Functional Effects of a Tandem Duplication Polymorphism in the 5=Flanking Region of the DRD4 Gene Ursula M. D’Souza, Carsten Russ, Eda Tahir, Jonathan Mill, Peter McGuffin, Philip J. Asherson, and Ian W. Craig Background: Several polymorphisms have been identified in the 5=flanking region of the human dopamine D 4 receptor gene (DRD4), including a tandem duplication polymorphism. This comprises a 120-base-pair repeat sequence that is known to have different allele frequencies in various populations around the world. Furthermore, various studies have revealed evidence of linkage to attention- deficit/hyperactivity disorder and association with schizophrenia and methamphetamine abuse. The location of the polymorphism in the 5=regulatory region of the DRD4 gene and the fact that it consists of potential transcription factor binding sites suggest that it might confer differential transcriptional activity of the alleles. Methods: We investigated the functional effects of this gene variant with transient transfection methods in four human cell lines and then assessed transcriptional activity with luciferase reporter gene assays. Results: The longer allele has lower transcriptional activity than the shorter allele in SK-N-MC, SH-SY5Y, HEK293, and HeLa cell lines. Conclusions: This evidence suggests that the duplication might have a role in regulating the expression of the DRD4 gene and provides an understanding of the biological mechanisms underlying the etiology of neuropsychiatric disorders such as ADHD, schizophrenia, and metamphetamine abuse. Key Words: Dopamine receptors, DRD4, ADHD, tandem duplica- tion, polymorphism, regulatory T he human dopamine D 4 receptor gene (DRD4), which codes for a seven transmembrane domain protein belong- ing to the super family of G-protein-coupled receptors, was originally cloned by Van Tol et al (1991). The gene has been implicated in several neuropsychiatric and neurobehavioral dis- orders, and pharmacologic studies have revealed that the atypi- cal antipsychotic drug clozapine binds with high affinity to the dopamine D 4 receptor. High expression of DRD4 messenger ribonucleic acid (mRNA) was detected in the medulla, frontal cortex, midbrain, and amygdala, with lower levels of the gene expressed in the basal ganglia (Van Tol et al 1991). Multiple polymorphic variants of the DRD4 gene in the human population are known to exist as two-, four-, or seven-repeat sequences of a 48-base-pair (bp) region in exon 3, which codes for the third putative cytoplasmic loop of the receptor (Van Tol et al 1992). Van Tol et al also reported that the seven-repeat variants had different binding profiles with clozapine and spiperone com- pared with the two- and four-repeat variants, with respect to sodium chloride sensitivity. Similarly, the same group later found that the polymorphic repeat sequences showed small differences in DRD4 pharmacologic binding properties (Asghari et al 1994; Jovanovic et al 1999) and also in functional characteristics to inhibit cyclic adenosine monophosphate (Asghari et al 1995). Therefore, it was concluded from the evidence that there was no direct relationship between length of the polymorphism and changes in these particular activities. The DRD4 variants were also capable of coupling to several G protein (G i ) subtypes, but no evidence of any quantitative difference in G protein coupling related to the number of repeats was observed (Kazmi et al 2000). Additionally, evidence suggests that the seven-repeat variant originated as a rare mutational event that increased to high frequency in human populations by positive selection (Ding et al 2002; Wang et al 2004). The DRD4 gene has been implicated in other neurobehavioral disorders, such as novelty seeking, attention-deficit/hyperactivity disorder (ADHD), and substance abuse. Additionally, reports have described the association of the personality trait of novelty seeking (which comprises impulsive, exploratory, and sensation- seeking behavior) with the allele having seven repeats in both Israeli and American populations (Benjamin et al 1996; Ebstein et al 1996). More recently, many findings demonstrate the associa- tion and linkage between the seven-repeat allele in DRD4 and ADHD (Faraone et al 1999, 2001; Holmes et al 2000; LaHoste et al 1996; Langley et al 2004; Mill et al 2001; Rowe et al 1998; Smalley et al 1998; Swanson et al 1998; Tahir et al 2000); however, a smaller number of studies have reported no associ- ation of the seven-repeat allele with ADHD (Castellanos et al 1998; Eisenberg et al 2000; Hawi et al 2000; Kotler et al 2000; Todd et al 2001). Attention-deficit/hyperactivity disorder is a common syndrome that begins in childhood and is characterized by inattention, hyperactivity, and impulsivity and is also associ- ated with school failure, peer-relationship problems, early use of nicotine and alcohol, and an increased frequency of accidents (Todd and O’Malley 2001). The seven-repeat allele was also reported to be associated with opiate dependence (Kotler et al 1997) and with heroin abuse in a Han Chinese sample (Li et al 1997). The cloning and characterization of the 5=flanking region of the human DRD4 gene (Kamakura et al 1997) enabled the search for novel polymorphisms. One of these was the -521CT poly- morphism, which was identified in the promoter region of DRD4 and showed weak association to schizophrenic Japanese patients (Okuyama et al 1999). Functional effects of this promoter polymorphism were analyzed in human retinoblastoma cells (Y-79 cells), and transient transfection methods demonstrated that the T allele reduced transcriptional efficiency by 40% From the Medical Research Council Social, Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, King’s College London, United Kingdom. Address reprint requests to Ursula M. D’Souza, Social, Genetic and Develop- mental Psychiatry Centre, Box Number P082, Denmark Hill, London SE5 8AF, United Kindom; E-mail: spjgumd@iop.kcl.ac.uk. Received February 26, 2004; revised July 20, 2004; accepted August 25, 2004. BIOL PSYCHIATRY 2004;56:691– 697 0006-3223/04/$30.00 doi:10.1016/j.biopsych.2004.08.008 © 2004 Society of Biological Psychiatry