MBE Advance Access originally published online on October 13, 2007
Molecular Biology and Evolution 2008 25(1):18-28; doi:10.1093/molbev/msm219
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Research Articles |
Sequence Variation in the Primate Dopamine Transporter Gene and Its Relationship to Social Dominance
* Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh
E-mail: cbutterworth{at}hgen.pitt.edu.
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Dopaminergic activity differs between socially dominant and subordinate monkeys, and in humans, it correlates significantly with extraversion, a trait analogous to social dominance in monkeys. Furthermore, concentrations of monoamine metabolites within the cerebrospinal fluid are highly heritable. Dopaminergic activity is modulated by the dopamine transporter (DAT), and the gene encoding this transporter is therefore an excellent candidate for studies aiming to identify variants of functional or evolutionary significance. However, the majority of such research has focused exclusively on the human homologue and its most common polymorphism, a functional variable number tandem repeat in the 3' untranslated region. Cross-species comparisons provide valuable insights into genome evolution, speciation, and selection mechanisms and may highlight sites of evolutionary significance. To date, however, no comprehensive studies of the DAT gene have been performed simultaneously on multiple primate species. We therefore characterized sequence variation and extent of linkage disequilibrium (LD) across the DAT genes of cynomolgus macaques (Macaca fascicularis), rhesus macaques (Macaca mulatta), and humans. We identified 2 potentially functional variants, which are associated with social rank in cynomolgus monkeys and which correspond to a putative transcription factor–binding site. Although highly conserved across mammals, the DAT gene differs significantly between humans and macaques in levels of sequence variation and LD structure, with the monkeys displaying up to 3 times more sequence variability and significantly less LD than humans. This suggests that the DAT gene has followed different evolutionary trajectories during primate speciation.
Key Words: Macaca • dopamine transporter • linkage disequilibrium • dominance • sequence variation • primate
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Introduction |
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The long-term survival of most group-living species, in particular the primates, depends on cooperation and mutual support. In the context of competition for scarce resources, many species have evolved asymmetrical antagonistic relationships, leading to the formation of dominance or status hierarchies. Such hierarchies are suggested to maintain group cohesion by imposing predictability and reducing overt aggression. Although patterns of behavioral interaction between dominant and subordinate individuals may initially be influenced by the presence of relatives and subsequently modified by experience, behavior also has a strong genetic component. Genetic factors account for 40–60% of the variance in both human and nonhuman primate personality traits (Weiss et al. 2000
; Van Gestel and Van Broeckhoven 2003; Williamson et al. 2003; Fairbanks et al. 2004).
The dopaminergic system plays an important role in cognitive development and modulation of behavior (Montague et al. 2004). However, little is known about how functional polymorphisms in genes crucial to basic physiology, such as those of the dopaminergic system, influence neurotransmitter activity and associated behavior patterns. Synaptic availability of dopamine and duration of dopaminergic neurotransmission are regulated by the dopamine transporter (DAT), which actively removes excess neurotransmitter from the synaptic cleft. The gene encoding human dopamine transporter (SLC6A3 or hDAT) maps to chromosome 5p15.33 and spans 52.6 kb. There is a functional 40-bp variable number tandem repeat (VNTR) in the 3' untranslated region (UTR) of hDAT (Vandenbergh et al. 1992), certain alleles of which may affect translation of the transporter protein. Numerous studies have attempted, with mixed success, to associate alleles of this VNTR with personality traits and disorders, including attention deficit hyperactivity disorder, bipolar disorder, alcoholism, and schizophrenia (Grunhage et al. 2000; Greenwood et al. 2002; Ueno 2003; Fanous et al. 2004; Kim et al. 2005).
Correlations between dopaminergic activity, social rank, and related behaviors have been reported for several primate species (Yodyingyuad et al. 1985; Grant et al. 1998; Shively 1998; Kaplan et al. 2002; Morgan et al. 2002). In particular, dominant cynomolgus macaques (Macaca fascicularis) of both sexes tend to have higher levels of the dopamine metabolite, homovanillic acid in cerebrospinal fluid than do subordinates (Kaplan et al. 2002). Subordinate females also exhibit diminished prolactin responses to the dopamine antagonist, haloperidol (Shively 1998), possibly due to decreased dopamine D2 receptor function. In humans, dopamine levels correlate significantly with extraversion (Depue and Collins 1999), the personality trait analogous to social dominance in monkeys (Kaplan et al. 2002; Capitanio and Widaman 2005).
It should be noted that an animal's social rank does not represent a particular behavioral trait (Bernstein 1981). Rather, it is an outcome that depends on multiple factors, including personality, early life experiences, physiology, and the immediate social environment (Bernstein 1981). However, individuals of particular social ranks consistently exhibit characteristic behaviors and personality traits (Capitanio 1999). Furthermore, captive cynomolgus macaques of both sexes tend to retain their relative dominance status even when assigned to different social groups (Kaplan et al. 1990; Shively and Kaplan 1991). In addition, many behavioral, personality, and physiological traits have strong genetic components, including those associated with the dopaminergic system (Weiss et al. 2000; Van Gestel and Van Broeckhoven 2003; Williamson et al. 2003; Montague et al. 2004; Fairbanks et al. 2004).These, in turn, may predispose an individual to assume a particular rank under a given social situation.
Concentrations of monoamine metabolites within the cerebrospinal fluid are also highly heritable (Rogers et al. 2001). To date, however, the majority of DAT studies have focused solely on the human 3'UTR VNTR. Only a handful of systematic searches for additional potentially functional variants across DAT has been reported, and all focused exclusively on the human homologue (Grunhage et al. 2000; Vandenbergh et al. 2000; Greenwood et al. 2001, 2002, 2006). Cross-species comparisons provide valuable insights into genome evolution, speciation, and selection mechanisms and can identify common variants, thereby highlighting sites that are potentially of functional or evolutionary importance. However, no comprehensive studies of DAT have been performed simultaneously on multiple primate species.
This study, therefore, aimed to compare sequence variation and linkage disequilibrium (LD) structure across the entire coding and regulatory regions of the DAT genes of macaques and humans. Furthermore, the relationship between social rank differences and polymorphisms in candidate genes of the dopaminergic pathways has not been addressed adequately. We therefore also aimed to determine whether variation in the macaque DAT gene is significantly associated with social rank differences between the monkeys.
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Materials and Methods |
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Samples
The primary study animals were 523 unrelated cynomolgus macaques, M. fascicularis (204 males and 319 females) imported as adults from Indonesia (Institute Pertanian Bogor, Bogor, Indonesia), where they were captured in the wild. These monkeys formed part of several ongoing studies conducted from 1985 to 2005. Venous blood specimens were collected in ethylenediaminetetraacetic acid (EDTA) vacutainers, and aliquots of whole blood were frozen at –70 °C. Liver samples were collected from animals when they were necropsied at the conclusion of the studies. All procedures were conducted in accordance with federal and state regulations and with the approval of the Wake Forest School of Medicine's Animal Care and Use Committee. Sections of liver were frozen in liquid nitrogen and stored at –70 °C. The Wizard Genomic DNA Purification Kit (Promega, Madison, WI) or the QIAamp DNA mini kit (Qiagen, Valencia, CA) were used to extract DNA, according to the manufacturers’ instructions. Extracted DNA samples were whole genome amplified using the Repli-g Kit (Qiagen), quantitated with the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Eugene, OR), and diluted to 40 ng/µl with molecular grade water.
Samples were also obtained from 243 rhesus macaques, Macaca mulatta (DNA kindly supplied by Leslie Lyons, University of California, Davis, CA), and from 23 human volunteers (91% Caucasian), who were members of our department and from whom we obtained informed consent. The rhesus monkeys were founder individuals of the UC Davis colony and include individuals of both full Indian and Chinese ancestry, as well as some of mixed ancestry, and are presumed to be largely unrelated. All DNA samples were originally obtained from blood sources. For each of the human subjects, 10–25 ml blood were collected into EDTA vacutainer tubes and centrifuged for 15 min at 2,800 rpm to remove the buffy coat. DNA was extracted from buffy coat cells by a salting-out protocol (Miller et al. 1988). DNA pellets were resuspended in 1x Tris–EDTA, quantitated, and diluted to 40 ng/µl as described above.
Social Rank Data Collection
The methods used to collect social rank data from the monkeys have been described previously (Kaplan et al. 2002). Briefly, dominance observations were conducted as follows. Monkeys were housed in unisex groups composed of, on average, 5 individuals. Each group was observed twice per week for a minimum of 28 weeks. Social interactions such as fight wins or losses, grooming, and passive body contact were recorded with the aid of a handheld computer. Dominant animals are often more aggressive; they initiate fights, use attack gestures, actions, and vocalizations more frequently, and consistently defeat individuals of lower rank (subordinates) during such agonistic encounters (Kaplan and Manuck 1998; Capitanio 1999; Manuck et al. 2006). In contrast, subordinates display more gestures and vocalizations associated with submission and will cower or flee from dominant animals (Capitanio 1999). Social rank for each observational event was therefore assigned according to the outcome of dyadic agonistic encounters. Animals that won all such interactions were assigned the highest social rank of 1; individuals that defeated all but the first ranking animals were assigned second rank, and so forth. Animals categorized as dominant were those that, on average, occupied a social rank of 1 or 2, whereas subordinate animals occupied ranks of 3–5. The monkeys were maintained in the same social groups throughout the rank assessment period. However, previous studies have shown that these rankings remain stable over time, even when animals are reorganized into different groups (Kaplan et al. 1990; Shively and Kaplan 1991; Shively et al. 2005). For the purposes of this study, DNA was obtained from only the highest (n = 242) and lowest (n = 281) ranking animals, drawn from a parent cohort of 986 animals, which included all intermediate ranks. No social rank data were available for the rhesus macaques, so it was not possible to test for association in this species.
Primer Design
Fifty cynomolgus macaques (23 dominant and 27 subordinate) were selected initially to characterize genetic variation in the coding region and UTR of the cynomolgus DAT gene (mfaDAT). Each of the 15 exons was amplified individually by the polymerase chain reaction (PCR) and then sequenced to identify and characterize polymorphisms. We focused on coding and regulatory regions because they were more likely to contain functional variation. Primers for PCR and sequencing (supplementary table S1 and fig. S1, Supplementary Material online) were designed based on the M. mulatta genome trace sequence. The complete genomic sequence (ca. 60 kb) of the hDAT gene was downloaded from the University of California, Santa Cruz, Human Genome Web site, hg16 assembly. Each exon of hDAT was individually compared with the M. mulatta trace sequence using a discontiguous MegaBlast (Altschul et al. 1997) search for cross-species comparisons. All returned sequences that aligned significantly with the hDAT exons were aligned using Sequencher v.4.6 (Gene Codes Corporation, Ann Arbor, MI) and a consensus M. mulatta sequence obtained. This was used to design intronic primers spanning each exon using Primer3 (Rozen and Skaletsky 2000) and NetPrimer (Premier Biosoft International, Palo Alto, CA). Where necessary, additional human-specific primers (supplementary table S1, Supplementary Material online) were designed based on the hDAT sequence from the hg16 assembly.
PCR and Sequencing
PCRs were performed on MJ Research PTC-100 and PTC-225 DNA Engine Tetrad Peltier Thermal Cyclers (Bio-Rad Laboratories, Waltham, MA) in 25 µl volumes. Most PCRs contained 0.5 U DNA polymerase (consisting of a 9:1 mix of MasterTaq [Eppendorf, Westbury, NY] to Native Pfu DNA polymerase [Stratagene, La Jolla, CA]), 1x MasterTaq reaction buffer (final concentrations: 1.5 mM Mg(OAc)2, 50 mM KCl, 10 mM Tris–HCl), 0.2 mM each deoxynucleotide triphosphate (dNTP) (Invitrogen, Carlsbad, CA), 0.4 µM each primer, and 40–80 ng template DNA. Amplification of some exons required use of HotMaster Taq DNA polymerase (Eppendorf) or of additives such as TaqMaster PCR Enhancer (Eppendorf), 5% dimethyl sulfoxide, or 20 ng/µl bovine serum albumin (supplementary table S1, Supplementary Material online). A typical PCR cycle consisted of 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at the optimal annealing temperature (supplementary table S1, Supplementary Material online), and 60–90 s at 72 °C, with a final extension step of 7 min at 72 °C. In some cases (supplementary table S1, Supplementary Material online), a touchdown PCR protocol was used, consisting of 3 min at 94 °C, followed by 10 cycles in which the annealing temperature was reduced by 2 °C every 2 cycles (30 s at 94 °C, 30 s at the annealing temperature, and 60 s at 72 °C), followed by 30 cycles at the lowest annealing temperature, and a final extension step of 7 min at 72 °C.
The PCR products were subjected to electrophoresis through a 1.5% agarose gel in 1x sodium hydroxide/boric acid buffer (Brody and Kern 2004) to ensure a single, clean product of the correct size had been amplified. Length alleles of the hDAT 3'UTR VNTR were identified by electrophoresis of PCR products (amplified with primers 15F10 and 15L12; supplementary table S1 and fig. S1, Supplementary Material online) through a 4% agarose gel. Unincorporated dNTPs were removed from amplified products by an "ExoSAP" reaction containing 2.5 µl PCR product, 0.5 U shrimp alkaline phosphatase (SAP) (USB Corporation, Cleveland, OH), 0.5x SAP reaction buffer, and 0.5 U exonuclease I (USB Corporation). The 10 µl reaction mix was incubated at 37 °C for 35 min, followed by 88 °C for 15 min to inactivate the enzymes. Products were cycle sequenced in both directions with the same primers used for PCR, using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), according to the manufacturer's instructions. They were then subjected to electrophoresis through an ABI 3730 DNA analyzer (Applied Biosystems).
Each sequence was independently examined by 2 people. These scorers disagreed on the validity of only 6 polymorphic loci in macaques, and these were subsequently eliminated from the data set. Genotype concordance across the remaining variants was 99.73%. Although no pseudogene or paralogue has been reported for DAT, it is possible that the high number of polymorphisms we identified in mfaDAT and mmuDAT was due to erroneous amplification of a pseudogene. To confirm that this was not the case, we performed a BLAST (Altschul et al. 1997) search on every exon, which matched each of them with very high likelihood to DAT. In addition, there were no stop codons or frameshift mutations in the coding sequences and no excess of nonsynonymous mutations over synonymous ones. Furthermore, each of the 15 exons was amplified independently, producing only a single PCR band.
Genotyping in the Full Cohort
The majority of the variants that we identified in the macaque DAT gene were synonymous, were located in the noncoding 3'UTR, or were considered too rare to be able to influence social rank significantly. However, 2 sites in the 5'UTR were identified as potentially functional variants and showed a trend toward association with social rank in the subsample of 50 animals. These 2 single nucleotide polymorphisms (SNPs) were therefore genotyped in the full cohort by custom TaqMan genotyping assays (Applied Biosystems) or fluorescence polarization (FP). TaqMan assays were conducted according to the manufacturer's instructions. Initial PCRs for the FP assays were performed in 10 µl reaction volumes containing 60 ng template DNA, 25 µM dNTPs, 50 nM each primer (supplementary table S1, Supplementary Material online), 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), and 0.5 U Taq DNA polymerase (Invitrogen) under the following reaction conditions: 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, and a final extension of 72 °C for 5 min. Unincorporated dNTPs and primers were removed by an "ExoSAP" reaction, as described above. The detection reaction (20 µl) contained 0.4 U ThermoSequenase DNA polymerase (Amersham Biosciences, Piscataway, NJ), 1x ThermoSequenase buffer, 1 µM detection primer, 125 nM dye dNTPs (1:16 dilution of Rhodamine 110 [A allele]:Tamra [G allele]), and 10 µl PCR product. Cycling was performed on a TaqMan 7900HT (Applied Biosystems) under the following conditions: 94 °C for 1 min, 35 cycles of 94 °C for 10 s, and 47 °C for 30 s. Products were genotyped using Allele Caller software (deCODE Genetics, Reykjavik, Iceland).
Quantitative PCR
Thirty cynomolgus females were selected according to their diplotypes at the two 5'UTR SNPs that were associated with social rank (ACGC: n = 15, ACAT: n = 6, and GCGC: n = 9). Unfortunately, many of the original cohorts of 523 animals had died prior to initiation of this component of the study; therefore, additional individuals with these and other diplotypes were not available for quantitative PCR analysis. Blood (3 ml) was collected from each female using Tempus vacutainer tubes (Applied Biosystems), and RNA was extracted according to the manufacturer's instructions. Reverse transcription into cDNA was performed with the High Capacity cDNA Archive Kit (Applied Biosystems), followed by preamplification of DAT (necessitated by its low expression in blood) and the endogenous control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the TaqMan PreAmp Master Mix Kit (Applied Biosystems). TaqMan assays were performed on a TaqMan 7900HT instrument, according to the manufacturer's instructions, using custom-designed primers and probe for mfaDAT (supplementary table S1, Supplementary Material online) and an existing assay for human GAPDH (Hs00266705_gl, Applied Biosystems).
Statistical Analysis
Sequencher v.4.6 (Gene Codes Corporation) was used to edit and produce sequence contigs for each species and to identify SNPs and insertions/deletions (indels). Multispecies sequence alignments were performed in ClustalX (Thompson et al. 1997) and edited manually with BioEdit (Hall 1999). Allele frequencies were tested for deviations from normality with Anderson–Darling tests and for equal variances with both an F-test and Levene's test, implemented in Minitab v.14.2 (Minitab Inc, State College, PA). Genotypes were normally distributed and had equal variances.
Logistic regression, implemented in R v.2.1.0 (R Development Core Team 2005), was used to determine whether genotype at each of the 2 cynomolgus macaque SNPs of interest was significantly correlated with social rank and whether there was a significant interaction between sex and genotype. The SNPs were examined separately because they were not in complete LD with one another. A significant interaction between sex and genotype was identified at SNP 5U+93 (see Results); therefore, the logistic regressions were repeated for each sex independently at this site.
Our cynomolgus macaques were captured in the wild on multiple independent occasions, over a period of 
20 years, and therefore, the likelihood of a significant number of them being related is low. However, because the exact origins of most of the individuals are unknown, there remains a possibility that a subset of individuals may be related or that there may be unidentified substructure in our sample. Both relatedness and substructure can confound association studies, leading to false-positive associations (Bacanu et al. 2002). We therefore also subjected our data set to genomic control (GC) analysis (Devlin and Roeder 1999), which controls for false positives due to the effects of relatedness, population heterogeneity, and multiple testing (Devlin and Roeder 1999; Bacanu et al. 2002; Devlin et al. 2004). We employed 2 GC approaches. Frequentist GC (GCF) estimates one or more inflation factors from a set of "null" loci and then uses these estimates to test for effects at 1 or 2 loci, in relation to a response variable, which in this case is social rank status (dominance/subordinance). The second approach, Bayesian GC (GCB), uses a Bayesian outlier test to identify loci that display significant LD with the trait of interest (Devlin and Roeder 1999; Bacanu et al. 2002; Devlin et al. 2004). Because the SNPs identified in this study are all located within a single gene, they are likely to be correlated with one another. We therefore used only those SNPs found to be in weakest LD with one another (n = 38) for the GC analyses, as well as an additional 10 SNPs identified in the macaque serotonin transporter gene (Miller-Butterworth et al., 2007).
Haplotypes and diplotypes were reconstructed using PHASE v.2.1 (Stephens et al. 2001). Haplotype structure and extent of LD across the gene were examined using Haploview v.3.32 (Barrett et al. 2005) and Entropy Blocker (Rinaldo et al. 2005). Only polymorphisms with minor allele frequencies (MAFs) 
10% were included in these analyses. All mmuDAT and hDAT variants and all but 2 mfaDAT polymorphisms were in Hardy–Weinberg equilibrium (HWE), and these 2 were excluded from the LD analyses. Searches for transcription factor–binding sites were conducted using MatInspector (Genomatix Software Ann Arbor, MI).
An omnibus test was implemented in eHap (Seltman et al. 2001, 2003) to determine whether haplotype status was significantly associated with social rank. This software package utilizes generalized linear models to account fully for haplotype phase ambiguity (Seltman et al. 2003). Chi-square analyses, implemented in R, were used to determine whether each of the 6 diplotypes (ACAT, ACAC, ACGC, ATAT, ATGC, and GCGC; supplementary fig. S3, Supplementary Material online) was significantly associated with social rank. To correct for multiple testing, adjusted P values were obtained by permutation testing (10,000 replicates).
Quantitative PCRs
One sample was included as a positive control on all plates. Standard curves covering 6-log dilution series confirmed that the mfaDAT and GAPDH assays had equal PCR efficiencies. Expression differences of each sample relative to the control were therefore calculated according to the 
CT method (Applied Biosystems 2001). They were tested for deviations from normality with Anderson–Darling tests and for equal variances with both an F-test and Levene's test, implemented in Minitab. Relative expression differences were compared among diplotypes with 1-way analysis of variance or, where necessary, with a nonparametric Kruskal–Wallis test, again using Minitab.
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Results and Discussion |
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Sequence Variation and LD
We identified 78 polymorphisms in 3,917 bp of mfaDAT exonic sequence, including 3 indels and 75 SNPs, producing an average of one SNP every
50 bp (table 1; supplementary table S2 and fig. S2, Supplementary Material online). Only 2 coding SNPs were nonsynonymous, located at amino acids 54 (proline to threonine) and 242 (alanine to serine); however, only a single heterozygote was identified in each case. The VNTR present in the 3'UTR of hDAT (Vandenbergh et al. 1992) was monomorphic (12 repeats) in cynomolgus macaques.
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These high levels of variation in mfaDAT contrast with previous reports (Grunhage et al. 2000
) and GenBank records (The Single Nucleotide Polymorphism database, [dbSNP], build 127) for the human homologue. There are currently 48 exonic SNPs reported for hDAT, of which 27 are validated, and 16 of these are in the 3'UTR. To determine whether these comparatively low levels of hDAT variation are accurate, we resequenced the entire exonic region of hDAT from 23 people and, as a further source of comparison, from 23 rhesus macaques (mmuDAT).
Sequence identity between mfaDAT and mmuDAT was 99.67%, whereas that between hDAT and mfaDAT and between hDAT and mmuDAT were 89.90% and 89.92%, respectively (supplementary fig. S2, Supplementary Material online). Variation in mmuDAT was similar to mfaDAT. The 23 rhesus monkeys had 53 polymorphisms (49 SNPs and 4 indels) in 3,920 bp, giving an average of 1 SNP every 75 bp (table 1; supplementary table S2, Supplementary Material online). One SNP at amino acid 198 was nonsynonymous, producing a neutral ionic change from glycine to serine. As with mfaDAT, all individuals had 12 repeats in the 3'UTR VNTR. The macaque species shared 1 indel and 10 SNPs, 7 of which were in the coding region.
In contrast, the 23 hDAT sequences contained only 13 SNPs and 1 VNTR (9 and 10 repeat alleles were identified) in 3,933 bp, giving an average of 1 SNP every 300 bp (table 1; supplementary table S2, Supplementary Material online). Three coding SNPs, the VNTR, and five 3'UTR SNPs match existing dbSNP records; the remaining 5 SNPs have not been reported previously. Our consensus messenger RNA (mRNA) sequence differed from the GenBank reference (NM_001044) at 8 invariant sites (table 1; supplementary fig. S2, Supplementary Material online). Accordingly, if these sites are considered variable, the number of hDAT polymorphisms increases to 1 SNP per 180 bp, but this remains less than one-third to one-half that identified in the monkeys. Humans shared one coding SNP (amino acid 38, dbSNP: 6350) and one 3'UTR SNP with cynomolgus macaques (not in dbSNP) and two 3'UTR SNPs with rhesus monkeys, one of which is in dbSNP (3797200). No SNPs were common to all 3 species (supplementary fig. S2 and table S2, Supplementary Material online).
There was no significant long-range LD in mfaDAT (fig. 1; supplementary table S3, Supplementary Material online). The single indel and 26 SNPs with MAFs 10% and in HWE produced 85 different haplotypes, with maximum frequency of 3.0% in the 50 animals. There were 2 short-range blocks of moderate to strong LD (fig. 1; supplementary table S3, Supplementary Material online). One linkage block of approximately 2.4 kb extended from the 5'UTR to exon 2 (logarithm of odds [LOD] = 3.810, D’ = 0.780, r2 = 0.343) and a second of approximately 1.7 kb corresponded to the 3'UTR (mean LOD = 6.028 ± 3.574, mean D’ = 0.814 ± 0.178, r2 = 0.403 ± 0.213). With the exception of a few high scores between individual SNP pairs (supplementary table S3, Supplementary Material online), there was no significant LD across the coding part of the gene. Mean scores for this region were LOD = 0.381 ± 0.448 (range 0–1.980), D’ = 0.377 ± 0.345 (range 0–1.000), r2 = 0.027 ± 0.031 (range 0–0.122).
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Even less LD was evident in mmuDAT (fig. 1; supplementary table S4, Supplementary Material online). Based on 22 SNPs with MAF
10%, only 30 of 253 pairwise analyses produced LOD scores 2.0, 14 of which were in the coding region. No linkage blocks were evident, and pairwise comparisons producing relatively high LOD scores were distributed randomly across the gene. The highest scores (LOD = 7.420, D’ = 1.000, r2 = 1.000) were between SNPs located only 380 bp apart in the 3'UTR. In the coding region, the highest scores were between 2 SNPs in exons 8 and 12, approximately 12 kb apart (LOD = 5.060, D’=1.000, r2 = 0.711), but there was minimal LD between any intervening SNPs.
In contrast, hDAT displayed strong LD, particularly in the 3'UTR (fig. 1). However, only one SNP in exon 2 had MAF 10%, and although it was in moderate LD with most of those in the 3'UTR (mean LOD = 2.840 ± 1.165, maximum LOD = 3.480, mean D’ = 0.785 ± 0.138, mean r2 = 0.403 ± 0.158), there are insufficient data to obtain a detailed representation of the LD across the remaining coding regions of hDAT. With the exception of 3U+2191, all SNPs within the 3'UTR were in strong LD, extending across 1,753 bp (mean LOD = 7.908 ± 1.193, maximum LOD = 9.060, mean D’ = 1.000 ± 0.00, mean r2 = 0.914 ± 0.093).
Previous studies (Greenwood et al. 2002, 2006) have reported strong segmental LD across hDAT, with linkage blocks extending over at least 5 kb in the 5' end and 18 kb from exon 9 through the 3'UTR. These are supported by data from the HapMap Project (The International HapMap Consortium 2003), which indicate that in all populations examined, hDAT displays a large haplotype block of approximately 20 kb between introns 3 and 6. In addition, European and African populations have a second block of 9–14 kb extending from exon 9 to the 3'UTR.
Our results are consistent with dbSNP records and previous reports (Grunhage et al. 2000) of low hDAT variability and suggest that hDAT contains significantly less variation than Old World monkey (OWM) homologues. Our results also agree with previous reports that chimpanzees display less LD than humans (Fischer et al. 2004). Furthermore, other studies of OWMs, such as baboons (Rogers and Kidd 1993; Wang et al. 2004), and of great apes (Ruvolo 1997; Kaessmann et al. 1999, 2001; Jensen-Seaman et al. 2001; Fischer et al. 2004) have similarly reported that these species display 2–3 times greater nucleotide sequence diversity than humans. Hernandez et al. (2007) found slightly lower levels of variability in rhesus macaques compared with our study (7.25 SNPs/kb in Chinese macaques), but this was based on only 9 individuals and remains considerably higher than humans. Furthermore, although Indian rhesus macaques reportedly display longer range LD than humans, possibly due to admixture, Hernandez et al. (2007) found that Chinese rhesus macaques display very little LD, even among closely positioned SNPs. Our results are consistent with these findings. Of the 23 rhesus macaques included in the LD component of our study, 10 are known to be of full Chinese or mixed ancestry. The remainder is of either full Indian or unknown origin, and our findings of very low LD suggest that these unknown individuals may have at least partial Chinese ancestry.
The differences in levels of variation and extent of LD may be an occurrence specific to this gene; however, we have also found that OWM display greater variation and weaker LD across their serotonin transporter gene than do humans (Miller-Butterworth et al. 2007). This may therefore be a genome-wide phenomenon, possibly reflecting lower variability and slower substitution rates in the human genome compared with OWM, as has previously been suggested (Yi et al. 2002). In addition, early humans are believed to have experienced a severe population bottleneck, subsequent to the "Out of Africa" event (Takahata et al. 1995; Harpending et al. 1998; Chen and Li 2001; Jensen-Seaman et al. 2001; Kaessmann et al. 2001; Reich et al. 2001; Wall 2003).The resulting smaller effective population size of humans, particularly Caucasians, may have led to reduced genetic variability and longer range LD in comparison to macaques, which did not undergo such dramatic reductions in their effective population size (Hernandez et al. 2007).
Sequence Variation Is Associated with Social Rank
One SNP in the 5'UTR (5U+93) was shared by both macaque species, which suggested that it could be a functional variant. A second SNP (5U+119), located 26 bp downstream from 5U+93, was monomorphic in rhesus macaques but deviated from HWE expectations (
2 = 7.007, P = 0.014) in cynomolgus macaques, suggesting that it may be under selective pressure in this species. Both SNPs were therefore genotyped in the full cohort of monkeys (table 2). Logistic regression revealed that genotype at each individual SNP was significantly correlated with social rank in cynomolgus monkeys. Individuals possessing at least one copy of the minor allele, G at 5U+93 were significantly more likely to be of subordinate rank than those who were homozygous for the major allele, A (n = 483; heterozygotes: β ± standard error [SE] = 0.373 ± 0.203, P = 0.065, odds ratio [OR] = 1.452; GG homozygotes: β ± SE = 0.554 ± 0.265, P = 0.037, OR = 1.740). In other words, the odds that a subordinate individual possesses at least one copy of the minor allele, G are one and a half to nearly twice the odds of it being homozygous, AA. In contrast, subordinates were significantly less likely to be heterozygous than homozygous for either the major (C) or the minor (T) alleles at the second SNP, 5U+119 (n = 471, β ± SE = –0.599 ± 0.211, P = 0.004, OR = 0.5489).
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Our analyses suggested an interaction between sex and genotype at 5U+93 (β ± SE = 0.783 ± 0.417, P = 0.061, OR = 2.187), and we therefore continued with further exploratory analyses to examine each sex independently at this site. No sex x genotype interaction was found for 5U+119 (P = 0.533). The odds of subordinate females being heterozygous at 5U+93 were nearly twice that of them being homozygous for either allele, AA or GG (n = 297, β ± SE = 0.677 ± 0.262, P = 0.009, OR = 1.968). No association with social rank was found for males at this site (n = 186, P = 0.746).
GC analyses did not detect any confounding of the relationship of genotype and social rank with unidentified relatedness or population heterogeneity in our sample. Both GCF and GCB approaches independently identified SNP 5U+93 as being significantly associated with social rank in females [(1) GCF: (a) all individuals, P = 0.022; (b) females only, P = 0.006 (Bonferroni-adjusted 
= 0.025); (2) GCB: (a) all individuals, probability = 0.719, 2 = 3.227; (b) females only, probability = 0.996, 2 = 6.597]. In addition, GCB confirmed that the probability of 5U+119 being associated with social rank in both sexes is greater than 95% (probability = 0.968, 2 = 6.434).
Because both SNPs were individually associated with social rank, we also examined whether their combination in the form of haplotypes or diplotypes was similarly associated with dominance. A haplotype is the combination of SNPs along a single chromosome, whereas a diplotype incorporates variation from both chromosomes (i.e., it is the pair of haplotypes; supplementary fig. S3, Supplementary Material online). The 4 possible haplotypes derived from the 2 SNPs (AC, AT, GC, and GT) had estimated population frequencies 0.401, 0.192, 0.405, and 0.002, respectively. Due to its rarity, haplotype GT was excluded from further analysis. After accounting for phase uncertainty, haplotype analysis did not provide any better support for association with social rank than did individual genotypes (omnibus test 2 = 5.484, degrees of freedom [df] = 2, P = 0.064).
Seven diplotypes were reconstructed in PHASE and could be assigned with 99.1–100% probability to each individual (table 3). Diplotype ATGT was found in only one monkey and so was excluded from further analysis. In the combined sample of males and females, diplotype status was significantly associated with social rank (2 = 12.541, df = 5, P = 0.028). Five diplotypes were approximately equally distributed between dominant and subordinate individuals or were slightly more common in subordinates (table 3). However, diplotype ACAT occurred significantly more frequently in dominant than subordinate individuals (2 = 10.549, df = 1, P = 0.001), and this association remained statistically significant after permutation testing (adjusted = 0.003). The odds of a dominant individual having diplotype ACAT (OR = 2.448) were two and a half times that of a subordinate monkey. This association was not statistically significant in males alone (P = 0.293) but remained significant in females (ACAT vs. all other diplotypes: 2 = 7.016, OR = 2.638, confidence interval = 1.262–5.733, P = 0.008), even after correction by permutation testing (adjusted = 0.022).
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The precise biological mechanism by which these SNPs may exert their effects is unknown; therefore, it is not possible to speculate at length on the reasons for the observed sex differences. It is possible that the smaller sample size of males reduced the power available for detecting significant associations. Alternatively, it is likely that these variants interact with or are modulated by other, as yet unidentified loci, which may themselves be influenced by the sex of the individual.
To assess whether diplotype ACAT influences expression of mfaDAT, we selected 30 females (17 dominant and 13 subordinate) having diplotypes ACAT, GCGC, and ACGC for quantitative PCR analysis. One individual was arbitrarily chosen as a reference, and the expression levels of mfaDAT in the others were compared with this sample. Relative expression varied widely within each diplotype category (table 4), from less than half to nearly 20 times that of the reference sample. No significant difference in mfaDAT expression was found among diplotypes or between dominant and subordinate females; however, this may be due to the small sample size available for each diplotype.
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The 2 SNPs of interest are located in the 5'UTR regulatory region. Therefore, it remains possible that one or both SNPs is a functional variant. The SNP shared by the macaque species, 5U+93 corresponds to a putative binding site for the transcription factor, nuclear factor of activated T cells (NFAT; core sequence match = 1.000, nucleotide matrix similarity = 0.988, random expectation = 0.54 matches/1,000 bp). The SNP site (underlined) is located within the core sequence (in upper case) of the putative binding site, agaGGAAagaa. The presence of the minor allele abolishes the core sequence. This putative binding site is not found in the homologous region of hDAT (supplementary fig. S2, Supplementary Material online). Originally thought to be expressed only in immune cells, NFAT proteins are now known to regulate gene expression in many cell types and tissues, including the brain (Graef et al. 1999
) and neuronal cells, such as astrocytes (Jones et al. 2003), which express receptors for neurotransmitters and other signaling molecules. These transcription factors thus play a crucial role in shaping long-term changes in neuronal function (Groth and Mermelstein 2003). They are also sensitive to secondary messenger systems activated by brain-derived neurotrophic factor (BDNF) (Groth and Mermelstein 2003), which regulates expression of the dopamine D3 receptor (Guillin et al. 2001). It is thus possible that NFAT and/or BDNF also modulates expression of DAT. In conclusion, cross-species comparisons reveal that the DAT genes of OWM and humans differ significantly in levels of variation and LD structure, suggesting that differing evolutionary processes may have influenced the evolution of this gene in each primate lineage. The dopaminergic system is known to play a crucial role in modulating behavior, and we have identified 2 variants in the 5'UTR regulatory region of the macaque DAT gene that are significantly associated with social rank differences in female cynomolgus monkeys. One of these is shared by rhesus macaques and may correspond to a putative transcription factor–binding site. The moderate significance of some of our association statistics may be due to small sample size, and our results need to be confirmed by future studies.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary figures S1–S3 and tables S1–S4 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/). Sequences generated in this study have been deposited in GenBank under accession numbers EF174601-EF174603. The most common SNPs have been deposited in dbSNP, under the following accession numbers: M. fascicularis, 69357469–69357512; M. mulatta, 71649272–71649303; H. sapiens, 69355476–69355488, as well as in MonkeySNP (http://monkeysnp.ohsu.edu/snp/BatchList.jsp?batchid=2).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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The authors thank Dewayne Cairnes, Melissa Ayers, and Leslie Lyons for assistance with obtaining samples; Heather Brockway, Yvette Conley, Liane Fairfull, Elizabeth Lawrence, Ling Mei Yu, Marisa Winkler, Nancy Petro, Christopher Kline, Mark Kimak, and the staff of the University of Pittsburgh Genomics and Proteomics Core Laboratories for their facilities and assistance with laboratory work. We also thank Michael Barmada, Candace Kammerer, Susan Miller, Heather Norton, Carlo Pearson, and Shawn Wood for their statistical expertise. This work was supported by a National Research Service Award (# 5 F32 MH073397) to C.M.M.B. from the National Institutes of Mental Health.
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Footnotes |
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1 Present address: Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh.
2 Present address: Department of Pathology, Section of Comparative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC.
3 Present address: Computational Genetics Laboratory, Department of Psychiatry, University of Pittsburgh School of Medicine.
4 Present address: Behavioral Physiology Laboratory, Department of Psychology, University of Pittsburgh.
Connie Mulligan, Associate Editor
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