RESEARCH ARTICLE Family-based association study of DRD4 gene in methylphenidate-responded Attention Deficit/Hyperactivity Disorder Patrick Wing-leung Leung1*, Janice Ka Yan Chan2, Lu Hua Chen1,3, Chi Chiu Lee4, Se Fong Hung5, Ting Pong Ho3, Chun Pan Tang4, Robert K. Moyzis6,7, James M. Swanson7 1 Department of Psychology, The Chinese University of Hong Kong, Hong Kong, PRC, 2 The Duchess of Kent Children's Hospital at Sandy Bay, Hospital Authority, Hong Kong, PRC, 3 Department of Psychiatry, University of Hong Kong, Hong Kong, PRC, 4 Kwai Chung Hospital, Hospital Authority, Hong Kong, PRC, a1111111111 5 Department of Psychiatry, The Chinese University of Hong Kong, Hong Kong, PRC, 6 Department of a1111111111 Biological Chemistry, University of California, Irvine, California, United States of America, 7 Department of a1111111111 Pediatrics, University of California, Irvine, California, United States of America a1111111111 a1111111111 * [email protected] Abstract OPEN ACCESS The 48-basepair (48-bp) variable number tandem repeat (VNTR) polymorphism in exon 3 of Citation: Leung PW-l, Chan JKY, Chen LH, Lee CC, the dopamine receptor D4 gene (DRD4) is implicated in the etiology of attention-deficit/ Hung SF, Ho TP, et al. (2017) Family-based hyperactivity disorder (ADHD). In particular, ADHD in European-ancestry population is association study of DRD4 gene in associated with an increased prevalence of the 7-repeat (7R) allele of the exon 3 VNTR. methylphenidate-responded Attention Deficit/ Hyperactivity Disorder. PLoS ONE 12(3): However, it is intriguing to note that the 7R allele has been found to be of very low preva- e0173748. doi:10.1371/journal.pone.0173748 lence in the Chinese general population. In a previous case-control study, our research Editor: Chandan Vaidya, Georgetown University, team had found that the 7R allele was similarly absent in Chinese ADHD children in Hong UNITED STATES Kong. Instead, there was an increased prevalence of the 2R allele in Chinese ADHD chil- Received: November 26, 2016 dren. Interestingly, in Asian samples, the 2R allele had been found to be an evolutionary derivative of the 7R allele with equivalent biochemical functionality. So, the finding of an Accepted: February 24, 2017 association between ADHD and 2R allele in Chinese population does not exactly contradict Published: March 10, 2017 the original 7R allele finding in European-ancestry population. However, given the potential Copyright: © 2017 Leung et al. This is an open pitfall of population stratification in the previous case-control design, this current study access article distributed under the terms of the tested the 2R allele and ADHD association using a methodologically more rigorous family- Creative Commons Attribution License, which permits unrestricted use, distribution, and based approach on 33 Chinese ADHD probands who had favorable clinical responses to reproduction in any medium, provided the original stimulant medication (methylphenidate). Haplotype Relative Risk (HRR) analysis and author and source are credited. Transmission Disequilibrium Test (TDT) both showed a significant preferential transmission Data Availability Statement: All relevant data are of the 2R allele from the biological parents to ADHD probands (pone-tailed = 0.038, OR = 2.04; within the paper and its Supporting Information pone-tailed = 0.048, OR = 2.29, respectively). A second hypothesis speculates that it is the file. deviation, including 7R and 2R alleles, from the conserved ancestral 4R allele which confers Funding: This study is funded by a General risk to ADHD. Thus, a preferential transmission of non-4R alleles, against the 4R allele, from Research Fund (GRF) grant to the first/ biological parents to their ADHD probands is predicted. Both HRR analysis and TDT con- corresponding author, Patrick WL Leung, from the Research Grants Council (RGC) (RGC 449511) in firmed such prediction (pone-tailed = 0.029, OR = 2.07; pone-tailed = 0.032, OR = 2.43, respec- Hong Kong (http://www.ugc.edu.hk/eng/rgc/index. tively). This study re-confirmed the original finding of a previous study that in Chinese htm). The funder had no role in study design, data population, the 2R allele of the DRD4 exon 3 VNTR was related to ADHD. This endorses collection and analysis, decision to publish, or preparation of the manuscript. the general thesis that DRD4 exon 3 VNTR polymorphism is related to ADHD, despite that PLOS ONE | DOI:10.1371/journal.pone.0173748 March 10, 2017 1 / 11 DRD4 gene in Attention Deficit/Hyperactivity Disorder Competing interests: The authors have declared the exact length or number of repeats of the associated alleles varies across ethnicity. This that no competing interests exist. in turn supports the dopamine dysregulation theory of ADHD. Introduction Attention-deficit/hyperactivity disorder (ADHD), a neuropsychiatric disorder, is character- ized by age-inappropriate inattention, hyperactivity and impulsivity, with male to female ratio ranging from 3:1 to 9:1 [1]. It is among the most prevalent mental health problems of children with a worldwide prevalence of about 5% [2]. Previously, ADHD was once thought to be a Western condition, encouraged by the permissiveness of the Western culture [3]. This previ- ous view implies that ADHD may be absent in Chinese culture, in which there is more empha- sis on orderly and placid behaviour. However, studies find that ADHD is no less prevalent in Chinese population with prevalence estimates of around 4%Ð6% in Chinese communities of Hong Kong and Taiwan [4,5]. Currently, ADHD is considered as a complex, multifactorial disorder with multiple biological etiologies, including genetics. The latter's involvement is con- firmed by various family, twin and adoption studies, with heritability estimate being 76% in one meta-analysis [6]. The biological etiologies, including genetics, underline the universality of this disorder, ADHD. Despite the high estimated heritability of ADHD, genome-wide association studies (GWAS) have so far failed to detect any consistent polymorphism related to ADHD at the genome-wide significant level [7±12]. Experiences with the more successful GWAS studies involving such psychiatric and medical disorders as bipolar disorder and diabetes suggest a required sample size of over 4,000 to implicate a few loci and up to 60,000 to detect a larger set of genes [13,14]. Such sample sizes do not seem to be achievable for ADHD studies in the near future. Multi- centre collaboration may be required. However, given the modest inter-rater reliability in psy- chiatric diagnosis, variability in diagnostic practice and referral bias across multiple sites, multi- centre collaboration will lead to additional phenotypic (and possibly genotypic) heterogeneity between datasets, resulting in increased ªnoiseº and reduced statistical power [15]. Instead, the traditional candidate gene approach, which is theory-driven and requires much smaller sample sizes to achieve adequate statistical power, has proposed genes associated with ADHD, based upon the dopamine (DA) dysregulation theory [16]. The stimulant medication (e.g., by methylphenidate), effectively treating ADHD symptomatically, binds to the dopamine transporters in the presynaptic membrane and blocks the transporter's ability to clear DA from the synaptic space. This ªmechanism of actionº of the stimulant medication implicates that DA as a neurotransmitter may be involved in the etiology of ADHD. DA neurons transmit DA from mid-brain regions to the other parts of the brain via three major pathways, i.e., the nigrostriatal, mesocorticolimbic and tuberoinfundibular pathways. DA within these pathways regulates functionally and structurally various cortical and basal ganglia loops, the disruption of which produces attentional and motivational difficulties characteristics of ADHD. Animal models of ADHD also show dysregulation of DA functions. The behaviour of ªADHDº mice is normalized upon administration of stimulant medication. Brain imaging studies similarly suggest altered regulation of striatal DA levels. Two neuroimaging studies with Chinese ADHD children have identified frontal cortical regions, which are rich in dopamine, as sites related to ADHD [17,18]. Among the dopamine system genes, dopamine receptor D4 gene (DRD4) is the most exten- sively investigated with three quarters of its studies producing consistently positive results PLOS ONE | DOI:10.1371/journal.pone.0173748 March 10, 2017 2 / 11 DRD4 gene in Attention Deficit/Hyperactivity Disorder [19]. Dopamine receptor D4 is a G protein-coupled receptor and belongs to the dopamine receptors family (D1-D5). Interestingly, it is expressed in the brain prefrontal cortex, including anterior cingulate and orbitofrontal cortex, which involves regions also predominantly affected in ADHD [20,21]. The DRD4 carries a polymorphism, which is a 48-basepair (48-bp) variable number tandem repeat (VNTR) and is located at the exon 3 of the gene. The alleles of this polymorphism range from 2 to 11 repeats with the 4-repeat (4R) allele being the most preva- lent [22,23]. Following the first association study between ADHD and DRD4 exon 3 7R allele published in 1996 [24], several subsequent meta-analyses have consistently confirmed an increase of the 7R allele in ADHD probands, based upon both case-control and family studies in European-ancestry population [6,25,26]. Functionally, the 7R allele, compared to the more common 4R allele, exhibits a blunted ability to reduce cyclic adenosine monophosphate (cAMP) level, thus