Dopaminergic candidate genes in Tourette syndrome Association between tic severity and 3 UTR polymorphism of the dopamine transporter gene.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:900 –905 (2007) Dopaminergic Candidate Genes in Tourette Syndrome: Association Between Tic Severity and 30 UTR Polymorphism of the Dopamine Transporter Gene Zsanett Tarnok,1 Zsolt Ronai,2 Judit Gervai,3 Eva Kereszturi,2 Julia Gadoros,1 Maria Sasvari-Szekely,2 and Zsofia Nemoda2* 1 Vadaskert Child and Adolescent Psychiatric Clinic, Budapest, Hungary Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary 3 Institute for Psychology of the Hungarian Academy of Sciences, Budapest, Hungary 2 Multiple evidence suggests an involvement of the dopamine neurotransmitter system in Tourette syndrome (TS). Therefore, dopaminergic candidate genes are in the center of genetic association analyses of TS. In this study, 103 TS patients and their parents have been characterized for different dopamine-related polymorphisms including the 48 bp variable number of tandem repeats (VNTR) of the dopamine D4 receptor (DRD4) gene, the 40 bp VNTR of the dopamine transporter (DAT1, SLC6A3) gene and the Val158Met polymorphism of the catechol-O-methyltransferase (COMT) gene. In addition, the 120 bp duplication and three single nucleotide polymorphisms (SNPs) were assessed in the promoter region of the DRD4 gene. The 616G allele and the 2-G-A-C haplotype (i.e., the 2-repeat form of the 120 bp sequence 616G 615A 521C combination) were preferentially transmitted, however, these results did not remain significant after correction for multiple testing. Case-control analyses have also been carried out, resulting in negative findings. On the other hand, using a dimensional approach, the DAT1 40 bp VNTR showed an association with the peak tic-severity as measured by the Yale Global Tic Severity Scale. Patients with at least one copy of the 9-repeat allele had significantly more severe symptoms than individuals with the homozygous 10/10 genotype (P ¼ 0.002). In summary, allele frequencies did not differ between cases and controls, but DAT1 genotype accounted for variations of tic severity within the TS group. ß 2007 Wiley-Liss, Inc. KEY WORDS: variable number of tandem repeats (VNTR); dopamine D4 receptor (DRD4); promoter; polymorphism; haplotype Grant sponsor: Hungarian Fund OTKA; Grant number: F042730. *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 21 August 2006; Accepted 29 January 2007 DOI 10.1002/ajmg.b.30517 ß 2007 Wiley-Liss, Inc. Please cite this article as follows: Tarnok Z, Ronai Z, Gervai J, Kereszturi E, Gadoros J, Sasvari-Szekely M, Nemoda Z. 2007. Dopaminergic Candidate Genes in Tourette Syndrome: Association Between Tic Severity and 30 UTR Polymorphism of the Dopamine Transporter Gene. Am J Med Genet Part B 144B:900–905. INTRODUCTION Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by multiple chronic tics, that is, involuntary, rapid, non-rhythmic skeletal movements and vocalizations. The prevalence of TS varies in different agegroups, and is presently estimated as 1% among school-age children [Robertson, 2003]. TS is often accompanied by obsessive-compulsive disorder (OCD) or attention deficit hyperactivity disorder (ADHD). A common neurobiological origin is hypothesized suggesting a dysfunction of the basal ganglia—thalamico-cortical loops in these neurodevelopmental disorders [Sheppard et al., 1999]. Tic severity shows great individual variation ranging from mild to severe. In addition, it is characteristic for the disorder that the symptoms are waxing and waning; their number, frequency, and complexity change during development [Leckman, 2002]. Family and twin studies demonstrated a significant contribution of genetic factors in the etiology of TS. Searching for the susceptible genetic regions, chromosomal mapping and analyses of candidate genes have been employed in this polygenic disorder [Pauls, 2003]. Based on pharmacological evidence, candidate genes investigated to date in TS belong primarily to the dopaminergic system. Dopamine D2 receptor antagonists can effectively suppress tics; while dopaminergic agonist stimulants can occasionally exacerbate the symptoms [McCracken, 2000]. Therefore, the first association studies targeted the D2 family dopamine receptors (D2, D3, and D4 receptors). The dopamine D4 receptor (DRD4) gene, which contains a 48 base pair variable number of tandem repeats (48 bp VNTR) in the third exon, has been in the center of genetic association studies of TS. The first association study of the 48 bp VNTR with TS showed preferential transmission of the 7-repeat allele [Grice et al., 1996], but the subsequent family-based study did not confirm this finding [Hebebrand et al., 1997]. Two other case-control studies also led to contradictory results [Cruz et al., 1997; Comings et al., 1999]. Recently, Diaz-Anzaldua et al.  demonstrated excessive transmission of the 7-repeat allele in a French Canadian TS population. The controversial results might be clarified by studying further DRD4 polymorphisms. A 120 bp duplication (120 bp dup) located 1.2 kb upstream of the transcription start site was identified by Seaman et al. . In addition to this duplication, there are several single nucleotide polymorphisms (SNPs) in the 50 region of the DRD4 gene, of which the 616C/G The P-values are presented for the allele (df ¼ 1) and genotype (df ¼ 2) distributions. DRD4 48 bp VNTR genotypes were grouped according to the presence of the 7-repeat allele, resulting in the 7-present (7þ) and 7-absent (7) groups. a df ¼ 3 for the four most frequent genotype (2/4, 4/4, 4/7, 7/7) categories. 0.624 0.419a 177 62.3 107 37.7 90 31.7 139 48.9 0.875 0.707 55 19.4 4 1.4 62 21.8 0.800 0.827 218 76.8 72 25.4 131 46.1 0.744 0.255 81 28.5 74 26.1 0.473 0.740 9 3.2 201 70.8 67 65.0 36 35.0 31 30.1 55 53.4 17 16.5 2 1.9 20 19.4 81 78.6 20 19.4 57 55.3 26 25.2 77 74.8 23 22.3 3 2.9 Tourette syndrome n (total 103) Frequency (%) Control n (total 284) Frequency (%) P (df ¼ 1) P (df ¼ 2) 7 7þ TT CT Genotype 1/1 1/2 2/2 CC CG GG AA AG GG CC 521 615 616 120 bp dup A group of 103 TS patients (mean age: 13 4.5 years; 87.4% male and 12.6% female) from the Vadaskert Child and Adolescent Psychiatric Clinic participated in this study. Diagnosis was based on DSM-IV criteria [American Psychiatric Association, 1994], and was conducted by two independent psychiatrists (inter-rater reliability reached 0.95). Comorbid conditions were assessed by the Hungarian child version of the Mini-International Neuropsychiatric Interview [MINI-Kid; Balazs et al., 2004]. The following conditions were found in the sample: ADHD 38.8%; OCD 29.1%; anxiety or mood disorder 23.3%; learning disorder: 15.5%; conduct or oppositional defiant disorder 10.7%. Children with IQ < 80 (estimated from the Raven progressive matrices test; Raven, 1965), as well as those who had severe medical or neurological conditions, or pervasive psychiatric disorder were excluded. In the analysis, peak tic severity (most severe condition in tic symptoms) was measured by the YGTSS score. The Yale Global Tic Severity Scale is a clinician-completed rating scale used to assess the variety, frequency, duration, intensity, and complexity of motor and vocal tics. In addition, it measures the degree of overall impairment. These scales produce a total severity score that ranges from 0 to 100; in normative clinical samples, the mean total severity score of the YGTSS is 44.8 17.7. The YGTSS has demonstrated acceptable internal consistency, good inter-rater reliability and acceptable convergent and divergent validity [Leckman et al., 1998]. The study was approved by the Local Ethics Committee (TUKEB). All children and their parents provided written informed consent for their participation. The sex-matched Polymorphic sites METHODS Subjects and Measures TABLE I. Genotype Frequencies of the DRD4 Polymorphisms in TS Patients and Controls and 521C/T SNPs have been extensively studied in ADHD [Mill et al., 2003; Lowe et al., 2004]. The dopamine transporter is another key element in the dopamine neurotransmission, as it removes dopamine from the synaptic cleft thus influencing the magnitude and duration of the post-synaptic receptor-mediated signaling. A common 40 bp VNTR in the 30 untranslated region has been widely studied in relation to ADHD; the 10-repeat allele has been suggested as a genetic risk factor for this disorder [Faraone et al., 2005]. The 10/10 genotype was also more frequent in a TS group [Comings et al., 1996]; and tendency for preferential transmission of the 10-repeat allele was observed in a familybased study [Diaz-Anzaldua et al., 2004]. The 10-repeat allele has been associated with internalizing disorders [Rowe et al., 1998], whereas externalizing behavior problems were linked to the 9-repeat allele [Young et al., 2002]. The dopamine catabolizing enzyme, catechol-O-methyltransferase (COMT) has been also studied in child psychiatric disorders. A G-to-A transition in the 158th codon of the membrane-bound enzyme form causes a valine–methionine substitution (Val158Met). In the soluble COMT form this polymorphism is located in the 108th codon. The Met form (A allele) has 20–25% reduced enzyme activity compared to the Val variant coded by the G allele [Lachman et al., 1996]. Since COMT catabolizes dopamine, this variation might cause significant differences in the brain dopamine level. Assessing the complexity of dopamine homeostasis, association studies started to use allele-combinations reflecting high and low dopamine levels [Fossella et al., 2002]. In the present study, we performed an association analysis between TS and seven dopaminergic polymorphisms located in the DRD4, DAT1, and COMT genes using both case-control and family-based approaches. Beside the diagnostic categories, we used a dimensional measure of tic-severity assessed by the Yale Global Tic Severity Scale [YGTSS; Leckman et al., 1998]. 48 bp VNTR DAT1 Polymorphism and Tic Severity 901 902 Tarnok et al. 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 and of Caucasian origin. Genotyping Non-invasive DNA sampling was applied as described elsewhere [Boor et al., 2002]. DNA was isolated from buccal cells using phenol-extraction and alcohol precipitation [Sambrook et al., 1989] or by the DNA-purification kit obtained from Gentra (Minneapolis, MN). Genotyping procedures for the DRD4 48 bp VNTR and the DAT1 30 VNTR were carried out using published protocols [Vandenbergh et al., 1992; Ronai et al., 2000]. Genotyping and haplotyping of the four DRD4 promoter polymorphisms were performed as described previously [Szantai et al., 2005]. The COMT Val158Met polymorphism was genotyped by a single-step allele-specific amplification using two allele-specific and two outer primers (comt-a: TGG TGG ATT TCG CTG GCA, comt-g: CAC ACC TTG TCC TTC AC; comt-k2: ACA CCC ATA CAA GCA TTC ATC, comt-k1: TGC TCA CCT CTC CTC CGT). The HotStarTaq DNA-polymerase kit (Qiagen, Valencia, CA) was used applying 0.125 U enzyme in a total volume of 10 ml, in the presence of 1x buffer and 1x Q solution supplied, 1 mM primers, 200 mM of dATP, dCTP, dTTP, and 100 mM of dGTP and dITP. The thermocycling was initiated by 15 min 958C denaturation step; it was followed by 40 cycles of 948C 1 min, 568C 30 sec, 728C 1 min; and 10 min 728C final extension. Data Analysis SPSS 10.0 for Windows was used for all statistical analyses. DRD4 48 bp VNTR genotypes were grouped according to the presence of the 7-repeat allele, resulting in 7-present (7þ) and 7-absent (7) groups. In the case of the DAT1 30 VNTR, the rare 10/11 genotype was grouped together with the 10/10 genotype, because a study investigating the effect of this repeat sequence on gene expression did not find significant difference between the 11-repeat and 10-repeat alleles [Inoue-Murayama et al., 2002]. Since no functional data existed for the 8-repeat allele, a single subject with 8/10 genotype was omitted from the association analyses. For the family-based approach the extended Transmission Disequilibrium Test [ETDT, Sham and Curtis, 1995] was used. RESULTS In the case-control analysis, 103 TS patients and 284 sexmatched control individuals were included. In addition to the DRD4 48 bp VNTR, the 120 bp duplication, the 616C/G SNP (rs74302), the 521C/T SNP (rs1800955), and the recently identified 615A/G SNP (rs936462) were included to evaluate potential effects of the DRD4 promoter variants. Neither allele nor the genotype frequencies of the DRD4 polymorphisms differed significantly between the case and the control groups (Table I). Haplotypes of the DRD4 promoter polymorphisms were also assessed, but there was no significant difference detected in the case-control study (Table II). Family genotype data were available for 71 trios and 23 duos. The TDT analysis showed a bias for the DRD4 promoter haplotypes (Table II). The 2-repeat allele of the 120 bp sequence 616G 615A 521C (2-G-A-C) combination was preferentially transmitted to TS patients (26 vs. 7 times; TDT w2 ¼ 10.939, df ¼ 1, P ¼ 0.0009). However, the P-value of the overall allele-wise TDT was only marginally significant (TDT w2 ¼ 18.064, df ¼ 10, P ¼ 0.054). A tendency of overtransmission of the 616G allele was also detected (39 vs. 24 times; TDT w2 ¼ 3.606, df ¼ 1, P ¼ 0.058). No preferential allele transmission has been observed for the other DRD4 polymorphisms (allele-wise TDT P values were the followings: 48 bp VNTR: 0.173; 120 bp dup: 0.516; 616A/G: 0.724; 521C/ T: 0.257). The same approach was used for the DAT1 3’ VNTR and the COMT Val158Met polymorphisms. Neither the casecontrol (Table III), nor the TDT analysis yielded significant association (the allele-wise TDT P values were 0.188 and 0.541, for the DAT1 and COMT polymorphisms, respectively). Finally, we tested the potential effects of the dopaminergic polymorphisms on the Yale Global Tic Severity Scale scores. Since TS symptoms are waxing and waning in time, we used the peak tic severity scores to obtain an overall measurement of tics. Preliminary analyses showed that the sex of subjects did not affect tic severity (females: 52.46 20.47, males: 51.73 18.58, F ¼ 0.02 (df ¼ 1,100) P ¼ 0.888), but the age of subjects had a nearly significant effect (F ¼ 3.72 (df ¼ 1,100) P ¼ 0.057). Therefore, age was entered as a covariant in all subsequent ANOVAs testing genetic effects. Table IV summarizes the results for the individual polymorphisms. Only the DAT1 3’ VNTR showed a significant association with the total tic severity score (F ¼ 4.784 (df ¼ 2,98) P ¼ 0.010). Since the YGTSS mean score of the 9/9 group was similar to that of the 9/ 10 group, we grouped the genotypes according to the presence or absence of the 9-repeat allele (9/9 þ 9/10 vs. 10/10). Patients carrying at least one copy of the 9-repeat allele had more severe tic symptoms on average than individuals with the 10/10 genotype (9-present: n ¼ 49, 57.80 16.83; 9-absent: n ¼ 53, 46.51 19.01; F ¼ 9.664 (df ¼ 1,99) P ¼ 0.002, effect size: etasquare ¼ 0.089). After the stringent Bonferroni correction for multiple testing the DAT1 VNTR—YGTSS association was still significant (P < 0.0073). DISCUSSION The aim of the present study was to identify specific dopaminergic gene variants as genetic risk factors for TS. In our TS group neither the case-control analysis nor the TDT supported an association between the DRD4 48 bp VNTR and TABLE II. Genotype Frequencies and Transmission Disequilibrium Test of the DRD4 Promoter Haplotypes Haplotype Tourette syndrome n (total 103) Frequency (%) Control n (total 284) Frequency (%) TDT Passed Not passed 1CAC 1CAT 1GAC 1GAT 1GGC 1GGT 2CAC 2CAT 2GAC 2GAT 2GGC 2GGT 7 3.4 10 4.9 2 1.0 8 3.9 — — 2 1.0 32 15.5 60 29.1 37 18.0 26 12.6 11 5.3 11 5.3 22 3.9 23 4.0 17 3.0 24 4.2 3 0.5 3 0.5 86 15.1 162 28.5 76 13.4 88 15.5 46 8.1 18 3.2 2 4 7 2 — — 2 2 18 25 26 7 14 19 8 10 9 5 4 5 5 10 27 33 The overall P-value in the case-control study was P ¼ 0.402 (df ¼ 9). In the family-based analysis the overall allele-wise TDT (df ¼ 10) yielded P ¼ 0.054. DAT1 Polymorphism and Tic Severity 903 TABLE III. Genotype Frequencies of the DAT1 and COMT Polymorphisms in TS Patients and Controls DAT1 30 VNTR Polymorphic sites Genotype Tourette syndrome n (total 103) Frequency (%) Control n (total 284) Frequency (%) P (df ¼ 1) P (df ¼ 2) COMT 9/9 9/10 10/10 Othersa Met/Met 10 9.7 39 37.9 51 49.5 3 2.9 28 27.2 51 49.5 24 23.3 28 9.9 100 35.2 150 52.8 6 2.1 80 28.2 151 53.2 0.488 0.595 53 18.7 0.797 0.859 Met/Val Val/Val The P-values are presented for the allele (df ¼ 1) and genotype (df ¼ 2) distributions. a Rare genotypes included one 8/10 and two 10/11 genotypes in the TS group; 10/11, 10/13, 10/14, 9/15, 6/10, and 7/9 genotypes in the control group. TS. To analyze the possible interactive effect of promoter polymorphisms, we also assessed the 120 bp duplication and three SNPs in the 50 non-coding region of the DRD4 gene. The 616G allele and the 2-G-A-C haplotype (i.e., the 2-repeat form of the 120 bp sequence 616G 615A 521C combination) was preferentially transmitted to TS patients. However, after correcting for multiple testing these results did not remain significant. In addition to the DRD4 polymorphisms, two other dopaminergic polymorphisms were tested. Although both case-control and TDT analyses have been carried out, neither the DAT1 30 VNTR, nor the COMT Val158Met polymorphisms were associated with TS using the diagnostic categories. Regarding the COMT polymorphism, our findings are consistent with previous studies [Barr et al., 1999; Cavallini et al., 2000]. In the case of the DAT1 VNTR, we cannot confirm the possible role of the 10-repeat allele as a risk allele. A different picture emerged when we analyzed tic severity of the TS patients. The 40 bp VNTR located in the 30 non-coding region of the dopamine transporter gene was associated with peak tic severity measured by the Yale Global Tic Severity Scale. Assessing the total tic severity score, subjects with a 9repeat variant had 25% higher scores than those possessing the long allele only. There was no difference in the mean tic severity scores between the 9/9 and 9/10 genotype groups, suggesting that the 9-repeat allele might act as a dominant allele in this neurobiological correlation. Several lines of evidence suggest that the DAT1 30 VNTR affect gene expression, however, results obtained from the in vitro experiments as well as from the in vivo neuroimaging studies are controversial. Using luciferase reporter gene expression system, Fuke et al.  showed that the 10repeat allele had higher transcriptional activity compared to the 9-repeat form, but other studies resulted in contradictory TABLE IV. Effect of the Dopaminergic Gene Polymorphisms on the YGTSS Total Tic Severity Score Polymorphism DRD4 120 bp dup 616C/G SNP 615A/G SNP 521C/T SNP 48 bp VNTR COMT Val158Met DAT 30 VNTR Genotype YGTSS score SD 1/2 2/2 CC CG GG AA AG CC CT TT 7 7þ 54.17 20.75 50.51 18.12 55.38 19.92 50.42 17.20 51.20 21.53 50.63 19.12 53.90 15.82 52.29 13.25 53.82 21.33 48.03 16.01 51.57 19.45 52.31 17.53 Met/Met Met/Val Val/Val 51.50 20.41 53.06 17.37 49.58 20.02 9/9 9/10 10/10 9/9þ9/10 10/10 57.00 16.01 58.00 17.23 46.51 19.00 57.80 16.83 46.51 19.00 F df P 1.198 1,97 0.276 0.326 2,99 0.722 0.750 1,98 0.388 1.020 2,99 0.364 0.009 1,100 0.926 0.570 2,99 0.568 4.784 2,98 0.010 9.664 1,99 0.002 Due to n < 5 group sizes, the 120 bp 1/1 and 615 GG genotypes were omitted from these analyses. In case of the DAT1 30 VNTR the 10/11 genotype was grouped together with the 10/10 genotype; the single 8/10 genotype was left out (see Methods Section). 904 Tarnok et al. findings [Miller and Madras, 2002; Mill et al., 2005]. Regarding the SPECT studies, one group showed significant differences in the protein density between various genotype groups of a healthy population: those with at least one copy of the 9-repeat allele (9/9 and 9/10 genotype) had higher transporter density compared to the 10/10 genotype group [van Dyck et al., 2005]. Two other studies did not find any significant difference between the genotype groups [Heinz et al., 2000; Martinez et al., 2001]. SPECT analyses of TS patients have produced more consistent results. Most studies showed increased dopamine transporter density in the striatum of TS patients compared to controls [Muller-Vahl et al., 2000; Cheon et al., 2004; SerraMestres et al., 2004]. When tic severity was assessed, there was no significant association between the dopamine transporter binding ratios and YGTSS scores (Serra-Mestres et al., 2004; Cheon et al., 2004). However, these analyses were carried out on very small samples (n 15), therefore, they might be unable to detect moderate effects of complex interactions. Based on the results of the largest SPECT study [n ¼ 96; van Dyck et al., 2005], where the 9-repeat allele was linked to higher dopamine transporter density, we may speculate about this allele as a risk factor for TS and/or for tic severity. Still, we need to be cautious in interpreting our result of the association between the DAT1 40 bp VNTR and tic severity until independent replication. In addition, functional studies clarifying the effect of the 40 bp VNTR in the 30 non-coding region of the dopamine transporter gene are still needed. It is important to note that using the dichotomous DSM-IV diagnostic variable, we did not find association between the DAT1 30 VNTR and TS. However, when we used the continuous YGTSS variable, the 9-repeat allele was associated with more severe tic symptoms. We propose that this dopamine transporter polymorphism is a genetic risk factor for tic severity in TS, but it contributes to the overall syndrome in a minor, sometimes undetectable way. The dopamine transporter— being a key component in the ‘‘uptake-dominated’’ dopamine transmission of basal ganglia—might contribute to the development of tics on a larger scale. In addition, our data support the recent guidelines in psychiatric genetics, which suggest that dimensional models, such as severity-scales, might better describe a disorder than the categorical ones. Using quantitative dimensions could also facilitate the detection of small genetic effects. 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