Association of the DAT1 polymorphism with attention deficit hyperactivity disorder (ADHD) A family-based approach.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:309 –311 (2006) Association of the DAT1 Polymorphism With Attention Deficit Hyperactivity Disorder (ADHD): A Family-Based Approach Myung-Ho Lim,1* Hyun Woo Kim,1 Ki-Chung Paik,1 Soo Churl Cho,2 Do Young Yoon,3 and Heon-Jeong Lee4 1 Department of Psychiatry, College of Medicine, Dankook University, Cheonan, Korea Department of Child and Adolescent Psychiatry, Seoul National University Hospital, Seoul, Korea 3 Cell Biology Laboratory, Korean Research Institute of Bioscience and Biotechnology, Daejeon, Korea 4 Department of Psychiatry, Korea University College of Medicine, Seoul, Korea 2 The dopamine transporter gene (DAT1) is of particular interest in the genetic study of attention deficit hyperactivity disorder (ADHD), because psychostimulants interact directly with the dopamine transporter protein. Association between ADHD and the 10-repeat allele of a 40 base pair (bp) variable number of tandem repeats (VNTR) polymorphism of DAT1 was first reported in 1995 [Cook et al. (1995); Am J Hum Genet 56:993–998]. Subsequently, several investigators have also confirmed this association, although others reported conflicting results. We analyzed the DAT1 polymorphism in a sample of 33 Korean probands with a Diagnostic and Statistical Manual of Mental Disorders version IV (DSM-IV) diagnosis of ADHD and found evidence of increased transmission of the 10-repeat allele using transmission disequilibrium test (TDT) (P ¼ 0.001; OR ¼ 7.88, CI ¼ 2.20–28.29). These data support the role of DAT1 in ADHD susceptibility among Asian populations. ß 2006 Wiley-Liss, Inc. KEY WORDS: association; attention deficit hyperactivity disorder(ADHD); DAT1; dopamine transporter gene; genetics; polymorphism; transmission disequilibrium test INTRODUCTION Attention deficit hyperactivity disorder (ADHD) is a common disorder that has about 3–7% prevalence rate among prepubertal elementary school children. Recent family study shows that ADHD has an 80–90% hereditability [Eaves et al., 1997]. Accordingly molecular genetic studies are being actively carried out. Most of the studies are being done on the enzyme or receptor related genes in the dopaminergic neurotransmission pathways. Recently, genetic studies of ADHD have focused on dopaminergic neurotransmission related genes. Symptoms of *Correspondence to: Myung-Ho Lim, M.D., Ph.D., Department of Psychiatry, College of Medicine, Dankook University, Cheonan, Chungnam Province, Korea. E-mail: email@example.com Received 16 November 2004; Accepted 21 April 2005 DOI 10.1002/ajmg.b.30282 ß 2006 Wiley-Liss, Inc. ADHD show a 70% improvement upon treatment with the psychostimulants such as methylphenidate [Elia et al., 1999]. These medications act on the dopamine transporter gene (DAT1), which is believed to prolong dopamine activity within the synapses. In a knockout-gene study in mice, Giros et al.  reported that mice that were homozygous for deactivation of the DAT1 allele were 5–6 times more active and had dopamine remaining active in the synaptic cleft 100 times longer than heterozygous and wild-type mice. Vandenbergh et al.  reported a 40 base pair (bp) variable number of tandem repeats (VNTR) polymorphism in the 30 -untranslated region (UTR) of the DAT1 gene located at 5q15.3. Association of DAT1 VNTR polymorphism and ADHD has been reported in several studies [Cook et al., 1995; Gill et al., 1997; Waldman et al., 1998; Daly et al., 1999; Barr et al., 2001; Chen et al., 2003], they indicated the allele with 10 copies of the 40-bp sequence (10-repeat allele) as the risk allele for ADHD. However, this association was not replicated in some studies [Palmer et al., 1999; Holmes et al., 2000; Curran et al., 2001; Roman et al., 2001; Qian et al., 2004]. In Korea, ADHD is a common disorder with a mortality rate of about 5%, recently there has been a few research on its imaging study and genetic study. [Cho, 1999]. Recently, using [123I]IPT SPECT, Cheon et al.  reported that ADHD in seven children with 10/10 genotype had a significantly higher DAT density in basal ganglia than four children without 10/10 genotype. [Cheon et al., 2004]. By using extended transmission disequilibrium test (TDT), we studied 33 Korean trio families to test the allelic association between ADHD and DAT1 VNTR polymorphism. METHODS Subjects The subjects were recruited in the ADHD outpatient clinic at the Child and Adolescent Psychiatric Division of the Psychiatric Department of Dankook University Hospital, Cheonan, Korea, between March and December 2001. We diagnosed ADHD by the Diagnostic and Statistical Manual of Mental Disorders version IV (DSM-IV) [American Psychiatric Association, 1994]. All subjects were Korean. Subjects were excluded if there was any evidence of conduct disorder, mood disorder, anxiety disorder, Tourette disorder, pervasive development disorder, mental retardation (IQ < 70), and neurological conditions including epilepsy. All the interviews were done by a well-trained child psychiatrist (M.H. Lim). The ADHD patients were administrated the attention deficit diagnosis system (ADS) [Shin et al., 2000], a Korean version of modified 310 Lim et al. computerized test of variables of attention (TOVA) [Greenberg and Waldman, 1993] for the accurate diagnosis. The Korean version of the child behavior checklist (CBCL) [Oh and Lee, 1990; Achenbach, 1991] and the Korean version of Conners rating scales [Conners and Barkley, 1985; Oh et al., 1997] were used in the present study to obtain parental reports of child behavior. Subjects were included from our sample if the subjects performed over two standard deviation of norm in the tests of ADS. Subjects with high-anxiety scores (Spielberger trait/state anxiety scale score >47/49) in Korean version of Spielberger trait/state anxiety scale for children [Spielberger, 1973; Cho and Choi, 1989] were also excluded. Finally, 33 families consisting of father, mother, and affected child (trios) were included in our study. The parents of subjects provided written informed consent. The study was approved by the Institutional Review Board of the Dankook University Hospital. Genotypings Standard high salt extraction methods were used to isolate DNA from whole blood lymphocytes. The primer sequence used to amplify the 40-bp sequence of the VNTR polymorphic loci was as follows [Cook et al., 1995; Holmes et al., 2000]: PCR amplification of the DAT1 40-bp VNTR in the 30 -UTR was carried out using the following pair of primers, upstream: 50 -TGT GGT GTA GGG AAC GGC CTG AG-30 and downstream: 50 -CTT CCT GGA GGT CAC GGC TCA AGG-30 . Thirty-five cycles were conducted consisting of denaturation at 948C for 60 sec, annealing at 608C for 60 sec, and extension at 728C for 60 sec. An initial denaturing step at 958C for 10 min and a last extension step at 728C for 9 min were also added. The reactions were 50 ml that consisted of 0.1 mg of genomic DNA, 20 pmol of each primer, 10 mM dNTPs, 2.5 U Taq polymerase, and 10 Taq buffer, and distilled water. Ten-microliters of PCR products were run on 1.5% agarose gel. A 50-bp DNA ladder was used to identify the various repeat alleles by size: 7-repeat (360 bp), 9-repeat (440 bp), 10-repeat (480 bp), and 11-repeat (520 bp). Statistical Analyses Allele and genotype frequencies were compared using the Chi-square test. For the family-based analyses, we used the TDT [Sham and Curtis, 1995] instead of haplotype-based haplotype relative risk (HHRR) method to have any potential population stratification [Terwilliger and Ott, 1992]. RESULTS Of the total 33 cases, 24 (72.7%) were boys and 9 (27.3%) girls. Their ages ranged from 7 to 13 years with a mean age of 9.3 2.4 (SD) years. CBCL mean score of ADHD patients was 68.4 9.3 (SD), while Connors scores was 47.8 18.2 (SD), Spielberger trait/state anxiety scale scores was 31.8 8.19/ 33.79 7.45. Allele frequencies in the ADHD sample were 1.5% 360-bp alleles (7 repeats), 1.5% 440-bp alleles (9 repeats), 1.5% 520-bp alleles (11 repeats), and 95.5% 480-bp alleles (10 repeats). Allele frequencies of the parents’ sample were 6.1% 360-bp alleles, 6.1% 440-bp alleles, 3.8% 520-bp alleles, and 84.1% 480bp alleles. The transmission of DAT1 VNTR 10-repeat allele in 33 ADHD trios is presented in Table I. The HHRR result suggested that the 10-repeat allele was preferentially transmitted to ADHD children. (Fisher exact test, P ¼ 0.001; OR ¼ 7.88, CI ¼ 2.20–28.29). DISCUSSION The allele frequency of the DAT1 10-repeat allele are 95.5% in our ADHD sample and 84.1% in their parents, and this TABLE I. Transmission Tests for Individual Alleles of DAT1 VNTR Polymorphism Transmission in 33 ADHD Triads Using the ETDT Program T NT X2 P value 360 440 480 520 1 7 4.50 0.036 1 7 4.50 0.036 18 3 10.71 0.0011 1 4 1.80 NS T, Transmitted Number; NT, Nontransmitted Number. result is quite different from the frequency around 70% in the Caucasian population [Doucette-Stamm et al., 1995]. However, ours is similar to those observed in Asian populations [Chen et al., 2003; Qian et al., 2004]. Genetic variation of DAT1 is of particular interest in the study of ADHD, since stimulant drugs for ADHD inhibit directly dopamine transporter function. Association between ADHD and the 10-repeat allele of a 40-bp VNTR polymorphism that lies within the 30 -UTR of DAT1 was first reported in 1995 [Cook et al., 1995], To date, in Caucasian population, at least five studies supported the positive association between DAT1 10-repeat allele and ADHD [Gill et al., 1997; Waldman et al., 1998; Daly et al., 1999; Barr et al., 2001; Curran et al., 2001]. However, at least five did not support an association [Swanson et al., 1998; Palmer et al., 1999; Holmes et al., 2000; Curran et al., 2001; Roman et al., 2001]. In Asian population, Chen et al.  have analyzed the same association in a sample of 110 Taiwanese probands with ADHD and found evidence of increased transmission of the 10-repeat allele. However, Qian et al.  did not replicate prior findings of an association between the DAT1 10-repeat allele and ADHD. Instead, they found that long repeat alleles (11–12 repeats) were associated with the disorder. However, our data did not show this finding. Therefore, our result confirmed the previous reports of the association between the DAT1 10-repeat allele and ADHD. Here we wish to acknowledge some study limitations. First, although we excluded comorbid major psychiatric illness, it is difficult to convince that controlling of comorbidity was complete. Second, we could not consider the ADHD subtype, gender difference, and stimulant treatment response. Recently, Kirley et al.  reported an association between the DAT1 10-repeat VNTR and favorable response to methylphenidate in ADHD children. Considering those variables might give more informative data. Third, we could not consider association of ADHD gene using haplotypes other than single VNTR. Barr et al.  analyzed using a TDT on the dopamine transporter gene whose haplotype is made of the three parts VNTR and exon 9, intron. The results showed that in ADHD patients, a haplotype made of a specific allele existing in the 10 VNTR and exon 9, intron 9 was highly transmitted. Similar to our study, it is believed that only following the VNTR in studies of ADHD will have varying results depending on the haplotype of the control group. However, larger sample size is necessary for controlling the confounding factors. Lastly, the relatively small sample size limits the generalizability of our findings. Our study showed a high-odds ratio and wide confidence interval compared to previous studies [Daly et al., 1999; Holmes et al., 2000]. This is most likely due to the small effect size as a result of the small number of samples. Taking these problems into consideration, further study with a larger sample size whilst controlling for subtype, gender difference, and drug response is needed. It is also necessary to evaluate the possible involvement of as-yet-uncovered gene(s) that influence ADHD other than DAT1, and also the possibility of gene–gene and gene–environment interactions. Association Study of DAT1 Polymorphism With ADHD REFERENCES Achenbach TM. 1991. Manual for the child behavior checklist. Burlington: Department of Psychiatry, University of Vermont. American Psychiatric Association. 1994. Diagnostic and statistical manual of mental disorders, 4th edition. Washington, DC: American Psychiatric Association. Barr CL, Xu C, Kroft J, Feng Y, Wigg K, Zai G, Tannock R, Schachar R, Malone M, Roberts W, Nothen MM, Grunhage F, Vandenbergh DJ, Uhl G, Sunohara G, King N, Kennedy JL. 2001. 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