferase reporter system. This experiments revealed a putative silencer region located between positions -1571 and -800.
Six overlapping fragments were amplified by PCR from the promoter of the DRD4 gene using the following primers containing either an Xho I or an Hind III recognition site: DR4-S1 (5' acc act cga g tg ggc tgg act cgc cgt ttg gc 3', corresponding to region -1571/-1550; translation start site is referred to as position +1), DR4-S2 (5' aag gct cga g cc cgg gac ccc ctg ccc agg 3', -799/-780), DR4-S3 (5' ggt tct cga g aa ctg tgc aac ggg tgg ggc cg 3', -668/-647), DR4-AS1 (5' aag gaa gct t cc ctc ggg cgc tca ccc tag tcc 3', -389/-411), and DR4-AS2 (5' aag gaa gct t cc cgc ggc gct cca ccg tga 3', -127/-146). The reaction mixture contained 10 ng of genomic DNA, 1-1 μM of each primer, 1 U Pfu DNA polymerase (MBI Fermentas), 1x Pfu PCR buffer, 0.2 mM dNTP-mix, 1x solution Q (Qiagen) and the final MgSO4 concentration was set to 2.5 mM in a final volume of 10 μl. Amplification began with a 3 min denaturation step at 95°C followed by 35 cycles of 1 min denaturation at 94°C and 3 min annealing/synthesis at 72°C. A final extension step was applied at 72°C for 10 min. The amplified products were digested with Hind III and Xho I and cloned into the luciferase reporter plasmid pGL3-Basic (Promega). After transformation into E. coli DH5α, all the plasmids were purified with Qiagen Plasmid Midi Kit. Constructs were verified by sequencing. The -521 C/T alleles were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The primers used were 5' ggc gtg gag ggt gcg cac gag gtc 3' (forward, -532/-509) and 5' gac ctc gtg cgc a cc ctc cac gcc 3' (reverse, -509/-532) for -521C site to T and 5' ggc gtg gag ggc gcg cac gag gtc 3' (forward, -532/-509) and 5' gac ctc gtg cgc g cc ctc cac gcc 3' (reverse, -509/-532) for -521T to C site. The mutations were confirmed by sequencing.
Total RNA isolation, RT-PCR
Total RNA was isolated from neural and HeLa cells using the TRIzol reagent (Invitrogen) according to the manufacturer's procedure. The reaction contained 3 μg of total RNA, 0.2 μg random hexameric primers (Promega), 8 U RNasin (Promega) and DEPC-treated water in a final volume of 10 μl. The reaction mixture was heated to 70°C for 4 min, followed by the addition of 4 μl M-MuLV of reverse transcriptase 5x reaction buffer (Fermentas), 24 U RNasin, 0.5 mM dNTPs, 100 U RevertAid M-MuLV reverse transcriptase (Fermentas) and DEPC-treated water to final volume of 20 μl total. The reaction was incubated at 37°C for 40 min followed by 42°C for 20 min and 99°C for 5 min. Single-stranded cDNAs were amplified by DRD4 specific primers DART-3 (5' gca ccg cct cca tct tca acc 3') and DART-4 (5' cgg aac gtg gcc cag tag agc 3') in a final volume of 10 μl containing 1 μl of RT reaction mix, 1 μM of each primer, 5 U HotStarTaq DNA-polymerase, 1x HotStar PCR buffer, 1x Q solution and 0.2 mM dNTPs (Qiagen). Thermocycling conditions were as follows: incubation at 95°C for 15 min followed by 32 cycles of 94°C for 1 min, 68°C for 1 min and 72°C for 1 min completed with a final extension step at 72°C for 10 min. As described earlier, human β-actin gene served as an internal control . PCR products were electrophorised in a 2% agarose gel.
The human neuroblastoma cell line IMR32 and HeLa cells were cultured in minimal essential medium Eagle (Gibco) supplemented with 10 % fetal bovine serum, 1% nonessential amino acids and 1% Na-pyruvate. The human retinoblastoma Y79 cells were maintained in RPMI 1640 medium (Sigma) supplemented with 20 % fetal bovine serum and 1% Na-pyruvate. SK-N-F1 (neuroblastoma) cells were grown in Dulbecco's modified Eagle's medium, High Glucose (Gibco) supplemented with 10 % fetal bovine serum and 1% nonessential amino acids. All cell lines were grown at 37°C with 5% CO2.
SK-N-F1 transfection was performed by the calcium phosphate-DNA coprecipitation method. A total of 1.2 × 106 cells were seeded 24 h prior to transfection in six-well plates. Equimolar amounts of reporter plasmids (~ 5 μg) together with 2.5 μg internal control plasmid pCMV-β-gal were introduced into the cells. The calcium phospate precipitate was washed out 14 h after transfection and the cells were harvested 36 hours later and lysed in 100 μl of 250 mM Tris-HCl pH 8.0. HeLa cells were transfected with Lipofectamine 2000 (Invitrogen). A mixture of 1.7–2.0 μg reporter construct and 0.2 μg pCMV-β-gal and 6 μl Lipofectamine 2000 was used to transfect 0.4 × 106 cells plated 24 h before transfection in six-well plates. Y79 and IMR32 transfections were performed in 12 well plates. Approximately 1.6 × 106 cells (Y79) or 1.35 × 106 cells (IMR32) were plated 24 h before transfection and transfected with equimolar amounts of reporter plasmids (1.7–2.0 μg), 1 μg pCMV-β-gal combined with 4 μl Lipofectamine Reagent and 4 μl Plus Reagent (Invitrogen). In the case of HeLa, Y79 and IMR32 cells, 1–1.2 ml fresh medium was added after 5 hours of incubation, and the cells were allowed to grow for 48 h before harvesting.
Luciferase and β-galactosidase assays
Cells were extracted by three consecutive freeze-thaw cycles and subsequent centrifugation. Supernatants were used for enzyme activity determinations. Luciferase and β-galactosidase activities were detected using the Luciferase Assay System kit (Promega) and by ONPG (o-nitrophenyl-β-D-galactopyranoside) cleavage rate, respectively. Luminescence was measured using a Mithras LB940 multilabel reader (Berthold Technologies, Bad Wildbad, Germany). Luciferase promoter activities were normalized to β-galactosidase activities. Three paralels were used in all transfections and all experiments were performed in triplicate.
Statistical analysis was performed with one-way ANOVA followed by the Tukey-Kramer Multiple Comparison Test (GraphPad InStat).
dopamine D4 receptor
single nucleotide polymorphism
reverse transcriptase polymerase chain reaction
- 5'- RACE:
5' rapid amplification of cDNA ends
attention deficit hyperactivity disorder
- 5' UTR:
5' untranslated region
This work was supported by Hungarian funds OTKA T 048576, D048430 and F46788. The luminometer used for the luciferase assay was provided by OTKA M045341. The authors thank Gergely Keszler for his valuable comments regarding the manuscript.
- Strange PG: New insights into dopamine receptors in the central nervous system. Neurochem Int. 1993, 22: 223-236. 10.1016/0197-0186(93)90050-FView ArticlePubMedGoogle Scholar
- Van Tol HH, Bunzow JR, Guan HC, Sunahara RK, Seeman P, Niznik HB, Civelli O: Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature. 1991, 350: 610-614. 10.1038/350610a0View ArticlePubMedGoogle Scholar
- Kamakura S, Iwaki A, Matsumoto M, Fukumaki Y: Cloning and characterization of the 5'-flanking region of the human dopamine D4 receptor gene. Biochem Biophys Res Commun. 1997, 235: 321-326. 10.1006/bbrc.1997.6770View ArticlePubMedGoogle Scholar
- Cohen AI, Todd RD, Harmon S, O'Malley KL: Photoreceptors of mouse retinas possess D4 receptors coupled to adenylate cyclase. Proc Natl Acad Sci U S A. 1992, 89: 12093-12097.PubMed CentralView ArticlePubMedGoogle Scholar
- Oak JN, Oldenhof J, Van Tol HH: The dopamine D(4) receptor: one decade of research. Eur J Pharmacol. 2000, 405: 303-327. 10.1016/S0014-2999(00)00562-8View ArticlePubMedGoogle Scholar
- Szantai E, Szmola R, Sasvari-Szekely M, Guttman A, Ronai Z: The polymorphic nature of the human dopamine D4 receptor gene: a comparative analysis of known variants and a novel 27 bp deletion in the promoter region. BMC Genet. 2005, 6: 39- 10.1186/1471-2156-6-39PubMed CentralView ArticlePubMedGoogle Scholar
- Kustanovich V, Ishii J, Crawford L, Yang M, McGough JJ, McCracken JT, Smalley SL, Nelson SF: Transmission disequilibrium testing of dopamine-related candidate gene polymorphisms in ADHD: confirmation of association of ADHD with DRD4 and DRD5. Mol Psychiatry. 2004, 9: 711-717.PubMedGoogle Scholar
- Leung PW, Lee CC, Hung SF, Ho TP, Tang CP, Kwong SL, Leung SY, Yuen ST, Lieh-Mak F, Oosterlaan J, Grady D, Harxhi A, Ding YC, Chi HC, Flodman P, Schuck S, Spence MA, Moyzis R, Swanson J: Dopamine receptor D4 (DRD4) gene in Han Chinese children with attention-deficit/hyperactivity disorder (ADHD): increased prevalence of the 2-repeat allele. Am J Med Genet B Neuropsychiatr Genet. 2005, 133: 54-56.View ArticleGoogle Scholar
- McCracken JT, Smalley SL, McGough JJ, Crawford L, Del'Homme M, Cantor RM, Liu A, Nelson SF: Evidence for linkage of a tandem duplication polymorphism upstream of the dopamine D4 receptor gene (DRD4) with attention deficit hyperactivity disorder (ADHD). Mol Psychiatry. 2000, 5: 531-536. 10.1038/sj.mp.4000770View ArticlePubMedGoogle Scholar
- Todd RD, Neuman RJ, Lobos EA, Jong YJ, Reich W, Heath AC: Lack of association of dopamine D4 receptor gene polymorphisms with ADHD subtypes in a population sample of twins. Am J Med Genet. 2001, 105: 432-438. 10.1002/ajmg.1403View ArticlePubMedGoogle Scholar
- Rogers G, Joyce P, Mulder R, Sellman D, Miller A, Allington M, Olds R, Wells E, Kennedy M: Association of a duplicated repeat polymorphism in the 5'-untranslated region of the DRD4 gene with novelty seeking. Am J Med Genet B Neuropsychiatr Genet. 2004, 126: 95-98. 10.1002/ajmg.b.20133View ArticleGoogle Scholar
- Seaman MI, Fisher JB, Chang F, Kidd KK: Tandem duplication polymorphism upstream of the dopamine D4 receptor gene (DRD4). Am J Med Genet. 1999, 88: 705-709. 10.1002/(SICI)1096-8628(19991215)88:6<705::AID-AJMG22>3.0.CO;2-FView ArticlePubMedGoogle Scholar
- Ronai Z, Guttman A, Keszler G, Sasvari-Szekely M: Capillary electrophoresis study on DNA-protein complex formation in the polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene. Curr Med Chem. 2004, 11: 1023-1029. 10.2174/0929867043455503View ArticlePubMedGoogle Scholar
- D'Souza UM, Russ C, Tahir E, Mill J, McGuffin P, Asherson PJ, Craig IW: Functional effects of a tandem duplication polymorphism in the 5'flanking region of the DRD4 gene. Biol Psychiatry. 2004, 56: 691-697. 10.1016/j.biopsych.2004.08.008View ArticlePubMedGoogle Scholar
- Ronai Z, Szantai E, Szmola R, Nemoda Z, Szekely A, Gervai J, Guttman A, Sasvari-Szekely M: A novel A/G SNP in the -615th position of the dopamine D4 receptor promoter region as a source of misgenotyping of the -616 C/G SNP. Am J Med Genet B Neuropsychiatr Genet. 2004, 126: 74-78. 10.1002/ajmg.b.20112View ArticleGoogle Scholar
- Barr CL, Feng Y, Wigg KG, Schachar R, Tannock R, Roberts W, Malone M, Kennedy JL: 5'-untranslated region of the dopamine D4 receptor gene and attention-deficit hyperactivity disorder. Am J Med Genet. 2001, 105: 84-90. 10.1002/1096-8628(20010108)105:1<84::AID-AJMG1068>3.0.CO;2-QView ArticlePubMedGoogle Scholar
- Lowe N, Kirley A, Mullins C, Fitzgerald M, Gill M, Hawi Z: Multiple marker analysis at the promoter region of the DRD4 gene and ADHD: evidence of linkage and association with the SNP -616. Am J Med Genet B Neuropsychiatr Genet. 2004, 131: 33-37. 10.1002/ajmg.b.30071View ArticleGoogle Scholar
- Mitsuyasu H, Hirata N, Sakai Y, Shibata H, Takeda Y, Ninomiya H, Kawasaki H, Tashiro N, Fukumaki Y: Association analysis of polymorphisms in the upstream region of the human dopamine D4 receptor gene (DRD4) with schizophrenia and personality traits. J Hum Genet. 2001, 46: 26-31. 10.1007/s100380170120View ArticlePubMedGoogle Scholar
- Bookman EB, Taylor RE, Adams-Campbell L, Kittles RA: DRD4 promoter SNPs and gender effects on Extraversion in African Americans. Mol Psychiatry. 2002, 7: 786-789. 10.1038/sj.mp.4001075View ArticlePubMedGoogle Scholar
- Fan J, Fossella J, Sommer T, Wu Y, Posner MI: Mapping the genetic variation of executive attention onto brain activity. Proc Natl Acad Sci U S A. 2003, 100: 7406-7411. 10.1073/pnas.0732088100PubMed CentralView ArticlePubMedGoogle Scholar
- Okuyama Y, Ishiguro H, Nankai M, Shibuya H, Watanabe A, Arinami T: Identification of a polymorphism in the promoter region of DRD4 associated with the human novelty seeking personality trait. Mol Psychiatry. 2000, 5: 64-69. 10.1038/sj.mp.4000563View ArticlePubMedGoogle Scholar
- Ronai Z, Szekely A, Nemoda Z, Lakatos K, Gervai J, Staub M, Sasvari-Szekely M: Association between Novelty Seeking and the -521 C/T polymorphism in the promoter region of the DRD4 gene. Mol Psychiatry. 2001, 6: 35-38. 10.1038/sj.mp.4000832View ArticlePubMedGoogle Scholar
- Ekelund J, Suhonen J, Jarvelin MR, Peltonen L, Lichtermann D: No association of the -521 C/T polymorphism in the promoter of DRD4 with novelty seeking. Mol Psychiatry. 2001, 6: 618-619. 10.1038/sj.mp.4000943View ArticlePubMedGoogle Scholar
- Strobel A, Lesch KP, Hohenberger K, Jatzke S, Gutzeit HO, Anacker K, Brocke B: No association between dopamine D4 receptor gene exon III and -521C/T polymorphism and novelty seeking. Mol Psychiatry. 2002, 7: 537-538. 10.1038/sj.mp.4001027View ArticlePubMedGoogle Scholar
- Schinka JA, Letsch EA, Crawford FC: DRD4 and novelty seeking: results of meta-analyses. Am J Med Genet. 2002, 114: 643-648. 10.1002/ajmg.10649View ArticlePubMedGoogle Scholar
- Lakatos K, Nemoda Z, Toth I, Ronai Z, Ney K, Szekely M, Gervai J: Further evidence for the role of the dopamine D4 receptor (DRD4) gene in attachment disorganization: interaction of the exon III 48-bp repeat and the -521 C/T promoter polymorphisms. Mol Psychiatry. 2002, 7: 27-31. 10.1038/sj.mp.4001986View ArticlePubMedGoogle Scholar
- Gervai J, Nemoda Z, Lakatos K, Ronai Z, Toth I, Ney K, Sasvari-Szekely M: Transmission disequilibrium tests confirm the link between DRD4 gene polymorphism and infant attachment. Am J Med Genet B Neuropsychiatr Genet. 2005, 132: 126-130.View ArticleGoogle Scholar
- Bakermans-Kranenburg MJ, Van Ijzendoorn MH: No association of the dopamine D4 receptor (DRD4) and -521 C/T promoter polymorphisms with infant attachment disorganization. Attach Hum Dev. 2004, 6: 211-218. 10.1080/14616730412331281584View ArticlePubMedGoogle Scholar
- Okuyama Y, Ishiguro H, Toru M, Arinami T: A genetic polymorphism in the promoter region of DRD4 associated with expression and schizophrenia. Biochem Biophys Res Commun. 1999, 258: 292-295. 10.1006/bbrc.1999.0630View ArticlePubMedGoogle Scholar
- Stefanis NC, Bresnick JN, Kerwin RW, Schofield WN, McAllister G: Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain. Brain Res Mol Brain Res. 1998, 53: 112-119. 10.1016/S0169-328X(97)00285-4View ArticlePubMedGoogle Scholar
- Beischlag TV, Marchese A, Meador-Woodruff JH, Damask SP, O'Dowd BF, Tyndale RF, Van Tol HH, Seeman P, Niznik HB: The human dopamine D5 receptor gene: cloning and characterization of the 5'-flanking and promoter region. Biochemistry. 1995, 34: 5960-5970. 10.1021/bi00017a025View ArticlePubMedGoogle Scholar
- D'Souza UM, Wang W, Gao DQ, Kanda S, Lee G, Junn E, Hwang CK, Jose PA, Mouradian MM: Characterization of the 5' flanking region of the rat D(3) dopamine receptor gene. J Neurochem. 2001, 76: 1736-1744. 10.1046/j.1471-4159.2001.00155.xView ArticlePubMedGoogle Scholar
- Lee SH, Wang W, Yajima S, Jose PA, Mouradian MM: Tissue-specific promoter usage in the D1A dopamine receptor gene in brain and kidney. DNA Cell Biol. 1997, 16: 1267-1275.View ArticlePubMedGoogle Scholar
- Minowa MT, Minowa T, Monsma FJJ, Sibley DR, Mouradian MM: Characterization of the 5' flanking region of the human D1A dopamine receptor gene. Proc Natl Acad Sci U S A. 1992, 89: 3045-3049.PubMed CentralView ArticlePubMedGoogle Scholar
- Minowa T, Minowa MT, Mouradian MM: Analysis of the promoter region of the rat D2 dopamine receptor gene. Biochemistry. 1992, 31: 8389-8396. 10.1021/bi00151a001View ArticlePubMedGoogle Scholar
- Valdenaire O, Vernier P, Maus M, Dumas Milne Edwards JB, Mallet J: Transcription of the rat dopamine-D2-receptor gene from two promoters. Eur J Biochem. 1994, 220: 577-584. 10.1111/j.1432-1033.1994.tb18658.xView ArticlePubMedGoogle Scholar
- Kehren V, Baumann A: Characterization of the 5' regulatory region of the Drosophila Dmdop1 dopamine receptor-gene. Arch Insect Biochem Physiol. 2005, 59: 118-131. 10.1002/arch.20062View ArticlePubMedGoogle Scholar
- Minowa T, Minowa MT, Mouradian MM: Negative modulator of the rat D2 dopamine receptor gene. J Biol Chem. 1994, 269: 11656-11662.PubMedGoogle Scholar
- Todd RD, O'Malley KL: The dopamine receptor DRD4 gene: are duplications distracting?. Trends Pharmacol Sci. 2001, 22: 55-56. 10.1016/S0165-6147(00)01612-6View ArticlePubMedGoogle Scholar
- Matsumoto M, Hidaka K, Tada S, Tasaki Y, Yamaguchi T: Low levels of mRNA for dopamine D4 receptor in human cerebral cortex and striatum. J Neurochem. 1996, 66: 915-919.View ArticlePubMedGoogle Scholar
- Tenhunen J, Salminen M, Lundstrom K, Kiviluoto T, Savolainen R, Ulmanen I: Genomic organization of the human catechol O-methyltransferase gene and its expression from two distinct promoters. Eur J Biochem. 1994, 223: 1049-1059. 10.1111/j.1432-1033.1994.tb19083.xView ArticlePubMedGoogle Scholar
- O'Malley KL, Mack KJ, Gandelman KY, Todd RD: Organization and expression of the rat D2A receptor gene: identification of alternative transcripts and a variant donor splice site. Biochemistry. 1990, 29: 1367-1371. 10.1021/bi00458a003View ArticlePubMedGoogle Scholar
- Rebbeck TR, Spitz M, Wu X: Assessing the function of genetic variants in candidate gene association studies. Nat Rev Genet. 2004, 5: 589-597. 10.1038/nrg1403View ArticlePubMedGoogle Scholar
- Raff T, van der GM, Endemann D, Wiederholt T, Paul M: Design and testing of beta-actin primers for RT-PCR that do not co-amplify processed pseudogenes. Biotechniques. 1997, 23: 456-460.PubMedGoogle Scholar
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