The dopamine transporter (DAT) plays a central role in dopaminergic neurotransmission in the human brain. Genetic association studies have used a variable number of tandem repeat (VNTR) polymorphism in the 3′-flanking region of the dopamine transporter gene (DAT1) to implicate the DAT in the development of various neuropsychiatric disorders. In this study, we have examined the possibility that a mutation exists in the coding region of the DAT1 gene which through linkage disequilibrium accounts for the observed associations. The complete coding region, as well as exon–intron boundaries, was screened in 91 unrelated individuals including 45 patients with bipolar affective disorder and 46 healthy control individuals by the means of single strand conformation analysis. Our findings suggest that the DAT1 gene is highly conserved since we detected only two rare missense substitutions (Ala559Val, Glu602Gly) and three silent mutations (242C/T, 1342A/G, and 1859C/T) in the whole coding region. Five sequence variants were observed in intronic sequences but none affects known splice sites. The lack of frequent variants of possible functional relevance indicates that genetic variation in the coding region of the DAT1 gene is not responsible for the previously observed associations with neuropsychiatric disorders. The two rare missense substitutions were found in single bipolar patients but not in controls. Investigation of the patients’ families revealed independent segregation between the Ala559Val variant and affective disorder. The Glu602Gly variant was inherited by the proband from an affected father. It therefore remains possible that Glu602Gly may be a rare cause of bipolar affective disorder.
The dopamine transporter (DAT) is a member of the family of Na+- and Cl−-dependent neurotransmitter transporters.1, 2, 3 The DAT is believed to control the temporal and spatial activity of released dopamine by rapid reuptake of the neurotransmitter into presynaptic terminals. It is therefore an important element in regulating the action of dopamine on locomotion, cognition, affect and neuroendocrine functions. The DAT is the initial site of action of psychostimulants such as cocaine and amphetamines;4, 5 it is also involved in the uptake of toxins generating Parkinson's disease.6
The DAT shares the characteristic structure of the highly conserved family of Na+- and Cl−-dependent neurotransmitter transport proteins. The human DAT protein consists of 620 aminoacids forming 12 putative transmembrane domains (TMD).7 Cloning and characterization of the human dopamine transporter gene (DAT1) showed that the gene maps to chromosome 5q15.3, contains 15 exons and carries a variable number of tandem repeat (VNTR) polymorphism in the 3′-untranslated region (3′-UTR).8, 9, 10, 11, 12, 13
Genetic association studies have implicated the DAT1 gene in the development of various neuropsychiatric disorders including attention deficit hyperactivity disorder,14, 15, 16, 17 bipolar affective disorder,18 schizoid/avoidant behavior,19 cocaine-induced paranoia,20 and both the presence of alcohol-withdrawal seizures or delirium21 and the severity of uncomplicated alcohol withdrawal.22 All published studies employed the VNTR polymorphism for testing of the DAT1 gene. The VNTR polymorphism has been used so widely because it is highly polymorphic with eight different alleles,23 and because no other polymorphisms with a definite location within the DAT1 gene have so far been identified. However, no data have been published to support a direct effect of the VNTR polymorphism on the function of the DAT1 gene. It is therefore likely that this polymorphism is not itself involved in the etiology of disease but is in linkage disequilibrium with the true pathogenic variant(s) lying within a functionally relevant region of the DAT1 gene. Accordingly, in the present study we set out to screen the complete coding region as well as exon-intron boundaries for the presence of genetic variants using single strand conformation analysis (SSCA). The screening sample consisted of 45 patients with bipolar affective disorder and 46 healthy individuals.
Materials and methods
The screening sample included 45 patients with bipolar affective disorder and 46 healthy control individuals. Thirty-six bipolar patients were derived from affected sib pairs and multiple affected pedigrees chosen at random prior to genotyping. The remaining subjects were recruited from inpatient and day hospital facilities. All patients were interviewed by an experienced psychiatrist using the Schedule for Affective Disorders and Schizophrenia-Lifetime Version (SADS-L).24 Best estimate diagnoses were assigned on the basis of the interview, review of all available clinical records and family history information according to DSM-III-R criteria.25 The same diagnostic procedures were applied to subjects II:1, II:2, II:3, II:4, II:5, III:1, and III:2 from family Wue-30, and subjects I:1, I:2, and II:4 from family BN-02. In family BN-02, information on disease status of individuals II:1, II:2, and II:3 was obtained from individuals I:1, I:2, and II:4 by using the Family Informant Schedule and Criteria (FISC).26
All index patients were unrelated and of German descent. Informed consent was obtained from all individuals participating in this study. The study was approved by the Ethical Committee of the University of Bonn Medical Center.
Characterization of exon-flanking intronic sequences
Limited information on intronic sequences was available from the papers reporting the genomic organization of the DAT1 gene13, 27 and DNA sequence databases. Because we wanted to include all exon-intron junctions in our mutation screening, we needed to determine exon-flanking intronic sequences for introns 1 (3′), 3 (3′), 5 (3′), 6 (3′), 9 (3′), 12 (3′), 13 (5′ and 3′), and 14 (5′ and 3′) for selection of primers. Sequences of introns 1, 3, 5, 6, 9, and 14 were obtained using the Human Genome Walker™ kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's recommendations. Sequences of introns 12 (3.6 kb) and 13 (2.5 kb) were obtained by intron-spanning long-distance amplification with exon-specific primers using the GeneAmp™ kit (Perkin Elmer Cetus, Foster City, CA, USA). PCR fragments were sequenced after blunt-end cloning using the SureClone™ Ligation Kit (Pharmacia, Uppsala, Sweden).
PCR amplification of genomic DNA
EDTA anticoagulated venous blood samples were collected from all individuals. Lymphocyte DNA was isolated by salting out with saturated NaCl solution.28
Since sensitivity of SSCA is optimal for PCR products of 150–300 bp length, sets of primers were chosen resulting in fragment sizes from 146 bp to 259 bp (Table 1). For exons 1, 3, and 5–15 fragments encompassed the whole exon and adjacent exon-intron junctions. Exon 2 and exon 4 were screened using two overlapping fragments for each exon.
Standard PCR was carried out in a 25-μl volume containing 40 ng genomic DNA, 10 pmol of each primer, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatine, 200 μM of each dNTP, and 0.5 U Taq DNA polymerase (Life Technologies, Gaithersburg, MD, USA). For optimizing purposes, glycerol and DMSO were added in various quantities (Table 1). Samples were amplified in a Cetus DNA Gene Amp 9600 (Perkin Elmer Cetus). After an initial 5-min denaturation at 94°C, 35 temperature cycles were carried out for fragments 1, 4, 6, 7, 8, 9, 12, 13, 15, 16, and 17, consisting of 30 s at 94°C, 30 s at optimal annealing temperature (Table 1), and 30 s at 72°C. For optimal results in fragments 2, 3, 5, 10, 11, and 14 a touchdown PCR program had to be performed with two different annealing temperatures and eight cycles with temperature one followed by 30 cycles with temperature two (Table 1). All PCR reactions ended with a final extension step of 5 min at 72°C.
Single strand conformation analysis (SSCA) and sequencing
For non-radioactive SSCA, 8 μl of the PCR product were mixed with 12 μl formamide containing 0.0125% bromphenol blue and 0.75% Ficoll 400 in 1 × TBE, denaturated for 5 min at 94°C and subsequently chilled on ice; 7 μl of the product were loaded on 10% polyacrylamide (PAA) gels (acrylamide:bisacrylamide = 49:1; 110 × 130 × 1.0 mm, Multigel-Long/Biometra, Göttingen, Germany) containing 0.5 × TBE. PAA gels were run for 12–15 h under two conditions: 6 V cm−1 at room temperature and 7 V cm−1 at +4°C. Bands were visualized by silver-staining.29 PCR products from individuals displaying variant banding patterns in SSCA were purified using the QIAquick™ PCR Purification Kit (Quiagen, Hilden, Germany). Nucleotide sequences were determined with internal sequencing primers using a model 377 Applied Biosystems automatic sequencer.
To verify genotypes and to determine the frequency of the respective variants, PCR-based restriction fragment length polymorphism (RFLP) assays were established (Table 2). Original primer sets were used when the sequence variant altered a natural restriction site. In the case of the 242C/T and the 1398−21G/A variants we used mutagenic primers to introduce a base substitution adjacent to the sequence of interest, creating an artificial restriction site with only one allelic form. For detection of the 242C/T variant we additionally had to perform a seminested PCR reaction to achieve a specific PCR product for RFLP analysis. The first round was performed with 2aF as forward and 2CpoIR 5′-CTGGTGAGCTGCGGTCC-3′ as reverse primer to introduce a CpoI restriction site. Then, the semi-nested PCR was carried out with 2CpoIR as reverse and 2CpoIF 5′-AAGAGGGAAGAAGCACAGAA-3′ as forward primer. As template for the semi-nested PCR reaction we used a 1:100 dilution of the PCR product derived from the first reaction. For detection of the 1398-21G/A variant, primer 10Tth111IF 5′-GTCGTGCCGCCATAGAAG-3′ was used in combination with primer 10Tth111IR 5′- CTGCACACAGAGGACAGGGT-3′ (nucleotide substitution is underlined) to introduce a Tth111I restriction site.
For restriction digest, 5 μl of PCR product were incubated with 5 U of the appropriate restriction enzyme (Table 2) according to the manufacturers’ recommendations. Fragments were resolved on 10% PAA gels (acrylamide:bisacrylamide = 49:1) containing 1 × TBE at 15 V cm−1. Restriction profiles were visualized by silver staining.29
Despite performing numerous experiments with mutagenic primers, we have not been able to establish RFLP assays for detection of variants 547−12C/A and 1967+29insTC. In these cases, genotyping was performed using SSCA.
Genotypes of the VNTR polymorphism in the 3′-untranslated region were determined as described elsewhere.9
We applied Fisher's exact test (2-tailed) for comparing genotype distributions and allele frequencies between affected and control individuals from the screening sample. The χ2-goodness-of-fit test, comparing the observed genotype frequencies with those expected under Hardy–Weinberg equilibrium, was used to test for deviations from equilibrium. To describe linkage disequilibrium between the variants, we used the measure D′ introduced by Lewontin.30 The likelihood ratio test as implemented in the EH program31 was used as a test for linkage equilibrium.
Variation in the dopamine transporter gene
For the present analysis, the whole coding region of the human DAT1 gene consisting of 15 exons and adjacent exon-intron boundaries was systematically screened for nucleotide sequence variation. A total of 91 individuals (including 45 bipolar patients and 46 controls) were investigated. Among the 182 DAT1 alleles 10 sequence changes were identified (Table 2). Two sequence variants resulted in amino-acid substitutions (Ala559Val, Glu602Gly), three were silent mutations (242C/T, 1342A/G, and 1859C/T), and five were mutations in exon-flanking intronic sequences (547−12C/A, 1398−56A/G, 1398−21G/A, 1626+14G/A, and 1967+29insTC) (Figure 1). The three silent mutations are probably without consequences on transporter function, since they should not introduce aberrant splice sites according to their Senapathy score;32 similarly, the five intron variants do not affect known splice sites.
Genotype and allele distributions
Genotype and allele distributions for the 10 variants observed in the screening sample are given in Table 3. The genotype distributions did not differ significantly from the expected numbers calculated on the basis of the observed allele frequencies according to the Hardy–Weinberg equilibrium (data not shown). Comparisons of genotypes and alleles between patients and controls revealed no significant differences. Missense substitutions Ala559Val and Glu602Gly were observed in single bipolar patients. The VNTR polymorphism also showed no significant differences in genotype and allele distributions between patients and controls (data not shown).
Examination of pedigrees
The bipolar probands heterozygous for the amino acid substitutions were derived from families segregating affective disorder. We therefore had the opportunity to examine the relationship of these variants to the psychiatric phenotype over two generations in each family. The pedigree structure for family Wue-30 segregating the Ala559Val variant is shown in Figure 2, and the pedigree structure for family BN-02 segregating the Glu602Gly variant is shown in Figure 3, respectively. Examination of pedigree Wue-30 showed that Ala559Val does not segregate with the disease. In contrast, the bipolar proband from family BN-02 inherited the Glu602Gly variant from her affected father while the mother is homozygous for glutamic acid in position 602. Unfortunately, the information which can be gained from family BN-02 is limited since neither the proband's sister and two brothers nor any other family members were available for a personal interview and genotyping.
The variants with an allele frequency >5% in controls or bipolars (242C/T, 547−12C/A, 1342A/G, and 1398−21G/A) were tested for the presence of linkage disequilibrium. Significant linkage disequilibrium was observed between variants 242C/T and 547−12C/A (controls: D′ = 0.845, P = 0.00007; bipolars: D′ = 1.000, P = 0.008), and 1342A/G and 1398−21G/A (controls: D′ = 0.628, P = 0.00009; bipolars: D′ = 1.000, P <0.00001), respectively. Since most previous association studies have used the 3′ VNTR polymorphism as DAT1 marker, we tested all frequent variants for linkage disequilibrium with the VNTR polymorphism. Genotyping our sample with the VNTR polymorphism revealed four different alleles (8, 9, 10, and 11 repeats) among patients and controls. Alleles 9 and 10 occurred frequently while alleles 8 and 11 were rare. For analysis of linkage disequilibrium the four alleles were reduced to two different two-allele systems by either collapsing allele 9 or 10 with the rare alleles, respectively (9 vs 8, 10, and 11 [VNTR1]; 10 vs 8, 9, and 11 [VNTR2]). Significant linkage disequilibria were observed between 1342A/G and the VNTR polymorphism (controls: VNTR1 D′ = −0.599, P = 0.001; VNTR2 D′ = 0.521, P = 0.019; bipolars: VNTR1 D′ = −0.841, P <0.00001; VNTR2 D′ = 0.836, P <0.00001), and 1398−21G/A and the VNTR poymorphism (controls: VNTR1 D′ = −0.897, P <0.00001; VNTR2 D′ = 0.879, P = 0.00002; bipolars: VNTR1 D′ = −1.000, P <0.00001; VNTR2 D′ = 1.000, P <0.00001), respectively. No significant linkage disequilibria were observed between variant 242C/T and the VNTR polymorphism (controls: VNTR1 D′ = −0.069, P = 0.722; VNTR2 D′ = −0.087, P = 0.867; bipolars: VNTR1 D′ = −0.99994, P = 0.539; VNTR2 D′ = −0.99998, P = 0.491), and 547−12C/A and the VNTR polymorphism (controls: VNTR1 D′ = 0.121, P = 0.766; VNTR2 D′ = −0.281, P = 0.425; bipolars: VNTR1 D′ = −0.178, P = 0.351; VNTR2 D′ = 0.148, P = 0.455), respectively.
Comparing haplotype frequencies between affected and control individuals by means of an appropriate likelihood ratio test revealed no statistically significant differences (data not shown).
Genetic association studies have used the VNTR in the 3′ region of the DAT1 gene to implicate the DAT in the development of various neuropsychiatric disorders.8, 14, 15, 16, 18, 19, 20, 21, 22 In this study, we have examined the possibility that a mutation exists in the coding region of the DAT1 gene which through linkage disequilibrium accounts for the observed associations. Our findings suggest that the DAT1 gene is highly conserved since we detected only two rare missense substitutions (Ala559Val, Glu602Gly) and three silent mutations (242C/T, 1342A/G, and 1859C/T) in the whole coding region. In addition, our study indicates that linkage disequilibrium with the 3′ VNTR does not extend to the 5′ part of the gene since significant linkage disequilibrium was observed with variants 1342A/G and 1398−21G/A, but not with 242C/T and 547–12C/A (variants 1342A/G and 547–12C/A are separated by ≥31.3 kb of genomic sequence, see Figure 1). Therefore, it is unlikely that a causative mutation, which is detected through linkage disequilibrium with the 3′ VNTR, is located in 5′ regulatory regions of the DAT1 gene.
Previous studies have suggested a role of the DAT1 in bipolar disorder by evidence of linkage and family-based association studies.18, 33, 34 However, there have also been a number of negative reports.35, 36, 37 In our study, we found variants of possible functional significance (Ala559Val, Glu602Gly) in two single bipolar individuals. These variants were not found among the control individuals. Influence on transporter function is unlikely for the conservative Ala559Val substitution. Alanine in position 559 is conserved in bovine and rat dopamine transporters but not within the transporter family. Examination of the pedigree showed that the Ala559Val variant does not segregate with the disorder (Figure 2). The affected brother, the affected mother, and the affected maternal aunt were Ala-559/Ala-559. The observed non-cosegregation of Ala559Val with affective disorder suggests it is non-pathogenic. However, it still remains possible that the variant makes an oligogenic contribution to the psychiatric phenotype. As the Ala559Val substitution was detected only in one single individual, if it is has a role it must be limited to a small proportion of cases. The role of the Glu602Gly variant remains largely unclear. The glutamic acid in position 602 is only conserved in the bovine dopamine transporter but not in the rat dopamine transporter and other members of the transporter family suggesting that this amino acid residue might not be crucial for proper protein function. However, the glutamic acid to glycine substitution results in a loss of a negative charge. On the level of the transporter protein the Glu602Gly variant is localized in the intracellular carboxyl-terminal tail. It has been shown that truncation/substitution of the carboxyl tail not only confers high affinity dopamine uptake mimicking that seen in native synaptosomes, but in addition abolishes appropriate and pharmacologically relevant binding interactions of the radiolabeled ligand [3H]CFT.38 Moreover, the experiments suggest that possibly the DAT carboxyl-terminal tail is also involved in the formation of DAT oligomeric complexes.38 Examination of the pedigree showed that the index patient, who suffered from bipolar I disorder, had inherited the Glu602Gly variant from her father who was in his seventies, still alert and since early adulthood had experienced recurrent hypomanic episodes fulfilling DSM-III-R criteria25 of bipolar disorder not otherwise specified (Figure 3). The mother was always mentally healthy and died in her sixties. Unfortunately, no additional family members were available for a personal interview. According to family history information obtained from the proband and her parents, none of the proband's siblings or other members of the family had ever shown signs of affective disorder or any other psychiatric disorder. However, because of the limited availability of family members for personal interview and genotyping, no definite conclusions can be drawn. Screening of larger samples will be necessary to establish association with bipolar affective disorder.
Using a systematic mutation screening approach similar to the present one, the coding regions of other monoamine transporter genes were also found to be highly conserved. Investigation of the serotonin transporter gene in a total of 151 unrelated individuals led to the identification of only a single individual bearing a missense substitution.39, 40 We have previously studied the norepinephrine transporter gene in 180 unrelated individuals and found five missense substitutions only one of which had a frequency of >1%.41, 42 These results suggest that low variability is a general feature of the monoamine transporter family. However, genetic variability is not restricted to the amino acid composition of the protein, it may be present in regulatory regions and thereby alter protein expression. This was demonstrated for the serotonin transporter43 and should be a matter of future studies for other monoamine transporters including the DAT. Recently, at least 4 kb of the 5′-flanking region of the DAT1 gene have been characterized, regulatory sequences have been identified therein, and a region involved in regulation of DAT neurospecific expression has been identified in intron 1.44 Because sequences with regulatory relevance extend over large genomic regions (>1.8 kb) we did not include these sequences in the present study. However, these sequences should be carefully examined for the presence of genetic variation in future experiments.
There remains the possibility that we have missed a mutation by relying on SSCA as a mutation screening procedure because the sensitivity of SSCA is not 100%. The possibility has been reduced by performing SSCA under two partly different conditions. However, the existence of undetected variants cannot be completely excluded. Finally, the 3′ VNTR remains a candidate for bringing about changes in expression of the DAT but this has not been formally tested.
In conclusion, we have undertaken mutational analysis of the complete coding region and exon–intron boundaries of the DAT1 gene and have found no evidence of genetic variation that could explain previously reported associations with neuropsychiatric disorders. We identified two rare missense substitutions, one of which may be a rare cause of bipolar affective disorder.
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This study was supported by the Deutsche Forschungsgemeinschaft (SFB 400 ‘Molekulare Grundlagen zentralnervöser Erkrankungen’, Teilprojekte A3 and D3).
About this article
- genetic variation
- candidate gene
- single nucleotide polymorphism
- bipolar affective disorder
- manic depression
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