32JOURNAL OF M.I. EXPERIMENTAL SEAMAN ET AL. ZOOLOGY (MOL DEV EVOL) 288:32–38 (2000)

Evolution of Exon 1 of the D4 Receptor (DRD4) Gene in Primates MICHAEL I. SEAMAN,1 FONG-MING CHANG,2,3 AMOS S. DEINARD,1 2 2,4 ANA T. QUIÑONES, AND KENNETH K. KIDD * 1Department of Anthropology, Yale University, New Haven, Connecticut 06520-8277 2Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520-8005 3Department of Obstetrics and Gynecology, National Cheng Kung University Medical College, Taiwan 4Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut 06250

ABSTRACT The dopamine D4 receptor (DRD4) gene exhibits a large amount of expressed polymorphism in humans. To understand the evolutionary history of the first exon of DRD4— which in humans contains a polymorphic 12bp tandem duplication, a polymorphic 13bp deletion, and other rare variants—we examined the homologous exon in thirteen other primate species. The great apes possess a variable number of tandem repeats in the same region as humans, both within and among species. In this sense, the 12bp tandem repeat of exon 1 is similar to the 48bp VNTR of exon 3 of DRD4, previously shown to be polymorphic in all primate species examined. The Old World monkeys show no variation in length, and a much higher conservation of amino acid sequence than great apes and humans. The New World monkeys show interspecific differ- ences in length in the region of the 12bp polymorphism, but otherwise show the higher conserva- tion seen in Old World monkeys. The different patterns of variation in monkeys compared to apes suggest strong purifying selective pressure on the exon in these monkeys, and somewhat different selection, possibly relaxed selection, in the apes. J. Exp. Zool. (Mol. Dev. Evol.) 288:32–38, 2000. © 2000 Wiley-Liss, Inc.

The dopamine D4 receptor is a 7-transmem- elty-seeking behavior (Benjamin et al., ’96; Ebstein brane G-protein-coupled found et al., ’96, ’97; Ono et al., ’97). in the limbic system, frontal cortex, and other ar- Among the numerous expressed polymorphisms eas of the brain (for reviews, see Strange, ’94; at DRD4, exon 3 contains a 48bp expressed VNTR, Seeman, ’95; Van Tol, ’96). It is one of three “D2- corresponding to a 16-amino-acid repeat in the like” dopamine receptors. The D4 receptor is be- third cytoplasmic loop (Lichter et al, ’93). Also in lieved to play a role in higher brain functions, such exon 3, a Val→Gly polymorphism at codon 194 is as affection and personality, and is a candidate found in some African populations (Seeman et al.,

gene for several behavioral disorders. The DRD4 ’94); the Gly194 allele is believed to result in an gene is located on 11p15.5 (Gelernter essentially nonfunctional protein (Liu et al., ’96). et al., ’92), and has four exons (Fig. 1). It harbors The coding region of exon 1 contains a polymor- a large amount of expressed polymorphism in hu- phic 12 bp (4 amino acid) tandem repeat with mans. Some of these allelic variants have been three alleles in humans, a 13bp deletion polymor- claimed to be associated with psychiatric disor- ders such as Tourette syndrome, delusional dis- order, attention-deficit hyperactivity disorder, and Grant sponsor: USPHS; Grant numbers: MH30929, MH39239, AA09379, and GM57672; Grant sponsor: National Science Council, obsessive-compulsive disorder with tics (Catalano Taiwan; Grant numbers: NSC86-2331-B006-004Y and NSC87-2312- et al., ’93; Grice et al., ’96; LaHoste et al., ’96; B006-009Y. A.S. Deinard’s current address: School of Veterinary Medicine, Uni- Cruz et al., ’97); substance abuse, including alco- versity of California, Davis, CA 95616. holism and heroin (George et al., ’93; *Correspondence to: Kenneth K. Kidd, Department of Genetics, Yale University School of Medicine, P.O. Box 208005, New Haven, Muramatsu et al., ’96; Kotler et al., ’97); and varia- CT 06520-8005. E-mail: [email protected] tion in normal human behavior, especially nov- Received 19 May 1999; Accepted 15 October 1999 © 2000 WILEY-LISS, INC. EVOLUTION OF DRD4 EXON 1 IN PRIMATES 33

Fig. 1. Schematic representation of the DRD4 gene, based sequencing primers used in this study are shown below the on Van Tol et al. (’92) and Kamakura et al. (’97). Locations of gene. For exact locations of the primers, compare the se- the four expressed polymorphic sites and the two rare vari- quences given in Table 1 to the genomic DNA sequence of ants are shown above the gene; the locations of the PCR and humans (GenBank accession number L12397). phic in Europeans, a rare 21 bp deletion, and a ined the homologous regions in several non-human rare nonsynonymous transversion in codon 11 primate species in order to accurately determine (Catalano et al., ’93; Nöthen et al., ’94; Cichon et the ancestral human allele (Iyengar et al., ’98). al., ’95; Chang and Kidd, ’97). The 13 bp and the Length variation has been regenerated several 21 bp deletions presumably result in nonfunc- times in the great apes in exon one, similar to exon tional proteins (Nöthen et al., ’94). Of the exon 1 three. Also, the patterns of nucleotide substitution polymorphisms, only the 12 bp repeat is polymor- observed suggest different selective constraints on phic worldwide (Chang et al., unpublished data). this region in humans and great apes compared to This polymorphism consists of a 12 bp unit other primates. In this paper, we use the term present once, twice, or rarely thrice. The result- “hominid” to refer to the Family Hominidae, includ- ing four amino acid duplication (GASA) occurs ing humans (Homo), chimpanzees (Pan), gorillas near the junction of the extracellular domain of (Gorilla), and orangutans (Pongo) as per Groves the protein and the first transmembrane domain (’86). While we realize that this usage is not uni- (Seeman et al., ’94). Exon one of DRD4 codes for versally accepted, it appears to be increasing in the extracellular amino terminus domain and the popularity, and furthermore is preferable to the first two transmembrane domains (Seeman, ’95). more traditional—but paraphyletic—usage of The extracellular domain has been shown to be Pongidae to include Pan and Gorilla. functionally important for binding and sig- MATERIALS AND METHODS nal transduction (Schoots et al., ’96). The evolution of DRD4 has been studied by com- DNA samples parative sequencing of the VNTR in exon three Genomic DNA of chimpanzees (Pan troglodytes; in several non-human primates. This VNTR was n = 11), bonobos or pygmy chimpanzees (P. panis- found to be polymorphic in many primate species, cus; n = 6), gorillas (Gorilla gorilla; n = 6), oran- both for the number of repeat units as well as for gutans (Pongo pygmaeus; n = 8), and spider monkey sequence variation among units. No alleles ap- (Ateles sp.; n = 1) was extracted by standard tech- peared to be shared between species; rather, the niques from whole blood or lymphoblastoid cell lines length polymorphism was apparently newly regen- established from blood generously provided by At- erated in each independent lineage examined lanta Zoo/Yerkes Regional Primate Center, New Ibe- (Livak et al., ’95; Matsumoto et al., ’95; Inoue- ria Regional Primate Center, Arizona Primate Murayama et al., ’98). Foundation, Henry Doorly Zoo, Sacramento Zoo, Here we report on the comparative sequencing Milwaukee County Zoo, and Miami Metro Zoo. Ge- of the coding region of exon one in several primate nomic DNA of the following gibbon species was ex- species. As part of ongoing studies of the 12 bp tan- tracted by standard techniques from lymphoblastoid dem repeat polymorphism in exon one of DRD4 cell lines generously provided by Dr. Johannes (Chang et al., ’97; Chang and Kidd, ’97), we exam- Weinberg: lar gibbon (Hylobates lar; n = 1), klossi’s 34 M.I. SEAMAN ET AL. gibbon (H. klossi; n = 1), and siamang (H. syndac- 100-µl PCR using the same primers. The sequence tylus; n = 1). DNA from proboscis monkey (Nasalis of the remaining primates was determined by am- larvatus; n = 1) and silver langur (Presbytis cristata; plifying genomic DNA with the degenerate primer n = 1) was extracted from liver tissue generously D4UPmod and D4DW2. An aliquot of this product provided by Dr. George Amato of the Wildlife Con- was re-amplified and sequenced with D4UPmod and servation Society. Genomic DNA of baboons (Papio D4EX1R, or with D4EX1F and D4EX1R. At times, hamadryas; n = 2) and squirrel monkeys (Saimiri the PCR product was excised from the gel and pu- boliviensis; n = 2) was generously provided by Dr. rified prior to sequencing because several bands Jeffrey Rogers of the Southwest Foundation for were present. Additional sequence was determined Biomedical Research. Genomic DNA of rhesus by amplifying genomic DNA with D4EX1D and macaques (M. mulatta; n = 7) was extracted from D4EX2R, and sequencing with D4EX1D. For most whole blood generously provided by Dr. Goldman- species only a single individual was sequenced, Rakic of the Yale University School of Medicine. although all of the different length alleles in the great apes were sequenced. Sequences were PCR-based typing of the exon 1 manually edited and nucleotide positions were length variant determined to be homozygous or heterozygous af- The exon 1 12 bp tandem repeat polymorphism ter visual comparison of sequence traces in both was typed using the PCR primers D4EX1F and directions with other individuals (Kwok et al., D4EX1R (Table 1) as described in Chang and Kidd ’94). All sequencing was done with the ABI Prism (’97) using genomic DNA of all of the great apes, cycle sequencing kit and run on an ABI373 auto- Old World monkeys, and New World monkeys. mated sequencer. Sequences were deposited in GenBank (accession numbers AF010294-AF010- DNA sequencing of exon 1 alleles 301, AF125662-AF125670). The sequences and locations of all primers used are given in Table 1 and Figure 1, respectively. Sequence analysis DNAs from humans, chimps, bonobos, gorillas, and The MEGA software program (Kumar et al., ’93) orangutans determined to be homozygous for the was used to count the numbers of nonsynonymous 12 bp repeat polymorphism using the above typing and synonymous substitutions between haplo- method were amplified and sequenced with the types, and to calculate the Jukes-Cantor distances primers D4EX1E and D4EX1DW, or with D4UP2 and standard errors of nonsynonymous and syn- and D4EX1DW, which flank exon 1. Additional se- onymous substitutions. quencing with D4EX1C and D4EX1BK1 was per- formed to resolve ambiguities. Since we detected RESULTS no gorillas or orangutans who were homozygous for All hominid (Family Hominidae, meaning the the short allele, those alleles were sequenced by ex- family comprised of great apes and humans) gen- cising the band from a 4% low-melting point agar- era possess alleles of exon one of more than one ose gel, melting in 100 µl dH2O for 10 min at 65°C, size (Fig. 2). Alleles of three lengths were observed and re-amplifying 1 µl of the melted solution in a in orangutans, identical in sizes to the three hu-

TABLE 1. Oligonucleotide primers used in this study

Primer name Sequence (5′ to 3′) Purpose D4EX1F CGCCATGGGGAACCGCAG Typing exon 1 12bp polymorphism, sequencing D4EX1R CGGCTCACCTCGGAGTAGA Typing exon 1 12 bp polymorphism, sequencing D4-3 GCGACTACGTGGTCTACTCG Typing exon 3 48bp VNTR D4-42 AGGACCCTCATGGCCTTG Typing exon 3 48bp VNTR D4UP2 CGCTCAGCTGCCGCCCAGTTT PCR D4EX1E AGGGACTCCCCGGCTTGC PCR, sequencing D4DW GAGCAGGGAGGATGCTCG PCR, sequencing D4EX1C GGGGGCGTGCTGCTCATC Sequencing D4BK1 GAGCACCGCGCCGATGAG Sequencing D4UPmod CGYTGYCYGCGGTGCTCAG PCR, sequencing D4DW2 TGCGGCCGGACGCGGCTCAC PCR, sequencing D4EX1D GCGTGCTGCTCATCGGCG PCR, sequencing D4EX2R AGCCACRCGCCACCCTGGAC PCR EVOLUTION OF DRD4 EXON 1 IN PRIMATES 35 occurred within and on either side of the repeat, indicating that the length variation has been re- peatedly regenerated within each taxon, rather than the individual alleles having persisted across species. No length variation was observed within or between any of the cercopithecoid species (Old World monkeys), although—except for rhesus macaques (2n = 14 )—only one or two individuals per species were examined. Among the platyrrhines (New World primates), the spider monkey’s coding region of exon 1 is 12 bp shorter than the short allele in hominids, and the squir- rel monkey has 39 bp, or 13 amino acids, less than the hominid short allele. Fig. 2. PCR products obtained from primates using the Figure 3 gives the inferred amino acid sequence primer pair D4EX1F/D4EX1R, showing the sizes of all ob- of exon 1 for the 20 primate alleles identified, as served alleles. The upper band in the heterozygous gorilla and orangutan is composed of heteroduplex molecules. Gib- well as that inferred from the previously published bon DNA does not amplify with these primers because of a rat and mouse sequences (O’Malley et al., ’92; nucleotide substitution under the 3′ end of the forward primer. Fishburn et al., ’95; GenBank accession numbers M84009, U19880). All of the insertions/deletions and most of the amino acid substitutions occur in man alleles (hereafter referred to as short, long, the first half of the exon, corresponding to the and extra long). Gorillas were observed to possess amino-terminus extracellular tail. Many of the alleles identical in length to the human short and substitutions are nonconservative (e.g., G→W at long alleles, while common chimpanzees and codon 11 in orangutans), and at least three sites bonobos are each apparently monomorphic at exon (codons 9, 11, and 19) have been repeatedly one, but for different alleles, possessing only alle- changed. The P→Q substitution in the great apes les identical in length to the human long and short and humans at amino acid 37 in the alignment alleles, respectively. Sequencing of each different occurs at the point where the peptide chain pre- allele confirmed that all of the length variation sumably enters the cellular membrane (Seeman in all of the great apes occurred in the same re- et al., ’94). gion as the 12 bp tandem repeat polymorphism Outside of the tandem repeat region there are in humans. Several shared changes have more nonsynonymous than synonymous substitu-

Fig. 3. Amino acid translation of DNA sequence of exon identity to the human XL allele. Alignment in the duplica- 1 in primates and the rat and mouse. Allele sizes are abbre- tion region is somewhat arbitrary, and furthermore, not viated “S,” “L,” and “XL” for short, long, and extra long, re- meant to imply strict homology between aligned repeat units spectively. Dashes indicate deletions and dots indicate between species. 36 M.I. SEAMAN ET AL. tions between haplotypes in the hominids, while differently in different lineages. Hominids are among all other primate clades examined there characterized by relaxed selection, while Old are more synonymous than nonsynonymous sub- World and New World monkeys are characterized stitutions (Table 2). Only one amino acid position, by strong purifying selection on this exon. Most at codon 11, varies among the cercopithecoids (Fig. of the amino acid substitutions in the hominids 3). Taking into account the sequence composition are found in the amino-terminus of the protein, of the exon, the average ratio of the rate of corresponding to the extracellular domain—a re- nonsynonymous substitutions per nonsynonymous gion shown to be important for the binding of

site (dN) to the rate of synonymous substitution dopamine and signal transduction (Schoots et al., per synonymous site (dS) is 0.44 for all pairwise ’96). This apparent onset of relaxation of selec- comparisons between observed alleles in the homi- tion on the amino-terminus of this protein in nids, and only 0.13 in the cercopithecoids. The null hominids seems to have occurred concomitantly hypothesis of neutral evolution of exon 1 cannot with the P→Q amino acid substitution at the junc- be rejected for the hominids, but is rejected for tion of the extracellular and transmembrane do- the cercopithecoids (one-tailed t-test, P < 0.05; mains. As proline residues are often involved in Kumar et al., ’93), suggesting that purifying se- conferring rigid structural turns in peptide chains, lection has maintained the amino acid sequence the loss of a proline here in hominids may have in these monkeys. The repeat region is excluded repositioned the extracellular domain in a way in these calculations. that decreased its functional relevance to the over- all function of the protein. DISCUSSION It is tempting to speculate that DRD4 may be Exon one of the DRD4 gene exhibits polymor- responsible for some of the remarkable changes phism in humans, gorillas, and orangutans. Re- involved in the evolution of the hominid brain, as current, or parallel, have occurred it has been suggested that this gene in humans repeatedly in several primate lineages generat- may be involved in higher thought processes, in- ing homoplasy in both repeat length and in amino cluding personality (Benjamin et al., ’96; Ebstein acid substitutions. The recurrent in the et al., ’96, ’97). Other studies have not supported 12 bp repeat region makes reconstruction of the that hypothesis (Malhotra et al., ’96; Pogue-Geile evolutionary pathway between existing haplotypes et al., ’98). In any case, some domains of this re- problematic, if not impossible. Orangutans appear ceptor may be functioning differently in Old World to possess alleles that are identical by state in and New World monkeys than in humans and size, but not identical by descent. It is certainly other apes, as inferred from the different patterns possible that additional intraspecific variation in of natural selection observed. any of the species might be found if more indi- Finally, although only a portion of this gene was viduals were examined. examined, the effective use of many primate taxa Different patterns of nucleotide substitutions in here reinforces the advantages of the “bushy tree” the different primate lineages suggest that natu- approach to comparative sequencing, as exempli- ral selection on exon one of DRD4 is operating fied by previous studies of primate molecular evo-

TABLE 2. Numbers of nonsynonymous and synonymous substitutions observed in exon one1 Nonsynonymous Synonymous substitutions2 substitutions2 Comparison with hominids

Hominids 90 84 — Gibbons 0 6 χ2=6.21, P < 0.025 Cercopithecoids 7 23 χ2=8.27, P < 0.005 Platyrrhines 0 6 χ2=6.21, P < 0.025 Cercopithecoids and platyrrhines 45 96 χ2=12.48, P < 0.001 Non-hominids 109.5 234 χ2=19.20, P < 0.001 1The 12 bp repeat region is excluded in these calculations. The observed ratios did not differ significantly between gibbons and cercopithecoids, cercopithecoids and platyrrhines, or gibbons and platyrrhines. The significance values given in the table are for each individual two-by-two comparison. However, the comparisons between hominids and gibbons and between hominids and platyrrhines are no longer significant after applying Bonferroni techniques to adjust the critical values for each of the eight individual comparisons in order to create an experimentwise error rate of P < 0.05 (Sokal and Rohlf, ’95). This is not surprising given this conservative approach and the small numbers involved in comparisons with gibbons and platyrrhines. 2Total numbers of substitutions between all haplotypes observed within each group. EVOLUTION OF DRD4 EXON 1 IN PRIMATES 37 lution (Messier and Stewart, ’97; Wu et al., ’97). Lichtermann D, Minges J, Albus M, Borrmann M, Granzek The use of many taxa allows better definition of E, Stöber G, Weigelt B, Körner J, Rietschel M, Propping P. → 1995. Identification of two novel polymorphisms and a rare “conserved” amino acids. For example, a Gly Arg deletion variant in the human dopamine D4 receptor gene. variant at codon 11 of DRD4 was identified in hu- Psychiatr Genet 5:97–103. mans (Cichon et al., ’95). Since mice also have a Cruz C, Camarena B, King N, Páez F, Sidenberg D, Ramón glycine at codon 11, a human-mouse comparison de la Fuente J, Nicolini H. 1997. Increased prevalence of would suggest that this site has been highly con- the seven-repeat variant of the dopamine D4 receptor gene in patients with obsessive-complsive disorder with tics. served for over 70 million years, and therefore that Neurosci Lett 231:1–4. this amino acid was essential for proper dopamine Ebstein RP, Novick O, Umansky R, Priel B, Osher Y, Blaine receptor function. However, this glycine has been D, Bennet E, Nemanov L, Katz M, Belmaker R. 1996. substituted at least three times independently in Dopamine D4 receptor (D4DR) exon III polymorphism as- different primate lineages (Fig. 3), including a sociated with human personality trait of novelty seeking. → Nat Genet 12:78–80. Gly Arg change in two Old World monkey species Ebstein RP, Nemanov L, Klotz I, Gritsenko I, Belmaker RH. indicating that the rare Arg11 allele in humans is 1997. Additional evidence for an association between the more likely to be functionally normal. dopamine D4 receptor (D4DR) exon III repeat polymorphism The function of DRD4 has previously been ex- and the human personality trait of novelty seeking. Mol amined through the use of in vitro studies, his- Psychiatr 2:472–477. Fishburn CS, Carmon S, Fuchs S. 1995. Molecular cloning tology, laboratory animals, population genetics, and characterisation of the gene encoding the murine D4 and disease association studies. Here we employ dopamine receptor. FEBS Lett 361:215–219. a comparative sequencing approach toward un- Gelernter J, Kennedy JL, Van Tol HHM, Civelli O, Kidd KK. derstanding the function of exon 1 of DRD4, and 1992. The D4 dopamine receptor (DRD4) maps to distal 11p the evolutionary history that has shaped it. close to HRAS. Genomics 13:208–210. George SR, Cheng R, Nguyen T, Israel Y, O’Dowd BF. 1993. 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