Association between the 120-bp duplication of the dopamine D4 receptor gene and attention deficit hyperactivity disorder Genetic and molecular analyses.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:231 –236 (2007) Association Between the 120-bp Duplication of the Dopamine D4 Receptor Gene and Attention Deficit Hyperactivity Disorder: Genetic and Molecular Analyses Eva Kereszturi,1 Orsolya Kiraly,1 Zsolt Csapo,1 Zsanett Tarnok,2 Julia Gadoros,2 Maria Sasvari-Szekely,1 and Zsofia Nemoda1* 1 Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary Vadaskert Child and Adolescent Psychiatric Clinic, Budapest, Hungary 2 Abnormalities of the dopamine neurotransmission have been hypothesized to play an important role in the pathophysiology of attention deficit hyperactivity disorder (ADHD). Promoter variants of the dopamine D4 receptor gene (DRD4) have attracted particular interest due to their possible role in regulation of gene transcription. Here we describe the haplotype analysis of the 120 base pair duplication (120-bp dup) and three SNPs (616C/G, 615A/ G, 521C/T) in the 50 region of the DRD4 gene among children with ADHD. We observed a trend (x2 ¼ 14.905, df ¼ 9, P ¼ 0.093) in the four-locus haplotype distribution between ADHD probands (N ¼ 173) and controls (N ¼ 284). The homozygote genotype of the 1-repeat form of the 120-bp dup (1–1) had a significantly higher frequency among ADHD children than in controls (8.1% vs. 3.2%, x2 ¼ 5.526, df ¼ 1, P ¼ 0.019, Odds Ratio ¼ 2.71). In addition, a novel, 4-repeat allele was identified among ADHD patients. This particular allele has been cloned to the luciferase expression vector and its transcriptional activity has been compared to the 1- and 2-repeat allele. The number of repeats of the 120-bp dup was found to have an effect on transcriptional activity in both neuroblastoma and retinoblastoma cell lines in a dose-dependent manner (1-repeat > 2-repeat > 4-repeat). These results suggest that the 1-repeat form of the 120-bp dup might be a risk factor of ADHD, especially in the homozygous form and/or in the context of certain haplotypes. ß 2006 Wiley-Liss, Inc. KEY WORDS: promoter; haplotype; gene expression; tandem repeat; SNP Please cite this article as follows: Kereszturi E, Kiraly O, Csapo Z, Tarnok Z, Gadoros J, Sasvari-Szekely M, Nemoda Z. 2007. Association Between the 120-bp Duplication of the Dopamine D4 Receptor Gene and Attention Deficit Hyperactivity Disorder: Genetic and Molecular Analyses. Am J Med Genet Part B 144B:231–236. *Correspondence to: Zsofia Nemoda, Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, POB 260, H-1444, Hungary. E-mail: firstname.lastname@example.org Received 31 May 2006; Accepted 23 August 2006 DOI 10.1002/ajmg.b.30444 ß 2006 Wiley-Liss, Inc. INTRODUCTION Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent childhood-onset psychiatric syndromes affecting 3–5% of school-age children worldwide [Swanson et al., 1998]. It is characterized by hyperactivity, inattention, impulsivity, and distractibility. Significant genetic component with an estimated heritability of 0.7–0.8 has been demonstrated in the etiology of ADHD by family, twin, and adoption studies [Faraone and Biederman, 1998]. Genetic association analyses have been carried out to identify multiple genes of minor effects [Comings et al., 2005]. Based on neurobiological theories, most of the candidate genes belong to the dopamine neurotransmitter system. In particular, the highly polymorphic dopamine D4 receptor (DRD4) gene has attracted increasing interest. The most widely investigated polymorphism of the DRD4 gene is the 48-bp variable number of tandem repeats (VNTR) in the third exon. A meta-analysis of casecontrol and family-based studies concluded that there is an association between ADHD and the VNTR [Faraone et al., 2001]. Polymorphisms located in the 50 untranslated region of the DRD4 gene have also been studied. A 120-bp duplication (120-bp dup) located 1.2-kb upstream of the transcription start site was identified by Seaman et al. . The 2-repeat allele was twice as frequent in ADHD children compared to controls [McCracken et al., 2000]; these results were replicated with a larger sample size [Kustanovich et al., 2004]. However, another group found no association between the 120-bp dup and ADHD in a twin study [Todd et al., 2001]. There are several single nucleotide polymorphisms (SNPs) located in the 50 regulatory region of the DRD4 gene [Mitsuyasu et al., 1999], of which 616C/G and 521C/T were extensively studied in association analyses of ADHD [Barr et al., 2001; Mill et al., 2003; Lowe et al., 2004; Bellgrove et al., 2005]. Assessing the polymorphisms separately, only the 616C/G but not the 521C/T SNP was associated with ADHD in an Irish population [Lowe et al., 2004]. The cloning and characterization of the 50 regulatory region of the DRD4 gene [Kamakura et al., 1997] made it possible to test the functional effect of promoter variants. The 521T allele reduced promoter activity by 40% relative to the C allele in transiently transfected human retinoblastoma Y79 cells [Okuyama et al., 1999, 2000]. The 120-bp duplicated sequence was found to contain several transcription factor binding sites by in silico analysis [Seaman et al., 1999], suggesting that the duplication of this region might influence promoter activity. In a functional study, the 1-repeat form was found to have a higher transcriptional activity relative to the 2-repeat allele [D’Souza et al., 2004]. However, few molecular biological studies have been reported in the literature and the results have not yet been confirmed in independent laboratories. Previous studies from our group failed to detect any difference 232 Kereszturi et al. in transcriptional activity with the 521C/T SNP [Kereszturi et al., 2006]. In this study, we present supporting evidence on the functional effect of the 120-bp dup. In addition, we examined DRD4 promoter haplotypes found to be associated with ADHD. MATERIALS AND METHODS retinoblastoma Y79 cells were maintained in RPMI 1640 medium (Sigma, St. Louis, MO) 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 378C with 5% CO2. Subjects Transient Transfections In this study, 173 ADHD patients (mean age: 9.14 2.6; 87.3% male and 12.7% female) from the Vadaskert Child and Adolescent Psychiatric Clinic were included. Diagnosis of ADHD was based on DSM-IV criteria [American Psychiatric Association, 1994], (combined subtype: 72%; inattentive subtype: 13%; hyperactive-impulsive subtype: 15%) and was confirmed by the Child Behaviour Checklist [Achenbach, 1991], the Conners Rating Scale [Conners et al., 1998], and the ADHD Rating Scale [DuPaul, 1998]. The diagnostic procedure was conducted by two independent psychiatrists and inter-rater reliability (kappas) reached 0.95 for all diagnoses. Children with IQ < 80 were excluded, as well as those who had severe medical or neurological conditions or pervasive psychiatric disorder. IQ was estimated from the Raven progressive matrices test [Raven, 1965]. Comorbid conditions were assessed by a semi-structured interview, the Hungarian child version of the Mini-International Neuropsychiatric Interview [MINI-Kid; Balazs et al., 2004]. According to the MINI-Kid, the frequencies of comorbidities were the following: learning disorder: 30.6%; conduct disorder: 28.9%; anxiety disorder: 13.3%; Tourette syndrome: 12.1%. The study was approved by the Local Ethical Committee (TUKEB). All children and their parents provided written informed consent for their participation. The sex-matched control group was selected from the previously described healthy Hungarian population [Szantai et al., 2005]. Both the clinical and the control samples were ethnically homogenous, Caucasian and consisted of unrelated individuals. SK-N-F1 transfections were performed by the calcium phosphate-DNA coprecipitation method. Y79, IMR32, and HeLa cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The luciferase reporter constructs were cotransfected with control pCMV-regulated b-galactosidase vector to normalize the transcriptional activities. Luciferase and b-galactosidase activities were detected by the Luciferase Assay System kit (Promega) and by ONPG (O-nitrophenyl-b-Dgalactopyranoside) cleavage rate, respectively. Three parallels were used in all transfections and all experiments were performed in triplicate. Genotyping and Haplotyping Non-invasive DNA sampling was applied as described elsewhere [Boor et al., 2002]. Genotyping of the 616C/ G SNP was performed using methods not affected by the 615A/G SNP. The 521C/T SNP was genotyped with methods insensitive to the 603 Tdel polymorphism. Direct molecular haplotyping of the four promoter polymorphisms was performed as described previously [Szantai et al., 2005]. Plasmid Constructs The pGL3 luciferase reporter vector (Promega, Madison, WI) was used to clone the 1,571 to 389 region of the DRD4 50 UTR (translation start site is referred to as position þ1 according to AC021663) using primers containing either an XhoI or an HindIII recognition site: DR4-S1 (50 acc act cga gtg ggc tgg act cgc cgt ttg gc 30 , corresponding to region 1,571/1,550) and DR4-AS1 (50 aag gaa gct tcc ctc ggg cgc tca ccc tag tcc 30 , 389/ 411). The templates were genomic DNA samples with 1-, 2-, and 4-repeat alleles of the 120-bp dup. The mutagenesis in positions 616 and 521 was generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Constructs were verified by sequencing. Cell Culture The human neuroblastoma cell line IMR32 and HeLa cells were cultured in minimal essential medium Eagle (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1% nonessential amino acids and 1% Na-pyruvate. The human Statistical Analysis SPSS 10.0 for Windows was used in the association analyses. Statistical analysis for transcriptional data was performed with one-way ANOVA followed by the Tukey-Kramer Multiple Comparison Test (GraphPad InStat). RESULTS Genotype and Haplotype Frequencies We first performed a single-locus analysis of four polymorphisms in the 50 regulatory region of the DRD4 gene. The 120-bp dup, 616C/G SNP, 521C/T SNP, and the newly identified 615A/G SNP were assessed in 173 children with ADHD. The allele and genotype frequencies were compared to a sex-matched control population genotyped earlier [Szantai et al., 2005]. Despite the close proximity of the investigated polymorphisms, no linkage disequilibrium was detected between the 120-bp dup and any of the SNPs [Szantai et al., 2005]. No significant deviations from Hardy–Weinberg equilibrium were detected for any of the polymorphisms either in the case or in the sex-matched control population. There was no significant difference between the ADHD and control groups in the allele frequency of the investigated SNPs (Table I). However, a significant difference was detected in the allelic distribution of the 120-bp dup (w2 ¼ 3.928, df ¼ 1, P ¼ 0.047). The homozygote genotype of the 1-repeat form of the 120-bp dup (1–1) was twice as frequent in the ADHD group when compared to the pooled genotypes with at least one allele of the 2-repeat form (8.1% vs. 3.2%, w2 ¼ 5.526, df ¼ 1, P ¼ 0.019, Odds Ratio ¼ 2.71). Interestingly, a new, 4-repeat allele of the 120-bp dup has been detected in one ADHD patient. To confirm this allele, the PCR amplicon of the 4-repeat allele was sequenced. In addition, DNA samples were collected from the patient’s parents and were genotyped for the 120-bp dup (Fig. 1). Next, we analyzed the four-locus haplotype frequencies in cases and controls using direct haplotyping methods (Table II). There was a tendency for an unequal distribution (w2 ¼ 14.905, df ¼ 9, P ¼ 0.093). An accumulation of the 1-C-A-T haplotype (1-repeat allele of the 120-bp dup 616C 615A 521T) was detected among ADHD children (w2 ¼ 9.326, df ¼ 1, P ¼ 0.002, Odds Ratio ¼ 2.33). These results suggest that the 1-repeat form of the 120-bp dup might be a risk factor of ADHD, especially in the context of certain haplotypes. The association analyses were also carried out with estimated haplotypes using the EH program elaborated by Ott . The estimated haplotype frequencies of the control DRD4 Promoter Polymorphisms and ADHD 233 TABLE I. Genotype Frequencies of the DRD4 Promoter Polymorphisms in ADHD Patients and Controls Polymorphic sites 616 C/G SNP 120-bp dup 615 A/G SNP 521 C/T SNP Genotype 1–1 1–2 1–4 2–2 CC CG GG AA AG GG CC CT TT Control (N ¼ 284) Frequency (%) ADHD (N ¼ 173) Frequency (%) 9 3.2 14 8.1 73 25.7 45 26.0 0 0.0 1 0.6 202 71.1 113 65.3 81 28.5 43 24.9 131 46.1 86 49.7 72 25.4 44 25.4 218 76.8 121 69.9 62 21.8 49 28.3 4 1.4 3 1.7 55 19.4 27 15.6 139 48.9 92 53.2 90 31.7 54 31.2 1–1, 1–2, 1–4, and 2–2 refer to the genotypes; 1–1: homozygous for the 1-repeat allele, 1–2: heterozygous for the 1 and 2 repeat alleles, 1–4: heterozygous for the 1 and 4 repeat alleles, 2–2: homozygous for the 2-repeat allele of the 120-bp dup. The P-values for the allele distribution (df ¼ 1) were the following: 120-bp dup: 0.0475, 616C/G: 0.583, 615A/G: 0.127, 521C/T: 0.627. and the ADHD samples did not differ significantly from the original frequencies detected with direct haplotype methods (w2 ¼ 9.45, df ¼ 9, P ¼ 0.40 for the control group; w2 ¼ 4.91, df ¼ 10, P ¼ 0.90 for the ADHD group). On the other hand, comparison of the estimated haplotype frequencies resulted in a significant difference of haplotype distribution between the case and control groups (w2 ¼ 19.55, df ¼ 9, P ¼ 0.021), while the data originating from the direct haplotype method showed only a trend (P ¼ 0.093). This observation supports the importance of direct haplotyping methods. Functional Analysis of Promoter Polymorphisms and Haplotypes We also examined whether these promoter haplotypes are functionally different at the molecular level. To this end, we characterized the previously studied 120-bp dup and 521C/T SNP in two neuronal cell lines: IMR32 neuroblastoma and Y79 retinoblastoma. Since the 616C and 615A alleles are more frequent, reporter constructs contained haplotypes of the 120bp dup and 521C/T with a background of 616C and 615A (Fig. 2A). Transient transfection experiments were carried out using the luciferase reporter assay with the pGL3-Basic vector serving as a negative control. Figure 2B shows the relative transcriptional activity normalized to the activity of the promoter-less vector. In this experimental setup, there was no significant difference in the transcriptional activities of the four promoter haplotypes in either cell lines tested, although there was a trend for haplotypes with the 2-repeat allele of the 120-bp dup to have a lower activity (1-C-A-C > 2-C-A-C). To further investigate the effect of the 120-bp dup on gene expression, we constructed reporter plasmids containing different numbers of repeats with a background of 616C, 615A, and 521C. Since we detected a 4-repeat allele in our ADHD sample, we also constructed reporter plasmids harboring four repeats of the 120-bp sequence. The DRD4 regulatory region containing the single copy of the 120-bp sequence revealed a significantly higher transcriptional activity in Y79 (P < 0.01), SK-N-F1 (P < 0.001), and HeLa (P < 0.05) cell lines compared with that having 2-repeat allele (Fig. 3). Additionally, the 2-repeat allele showed significantly higher transcriptional activity than the 4-repeat form in SK-N-F1 and HeLa cells (P < 0.001, Fig. 3). All cell lines have been found to express DRD4 mRNA endogenously from previous studies [Kereszturi et al., 2006]. These results show that the 120-bp sequence represses gene expression and the increasing copy number results in a further decrease in transcriptional activity (1-repeat > 2-repeat > 4-repeat). DISCUSSION In the present study, we performed an association analysis of the DRD4 promoter polymorphisms and ADHD. In addition to the 521C/T SNP, the 616C/G SNP and the 120-bp dup, the newly described 615A/G SNP [Ronai et al., 2004a] was also studied in a Hungarian sample. Using a case-control approach, no association was found between ADHD and any of the SNPs mentioned above. We did observe, however, an overrepresentation of the 1-repeat form of the 120-bp dup in the clinical TABLE II. Haplotype Frequencies of the DRD4 Promoter Polymorphisms in ADHD Patients and Controls Control Haplotype 1-C-A-C 1-C-A-T 1-G-A-C 1-G-A-T 1-G-G-C 1-G-G-T 2-C-A-C 2-C-A-T 2-G-A-C 2-G-A-T 2-G-G-C 2-G-G-T 4-G-A-C Fig. 1. Genotyping for the 4-repeat allele of the 120-bp dup in an ADHD family. The gel-insert shows the newly identified 1–4 genotype of an ADHD patient (first lane). The 4-repeat allele was transmitted from his father (2– 4 genotype, second lane), while the 1-repeat allele came from his mother (1–2 genotype, third lane). Please note that the PCR product of the longer allele is faint because of the preferential amplification of the shorter allele. ADHD 2N ¼ 568 Frequency (%) 2N ¼ 346 Frequency (%) 22 23 17 24 3 3 86 162 76 88 46 18 0 3.9 4.0 3.0 4.2 0.5 0.5 15.1 28.5 13.4 15.5 8.1 3.2 0.0 14 31 11 8 5 5 40 87 42 57 33 12 1 4.0 9.0 3.2 2.3 1.4 1.4 11.6 25.1 12.1 16.5 9.5 3.5 0.3 The number of chromosomes (2N) is 568 and 346 for the control and the ADHD sample, respectively. The relative chromosomal localization of the alleles could be identified unambiguously by using direct haplotyping methods [Szantai et al., 2005]. The alleles in the haplotypes (in cis phase, i.e., on the same chromosome) are presented in the following order: 120-bp dup, 616C/G, 615A/G, and 521C/T. 234 Kereszturi et al. Fig. 2. Functional analysis of the DRD4 promoter haplotypes. A: Four haplotypes of the 120-bp dup and the 521C/T SNP were constructed in pGL3 luciferase reporter plasmid containing the human DRD4 gene promoter region (1,571 to 389) with the 616C and 615A background. The promoter polymorphisms are indicated in the following order: 120-bp dup (1- or 2-repeat), 616C/G, 615A/G, and 521C/T SNPs. Transient transfection was carried out in IMR32 and Y79 cells. B: Luciferase activity was normalized to the b-galactosidase activity. Data are presented as fold increments over the pGL3-Basic activity and shown as mean SD. Results of a representative experiment are shown as measured in triplicates. Similar data were obtained from three independent transfection experiments. group (Table I). One weakness of the present study is that population admixture was not controlled for (since the studied populations were ethnically homogenous, of Caucasian origin). Although an association between the promoter SNPs and ADHD was reported recently [Lowe et al., 2004], several negative findings have also been published [Barr et al., 2001; Mill et al., 2003]. Likewise, data presented in the literature regarding the 120-bp dup have been controversial. A few studies indicated the 2-repeat form as a risk factor for ADHD [McCracken et al., 2000; Kustanovich et al., 2004], while others did not [Todd et al., 2001; Barr et al., 2001; Mill et al., 2003; Brookes et al., 2005]. Our results suggest that the 1-repeat form of the 120-bp dup might be the risk allele in ADHD. This assumption is supported by an association between the 1repeat allele and novelty seeking, a personality trait sharing common characteristics with ADHD [Rogers et al., 2004]. Contradictory findings in the literature necessitate a broader approach in the analyses of these polymorphisms. Studies of separate polymorphisms are being replaced by haplotype analyses, given that the effect of each polymorphism is small and can be modulated by other variants [Ebstein, 2006]. Among association studies of ADHD, many used familybased approach to investigate DRD4 promoter haplotypes. Barr et al.  found no association between promoter haplotypes and ADHD using Transmission Disequilibrium Test. However, after including the 48-bp exon III VNTR in the haplotype, one of the combinations (2-repeat of the 120-bp dup, 616C, 521T, 7-repeat allele of the 48-bp VNTR) was found to be preferentially transmitted. Mill et al.  reported a weak but significant association with the haplotype of the 2-repeat allele of the 120-bp dup, 616C and 521C. Lowe et al.  studied variations of the 120-bp dup and 616 C/G SNP only, and observed preferential transmission of the 2-C haplotype. We could not replicate any of these findings; our case-control analysis showed an association between the 1-C-A-T haplotype and ADHD. It has to be noted that all previous haplotype studies used either statistics software or derived genotypes from family data to construct haplotypes. Statistical programs can only give probabilities for individual haplotypes. From family genotype data, not all haplotype combinations can be constructed, resulting in a loss of data if both parents are heterozygous for multiple polymorphisms. In this study, allele combinations were determined by molecular haplotyping methods previously developed in our laboratory [Ronai et al., 2004a; Szantai et al., 2005]. The haplotype variations in the DRD4 promoter may influence transcriptional activity, which in turn may result in different receptor densities. Thus, promoter polymorphisms that affect transcriptional activity can contribute to the molecular background of neuropsychiatric disorder. Characterization studies of the 50 regulatory region of the DRD4 gene were performed with transiently transfected SK-N-F1 and IMR32 (neuroblastoma) and Y79 (retinoblastoma) cell lines. These studies revealed the presence of a putative negative modulatory region [Kamakura et al., 1997; Kereszturi et al., 2006], but the exact position of this region is still unclear [Kereszturi et al., 2006]. Functional studies of the DRD4 promoter polymorphisms previously associated with psychiatric disorders have also been performed. Okuyama et al. [1999, 2000] found that the 521T allele had a 40% lower transcriptional activity compared to the C allele. Our group could not replicate these results as we found that promoter activities of the 521 alleles were essentially identical in three neuronal cell lines [Kereszturi et al., 2006]. The 120-bp dup contains consensus sequences of several transcription factor binding sites such as MEP-1, CEB/P, and Sp1 [Seaman et al., 1999]. The 2-repeat allele showed enhanced binding capacity for Sp1 in a mobility shift assay [Ronai et al., 2004b]. A study addressing the functional effect of the 120-bp dup found that the 1-repeat allele had a higher promoter activity [D’Souza et al., 2004]. Thus, several lines of evidence suggest that the 120-bp dup has a role in transcriptional regulation of the DRD4 gene. In the present study, a functional analysis of the DRD4 promoter haplotypes associated with ADHD was carried out. Transcriptional activity of the haplotypes was essentially identical in neuroblastoma as well as in retinoblastoma cell lines. However, there was a trend for combinations containing the 2-repeat allele of the 120-bp dup to have reduced promoter activity (Fig. 2). It has to be noted here that several other polymorphisms have been identified in the 50 regulatory region of the DRD4 gene [Mitsuyasu et al., 1999], some of which have not yet been included in either association or functional studies. Thus, it cannot be ruled out that further haplotype variations could eventually show functional differences. To test the effect of the 120-bp dup, we used reporter plasmids containing different numbers of these repeats. Since we DRD4 Promoter Polymorphisms and ADHD 235 ities in all three cell lines tested (Fig. 3). These results support the findings of D’Souza et al. , supplemented with the study of the 4-repeat variant. It should be noted that the study of D’Souza et al.  used a short DRD4 promoter fragment containing only the 120-bp dup to detect functional differences between alleles. In our experiments, we used reporter constructs comprising the major part of the 50 regulatory region which previously demonstrated promoter activity [Kereszturi et al., 2006]. In summary, we did not find an association between ADHD and three SNPs (616C/G, 615A/G, and 521C/T) in the promoter region of the DRD4 gene with a case-control approach. 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