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Association of intron 1 variants of the dopamine transporter gene with schizophrenia
Neuroscience Letters 513 (2012) 137–140
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Association of intron 1 variants of the dopamine transporter gene with schizophrenia Chunming Zheng, Yan Shen, Qi Xu ∗ National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, Beijing 100005, China
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Article history: Received 21 January 2012 Received in revised form 5 February 2012 Accepted 7 February 2012 Keywords: Association study Dopamine transporter gene Psychotic symptoms Schizophrenia
a b s t r a c t The dopamine transporter (DAT1) gene has been implicated in the pathogenesis of many neuropsychiatric disorders, including schizophrenia. The present study aimed to investigate association of the DAT1 gene polymorphisms with schizophrenia in a Han Chinese population. Two single nucleotide polymorphisms (SNPs) in the DAT1 gene (rs2975223 and rs2455391) were tested in 368 patients with schizophrenia and 420 healthy controls, of whom 293 patients underwent an assessment of psychotic symptoms through the positive and negative syndrome scale (PANSS). The chi-square test (2 ) showed disease association for rs2455391 (corrected p = 0.023 for allelic association and p = 0.034 for genotypic association, respectively). The rs2975223(G)–rs2455391(C) haplotype was associated with increased risk of the illness (p = 0.0012, OR = 2.09, 95% CI = 1.28–3.42). Quantitative trait analysis showed that rs2455391 was associated with positive symptoms, general symptoms and global symptoms but not with negative symptoms. The present results suggest that the DAT1 gene may be mainly involved in the development of the positive symptoms in the Chinese population. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Schizophrenia is a severe and chronic mental disorder with a 1% lifetime prevalence [31] characterized by positive symptoms, negative symptoms and cognitive dysfunctions [13]. Family, twin and adoption studies indicate that genetic factors account for 64–80% of the etiology of schizophrenia [6,7,26,27,32]. Genetic variants involved in the dopaminergic system have been extensively explored according to the dopaminergic hypothesis [21]. Accumulating evidence suggested that the imbalanced presynaptic activity of dopaminergic neurons in various brain regions contributes to the pathophysiology of schizophrenia [28,30]. The presynaptic dopamine balance is regulated by reuptake and degradation of released dopamine; dopamine transporter (DAT1), a crucial component of dopamine neurotransmission in the central nervous system (CNS), plays an important role in reuptake process [3]. DAT1 is believed to regulate the temporal and spatial activity of released dopamine by rapid reuptake of the neurotransmitter into presynaptic terminals [16]. Moreover, DAT1 is an important element in regulating the duration of dopamine activity [8]. Therefore,
∗ Corresponding author at: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, No.5 Dong Dan San Tiao, Beijing 100005, China. Tel.: +86 10 65296432; fax: +86 10 65263392. E-mail addresses: [email protected], [email protected] (Q. Xu). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.02.015
it is possible that the polymorphisms of the DAT1 gene (also known as SLC6A3) may be a risk factor predisposing to schizophrenia. The DAT1 protein is primarily expressed in midbrain dopaminergic neurons projecting to the substantia nigra and ventral tegmental regions as well as to the striatum, nucleus accumbens, prefrontal cortex, and hypothalamus [3,9]. Dysfunction of DAT1 could contribute to several neuropsychiatric disorders and this gene has been widely studied in a number of dopamine-related neuropsychiatric disorders, including attentiondeficit/hyperactivity disorder (ADHD) [1], Parkinson’s disease (PD) [24], bipolar disorder [18], and schizophrenia. The human DAT1 gene is mapped to chromosome 5p15.3 with 15 exons spanning about 65 kb [34]. The protein-coding domains start within exon 2 and end near the beginning of exon 15 [3]. Grünhage et al. [19] systemically screened for genetic variations across the coding region of the DAT1 gene and revealed that the coding region was highly conserved; they therefore hypothesized that variations in the DAT1 gene most from the noncoding region could contribute to the differences in gene expression levels rather than protein structures between individuals, and that mutations in promoter regulator regions of this gene could provide a significant impact on dopaminergic activity in the brain. Several studies analyzed the association between the DAT1 gene and schizophrenia, but the results reported to date have been inconsistent. Most association studies focused on testing a 40-bp variable number of tandem repeat (VNTR) located in the 3 untranslated region (UTR) in exon 15 although a meta-analysis failed to
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confirmed such an association [14]. The 5 regulatory region of the DAT1 gene has also been a hotspot. Systematic analysis of the 5 regulatory region indicated the presence of regulatory elements between the 5 UTR and intron 1 regions [17]. Several lines of evidence have recently suggested that the 5 promoter region of the DAT1 gene may play a role in increasing susceptibility to schizophrenia [22]. Because only a few studies of intron1 variants have been conducted in schizophrenia, it may be useful to re-confirm the association of DAT1 intron 1 variants with the disease in different populations. Accordingly, this study was designed to investigate whether the DAT1 intron 1 polymorphisms could contribute to the etiology of schizophrenia in a Han Chinese population.
All subjects were of Chinese Han origin from the northern area of China and gave written informed consent to participate in this study as approved by the Ethics Committee of the Chinese Academy of Medical Science and Peking Union Medical College. 2.2. SNPs selection and genotyping Genomic DNA was extracted from peripheral blood samples using standard phenol–chloroform extraction method. Two single nucleotide polymorphisms (SNPs), rs2975223 and rs2455391 in intron1 of the DAT1 gene were selected for genetic analysis from the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP). Polymerase chain reaction (PCR) amplification was performed with the primer sequences as follows: 5 -TGTATTCCATCCTGGGTGAC-3 (forward) and 5 -GCTTCTTCCCTCTTGGTCTT-3 (reverse). PCR was carried out with the GeneAmp PCR 2700 system (Applied Biosystems) in a 25-l reaction volume containing 200 M of each dNTP, 0.4 M of each primer, 2.5 l of 10× PCR buffer, 1 unit of Taq DNA polymerase (Tiangen, Beijing, China), and 60 ng of genomic DNA. The conditions used for PCR amplification consisted of initial denaturation at 94 ◦ C for 5 min, 35 cycles at 94 ◦ C for 30 s, 55 ◦ C for 30 s and 72 ◦ C for 30 s, followed by a final extension at 72 ◦ C for 10 min. The genotypes were then determined by use of bidirectional sequencing with the ABI 3700 DNA analyzer (Perkin-Elmer, Applied Biosystems, Foster City, CA, USA).
2. Materials and methods 2.1. Subjects In this study, we recruited a total of 368 unrelated patients with paranoid schizophrenia (219 males and 149 females), with a mean age of 31.97 ± 12.09 years. They were randomly sampled from the whole patient population through clinical settings in the Department of Psychiatry, First Hospital of Shanxi Medical University, Taiyuan, China, in the period between January 2006 and August 2009. Clinical diagnosis was made by at least two senior psychiatrists according to the criteria of the Diagnostic and Statistical Manual of mental disorders, fourth edition (DSM-IV) [2]. All patients recruited for this study were also assessed with the Chinese Version of the Modified Structured Clinical Interview for DSM-IV TR Axis I Disorders Patient Edition (SCID-I/P, 11/2002 revision). Since antipsychotic drugs can alter psychopathology status and regulate dopamine concentration, 293 patients, who were medication-free for at least 1 month (n = 154) or drug-naïve (n = 139) among the case group, were assessed by the positive and negative syndrome scale (PANSS) [23]. The PANSS assessment included three subscales for the measurements of positive symptoms (7 items), negative symptoms (7 items) and general psychopathological symptoms (16 items). The severity of each symptom was scored with 7 grades (1 = absent, 2 = minimal, 3 = mild, 4 = moderate, 5 = moderate severe, 6 = severe, and 7 = extreme). Some potential participants were excluded from this study if they had suffered from any organic brain disorders, mental retardation, epilepsy, head trauma, or the psychotic symptoms were due to medical conditions or treatments. The control group consisted of 420 unrelated healthy volunteers (248 males and 172 females) aged 29.53 ± 9.47 years. They were recruited from local communities. All control subjects were assessed using the SCID, and those who had a history of major psychiatric or neurological disorders, or family history of severe forms of psychiatric disorders, were excluded.
2.3. Statistical analysis The Haploview program (version 4.1, Broad Institute of MIT and Harvard, Cambridge, MA, USA) was applied to test the genotypic distributions of SNPs for Hardy–Weinburgh equilibrium and to estimate linkage disequilibrium (LD) between these two SNPs in which the LD strength was expressed by measurements D and r2 . Allelic, haplotypic and genotypic associations were analyzed using the UNPHASED program (version 3.1.5) [10], with calculation of odds ratio (OR) and 95% confidence interval (CI). The scored PANSSbased symptoms were used as quantitative traits for analysis of the association between the DAT1 gene and clinical presentation of the illness; an additive value (AddVal) was introduced to represent the change in expected trait value due to the minor allele, relative to the major allele whose expected trait value was defined as “0”. To circumvent the problem of multiple testing for disease association, the significance level was set at an adjusted p-value of 0.05 by 10,000 permutations. We also used the Benjamini–Hochberg false discovery rate (FDR) procedure to adjust for multiple testing, and used an FDR-adjusted p value (q value) threshold of 0.05 to determine significance [20]. Power analysis was performed with Quanto Program (version 1.2.4) [15], under the log additive mode, with each known risk allele frequency, and the prevalence of 0.01 in schizophrenia.
Table 1 Allelic and genotypic associations of the two DAT1 SNPs with schizophrenia. SNP
Group
N
A
G
rs2975223
Case Control
368 420
626(85.1) 718(85.5)
110(14.9) 122(14.5)
SNP
Group
N
Allele frequency (%)
rs2455391
Case Control
367 420
a b
p = 0.023 after 10,000 permutations. p = 0.034 after 10,000 permutations.
Allele frequency (%)
C
T
670(91.3) 735(87.5)
64(8.7) 105(12.5)
p
OR(95%CI)
0.81
1.03(0.78–1.37)
p
OR(95%CI)
0.015a
0.67(0.48–0.93)
Genotype frequency (%)
p
A/A
A/G
G/G
260(70.7) 306(72.9)
106(28.8) 106(25.2)
2(0.5) 8(1.9)
Genotype frequency (%)
0.12
p
C/C
C/T
T/T
304(82.8) 323(76.9)
62(16.9) 89(21.2)
1(0.3) 8(1.9)
0.018b
C. Zheng et al. / Neuroscience Letters 513 (2012) 137–140
139
Table 2 Association of the rs2975223–rs2455391 haplotypes with schizophrenia. Haplotype
Case(%)
Control(%)
2
p
OR(95%CI)
Global p
A-C A-T G-C G-T
620(84.7) 2(0.3) 48(6.6) 62(8.4)
709(84.4) 9(1.1) 26(3.1) 96(11.4)
0.027 3.645 10.42 3.798
0.869 0.056 0.0012 0.051
1.02(0.78–1.35) 0.27(0.06–1.23) 2.09(1.28–3.42) 0.74(0.53–1.04)
0.0006
Table 3 Genotypic association of the DAT1 gene with psychotic symptoms in schizophrenia. PNASS scores
Genotype rs2975223 (N = 293)
rs2455391 (N = 293)
AddValue
Positive symptoms Negative symptoms Gerenal symptoms Global symptoms
A/A (226)
A/G (65)
G/G (2)
0.00 0.00 0.00 0.00
−0.012 0.001 −0.008 −0.003
−0.16 −0.29 −0.14 −0.08
2
p
2.28 4.58 3.30 4.39
0.32 0.10 0.19 0.11
3. Results The genotypic distributions of these two SNPs did not deviate from Hardy–Weinberg equilibrium in the control group. Their genotype and allele distributions (compared between the patient and control groups) are summarized in Table 1. There was a statistically significant difference in allele frequency of rs2455391 between the patient group and the control group (p = 0.015). Such an association survived 10,000 permutations (adjusted p = 0.023) and the FDR q value = 0.03. The frequency of rs2455391-T allele was lower in the patient group (8.7%) than the control group (12.5%). Genotypic analysis also showed a disease association for rs2455391 (adjusted p = 0.034 after 10,000 permutations and the FDR q value = 0.036). However, rs2975223 failed to show either allelic or genotypic association with the illness. Power calculations showed that our samples had >80% power to detect OR = 1.5 for allelic association at a false rate of 0.05 (Supplementary Table 1). These two SNPs were in strong LD (D = 0.96, r2 = 0.89) and the rs2975223(G)–rs2455391(C) haplotype was associated with increased risk of schizophrenia (p = 0.0012). The detailed information regarding the haplotype analysis is given in Table 2. As shown in Table 3, rs2455391 showed genotypic association with positive symptoms, general symptoms and global symptoms but not with negative symptoms. There was no disease association for rs2975223. The association of the rs2975223–rs2455391 haplotypes with psychotic symptoms was also tested but failed to show significant (Supplementary Table 2). 4. Discussion The DAT1 gene has been one of the extensively tested candidate genes for schizophrenia although association studies of DAT1 in schizophrenia have yielded controversial results. It is worth noting that all previous studies almost focused on testing the 40-bp VNTR present in the 3 UTR. In fact, the VNTR association with schizophrenia has been ruled out by meta-analysis [14]. Given the central role of the dopamine transporter in dopaminergic neurotransmission as well as the dopamine hypothesis of schizophrenia, more attention has been paid to new variants across the DAT1 gene in association study of this illness. In the present study, we found that rs2455391 presented in DAT1 intron1 may confer a risk of schizophrenia. The rs2975223(G)–rs2455391(C) haplotype appeared to confer an increased risk of this disease. These findings are consistent with previous reports that the variants in the 5 upstream region and
AddValue C/C (241)
C/T (51)
T/T (1)
0.00 0.00 0.00 0.00
−0.023 −0.001 −0.023 −0.009
−16.63 −0.56 −15.14 −0.49
2
p
7.26 4.25 10.2 8.91
0.03 0.12 0.006 0.01
intron 1 of the DAT1 gene are very likely to contribute to the etiology of schizophrenia [22,35]. The DAT1 5 flanking promoter polymorphisms may lead to alteration of DAT1 gene expression [17] based on the correlation between the DAT1 polymorphisms and its gene expression in the striatum [33]. Interestingly, comprehensive screening of the 5 UTR variations showed that a common haplotype containing the rs2975223(G) and rs2455391 (C) alleles displayed the lowest transcriptional activity in luciferase reporter gene assay [25], suggesting that the rs2975223(G)–rs2455391(C) haplotype may confer the risk of schizophrenia due to decreased transcriptional activity. Moreover, in silico analysis shows that rs2455391 possibly harbors a transcription factor binding site (http://brainarray.mbni.med.umich.edu/), which may modify DAT1 expression and disease susceptibility by altering the binding affinity of transcription factors. To investigate the effect of the DAT1 variants on clinical presentation of the illness, we applied the PANSS-scored psychotic symptoms as quantitative traits to confirm whether the DAT1 gene could contribute to the severity of clinical symptoms. Association signal was then detected at rs2455391 for the positive symptoms, general psychopathological symptoms and global symptoms (Table 3). In addition, patients with the C/T and T/T genotypes at rs2455391 were more likely to have a lower subscale score for positive, general and global symptoms than those with the C/C genotype. It is possible that the C/T and T/T genotypes play a role in protecting individuals from the development of positive, general and global symptoms. However, our finding is not in line with previous reports that the DAT1 gene was neither involved in the severity of clinical symptoms [22] nor associated with negative symptoms [12]. To our knowledge, this is the first study to report on the genetic association between the DAT1 intron1 polymorphism and schizophrenia. In combination of our results and previous studies, the controversial findings from association studies of DAT1 in schizophrenia could be explained with the following reasons. First, ethnic background might be an important one because of different genetic architectures between subpopulations. For example, the 3 VNTR in the DAT1 gene may be associated with schizophrenia only in some populations but not in all. Therefore, the replication of initial finding should be conducted on a gene-based level rather than limited to a SNP reported to be associated with a disease. Second, genetic heterogeneity of schizophrenia may also handicap the replication of disease association [5]. While different genes are involved in the complex nature of schizophrenia, allelic heterogeneity, in which there are multiple variants involved in the
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development of the same disease, may also contribute to poor replication of an initial finding [4,29]. The association of rs2455391 with schizophrenia has been observed in our work, suggesting a novel disease-underlying allele in the DAT1 gene in the Chinese population. Third, in some previous studies, genetic polymorphisms related to the diseases are more likely to serve just as a marker which was co-segregated with a functional and/or causative variant nearby. Thus, the precise mechanisms of conferring risk of disease remain to be established. Fourth, some genes may contribute not only to susceptibility to schizophrenia but also to clinical features of the illness [11]. In order to increase the statistical power and representativeness, it is necessary to stratify the population into clinically homogeneous subgroups according to clinical symptoms or endophenotypes in future studies. In conclusion, the present study provides evidence that the DAT1 gene may confer susceptibility to schizophrenia as well as influence the severity of psychotic symptoms in the Chinese population. Further investigation with larger sample size is needed to confirm this initial finding and to explore the disease underlying mechanisms involved. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements This work was supported by the research grants from the National Basic Research Program of China (2010CB529603 and 2012CB517902), the National Natural Science Foundation of China (31021091 and 30971001), the Beijing Natural Science Foundation (7102109) and the Fok Ying Tong Education Foundation (121024). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2012.02.015. References [1] M. Althaus, Y. Groen, A.A. Wijers, R.B. Minderaa, I.P. Kema, J.D. Dijck, P.J. Hoekstra, Variants of the SLC6A3 (DAT1) polymorphism affect performance monitoring-related cortical evoked potentials that are associated with ADHD, Biol. Psychol. 85 (2010) 19–32. [2] A.P. Association, Diagnostic and Statistical Manual of Mental Disorders, 4th ed., American Psychiatric Press, Washington, DC, 2000. [3] M.J. Bannon, S.K. Michelhaugh, J. Wang, P. Sacchetti, The human dopamine transporter gene: gene organization, transcriptional regulation, and potential involvement in neuropsychiatric disorders, Eur. Neuropsychopharmacol. 11 (2001) 449–455. [4] K.J. Brookes, X. Xu, R. Anney, B. Franke, K. Zhou, W. Chen, T. Banaschewski, J. Buitelaar, R. Ebstein, J. Eisenberg, M. Gill, A. Miranda, R.D. Oades, H. Roeyers, A. Rothenberger, J. Sergeant, E. Sonuga-Barke, H.C. Steinhausen, E. Taylor, S.V. Faraone, P. Asherson, Association of ADHD with genetic variants in the 5 -region of the dopamine transporter gene: evidence for allelic heterogeneity, Am. J. Med. Genet. B: Neuropsychiatr. Genet. 147B (2008) 1519–1523. [5] M. Burmeister, M.G. McInnis, S. Zollner, Psychiatric genetics: progress amid controversy, Nat. Rev. Genet. 9 (2008) 527–540. [6] A.G. Cardno, I.I. Gottesman, Twin studies of schizophrenia: from bow-andarrow concordances to star wars Mx and functional genomics, Am. J. Med. Genet. 97 (2000) 12–17. [7] A.G. Cardno, K. Thomas, P. McGuffin, Clinical variables and genetic loading for schizophrenia: analysis of published Danish adoption study data, Schizophr. Bull. 28 (2002) 393–399. [8] L. Carvelli, R.D. Blakely, L.J. DeFelice, Dopamine transporter/syntaxin 1A interactions regulate transporter channel activity and dopaminergic synaptic transmission, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 14192–14197. [9] B.J. Ciliax, C. Heilman, L.L. Demchyshyn, Z.B. Pristupa, E. Ince, S.M. Hersch, H.B. Niznik, A.I. Levey, The dopamine transporter: immunochemical characterization and localization in brain, J. Neurosci. 15 (1995) 1714–1723.
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Association of intron 1 variants of the dopamine transporter gene with schizophrenia Chunming Zheng, Yan Shen, Qi Xu ∗ National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, Beijing 100005, China
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 5 February 2012 Accepted 7 February 2012 Keywords: Association study Dopamine transporter gene Psychotic symptoms Schizophrenia
a b s t r a c t The dopamine transporter (DAT1) gene has been implicated in the pathogenesis of many neuropsychiatric disorders, including schizophrenia. The present study aimed to investigate association of the DAT1 gene polymorphisms with schizophrenia in a Han Chinese population. Two single nucleotide polymorphisms (SNPs) in the DAT1 gene (rs2975223 and rs2455391) were tested in 368 patients with schizophrenia and 420 healthy controls, of whom 293 patients underwent an assessment of psychotic symptoms through the positive and negative syndrome scale (PANSS). The chi-square test (2 ) showed disease association for rs2455391 (corrected p = 0.023 for allelic association and p = 0.034 for genotypic association, respectively). The rs2975223(G)–rs2455391(C) haplotype was associated with increased risk of the illness (p = 0.0012, OR = 2.09, 95% CI = 1.28–3.42). Quantitative trait analysis showed that rs2455391 was associated with positive symptoms, general symptoms and global symptoms but not with negative symptoms. The present results suggest that the DAT1 gene may be mainly involved in the development of the positive symptoms in the Chinese population. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Schizophrenia is a severe and chronic mental disorder with a 1% lifetime prevalence [31] characterized by positive symptoms, negative symptoms and cognitive dysfunctions [13]. Family, twin and adoption studies indicate that genetic factors account for 64–80% of the etiology of schizophrenia [6,7,26,27,32]. Genetic variants involved in the dopaminergic system have been extensively explored according to the dopaminergic hypothesis [21]. Accumulating evidence suggested that the imbalanced presynaptic activity of dopaminergic neurons in various brain regions contributes to the pathophysiology of schizophrenia [28,30]. The presynaptic dopamine balance is regulated by reuptake and degradation of released dopamine; dopamine transporter (DAT1), a crucial component of dopamine neurotransmission in the central nervous system (CNS), plays an important role in reuptake process [3]. DAT1 is believed to regulate the temporal and spatial activity of released dopamine by rapid reuptake of the neurotransmitter into presynaptic terminals [16]. Moreover, DAT1 is an important element in regulating the duration of dopamine activity [8]. Therefore,
∗ Corresponding author at: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, No.5 Dong Dan San Tiao, Beijing 100005, China. Tel.: +86 10 65296432; fax: +86 10 65263392. E-mail addresses: [email protected], [email protected] (Q. Xu). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2012.02.015
it is possible that the polymorphisms of the DAT1 gene (also known as SLC6A3) may be a risk factor predisposing to schizophrenia. The DAT1 protein is primarily expressed in midbrain dopaminergic neurons projecting to the substantia nigra and ventral tegmental regions as well as to the striatum, nucleus accumbens, prefrontal cortex, and hypothalamus [3,9]. Dysfunction of DAT1 could contribute to several neuropsychiatric disorders and this gene has been widely studied in a number of dopamine-related neuropsychiatric disorders, including attentiondeficit/hyperactivity disorder (ADHD) [1], Parkinson’s disease (PD) [24], bipolar disorder [18], and schizophrenia. The human DAT1 gene is mapped to chromosome 5p15.3 with 15 exons spanning about 65 kb [34]. The protein-coding domains start within exon 2 and end near the beginning of exon 15 [3]. Grünhage et al. [19] systemically screened for genetic variations across the coding region of the DAT1 gene and revealed that the coding region was highly conserved; they therefore hypothesized that variations in the DAT1 gene most from the noncoding region could contribute to the differences in gene expression levels rather than protein structures between individuals, and that mutations in promoter regulator regions of this gene could provide a significant impact on dopaminergic activity in the brain. Several studies analyzed the association between the DAT1 gene and schizophrenia, but the results reported to date have been inconsistent. Most association studies focused on testing a 40-bp variable number of tandem repeat (VNTR) located in the 3 untranslated region (UTR) in exon 15 although a meta-analysis failed to
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confirmed such an association [14]. The 5 regulatory region of the DAT1 gene has also been a hotspot. Systematic analysis of the 5 regulatory region indicated the presence of regulatory elements between the 5 UTR and intron 1 regions [17]. Several lines of evidence have recently suggested that the 5 promoter region of the DAT1 gene may play a role in increasing susceptibility to schizophrenia [22]. Because only a few studies of intron1 variants have been conducted in schizophrenia, it may be useful to re-confirm the association of DAT1 intron 1 variants with the disease in different populations. Accordingly, this study was designed to investigate whether the DAT1 intron 1 polymorphisms could contribute to the etiology of schizophrenia in a Han Chinese population.
All subjects were of Chinese Han origin from the northern area of China and gave written informed consent to participate in this study as approved by the Ethics Committee of the Chinese Academy of Medical Science and Peking Union Medical College. 2.2. SNPs selection and genotyping Genomic DNA was extracted from peripheral blood samples using standard phenol–chloroform extraction method. Two single nucleotide polymorphisms (SNPs), rs2975223 and rs2455391 in intron1 of the DAT1 gene were selected for genetic analysis from the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP). Polymerase chain reaction (PCR) amplification was performed with the primer sequences as follows: 5 -TGTATTCCATCCTGGGTGAC-3 (forward) and 5 -GCTTCTTCCCTCTTGGTCTT-3 (reverse). PCR was carried out with the GeneAmp PCR 2700 system (Applied Biosystems) in a 25-l reaction volume containing 200 M of each dNTP, 0.4 M of each primer, 2.5 l of 10× PCR buffer, 1 unit of Taq DNA polymerase (Tiangen, Beijing, China), and 60 ng of genomic DNA. The conditions used for PCR amplification consisted of initial denaturation at 94 ◦ C for 5 min, 35 cycles at 94 ◦ C for 30 s, 55 ◦ C for 30 s and 72 ◦ C for 30 s, followed by a final extension at 72 ◦ C for 10 min. The genotypes were then determined by use of bidirectional sequencing with the ABI 3700 DNA analyzer (Perkin-Elmer, Applied Biosystems, Foster City, CA, USA).
2. Materials and methods 2.1. Subjects In this study, we recruited a total of 368 unrelated patients with paranoid schizophrenia (219 males and 149 females), with a mean age of 31.97 ± 12.09 years. They were randomly sampled from the whole patient population through clinical settings in the Department of Psychiatry, First Hospital of Shanxi Medical University, Taiyuan, China, in the period between January 2006 and August 2009. Clinical diagnosis was made by at least two senior psychiatrists according to the criteria of the Diagnostic and Statistical Manual of mental disorders, fourth edition (DSM-IV) [2]. All patients recruited for this study were also assessed with the Chinese Version of the Modified Structured Clinical Interview for DSM-IV TR Axis I Disorders Patient Edition (SCID-I/P, 11/2002 revision). Since antipsychotic drugs can alter psychopathology status and regulate dopamine concentration, 293 patients, who were medication-free for at least 1 month (n = 154) or drug-naïve (n = 139) among the case group, were assessed by the positive and negative syndrome scale (PANSS) [23]. The PANSS assessment included three subscales for the measurements of positive symptoms (7 items), negative symptoms (7 items) and general psychopathological symptoms (16 items). The severity of each symptom was scored with 7 grades (1 = absent, 2 = minimal, 3 = mild, 4 = moderate, 5 = moderate severe, 6 = severe, and 7 = extreme). Some potential participants were excluded from this study if they had suffered from any organic brain disorders, mental retardation, epilepsy, head trauma, or the psychotic symptoms were due to medical conditions or treatments. The control group consisted of 420 unrelated healthy volunteers (248 males and 172 females) aged 29.53 ± 9.47 years. They were recruited from local communities. All control subjects were assessed using the SCID, and those who had a history of major psychiatric or neurological disorders, or family history of severe forms of psychiatric disorders, were excluded.
2.3. Statistical analysis The Haploview program (version 4.1, Broad Institute of MIT and Harvard, Cambridge, MA, USA) was applied to test the genotypic distributions of SNPs for Hardy–Weinburgh equilibrium and to estimate linkage disequilibrium (LD) between these two SNPs in which the LD strength was expressed by measurements D and r2 . Allelic, haplotypic and genotypic associations were analyzed using the UNPHASED program (version 3.1.5) [10], with calculation of odds ratio (OR) and 95% confidence interval (CI). The scored PANSSbased symptoms were used as quantitative traits for analysis of the association between the DAT1 gene and clinical presentation of the illness; an additive value (AddVal) was introduced to represent the change in expected trait value due to the minor allele, relative to the major allele whose expected trait value was defined as “0”. To circumvent the problem of multiple testing for disease association, the significance level was set at an adjusted p-value of 0.05 by 10,000 permutations. We also used the Benjamini–Hochberg false discovery rate (FDR) procedure to adjust for multiple testing, and used an FDR-adjusted p value (q value) threshold of 0.05 to determine significance [20]. Power analysis was performed with Quanto Program (version 1.2.4) [15], under the log additive mode, with each known risk allele frequency, and the prevalence of 0.01 in schizophrenia.
Table 1 Allelic and genotypic associations of the two DAT1 SNPs with schizophrenia. SNP
Group
N
A
G
rs2975223
Case Control
368 420
626(85.1) 718(85.5)
110(14.9) 122(14.5)
SNP
Group
N
Allele frequency (%)
rs2455391
Case Control
367 420
a b
p = 0.023 after 10,000 permutations. p = 0.034 after 10,000 permutations.
Allele frequency (%)
C
T
670(91.3) 735(87.5)
64(8.7) 105(12.5)
p
OR(95%CI)
0.81
1.03(0.78–1.37)
p
OR(95%CI)
0.015a
0.67(0.48–0.93)
Genotype frequency (%)
p
A/A
A/G
G/G
260(70.7) 306(72.9)
106(28.8) 106(25.2)
2(0.5) 8(1.9)
Genotype frequency (%)
0.12
p
C/C
C/T
T/T
304(82.8) 323(76.9)
62(16.9) 89(21.2)
1(0.3) 8(1.9)
0.018b
C. Zheng et al. / Neuroscience Letters 513 (2012) 137–140
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Table 2 Association of the rs2975223–rs2455391 haplotypes with schizophrenia. Haplotype
Case(%)
Control(%)
2
p
OR(95%CI)
Global p
A-C A-T G-C G-T
620(84.7) 2(0.3) 48(6.6) 62(8.4)
709(84.4) 9(1.1) 26(3.1) 96(11.4)
0.027 3.645 10.42 3.798
0.869 0.056 0.0012 0.051
1.02(0.78–1.35) 0.27(0.06–1.23) 2.09(1.28–3.42) 0.74(0.53–1.04)
0.0006
Table 3 Genotypic association of the DAT1 gene with psychotic symptoms in schizophrenia. PNASS scores
Genotype rs2975223 (N = 293)
rs2455391 (N = 293)
AddValue
Positive symptoms Negative symptoms Gerenal symptoms Global symptoms
A/A (226)
A/G (65)
G/G (2)
0.00 0.00 0.00 0.00
−0.012 0.001 −0.008 −0.003
−0.16 −0.29 −0.14 −0.08
2
p
2.28 4.58 3.30 4.39
0.32 0.10 0.19 0.11
3. Results The genotypic distributions of these two SNPs did not deviate from Hardy–Weinberg equilibrium in the control group. Their genotype and allele distributions (compared between the patient and control groups) are summarized in Table 1. There was a statistically significant difference in allele frequency of rs2455391 between the patient group and the control group (p = 0.015). Such an association survived 10,000 permutations (adjusted p = 0.023) and the FDR q value = 0.03. The frequency of rs2455391-T allele was lower in the patient group (8.7%) than the control group (12.5%). Genotypic analysis also showed a disease association for rs2455391 (adjusted p = 0.034 after 10,000 permutations and the FDR q value = 0.036). However, rs2975223 failed to show either allelic or genotypic association with the illness. Power calculations showed that our samples had >80% power to detect OR = 1.5 for allelic association at a false rate of 0.05 (Supplementary Table 1). These two SNPs were in strong LD (D = 0.96, r2 = 0.89) and the rs2975223(G)–rs2455391(C) haplotype was associated with increased risk of schizophrenia (p = 0.0012). The detailed information regarding the haplotype analysis is given in Table 2. As shown in Table 3, rs2455391 showed genotypic association with positive symptoms, general symptoms and global symptoms but not with negative symptoms. There was no disease association for rs2975223. The association of the rs2975223–rs2455391 haplotypes with psychotic symptoms was also tested but failed to show significant (Supplementary Table 2). 4. Discussion The DAT1 gene has been one of the extensively tested candidate genes for schizophrenia although association studies of DAT1 in schizophrenia have yielded controversial results. It is worth noting that all previous studies almost focused on testing the 40-bp VNTR present in the 3 UTR. In fact, the VNTR association with schizophrenia has been ruled out by meta-analysis [14]. Given the central role of the dopamine transporter in dopaminergic neurotransmission as well as the dopamine hypothesis of schizophrenia, more attention has been paid to new variants across the DAT1 gene in association study of this illness. In the present study, we found that rs2455391 presented in DAT1 intron1 may confer a risk of schizophrenia. The rs2975223(G)–rs2455391(C) haplotype appeared to confer an increased risk of this disease. These findings are consistent with previous reports that the variants in the 5 upstream region and
AddValue C/C (241)
C/T (51)
T/T (1)
0.00 0.00 0.00 0.00
−0.023 −0.001 −0.023 −0.009
−16.63 −0.56 −15.14 −0.49
2
p
7.26 4.25 10.2 8.91
0.03 0.12 0.006 0.01
intron 1 of the DAT1 gene are very likely to contribute to the etiology of schizophrenia [22,35]. The DAT1 5 flanking promoter polymorphisms may lead to alteration of DAT1 gene expression [17] based on the correlation between the DAT1 polymorphisms and its gene expression in the striatum [33]. Interestingly, comprehensive screening of the 5 UTR variations showed that a common haplotype containing the rs2975223(G) and rs2455391 (C) alleles displayed the lowest transcriptional activity in luciferase reporter gene assay [25], suggesting that the rs2975223(G)–rs2455391(C) haplotype may confer the risk of schizophrenia due to decreased transcriptional activity. Moreover, in silico analysis shows that rs2455391 possibly harbors a transcription factor binding site (http://brainarray.mbni.med.umich.edu/), which may modify DAT1 expression and disease susceptibility by altering the binding affinity of transcription factors. To investigate the effect of the DAT1 variants on clinical presentation of the illness, we applied the PANSS-scored psychotic symptoms as quantitative traits to confirm whether the DAT1 gene could contribute to the severity of clinical symptoms. Association signal was then detected at rs2455391 for the positive symptoms, general psychopathological symptoms and global symptoms (Table 3). In addition, patients with the C/T and T/T genotypes at rs2455391 were more likely to have a lower subscale score for positive, general and global symptoms than those with the C/C genotype. It is possible that the C/T and T/T genotypes play a role in protecting individuals from the development of positive, general and global symptoms. However, our finding is not in line with previous reports that the DAT1 gene was neither involved in the severity of clinical symptoms [22] nor associated with negative symptoms [12]. To our knowledge, this is the first study to report on the genetic association between the DAT1 intron1 polymorphism and schizophrenia. In combination of our results and previous studies, the controversial findings from association studies of DAT1 in schizophrenia could be explained with the following reasons. First, ethnic background might be an important one because of different genetic architectures between subpopulations. For example, the 3 VNTR in the DAT1 gene may be associated with schizophrenia only in some populations but not in all. Therefore, the replication of initial finding should be conducted on a gene-based level rather than limited to a SNP reported to be associated with a disease. Second, genetic heterogeneity of schizophrenia may also handicap the replication of disease association [5]. While different genes are involved in the complex nature of schizophrenia, allelic heterogeneity, in which there are multiple variants involved in the
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development of the same disease, may also contribute to poor replication of an initial finding [4,29]. The association of rs2455391 with schizophrenia has been observed in our work, suggesting a novel disease-underlying allele in the DAT1 gene in the Chinese population. Third, in some previous studies, genetic polymorphisms related to the diseases are more likely to serve just as a marker which was co-segregated with a functional and/or causative variant nearby. Thus, the precise mechanisms of conferring risk of disease remain to be established. Fourth, some genes may contribute not only to susceptibility to schizophrenia but also to clinical features of the illness [11]. In order to increase the statistical power and representativeness, it is necessary to stratify the population into clinically homogeneous subgroups according to clinical symptoms or endophenotypes in future studies. In conclusion, the present study provides evidence that the DAT1 gene may confer susceptibility to schizophrenia as well as influence the severity of psychotic symptoms in the Chinese population. Further investigation with larger sample size is needed to confirm this initial finding and to explore the disease underlying mechanisms involved. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements This work was supported by the research grants from the National Basic Research Program of China (2010CB529603 and 2012CB517902), the National Natural Science Foundation of China (31021091 and 30971001), the Beijing Natural Science Foundation (7102109) and the Fok Ying Tong Education Foundation (121024). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2012.02.015. References [1] M. Althaus, Y. Groen, A.A. Wijers, R.B. Minderaa, I.P. Kema, J.D. Dijck, P.J. Hoekstra, Variants of the SLC6A3 (DAT1) polymorphism affect performance monitoring-related cortical evoked potentials that are associated with ADHD, Biol. Psychol. 85 (2010) 19–32. [2] A.P. Association, Diagnostic and Statistical Manual of Mental Disorders, 4th ed., American Psychiatric Press, Washington, DC, 2000. [3] M.J. Bannon, S.K. Michelhaugh, J. Wang, P. Sacchetti, The human dopamine transporter gene: gene organization, transcriptional regulation, and potential involvement in neuropsychiatric disorders, Eur. Neuropsychopharmacol. 11 (2001) 449–455. [4] K.J. Brookes, X. Xu, R. Anney, B. Franke, K. Zhou, W. Chen, T. Banaschewski, J. Buitelaar, R. Ebstein, J. Eisenberg, M. Gill, A. Miranda, R.D. Oades, H. Roeyers, A. Rothenberger, J. Sergeant, E. Sonuga-Barke, H.C. Steinhausen, E. Taylor, S.V. Faraone, P. Asherson, Association of ADHD with genetic variants in the 5 -region of the dopamine transporter gene: evidence for allelic heterogeneity, Am. J. Med. Genet. B: Neuropsychiatr. Genet. 147B (2008) 1519–1523. [5] M. Burmeister, M.G. McInnis, S. Zollner, Psychiatric genetics: progress amid controversy, Nat. Rev. Genet. 9 (2008) 527–540. [6] A.G. Cardno, I.I. Gottesman, Twin studies of schizophrenia: from bow-andarrow concordances to star wars Mx and functional genomics, Am. J. Med. Genet. 97 (2000) 12–17. [7] A.G. Cardno, K. Thomas, P. McGuffin, Clinical variables and genetic loading for schizophrenia: analysis of published Danish adoption study data, Schizophr. Bull. 28 (2002) 393–399. [8] L. Carvelli, R.D. Blakely, L.J. DeFelice, Dopamine transporter/syntaxin 1A interactions regulate transporter channel activity and dopaminergic synaptic transmission, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 14192–14197. [9] B.J. Ciliax, C. Heilman, L.L. Demchyshyn, Z.B. Pristupa, E. Ince, S.M. Hersch, H.B. Niznik, A.I. Levey, The dopamine transporter: immunochemical characterization and localization in brain, J. Neurosci. 15 (1995) 1714–1723.
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