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Similarities of Non-human Primates to Humans: Genetic Variations and Phenotypic Associations Common to Rhesus Monkeys and Humans
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Similarities of Non-human Primates to Humans: Genetic Variations and Phenotypic Associations Common to Rhesus Monkeys and Humans
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
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The New England Primate Research Center, Harvard Medical School, Southborough, MA 01772-9021, USA
Introduction Our understanding of the causes and pathogenesis of neuropsychiatric disorders is at an early phase. Based on family, twin, adoption studies and genetic linkage analysis, it is generally accepted that genetics is a significant contributor to the manifestation of the majority of spontaneously occurring or drug-induced neuropsychiatric disorders. Until recently, genetic components were not integrated into animal models designed to develop medications or to clarify the pathophysiology of these disorders. Impediments to this approach are clear: the genetic basis of definitive human traits in affected populations is largely unknown, even though the list of genetic variants associated with neuropsychiatric The Laboratory Primate Copyright 2005 Elsevier ISBN 0-1208-0261-9
disorders is mounting (Comings et al., 2000a; 2000b). Equally taxing is the need to digress from convenient genetically identical strains of rats, widely used in the majority of models, and attempts to create genetic strains reflective of human polymorphisms associated with disease states. Spontaneously occurring gene variants, common to humans and animal populations, offer an appealing, albeit elusive option. An animal species with a spontaneously occurring genetic variant that contributes to some identifiable phenotype, also found in a population of humans with a specific brain disorder, would present a unique opportunity and a naturalistic model to explore the role of genetic variants on physiology and behavior. An important caveat is that multiple variants, at diverse gene loci, interact with each other and with non-genetic factors to produce susceptibility
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DEFINITION OF THE PRIMATE MODEL
Gregory M. Miller and Bertha K. Madras
Polymorphisms Polymorphisms are natural differences occurring in DNA sequences that are distributed among the individuals of a species. These individual differences in the
% Homology
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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to brain disorders. Furthermore, the search for relevant gene variants in animal populations may be daunting if the incidence of certain diseases is very low in the population. Gene manipulation in mice (null mutations, gene insertions, transgenics) presents important and effective approaches to unraveling the contribution of specific genes to definable phenotypes (reviewed in van der Weyden et al., 2002). Because of the evolutionary, social and behavioral distance between mice and humans, there may not be analogous gene variants between humans and mice at loci relevant to neuropsychiatric disorders. Primates offer an intriguing choice for this quest. In our experience, the coding regions of genes that encode key therapeutic targets (receptors and transporters) in brain, share more than 95% homology with humans and contrast with the 75–92% homology common to humans and rodents (Figure 2.1). This chapter outlines an early phase in exploring functional genetic polymorphisms shared by rhesus monkeys and humans. Described herein are polymorphisms that appear to be associated with similar phenotypic characteristics in both species and are strongly implicated in neuropsychiatric disorders.
Transporters, receptors
Figure 2.1 Coding sequence percent homology of human, rhesus monkey and rat proteins of relevance to neuropsychiatric disorders. Sequences for the dopamine transporter (DAT), norepinephrine transporter (NET), serotonin transporter (SERT), cannabinoid CB-1 receptor (CB1), mu-opioid receptor (MOR), vesicular monoamine transporter-2 (VMAT2), and trace amine receptor 1 (TAR1) are derived from GenBANK for human and rat. The rhesus monkey sequences were determined in this laboratory and the majority are currently listed in GenBANK.
DNA sequence occur throughout the entire genome, within the protein coding and non-coding regions of genes, in introns and within the vast stretches of DNA that separate individual genes. Polymorphisms within the coding region can alter the amino acid sequence of the encoded proteins, resulting in structural changes (amino acids) that may or may not affect protein function. DNA variations in non-coding regions do not alter the structure of proteins but may result in functional changes by altering parameters of protein expression. Polymorphic DNA sequences range in form from large stretches of repeated DNA sequences to smaller di- or tri-nucleotide repeats or single nucleotide polymorphisms (SNPs). SNPs have become an important focus in biomedical research. The human genome project has revealed over three million SNPs and the functional aspects of these are likely to be directly involved in, or serve as markers for, a wide range of diseases, traits and physiological characteristics.
Rationale for specific SNP studies in monkey We currently focus on three genes that code for three brain membrane proteins: the mu-opioid receptor, the dopamine transporter and the serotonin transporter. Each encodes proteins implicated in neuropsychiatric disorders and is a primary target of psychotherapeutic drugs and drugs of abuse. The mu-opioid receptor plays a fundamental role in a variety of physiological effects, including analgesia, hormone release, gastric motility and anxiety. It is the principal mediator of opiate analgesics in brain and is implicated as the immediate site of action of heroin. The dopamine transporter is a key regulator of extracellular dopamine levels in the brain, thereby playing a pivotal role in regulating processes triggered by dopamine, including movement, cognition, and reward. The dopamine transporter in brain is a target of both anti-hyperactivity and select antidepressant medications, as well as the psychostimulant drugs of abuse, cocaine and amphetamine. As regulator of the extracellular brain serotonin concentrations, the serotonin transporter is implicated in influencing mood, sleep, and other affective states in the brain. It is the immediate site of action of the majority of antidepressant drugs. The therapeutic role of the serotonin transporter is also balanced by its capacity to transport the hallucinogenic agent MDMA (ecstasy) into serotonin neurons. The genes encoding these clinically relevant proteins in rhesus monkeys are described in detail because each is instructive and representative of genetic
Mu-opioid receptor
Human mu-opioid receptor gene The human mu-opioid receptor gene contains numerous SNPs, one of which, A118G, alters the structure of the N-terminal extracellular arm of the encoded receptor protein (Bond et al., 1998). This SNP results in enhanced β-endorphin affinity for the receptor and has
Rhesus monkey mu-opioid receptor gene Using these data as a lead, we investigated whether the expressed receptors bound an agonist or antagonist differently. The affinity of β-endorphin was 3.5-fold higher for membranes derived from HEK-293 cells transfected with a G77-containing clone versus a C77containing clone, whereas the affinities of naloxone and buprenorphine did not differ between the two cell lines. Two-site analysis revealed that the 3.5-fold difference in affinity for β-endorphin expanded to 100-fold if the high affinity component was compared with its affinity for the C77-derived receptor. Intriguingly, site-directed mutagenesis, to mimic a G118 allele of the human mu-opioid receptor gene, also resulted in a 3.5-fold increase in β-endorphin affinity for the human mu-opioid receptor but no differences in the affinities of other opioid agonists (Bond et al., 1998). As detailed analysis was not reported, parallel comparisons between our data and the A118G mutant data of the human mu-opioid receptor was not feasible. Nevertheless, the physiological relevance of this difference in β-endorphin affinity warrants further investigation in both human and rhesus monkey defined haplotypes. The higher affinity for β-endorphin by the G77-containing allele may result in altered mu-opioid receptor function (signal transduction, receptor trafficking, regulation, recycling, neurotransmitter and/or hormone release). The amino acid substitution in the monkey receptors was accompanied by other amino acid changes that could also contribute to modifying β-endorphin binding affinity. Accordingly, both the rhesus monkey and the human mu-opioid receptor genes are likely to be highly polymorphic, with numerous haplotypes that may impart distinct phenotypic determinants (Hoehe et al., 2000; Miller et al., 2004). The allelic frequencies of the C77G SNP in rhesus monkeys indicated that, of 32 animals, 94% had at least one C77-containing allele and only two animals were
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DEFINITION OF THE PRIMATE MODEL
Non-human primates are widely used as research models for studying neuroanatomical and behavioral parameters of human drug addiction. Investigation of genotype/ phenotype associations in non-human primates may lead to better predictive, diagnostic and clinical assessments with regard to drug addiction and pain management. Accordingly, we asked some fundamental questions specific to our current level of understanding of mu-opioid receptors: 1. Do humans and rhesus monkeys share a structural and functional similarity in the mu-opioid receptor gene? 2. Are there polymorphisms in rhesus monkeys that are analogous to those found in humans, associated with differences in drug binding profiles and other phenotypes? 3. Can rhesus monkeys provide a “naturalistic” model for deciphering genetic associations with behavioral and physiological parameters reported in humans?
been implicated in modulating hypothalamic-pituitaryadrenal axis activation (Wand et al., 2002). To explore whether genetic variations of the mu-opioid receptor gene exist in rhesus monkey, we cloned the rhesus monkey mu-opioid receptor coding region (Miller et al., 2004). The finding of a promising ∼98% homology to the human coding region was followed by the discovery of a C77G SNP that altered an amino acid in the same region (N-terminal arm) of the A118G SNP in the human mu-opioid receptor (Figure 2.2).
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
variation occurring in different parts of the gene: in a coding region (mu-opiate receptor), in the 3′-untranslated region (dopamine transporter), and the 5′-regulatory (promoter) region (serotonin transporter), respectively. All three genes contain polymorphisms that have been associated with distinct phenotypic parameters. Polymorphisms are described in rhesus monkeys for each of these genes that differ in exact DNA sequence from analogous human polymorphisms. They are, nevertheless, strikingly parallel to those found in humans with regard to type, location in the gene and functional consequence. Taken together, these studies provide intriguing leads to investigate the usefulness of rhesus monkeys as models for deciphering genotype/phenotype relationships relevant to human disorders. Equally significant, this approach may provide novel insights into the relevance of genetic differences between individuals and the resultant effects of these differences on disease susceptibility, treatment and prognosis.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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Figure 2.2 Schematic representation of the human and rhesus monkey mu-opioid receptor protein. The seven transmembrane structure of the rhesus monkey (left) and human (right) mu-opioid receptors is depicted with open circles representing amino acids. The similar location of amino acid changes in the mu-opioid receptor proteins resulting from single nucleotide polymorphisms in the rhesus monkey and human mu-opioid receptor genes are shown in filled circles. The schematic illustrates the seven alpha helices that are embedded in the plasma membrane, an intracellular carboxy terminus (COOH), and an extracellular amino terminus (H2N). The location of the proline-to-arginine (P26R) amino acid change that results from the C77G single nucleotide polymorphism in the rhesus monkey mu-opioid receptor gene, and the asparagine-to-aspartate (N40D) amino acid change that results from the A118G single nucleotide polymorphism in the human mu-opioid receptor gene are in the amino terminal arm of the proteins.
homozygous for G77-containing alleles. Noteworthy is the finding that analogous screening for G118 alleles in the human also demonstrated that homozygosity for the rarer allele is equally uncommon (Bond et al., 1998; Grosch et al., 2001).
Mu-opioid receptor gene: physiological, behavioral association In humans, the A118G polymorphism in the muopioid receptor gene is associated with enhanced HPA axis responses to opioid receptor blockade by naloxone (Wand et al., 2002). More broadly, it has been suggested that persons harboring a G118-containing allele may have abnormal HPA axis responses to stress (Kreek, 1996; Bond et al., 1998; Wand et al., 2002). Persons with enhanced stress responsivity are more prone to addictive disorders as well as to insulin resistance, immunosuppression, osteoporosis and hippocampal injury. If the mu-opioid receptor SNP in rhesus monkeys is relevant to the human SNP, then the physiological consequences should be parallel in both species. Accordingly, we compared the incidence of G77- and C77-containing alleles with plasma cortisol levels in 21 rhesus monkeys. Plasma cortisol levels were measured on two separate occasions, two years apart. Consistently, rhesus monkeys with a G77-containing allele had significantly lower plasma cortisol levels. Upon challenge with ACTH following dexamethasone suppression,
rhesus monkeys with a G77-containing allele had significantly lower plasma cortisol increases (Miller et al., 2004). These findings implicate the mu-opioid receptor genotype as a relevant factor in the duration and efficacy of the hormonal cascades occurring in response to stress. Moreover, the mu-opioid receptor polymorphisms in rhesus monkeys and humans appear to be functionally similar. In rodents, mu-opioid receptor activity is associated with aggression and locomotor activity (Gwynn and Domino, 1984; Benton, 1985; Becker et al., 1997). In rhesus monkeys, we found a statistically significant association between the C77G SNP and the early communicative aspect of aggression (termed aggressive threat which includes behaviors such as staring and an open-mouthed, teeth-baring, ear-flapping, facial display), but not with the physical manifestations of aggression (cage shaking, environment- and self-directed aggression). Animals with one G77-containing allele scored twice the average of animals with two C77containing alleles. Animals with two C77-containing alleles clustered with low aggression index scores, whereas animals with one G77-containing allele varied widely, perhaps due to a greater diversity of representative haplotypes. This trend intensified with the two animals that harbored two G77-containing alleles. Although not statistically significant, we also observed a trend towards lower total locomotor activity in animals harboring G77-containing alleles (Miller et al., 2004). Are mu-opioid receptor polymorphisms linked to cortisol levels and aggression in humans? Intriguingly,
The brain dopamine transporter (DAT) is a member of a superfamily of Na+/Cl− dependent neurotransmitter transporters. By actively sequestering extracellular dopamine to intracellular compartments, the DAT
Human dopamine transporter gene The DAT gene coding sequence is derived from 15 exons distributed across a >64 kb gene in humans. Whereas the human DAT coding region is of fixed length, the 3′-untranslated region (3′-UTR) varies in length due to a polymorphic variable number tandem repeat (VNTR) region (Vandenbergh et al., 1992). This VNTR consists of 3 to >11 copies of a 40-base repeat unit. Numerous reports have attempted to associate the presence or absence of particular DAT alleles, as defined by the size (number of repeats) of the VNTR, with the occurrence of dopamine-related disorders, including Parkinson’s disease, schizophrenia, delusional disorder, smoking cessation, polysubstance abuse and alcoholism. The most consistent finding among this literature has been an association of a ten copy allele with attention deficit hyperactivity disorder (ADHD). Although this lead has not clarified the pathophysiology of ADHD, a significant focus of ADHD research has converged on the DAT. The DAT is one principal target of anti-hyperactivity medications in brain and may be elevated in brains of adults with ADHD. High transporter levels can arise from a number of processes, including dysfunctional regulation of DAT protein expression by the transporter gene. Emergent from these findings is whether the number of repeat sequences, in the 3′-UTR of the DAT gene, influences DAT protein levels in the brain.
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DEFINITION OF THE PRIMATE MODEL
Dopamine transporter
plays a key role in adjusting dopamine availability and consequent dopamine-mediated behaviors. As dopamine is implicated in neuropsychiatric, neurodegenerative disorders and substance abuse, dysregulation of dopamine levels by the DAT may contribute to the etiology of, or susceptibility to, dopamine-related disorders. DAT protein levels vary in normal subjects, particularly as a function of age, and deviate from the normal range in pathological states. DAT is markedly reduced in Parkinson’s disease and Lesch-Nyhan syndrome and elevated levels are observed in attention deficit hyperactivity disorder (ADHD) (Dougherty et al., 1999, Dresel et al., 2000, Cheon et al., 2003, Krause et al., 2003, Madras et al., 2002) and Tourette’s Syndrome (Malison et al., 1995, Cheon et al., 2004). Chronic use of stimulant drugs such as cocaine leads to increases in DAT levels during withdrawal, whereas amphetamine results in DAT depletion, possibly a consequence of neurotoxicity or amphetamine-induced DAT internalization.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
the association between plasma cortisol levels and aggression are parallel in rhesus monkey and humans (Kalin et al., 1998; Kalin, 1999; McBurnett et al., 2000; Pajer et al., 2001; Miller et al., 2004). An inverse relationship between plasma cortisol levels and aggressive behavior that we discovered in male rhesus monkeys is mirrored by reports in human subjects. Decreased cortisol levels have been associated with antisocial behavior in girls, and early onset aggression in boys (McBurnett et al., 2000; Pajer et al., 2001). Taken together, these data suggest that variations in the mu-opioid receptor gene might contribute to and provide a common link between certain forms of aggression and HPA axis function. For both C77G and A118G SNPs, the degree to which other SNPs occur in tandem remains to be elucidated. The number of distinct haplotypes of the human mu-opioid receptor gene are unknown, if considering combinations of alleles at A118G and other SNPs. Mu-opioid receptor function may be subtly or profoundly influenced by particular haplotypes in an individual. Altered receptor structure may modify interaction with other receptors that heterodimerize with mu-opioid receptors (e.g. other opioid receptors and of particular emerging interest, cytokine receptors), or receptor function with consequent effects on a range of biochemical sequelae triggered by this receptor. Nevertheless, the significant associations of the C77G SNP with plasma cortisol levels and aggression, reported herein, are independent of the pharmacology of the clones studied. Taken together, mu-opioid polymorphisms in rhesus monkeys and humans demonstrate similarities in the consequences of SNPs on receptor affinity, cortisol levels and aggression. Although the specific nucleotide affected by the SNPs differs, the conserved location within the gene, the functional effects of the SNP at the level of receptor binding and the similar association of the SNPs with effects on the HPA axis have striking parallels. These data support the use of non-human primates to investigate the physiological and pathological significance of mu-opioid receptor polymorphisms and other functional polymorphisms of relevance to humans.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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The relationship between DAT genotype and phenotype was explored in studies that measured DAT levels in living human brain striatum with single photon emission computed tomography (SPECT) and genotyped DAT alleles, in the same subjects, by the number of tandem repeats in the VNTR region (Jacobsen et al., 2000; Heinz et al., 2000; Martinez et al., 2001). Opposite findings were reported as subjects with the nine-repeats either had lower or higher DAT levels compared with subjects with ten-repeats. We speculated that this discrepancy may arise from the existence of allele diversity independent of the length of the DAT 3′-UTR (Miller et al., 2001; Miller and Madras, 2002).
rhesus monkey genome, and investigated the following: 1. Does the rhesus monkey DAT gene contain a tandem repeat sequence? 2. If so, is it associated with levels of activity in rhesus monkeys? 3. Are other polymorphisms present in the monkey and are these associated with activity levels? 4. Can these polymorphisms in monkey or human influence levels of protein expression? We therefore sought to determine whether a tandem repeat sequence was present in the 3′-untranslated region of the DAT gene in rhesus monkeys, whether the number of repeat units varied between animals, and whether there was an association between the 3′-UTR of the DAT gene with hyperactivity in monkeys.
Rhesus monkey dopamine transporter gene
Dopamine transporter gene: functional, behavioral association
Although a species of spontaneously hypertensive rat displays hyperactivity and is considered a model for ADHD, rats and mice do not contain analogous repeat sequences in the 3′-UTR of the DAT gene. Accordingly, rodents are inappropriate for investigating the contribution of DAT alleles, of a particular length, to hyperactivity. In view of the evolutionary proximity of rhesus monkeys to humans, we hypothesized that a repeat sequence in the DAT gene may be present in the
Similar to human, but unlike other species previously studied, we found a fixed number tandem repeat (FNTR) sequence in the 3′-UTR of the monkey DAT gene (Figure 2.3). In the absence of an established animal model of ADHD, we compared, in rhesus monkeys, the five most active with the five most sedate animals from a behaviorally characterized cohort of 22 subjects (Miller et al., 2001). In contrast to the human gene sequence, the FNTR (39 bases/repeat and 12 repeats)
Figure 2.3 Schematic diagram depicting the human dopamine transporter (DAT) gene, implicated in ADHD, and a comparison of the human, rhesus monkey and rat DAT mRNAs. In the human DAT DNA, black boxes depict exons that make up the coding region, empty boxes depict non-coding exons, and the stippled box locates the position of a polymorphic variable number tandem repeat (VNTR) region within the portion of the gene that codes for the 3′-untranslated region. Human DAT mRNAs vary in length depending on how many repeated 40-base-pair sequences are present and each box represents one repeat sequence (e.g. 12 boxes = 12 repeats). Similar to human, the rhesus monkey DAT gene contains a series of tandem repeats in the 3′-untranslated region but, thus far, only a fixed number of tandem repeats (FNTR = 12 repeats) have been identified in >24 rhesus monkeys. The rat DAT gene does not contain analogous repeat sequences in the 3′-UTR.
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DEFINITION OF THE PRIMATE MODEL
These studies resulted in four major findings. First, a tandem repeat region, previously identified in human but not in rodent, was present in the 3′-UTR of the rhesus monkey DAT gene. Second, the length of the repeat region, which varies in human subjects, was of fixed length in the monkeys. Third, the sequence of the repeat region in the monkey DAT gene varied between animals and both the human and monkey DAT gene contain SNPs in this region. Finally, we related genetic variations in the DAT gene to differences in gene expression and levels of spontaneous activity in monkeys. Most relevant, these data led us to hypothesize that, between individuals, SNPs create a diversity of DAT alleles that extend beyond the length of the VNTR region, implying that sequence-defined haplotypes may differentially contribute to dopamine-related disorders. Is it possible to relate DAT gene polymorphisms with levels of DAT protein in brain? Imaging agents, that label the DAT non-invasively, have enabled quantification of DAT density in living brain. As described previously, contradictory data were reported when comparing individuals harboring nine- and/or ten-repeat length alleles (Jacobsen et al., 2000; Heinz et al., 2000; Martinez et al., 2001). As ADHD has a small but significant association with DAT ten-repeat length alleles, several groups investigated whether DAT levels in ADHD brains deviate from the normal range. In three of four SPECT studies of adults with ADHD, elevated levels of DAT protein were detected but genotyping was not performed in this cohort (Dougherty et al., 1999; Krause et al., 2003; Dresel et al., 2000; van Dyck et al., 2002). As ADHD is most likely polygenic, and the association of the ten-repeat length allele accounts for <4% of the variance in hyperactive-impulsive symptoms and about 1% of the variance in inattentive symptoms in ADHD (Waldman et al., 1998), it is unlikely that a robust association between DAT (length) genotype and DAT density would emerge. However, a detailed analysis of the frequency of SNPs, throughout human ten-repeat length-containing alleles, is needed to further investigate DAT gene polymorphisms and ADHD, as well as other dopamine-related disorders. Accordingly, the relationship between the sequence of the 3′-UTR, DAT gene expression and behavior is yet to be resolved. Other SNPs may also influence levels of gene expression. The relevance of these observations to DAT gene regulation in vivo warrants a careful determination of the location and frequency of SNPs in the human DAT 3′-UTR as well as the 5′-UTR and other non-coding regions. The recent discovery of SNPs, in the 5′-flanking region of the human DAT gene, raises the possibility that functional SNPs may exist in the
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
is present in both very active and sedate animals as well as other monkeys. Accordingly, this FNTR is unbefitting an association of DAT transcript length with hyperactivity. However, sequence analysis revealed potential SNPs, one of which affects a Bst1107I restriction site. We screened the entire cohort, confirmed that all the rhesus monkeys had repeat regions of the same length, and demonstrated that digestion with Bst1107I was sufficient to distinguish two distinct FNTR alleles. Bst1107I genotype was suggestive but not predictive of hyperactive behavior (Miller et al., 2001). Based on these data, we speculated that SNPs may exist in human DAT VNTR alleles. To support this hypothesis, we cloned a portion of a novel ten-repeat allele of the human DAT gene and discovered a DraI restriction site-sensitive SNP. If particular alleles of the DAT gene differentially contribute to altered levels of DAT protein, it is important to consider both types of polymorphisms as potential modulators. Based on these considerations, and using a reporter assay, we investigated whether both the number of repeat sequences and the particular SNPs, in the 3′-UTR of the human and rhesus monkey DAT genes, could modify levels of gene expression (Miller and Madras, 2002). In the human sequence, the number of tandem repeat sequences in the VNTR region of the DAT 3′-UTR was an important contributor to levels of reporter gene expression. Vectors containing the nine-repeat sequence yielded higher levels of reporter gene expression than vectors containing a ten-repeat sequence. SNPs also modified reporter gene expression as the human DAT 3′-UTR segment, containing an enzyme-sensitive or insensitive nucleotide (as determined by the allele), yielded significant differences in the levels of reporter gene expression. Interestingly, the effect of the SNP was dependent upon the promoter in the vector (Figure 2.4). This observation further illustrates the concept that the functional consequence of a particular SNP is context-dependent and helps to explain the commonality of discrepancies between SNP association studies in the literature and the need to define haplotypes. Although the frequency of the SNP in the human population is unknown, this SNP in the rhesus monkey, DAT 3′-UTR, occurs in about 68% of fixed-length allele PCR products sensitive to Bst1107I digestion. Thus, although the specific sequence of the DNA repeat region, the number of repeats and the specific SNP differed between the rhesus monkey and the human DAT 3′-UTR, the polymorphic structure, the location within the gene and, in particular, the functional effects of the polymorphisms, at the level of regulation of gene expression, had striking parallels.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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Figure 2.4 Effects of a single nucleotide polymorphism in the 3-untranslated region of the dopamine transporter gene on expression of reporter gene levels. A (top): Schematic comparison of the location of a single nucleotide polymorphism in a region of the DAT gene implicated in ADHD. Both the human DAT mRNA and rhesus monkey DAT mRNA have a tandem repeat region and single nucleotide polymorphisms. The coding region is shown in black and the 3′-untranslated region is shown in white. The location of the tandem repeat regions (open boxes) and the single nucleotide polymorphisms (T/C, human; T/G, monkey) that alter restriction endonuclease-sensitive sequences for DraI (human) and Bst1107I (rhesus monkey) are also shown. B (bottom): The effects of polymorphisms of human and rhesus monkey 3′-untranslated regions on luciferase reporter gene expression. Two (ten-repeat-containing) human DAT 3′-untranslated regions (DraIsensitive and DraI-insensitive, top) are compared to two rhesus monkey 3′-untranslated regions (Bst1107I-sensitive vs. Bst1107I-insensitive, bottom) are shown. Although the polymorphism in human and rhesus monkey differed in sequence, the magnitude and direction of the promoter-dependent reporter protein levels were parallel. Data is adapted from Miller and Madras, 2002.
Serotonin transporter
Variations in the SERT gene may contribute to specific behavioral and neuropsychiatric phenotypes, and may underlie individual responses to drugs.
Human serotonin transporter Lesch et al. (1996) reported that a short-length variant, in the promoter region of the SERT gene, reduces the transcriptional efficiency of the SERT gene promoter, resulting in decreased SERT expression and serotonin transport. The short allele accounted for 3 to 4% of total variation and 7 to 9% of inherited variance for anxiety-related personality traits. This finding launched many efforts to uncover a relationship between long and short alleles of the SERT gene and SERT-related neuropsychiatric disorders (Figure 2.5). Long and short variants of the promoter region of the SERT gene are readily discernible with PCR amplification of genomic DNA samples, followed by size fractionation of the PCR products on agarose gels. With this level of analysis, allelic variations in the promoter region of the SERT gene have been associated with anxiety, depression, aggression-related personality traits and affective disorders in some, but not all, human studies (reviewed in Veenstra-VanderWeele et al., 2000).
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
promoter or other 5′ regulatory elements in the DAT gene (Rubie et al., 2001). Our data may imply that a DAT 3′-UTR of a particular sequence might function differently, depending on the sequence of the DAT promoter or other regions of the DAT gene, in any given haplotype. The findings support the need to investigate the interaction between the native DAT promoter and 3′-UTR of relevant polymorphisms directly, or in an appropriate cell line that closely mimics dopamine neurons. Similar to the studies described previously, on muopioid polymorphisms in rhesus monkeys and humans, these studies on the DAT also demonstrate similarities between the two species at the level of genetic polymorphisms, their function and phenotypic associations. Although the specific DNA sequences may differ, the conserved location within the gene, the functional effects of the polymorphisms at the level of gene expression, and the similar association of the polymorphisms with behavior, have striking parallels.
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Rhesus monkey serotonin transporter gene The close evolutionary proximity of monkeys and humans is reflected by the observation that rhesus monkeys have a similar length polymorphism in the promoter region of the SERT gene to humans
Figure 2.5 Schematic representation of the human serotonin transporter (SERT) DNA, that encodes a protein which is a primary target of antidepressants in brain. mRNAs derived from long and short alleles illustrate the position of a region of the SERT gene reported to regulate SERT protein expression. Black boxes depict exons that make up the coding region, empty boxes depict non-coding exons, and the stippled box indicates a polymorphic promoter region of variable length, that gives rise to mRNAs with different 5′ regulatory regions and different size. Long and short alleles are also present in rhesus monkeys.
DEFINITION OF THE PRIMATE MODEL
The serotonin transporter (SERT) regulates the magnitude and duration of 5-HT signaling by terminating neurotransmission via cellular transport. SERT is the site of action of many centrally active drugs. Of the multiple chemical classes of antidepressants, serotoninselective re-uptake inhibitors (SSRIs) are the most widely used drugs in the treatment of depression.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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(Trefilov et al., 1999, 2000). Thus, rhesus monkeys provide an excellent model for deciphering genotype/ phenotype relationships between SERT gene polymorphisms and behavioral, pharmacological, environmental and endocrine parameters. This is illustrated in a recent study by Bennett et al. (2002), in which rhesus monkeys, with deleterious early rearing experiences, were differentiated, by genotype, in cerebrospinal fluid concentrations of the 5-HT metabolite, 5-hydroxyindoleacetic acid, while monkeys reared normally were not. The vast majority of association studies between monoamine transporter gene polymorphisms and monoamine-related phenotypes have focused on segregating genotypes according to length of polymorphisms, disregarding SNPs. With regard to the SERT polymorphism, Nakamura et al. (2000) examined the human SERT polymorphism in detail and identified ten novel sequence variants, concluding that the alleles reported as short and long can be divided into four and six kinds of allelic variant, respectively. We would speculate that SNPs impart a differential function in a haplotype-dependent manner. Again, discordance between studies that report associations, or lack of association, between polymorphic length variants and a phenotypic parameter may be explained by this variance in genetic structure. Accordingly, sequence-defined alleles are mandated for a consistent assessment of phenotypic associations across laboratories.
Conclusion Although relatively little of the rhesus monkey genome has been sequenced, it is probable that the close genetic proximity to human will underlie some degree of conservation of specific polymorphisms shared by humans and rhesus monkeys. An example is the tandem repeat region in the 3′-UTR of the DAT gene, present in both humans and rhesus monkeys, but absent in rodents. The studies, described above, demonstrate that polymorphisms of length or of single nucleotides in genes may contribute to the dynamic processes that regulate protein expression in the brain. Consequently, association studies that group together multiple haplotypes, solely on the basis of the number of repeat sequences (length) within the 5′ or 3′-UTR, may obliterate the relevance of other sequence variations which may be involved in gene regulation. An emerging concept is that polymorphisms, in the non-coding regions of transporter and receptor genes, should be investigated as contributors to the etiology of neuropsychiatric disorders,
neurodegenerative diseases and susceptibility to drug addiction. For the mu-opioid receptor gene and others, association studies that correlate variation at a single SNP with defined phenotypic variation, should consider that a single SNP will define two heterogeneous groups of haplotypes that differ at the two alleles of the SNP and at alleles at other SNPs. Although it is likely that many CNS disorders are polygenic in nature, detailed analysis of each gene implicated will reveal a comprehensive view of their etiology and expand the range of diagnostic and therapeutic targets for these disorders. The genetic similarity and common polymorphisms of humans and rhesus monkeys may be exploitable for developing effective and “naturalistic” models to explore, in non-human primates, associations between genotype and phenotype that are of relevance to humans. There will undoubtedly be, as has been found in the mu-opioid receptor gene, specific SNPs or other polymorphisms not shared by humans and rhesus monkeys. The functional consequences of a spontaneously occurring, but non-identical, SNP may be conserved between the species. For specific genes, rhesus monkeys may share a physiogenetic similarity to humans that can transcend identical genomic and proteomic structure. More generally, genotype/phenotype relationships, in closely related species, may not depend solely on a particular specific polymorphism that is shared by the two species, but rather on parallel effects of different polymorphisms on function. Non-human primates are promising for developing models to investigate the physiological and pathological consequences of genetic polymorphisms of relevance to humans.
Correspondence Any correspondence should be directed to Gregory Miller, The New England Primate Research Center, Harvard Medical School, Southborough, MA 017729021, USA. Email: [email protected]
References Bannon, M.J., Poosch, M.S., Xia, Y., Goebel, D.J., Cassin, B. and Kapatos, G. (1992). Proc. Natl. Acad. Sci. USA. 89, 7095–7099. Becker, A., Schroder, H., Brosz, M., Grecksch, G. and Schneider-Stock, R. (1997). Pharmacol. Biochem. Behav. 58, 763–766.
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Kreek, M.J. (1996). Neurochem. Res. 21, 1469–1488. Lesch, K.P., Bengel, D., Heils, A., Sabol, S.Z., Greenberg, B.D., Petri, S., Benjamin, J., Muller, C.R., Hamer, D.H. and Murphy, D.L. (1996). Science 274(5292), 1527–1531. Madras, B.K., Miller, G.M. and Fischman, A.J. (2002). Behav. Brain. Res. 130, 57–63. Malison, R.T., McDougle, C.J., van Dyck, C.H., Scahill, L., Baldwin, R.M., Seibyl, P., Price, L.H., Leckman, J.F. and Innis, R.B. (1995). Am. J. Psychiatry 152, 1359–1361. Martinez, D., Gelernter, J., Abi-Dargham, A., van Dyck, C.H., Kegeles, L., Innis, R.B. and Laruelle, M. (2001). Neuropsychopharmacology 24(5), 553–560. McBurnett, K., Lahey, B.B., Rathouz, P.J. and Loeber, R. (2000). Arch. Gen. Psychiatry 57, 38–43. Miller, G.M., Bendor, J., Tiefenbacher, S., Yang, D., Novak, M. and Madras, B.K. (2004). Mol. Psychiatry 9, 99–108. Miller, G.M., de la Garza, R. 2nd, Novak, M.A. and Madras, B.K. (2001). Mol. Psychiatry 6, 50–58. Miller, G.M. and Madras, B.K. (2002). Mol. Psychiatry 7, 44–55. Nakamura, M., Ueno, S., Sano, A. and Tanabe, H. (2000). Mol. Psychiatry 5(1), 32–38. Pajer, K., Gardner, W., Rubin, R.T., Perel, J. and Neal, S. (2001). Arch. Gen. Psychiatry 58, 297–302. Rubie, C., Schmidt, F., Knapp, M., Sprandel, J., Wiegand, C., Meyer, J., Jungkunz, G., Riederer, P. and Stober, G. (2001). Neurosci. Lett. 297(2), 125–128. Trefilov, A., Berard, J., Krawczak, M. and Schmidtke, J. (2000). Behav. Genet. 30(4), 295–301. Trefilov, A., Krawczak, M., Berard, J. and Schmidtke, J. (1999). Electrophoresis 20(8), 1771–1777. Vandenbergh, D.J., Persico, A.M., Hawkins, A.L., Griffin, C.A., Li, X. and Jabs, E.W. (1992). Genomics 14, 1104–1106. van der Weyden, L., Adams, D.J. and Bradley, A. (2002). Physiol. Genomics 11(3), 133–164. van Dyck, C.H., Quinlan, D.M., Cretella, L.M., Staley, J.K., Malison, R.T., Baldwin, R.M., Seibyl, J.P. and Innis, R.B. (2002). Am. J. Psychiatry 159(2), 309–312. Veenstra-VanderWeele, J., Anderson, G.M. and Cook, E.H. Jr. (2000). Eur. J. Pharmacol. 27, 410(2-3), 165–181. Waldman, I.D., Rowe, D.C., Abramowitz, A., Kozel, S.T., Mohr, J.H. and Sherman, S.L. (1998). Am. J. Hum. Genet. 63, 1767–1776. Wand, G.S., McCaul, M., Yang, X., Reynolds, J., Gotjen, D. and Lee, S. (2002). Neuropsychopharmacology 26, 106–114.
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Bennett, A.J., Lesch, K.P., Heils, A., Long, J.C., Lorenz, J.G., Shoaf, S.E., Champoux, M., Suomi, S.J., Linnoila, M.V. and Higley, J.D. (2002). Mol. Psychiatry 7(1), 118–122. Benton, D. (1985). Pharmacol. Biochem. Behav. 23, 871–876. Bond, C., LaForge, K.S., Tian, M., Melia, D., Zhang, S. and Borg, L. (1998). Proc. Natl. Acad. Sci. USA. 95, 9608–9613. Cheon, K.A., Ryu, Y.H., Kim, Y.K., Namkoong, K., Kim, C.H. and Lee, J.D. (2003). Eur. J. Nucl. Med. Mol. Imaging 30, 306–311. Cheon, K.A., Ryu, Y.H., Namkoong, K., Kim, C.H., Kim, J.J. and Lee, J.D. (2004). Psychiatry Res. 130, 85–95. Comings, D.E., Gade-Andavolu, R., Gonzalez, N., Wu, S., Muhleman, D., Blake, H., Chiu, F., Wang, E., Farwell, K., Darakjy, S., Baker, R., Dietz, G., Saucier, G. and MacMurray, J.P. (2000a) Clin. Genet. 58(1), 31–40. Comings, D.E., Gade-Andavolu, R., Gonzalez, N., Wu, S., Muhleman, D., Blake, H., Mann, M.B., Dietz, G., Saucier, G. and MacMurray, J.P. (2000b). Clin. Genet. 58(5), 375–385. Dougherty, D.D., Bonab, A.A., Spencer, T.J., Rauch, S.L., Madras, B.K. and Fischman, A.J. (1999). Lancet 354, 2132–2133. Dresel, S., Krause, J., Krause, K.H., LaFougere, C., Brinkbaumer, K., Kung, H.F., Hahn, K. and Tatsch, K. (2000). Eur. J. Nucl. Med. 27(10), 1518–1524. Frost, J.J., Rosier, A.J., Reich, S.G., Smith, J.S., Ehlers, M.D. and Snyder, S.H. (1993). Ann. Neurol. 34, 423–431. Grosch, S., Niederberger, E., Lotsch, J., Skarke, C. and Geisslinger, G. (2001). Br. J. Clin. Pharmacol. 52, 711–714. Gwynn, G.J. and Domino, E.F. (1984). J. Pharmacol. Exp. Ther. 231, 306–311. Heinz, A., Goldman, D., Jones, D.W., Palmour, R., Hommer, D., Gorey, J.G., Lee, K.S., Linnoila, M. and Weinberger, D.R. (2000). Neuropsychopharmacology 22(2), 133–139. Hoehe, M.R., Kopke, K., Wendel, B., Rohde, K., Flachmeier, C., Kidd, K.K., Berrettini, W.H. and Church, G.M. (2000). Hum. Mol. Genet. 9(19), 2895–2908. Jacobsen, L.K., Staley, J.K., Zoghbi, S.S., Seibyl, J.P., Kosten, T.R., Innis, R.B. and Gelernter, J. (2000). Am. J. Psychiatry 157(10), 1700–1703. Kalin, N.H., Larson, C., Shelton, S.E. and Davidson, R.J. (1998). Behav. Neurosci. 112, 286–292. Kalin, N.H. (1999). J. Clin. Psychiatry 60, Suppl 15, 29-32. Krause, K.H., Dresel, S.H., Krause, J., la Fougere, C. and Ackenheil, M. (2003). Neurosci. Biobehav. Rev. 27, 605–613.
Similarities of Non-human Primates to Humans: Genetic Variations and Phenotypic Associations Common to Rhesus Monkeys and Humans
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
CHAPTER
17
The New England Primate Research Center, Harvard Medical School, Southborough, MA 01772-9021, USA
Introduction Our understanding of the causes and pathogenesis of neuropsychiatric disorders is at an early phase. Based on family, twin, adoption studies and genetic linkage analysis, it is generally accepted that genetics is a significant contributor to the manifestation of the majority of spontaneously occurring or drug-induced neuropsychiatric disorders. Until recently, genetic components were not integrated into animal models designed to develop medications or to clarify the pathophysiology of these disorders. Impediments to this approach are clear: the genetic basis of definitive human traits in affected populations is largely unknown, even though the list of genetic variants associated with neuropsychiatric The Laboratory Primate Copyright 2005 Elsevier ISBN 0-1208-0261-9
disorders is mounting (Comings et al., 2000a; 2000b). Equally taxing is the need to digress from convenient genetically identical strains of rats, widely used in the majority of models, and attempts to create genetic strains reflective of human polymorphisms associated with disease states. Spontaneously occurring gene variants, common to humans and animal populations, offer an appealing, albeit elusive option. An animal species with a spontaneously occurring genetic variant that contributes to some identifiable phenotype, also found in a population of humans with a specific brain disorder, would present a unique opportunity and a naturalistic model to explore the role of genetic variants on physiology and behavior. An important caveat is that multiple variants, at diverse gene loci, interact with each other and with non-genetic factors to produce susceptibility
All rights of production in any form reserved
DEFINITION OF THE PRIMATE MODEL
Gregory M. Miller and Bertha K. Madras
Polymorphisms Polymorphisms are natural differences occurring in DNA sequences that are distributed among the individuals of a species. These individual differences in the
% Homology
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to brain disorders. Furthermore, the search for relevant gene variants in animal populations may be daunting if the incidence of certain diseases is very low in the population. Gene manipulation in mice (null mutations, gene insertions, transgenics) presents important and effective approaches to unraveling the contribution of specific genes to definable phenotypes (reviewed in van der Weyden et al., 2002). Because of the evolutionary, social and behavioral distance between mice and humans, there may not be analogous gene variants between humans and mice at loci relevant to neuropsychiatric disorders. Primates offer an intriguing choice for this quest. In our experience, the coding regions of genes that encode key therapeutic targets (receptors and transporters) in brain, share more than 95% homology with humans and contrast with the 75–92% homology common to humans and rodents (Figure 2.1). This chapter outlines an early phase in exploring functional genetic polymorphisms shared by rhesus monkeys and humans. Described herein are polymorphisms that appear to be associated with similar phenotypic characteristics in both species and are strongly implicated in neuropsychiatric disorders.
Transporters, receptors
Figure 2.1 Coding sequence percent homology of human, rhesus monkey and rat proteins of relevance to neuropsychiatric disorders. Sequences for the dopamine transporter (DAT), norepinephrine transporter (NET), serotonin transporter (SERT), cannabinoid CB-1 receptor (CB1), mu-opioid receptor (MOR), vesicular monoamine transporter-2 (VMAT2), and trace amine receptor 1 (TAR1) are derived from GenBANK for human and rat. The rhesus monkey sequences were determined in this laboratory and the majority are currently listed in GenBANK.
DNA sequence occur throughout the entire genome, within the protein coding and non-coding regions of genes, in introns and within the vast stretches of DNA that separate individual genes. Polymorphisms within the coding region can alter the amino acid sequence of the encoded proteins, resulting in structural changes (amino acids) that may or may not affect protein function. DNA variations in non-coding regions do not alter the structure of proteins but may result in functional changes by altering parameters of protein expression. Polymorphic DNA sequences range in form from large stretches of repeated DNA sequences to smaller di- or tri-nucleotide repeats or single nucleotide polymorphisms (SNPs). SNPs have become an important focus in biomedical research. The human genome project has revealed over three million SNPs and the functional aspects of these are likely to be directly involved in, or serve as markers for, a wide range of diseases, traits and physiological characteristics.
Rationale for specific SNP studies in monkey We currently focus on three genes that code for three brain membrane proteins: the mu-opioid receptor, the dopamine transporter and the serotonin transporter. Each encodes proteins implicated in neuropsychiatric disorders and is a primary target of psychotherapeutic drugs and drugs of abuse. The mu-opioid receptor plays a fundamental role in a variety of physiological effects, including analgesia, hormone release, gastric motility and anxiety. It is the principal mediator of opiate analgesics in brain and is implicated as the immediate site of action of heroin. The dopamine transporter is a key regulator of extracellular dopamine levels in the brain, thereby playing a pivotal role in regulating processes triggered by dopamine, including movement, cognition, and reward. The dopamine transporter in brain is a target of both anti-hyperactivity and select antidepressant medications, as well as the psychostimulant drugs of abuse, cocaine and amphetamine. As regulator of the extracellular brain serotonin concentrations, the serotonin transporter is implicated in influencing mood, sleep, and other affective states in the brain. It is the immediate site of action of the majority of antidepressant drugs. The therapeutic role of the serotonin transporter is also balanced by its capacity to transport the hallucinogenic agent MDMA (ecstasy) into serotonin neurons. The genes encoding these clinically relevant proteins in rhesus monkeys are described in detail because each is instructive and representative of genetic
Mu-opioid receptor
Human mu-opioid receptor gene The human mu-opioid receptor gene contains numerous SNPs, one of which, A118G, alters the structure of the N-terminal extracellular arm of the encoded receptor protein (Bond et al., 1998). This SNP results in enhanced β-endorphin affinity for the receptor and has
Rhesus monkey mu-opioid receptor gene Using these data as a lead, we investigated whether the expressed receptors bound an agonist or antagonist differently. The affinity of β-endorphin was 3.5-fold higher for membranes derived from HEK-293 cells transfected with a G77-containing clone versus a C77containing clone, whereas the affinities of naloxone and buprenorphine did not differ between the two cell lines. Two-site analysis revealed that the 3.5-fold difference in affinity for β-endorphin expanded to 100-fold if the high affinity component was compared with its affinity for the C77-derived receptor. Intriguingly, site-directed mutagenesis, to mimic a G118 allele of the human mu-opioid receptor gene, also resulted in a 3.5-fold increase in β-endorphin affinity for the human mu-opioid receptor but no differences in the affinities of other opioid agonists (Bond et al., 1998). As detailed analysis was not reported, parallel comparisons between our data and the A118G mutant data of the human mu-opioid receptor was not feasible. Nevertheless, the physiological relevance of this difference in β-endorphin affinity warrants further investigation in both human and rhesus monkey defined haplotypes. The higher affinity for β-endorphin by the G77-containing allele may result in altered mu-opioid receptor function (signal transduction, receptor trafficking, regulation, recycling, neurotransmitter and/or hormone release). The amino acid substitution in the monkey receptors was accompanied by other amino acid changes that could also contribute to modifying β-endorphin binding affinity. Accordingly, both the rhesus monkey and the human mu-opioid receptor genes are likely to be highly polymorphic, with numerous haplotypes that may impart distinct phenotypic determinants (Hoehe et al., 2000; Miller et al., 2004). The allelic frequencies of the C77G SNP in rhesus monkeys indicated that, of 32 animals, 94% had at least one C77-containing allele and only two animals were
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DEFINITION OF THE PRIMATE MODEL
Non-human primates are widely used as research models for studying neuroanatomical and behavioral parameters of human drug addiction. Investigation of genotype/ phenotype associations in non-human primates may lead to better predictive, diagnostic and clinical assessments with regard to drug addiction and pain management. Accordingly, we asked some fundamental questions specific to our current level of understanding of mu-opioid receptors: 1. Do humans and rhesus monkeys share a structural and functional similarity in the mu-opioid receptor gene? 2. Are there polymorphisms in rhesus monkeys that are analogous to those found in humans, associated with differences in drug binding profiles and other phenotypes? 3. Can rhesus monkeys provide a “naturalistic” model for deciphering genetic associations with behavioral and physiological parameters reported in humans?
been implicated in modulating hypothalamic-pituitaryadrenal axis activation (Wand et al., 2002). To explore whether genetic variations of the mu-opioid receptor gene exist in rhesus monkey, we cloned the rhesus monkey mu-opioid receptor coding region (Miller et al., 2004). The finding of a promising ∼98% homology to the human coding region was followed by the discovery of a C77G SNP that altered an amino acid in the same region (N-terminal arm) of the A118G SNP in the human mu-opioid receptor (Figure 2.2).
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
variation occurring in different parts of the gene: in a coding region (mu-opiate receptor), in the 3′-untranslated region (dopamine transporter), and the 5′-regulatory (promoter) region (serotonin transporter), respectively. All three genes contain polymorphisms that have been associated with distinct phenotypic parameters. Polymorphisms are described in rhesus monkeys for each of these genes that differ in exact DNA sequence from analogous human polymorphisms. They are, nevertheless, strikingly parallel to those found in humans with regard to type, location in the gene and functional consequence. Taken together, these studies provide intriguing leads to investigate the usefulness of rhesus monkeys as models for deciphering genotype/phenotype relationships relevant to human disorders. Equally significant, this approach may provide novel insights into the relevance of genetic differences between individuals and the resultant effects of these differences on disease susceptibility, treatment and prognosis.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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Figure 2.2 Schematic representation of the human and rhesus monkey mu-opioid receptor protein. The seven transmembrane structure of the rhesus monkey (left) and human (right) mu-opioid receptors is depicted with open circles representing amino acids. The similar location of amino acid changes in the mu-opioid receptor proteins resulting from single nucleotide polymorphisms in the rhesus monkey and human mu-opioid receptor genes are shown in filled circles. The schematic illustrates the seven alpha helices that are embedded in the plasma membrane, an intracellular carboxy terminus (COOH), and an extracellular amino terminus (H2N). The location of the proline-to-arginine (P26R) amino acid change that results from the C77G single nucleotide polymorphism in the rhesus monkey mu-opioid receptor gene, and the asparagine-to-aspartate (N40D) amino acid change that results from the A118G single nucleotide polymorphism in the human mu-opioid receptor gene are in the amino terminal arm of the proteins.
homozygous for G77-containing alleles. Noteworthy is the finding that analogous screening for G118 alleles in the human also demonstrated that homozygosity for the rarer allele is equally uncommon (Bond et al., 1998; Grosch et al., 2001).
Mu-opioid receptor gene: physiological, behavioral association In humans, the A118G polymorphism in the muopioid receptor gene is associated with enhanced HPA axis responses to opioid receptor blockade by naloxone (Wand et al., 2002). More broadly, it has been suggested that persons harboring a G118-containing allele may have abnormal HPA axis responses to stress (Kreek, 1996; Bond et al., 1998; Wand et al., 2002). Persons with enhanced stress responsivity are more prone to addictive disorders as well as to insulin resistance, immunosuppression, osteoporosis and hippocampal injury. If the mu-opioid receptor SNP in rhesus monkeys is relevant to the human SNP, then the physiological consequences should be parallel in both species. Accordingly, we compared the incidence of G77- and C77-containing alleles with plasma cortisol levels in 21 rhesus monkeys. Plasma cortisol levels were measured on two separate occasions, two years apart. Consistently, rhesus monkeys with a G77-containing allele had significantly lower plasma cortisol levels. Upon challenge with ACTH following dexamethasone suppression,
rhesus monkeys with a G77-containing allele had significantly lower plasma cortisol increases (Miller et al., 2004). These findings implicate the mu-opioid receptor genotype as a relevant factor in the duration and efficacy of the hormonal cascades occurring in response to stress. Moreover, the mu-opioid receptor polymorphisms in rhesus monkeys and humans appear to be functionally similar. In rodents, mu-opioid receptor activity is associated with aggression and locomotor activity (Gwynn and Domino, 1984; Benton, 1985; Becker et al., 1997). In rhesus monkeys, we found a statistically significant association between the C77G SNP and the early communicative aspect of aggression (termed aggressive threat which includes behaviors such as staring and an open-mouthed, teeth-baring, ear-flapping, facial display), but not with the physical manifestations of aggression (cage shaking, environment- and self-directed aggression). Animals with one G77-containing allele scored twice the average of animals with two C77containing alleles. Animals with two C77-containing alleles clustered with low aggression index scores, whereas animals with one G77-containing allele varied widely, perhaps due to a greater diversity of representative haplotypes. This trend intensified with the two animals that harbored two G77-containing alleles. Although not statistically significant, we also observed a trend towards lower total locomotor activity in animals harboring G77-containing alleles (Miller et al., 2004). Are mu-opioid receptor polymorphisms linked to cortisol levels and aggression in humans? Intriguingly,
The brain dopamine transporter (DAT) is a member of a superfamily of Na+/Cl− dependent neurotransmitter transporters. By actively sequestering extracellular dopamine to intracellular compartments, the DAT
Human dopamine transporter gene The DAT gene coding sequence is derived from 15 exons distributed across a >64 kb gene in humans. Whereas the human DAT coding region is of fixed length, the 3′-untranslated region (3′-UTR) varies in length due to a polymorphic variable number tandem repeat (VNTR) region (Vandenbergh et al., 1992). This VNTR consists of 3 to >11 copies of a 40-base repeat unit. Numerous reports have attempted to associate the presence or absence of particular DAT alleles, as defined by the size (number of repeats) of the VNTR, with the occurrence of dopamine-related disorders, including Parkinson’s disease, schizophrenia, delusional disorder, smoking cessation, polysubstance abuse and alcoholism. The most consistent finding among this literature has been an association of a ten copy allele with attention deficit hyperactivity disorder (ADHD). Although this lead has not clarified the pathophysiology of ADHD, a significant focus of ADHD research has converged on the DAT. The DAT is one principal target of anti-hyperactivity medications in brain and may be elevated in brains of adults with ADHD. High transporter levels can arise from a number of processes, including dysfunctional regulation of DAT protein expression by the transporter gene. Emergent from these findings is whether the number of repeat sequences, in the 3′-UTR of the DAT gene, influences DAT protein levels in the brain.
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DEFINITION OF THE PRIMATE MODEL
Dopamine transporter
plays a key role in adjusting dopamine availability and consequent dopamine-mediated behaviors. As dopamine is implicated in neuropsychiatric, neurodegenerative disorders and substance abuse, dysregulation of dopamine levels by the DAT may contribute to the etiology of, or susceptibility to, dopamine-related disorders. DAT protein levels vary in normal subjects, particularly as a function of age, and deviate from the normal range in pathological states. DAT is markedly reduced in Parkinson’s disease and Lesch-Nyhan syndrome and elevated levels are observed in attention deficit hyperactivity disorder (ADHD) (Dougherty et al., 1999, Dresel et al., 2000, Cheon et al., 2003, Krause et al., 2003, Madras et al., 2002) and Tourette’s Syndrome (Malison et al., 1995, Cheon et al., 2004). Chronic use of stimulant drugs such as cocaine leads to increases in DAT levels during withdrawal, whereas amphetamine results in DAT depletion, possibly a consequence of neurotoxicity or amphetamine-induced DAT internalization.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
the association between plasma cortisol levels and aggression are parallel in rhesus monkey and humans (Kalin et al., 1998; Kalin, 1999; McBurnett et al., 2000; Pajer et al., 2001; Miller et al., 2004). An inverse relationship between plasma cortisol levels and aggressive behavior that we discovered in male rhesus monkeys is mirrored by reports in human subjects. Decreased cortisol levels have been associated with antisocial behavior in girls, and early onset aggression in boys (McBurnett et al., 2000; Pajer et al., 2001). Taken together, these data suggest that variations in the mu-opioid receptor gene might contribute to and provide a common link between certain forms of aggression and HPA axis function. For both C77G and A118G SNPs, the degree to which other SNPs occur in tandem remains to be elucidated. The number of distinct haplotypes of the human mu-opioid receptor gene are unknown, if considering combinations of alleles at A118G and other SNPs. Mu-opioid receptor function may be subtly or profoundly influenced by particular haplotypes in an individual. Altered receptor structure may modify interaction with other receptors that heterodimerize with mu-opioid receptors (e.g. other opioid receptors and of particular emerging interest, cytokine receptors), or receptor function with consequent effects on a range of biochemical sequelae triggered by this receptor. Nevertheless, the significant associations of the C77G SNP with plasma cortisol levels and aggression, reported herein, are independent of the pharmacology of the clones studied. Taken together, mu-opioid polymorphisms in rhesus monkeys and humans demonstrate similarities in the consequences of SNPs on receptor affinity, cortisol levels and aggression. Although the specific nucleotide affected by the SNPs differs, the conserved location within the gene, the functional effects of the SNP at the level of receptor binding and the similar association of the SNPs with effects on the HPA axis have striking parallels. These data support the use of non-human primates to investigate the physiological and pathological significance of mu-opioid receptor polymorphisms and other functional polymorphisms of relevance to humans.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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The relationship between DAT genotype and phenotype was explored in studies that measured DAT levels in living human brain striatum with single photon emission computed tomography (SPECT) and genotyped DAT alleles, in the same subjects, by the number of tandem repeats in the VNTR region (Jacobsen et al., 2000; Heinz et al., 2000; Martinez et al., 2001). Opposite findings were reported as subjects with the nine-repeats either had lower or higher DAT levels compared with subjects with ten-repeats. We speculated that this discrepancy may arise from the existence of allele diversity independent of the length of the DAT 3′-UTR (Miller et al., 2001; Miller and Madras, 2002).
rhesus monkey genome, and investigated the following: 1. Does the rhesus monkey DAT gene contain a tandem repeat sequence? 2. If so, is it associated with levels of activity in rhesus monkeys? 3. Are other polymorphisms present in the monkey and are these associated with activity levels? 4. Can these polymorphisms in monkey or human influence levels of protein expression? We therefore sought to determine whether a tandem repeat sequence was present in the 3′-untranslated region of the DAT gene in rhesus monkeys, whether the number of repeat units varied between animals, and whether there was an association between the 3′-UTR of the DAT gene with hyperactivity in monkeys.
Rhesus monkey dopamine transporter gene
Dopamine transporter gene: functional, behavioral association
Although a species of spontaneously hypertensive rat displays hyperactivity and is considered a model for ADHD, rats and mice do not contain analogous repeat sequences in the 3′-UTR of the DAT gene. Accordingly, rodents are inappropriate for investigating the contribution of DAT alleles, of a particular length, to hyperactivity. In view of the evolutionary proximity of rhesus monkeys to humans, we hypothesized that a repeat sequence in the DAT gene may be present in the
Similar to human, but unlike other species previously studied, we found a fixed number tandem repeat (FNTR) sequence in the 3′-UTR of the monkey DAT gene (Figure 2.3). In the absence of an established animal model of ADHD, we compared, in rhesus monkeys, the five most active with the five most sedate animals from a behaviorally characterized cohort of 22 subjects (Miller et al., 2001). In contrast to the human gene sequence, the FNTR (39 bases/repeat and 12 repeats)
Figure 2.3 Schematic diagram depicting the human dopamine transporter (DAT) gene, implicated in ADHD, and a comparison of the human, rhesus monkey and rat DAT mRNAs. In the human DAT DNA, black boxes depict exons that make up the coding region, empty boxes depict non-coding exons, and the stippled box locates the position of a polymorphic variable number tandem repeat (VNTR) region within the portion of the gene that codes for the 3′-untranslated region. Human DAT mRNAs vary in length depending on how many repeated 40-base-pair sequences are present and each box represents one repeat sequence (e.g. 12 boxes = 12 repeats). Similar to human, the rhesus monkey DAT gene contains a series of tandem repeats in the 3′-untranslated region but, thus far, only a fixed number of tandem repeats (FNTR = 12 repeats) have been identified in >24 rhesus monkeys. The rat DAT gene does not contain analogous repeat sequences in the 3′-UTR.
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DEFINITION OF THE PRIMATE MODEL
These studies resulted in four major findings. First, a tandem repeat region, previously identified in human but not in rodent, was present in the 3′-UTR of the rhesus monkey DAT gene. Second, the length of the repeat region, which varies in human subjects, was of fixed length in the monkeys. Third, the sequence of the repeat region in the monkey DAT gene varied between animals and both the human and monkey DAT gene contain SNPs in this region. Finally, we related genetic variations in the DAT gene to differences in gene expression and levels of spontaneous activity in monkeys. Most relevant, these data led us to hypothesize that, between individuals, SNPs create a diversity of DAT alleles that extend beyond the length of the VNTR region, implying that sequence-defined haplotypes may differentially contribute to dopamine-related disorders. Is it possible to relate DAT gene polymorphisms with levels of DAT protein in brain? Imaging agents, that label the DAT non-invasively, have enabled quantification of DAT density in living brain. As described previously, contradictory data were reported when comparing individuals harboring nine- and/or ten-repeat length alleles (Jacobsen et al., 2000; Heinz et al., 2000; Martinez et al., 2001). As ADHD has a small but significant association with DAT ten-repeat length alleles, several groups investigated whether DAT levels in ADHD brains deviate from the normal range. In three of four SPECT studies of adults with ADHD, elevated levels of DAT protein were detected but genotyping was not performed in this cohort (Dougherty et al., 1999; Krause et al., 2003; Dresel et al., 2000; van Dyck et al., 2002). As ADHD is most likely polygenic, and the association of the ten-repeat length allele accounts for <4% of the variance in hyperactive-impulsive symptoms and about 1% of the variance in inattentive symptoms in ADHD (Waldman et al., 1998), it is unlikely that a robust association between DAT (length) genotype and DAT density would emerge. However, a detailed analysis of the frequency of SNPs, throughout human ten-repeat length-containing alleles, is needed to further investigate DAT gene polymorphisms and ADHD, as well as other dopamine-related disorders. Accordingly, the relationship between the sequence of the 3′-UTR, DAT gene expression and behavior is yet to be resolved. Other SNPs may also influence levels of gene expression. The relevance of these observations to DAT gene regulation in vivo warrants a careful determination of the location and frequency of SNPs in the human DAT 3′-UTR as well as the 5′-UTR and other non-coding regions. The recent discovery of SNPs, in the 5′-flanking region of the human DAT gene, raises the possibility that functional SNPs may exist in the
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
is present in both very active and sedate animals as well as other monkeys. Accordingly, this FNTR is unbefitting an association of DAT transcript length with hyperactivity. However, sequence analysis revealed potential SNPs, one of which affects a Bst1107I restriction site. We screened the entire cohort, confirmed that all the rhesus monkeys had repeat regions of the same length, and demonstrated that digestion with Bst1107I was sufficient to distinguish two distinct FNTR alleles. Bst1107I genotype was suggestive but not predictive of hyperactive behavior (Miller et al., 2001). Based on these data, we speculated that SNPs may exist in human DAT VNTR alleles. To support this hypothesis, we cloned a portion of a novel ten-repeat allele of the human DAT gene and discovered a DraI restriction site-sensitive SNP. If particular alleles of the DAT gene differentially contribute to altered levels of DAT protein, it is important to consider both types of polymorphisms as potential modulators. Based on these considerations, and using a reporter assay, we investigated whether both the number of repeat sequences and the particular SNPs, in the 3′-UTR of the human and rhesus monkey DAT genes, could modify levels of gene expression (Miller and Madras, 2002). In the human sequence, the number of tandem repeat sequences in the VNTR region of the DAT 3′-UTR was an important contributor to levels of reporter gene expression. Vectors containing the nine-repeat sequence yielded higher levels of reporter gene expression than vectors containing a ten-repeat sequence. SNPs also modified reporter gene expression as the human DAT 3′-UTR segment, containing an enzyme-sensitive or insensitive nucleotide (as determined by the allele), yielded significant differences in the levels of reporter gene expression. Interestingly, the effect of the SNP was dependent upon the promoter in the vector (Figure 2.4). This observation further illustrates the concept that the functional consequence of a particular SNP is context-dependent and helps to explain the commonality of discrepancies between SNP association studies in the literature and the need to define haplotypes. Although the frequency of the SNP in the human population is unknown, this SNP in the rhesus monkey, DAT 3′-UTR, occurs in about 68% of fixed-length allele PCR products sensitive to Bst1107I digestion. Thus, although the specific sequence of the DNA repeat region, the number of repeats and the specific SNP differed between the rhesus monkey and the human DAT 3′-UTR, the polymorphic structure, the location within the gene and, in particular, the functional effects of the polymorphisms, at the level of regulation of gene expression, had striking parallels.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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Figure 2.4 Effects of a single nucleotide polymorphism in the 3-untranslated region of the dopamine transporter gene on expression of reporter gene levels. A (top): Schematic comparison of the location of a single nucleotide polymorphism in a region of the DAT gene implicated in ADHD. Both the human DAT mRNA and rhesus monkey DAT mRNA have a tandem repeat region and single nucleotide polymorphisms. The coding region is shown in black and the 3′-untranslated region is shown in white. The location of the tandem repeat regions (open boxes) and the single nucleotide polymorphisms (T/C, human; T/G, monkey) that alter restriction endonuclease-sensitive sequences for DraI (human) and Bst1107I (rhesus monkey) are also shown. B (bottom): The effects of polymorphisms of human and rhesus monkey 3′-untranslated regions on luciferase reporter gene expression. Two (ten-repeat-containing) human DAT 3′-untranslated regions (DraIsensitive and DraI-insensitive, top) are compared to two rhesus monkey 3′-untranslated regions (Bst1107I-sensitive vs. Bst1107I-insensitive, bottom) are shown. Although the polymorphism in human and rhesus monkey differed in sequence, the magnitude and direction of the promoter-dependent reporter protein levels were parallel. Data is adapted from Miller and Madras, 2002.
Serotonin transporter
Variations in the SERT gene may contribute to specific behavioral and neuropsychiatric phenotypes, and may underlie individual responses to drugs.
Human serotonin transporter Lesch et al. (1996) reported that a short-length variant, in the promoter region of the SERT gene, reduces the transcriptional efficiency of the SERT gene promoter, resulting in decreased SERT expression and serotonin transport. The short allele accounted for 3 to 4% of total variation and 7 to 9% of inherited variance for anxiety-related personality traits. This finding launched many efforts to uncover a relationship between long and short alleles of the SERT gene and SERT-related neuropsychiatric disorders (Figure 2.5). Long and short variants of the promoter region of the SERT gene are readily discernible with PCR amplification of genomic DNA samples, followed by size fractionation of the PCR products on agarose gels. With this level of analysis, allelic variations in the promoter region of the SERT gene have been associated with anxiety, depression, aggression-related personality traits and affective disorders in some, but not all, human studies (reviewed in Veenstra-VanderWeele et al., 2000).
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
promoter or other 5′ regulatory elements in the DAT gene (Rubie et al., 2001). Our data may imply that a DAT 3′-UTR of a particular sequence might function differently, depending on the sequence of the DAT promoter or other regions of the DAT gene, in any given haplotype. The findings support the need to investigate the interaction between the native DAT promoter and 3′-UTR of relevant polymorphisms directly, or in an appropriate cell line that closely mimics dopamine neurons. Similar to the studies described previously, on muopioid polymorphisms in rhesus monkeys and humans, these studies on the DAT also demonstrate similarities between the two species at the level of genetic polymorphisms, their function and phenotypic associations. Although the specific DNA sequences may differ, the conserved location within the gene, the functional effects of the polymorphisms at the level of gene expression, and the similar association of the polymorphisms with behavior, have striking parallels.
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Rhesus monkey serotonin transporter gene The close evolutionary proximity of monkeys and humans is reflected by the observation that rhesus monkeys have a similar length polymorphism in the promoter region of the SERT gene to humans
Figure 2.5 Schematic representation of the human serotonin transporter (SERT) DNA, that encodes a protein which is a primary target of antidepressants in brain. mRNAs derived from long and short alleles illustrate the position of a region of the SERT gene reported to regulate SERT protein expression. Black boxes depict exons that make up the coding region, empty boxes depict non-coding exons, and the stippled box indicates a polymorphic promoter region of variable length, that gives rise to mRNAs with different 5′ regulatory regions and different size. Long and short alleles are also present in rhesus monkeys.
DEFINITION OF THE PRIMATE MODEL
The serotonin transporter (SERT) regulates the magnitude and duration of 5-HT signaling by terminating neurotransmission via cellular transport. SERT is the site of action of many centrally active drugs. Of the multiple chemical classes of antidepressants, serotoninselective re-uptake inhibitors (SSRIs) are the most widely used drugs in the treatment of depression.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS DEFINITION OF THE PRIMATE MODEL
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(Trefilov et al., 1999, 2000). Thus, rhesus monkeys provide an excellent model for deciphering genotype/ phenotype relationships between SERT gene polymorphisms and behavioral, pharmacological, environmental and endocrine parameters. This is illustrated in a recent study by Bennett et al. (2002), in which rhesus monkeys, with deleterious early rearing experiences, were differentiated, by genotype, in cerebrospinal fluid concentrations of the 5-HT metabolite, 5-hydroxyindoleacetic acid, while monkeys reared normally were not. The vast majority of association studies between monoamine transporter gene polymorphisms and monoamine-related phenotypes have focused on segregating genotypes according to length of polymorphisms, disregarding SNPs. With regard to the SERT polymorphism, Nakamura et al. (2000) examined the human SERT polymorphism in detail and identified ten novel sequence variants, concluding that the alleles reported as short and long can be divided into four and six kinds of allelic variant, respectively. We would speculate that SNPs impart a differential function in a haplotype-dependent manner. Again, discordance between studies that report associations, or lack of association, between polymorphic length variants and a phenotypic parameter may be explained by this variance in genetic structure. Accordingly, sequence-defined alleles are mandated for a consistent assessment of phenotypic associations across laboratories.
Conclusion Although relatively little of the rhesus monkey genome has been sequenced, it is probable that the close genetic proximity to human will underlie some degree of conservation of specific polymorphisms shared by humans and rhesus monkeys. An example is the tandem repeat region in the 3′-UTR of the DAT gene, present in both humans and rhesus monkeys, but absent in rodents. The studies, described above, demonstrate that polymorphisms of length or of single nucleotides in genes may contribute to the dynamic processes that regulate protein expression in the brain. Consequently, association studies that group together multiple haplotypes, solely on the basis of the number of repeat sequences (length) within the 5′ or 3′-UTR, may obliterate the relevance of other sequence variations which may be involved in gene regulation. An emerging concept is that polymorphisms, in the non-coding regions of transporter and receptor genes, should be investigated as contributors to the etiology of neuropsychiatric disorders,
neurodegenerative diseases and susceptibility to drug addiction. For the mu-opioid receptor gene and others, association studies that correlate variation at a single SNP with defined phenotypic variation, should consider that a single SNP will define two heterogeneous groups of haplotypes that differ at the two alleles of the SNP and at alleles at other SNPs. Although it is likely that many CNS disorders are polygenic in nature, detailed analysis of each gene implicated will reveal a comprehensive view of their etiology and expand the range of diagnostic and therapeutic targets for these disorders. The genetic similarity and common polymorphisms of humans and rhesus monkeys may be exploitable for developing effective and “naturalistic” models to explore, in non-human primates, associations between genotype and phenotype that are of relevance to humans. There will undoubtedly be, as has been found in the mu-opioid receptor gene, specific SNPs or other polymorphisms not shared by humans and rhesus monkeys. The functional consequences of a spontaneously occurring, but non-identical, SNP may be conserved between the species. For specific genes, rhesus monkeys may share a physiogenetic similarity to humans that can transcend identical genomic and proteomic structure. More generally, genotype/phenotype relationships, in closely related species, may not depend solely on a particular specific polymorphism that is shared by the two species, but rather on parallel effects of different polymorphisms on function. Non-human primates are promising for developing models to investigate the physiological and pathological consequences of genetic polymorphisms of relevance to humans.
Correspondence Any correspondence should be directed to Gregory Miller, The New England Primate Research Center, Harvard Medical School, Southborough, MA 017729021, USA. Email: [email protected]
References Bannon, M.J., Poosch, M.S., Xia, Y., Goebel, D.J., Cassin, B. and Kapatos, G. (1992). Proc. Natl. Acad. Sci. USA. 89, 7095–7099. Becker, A., Schroder, H., Brosz, M., Grecksch, G. and Schneider-Stock, R. (1997). Pharmacol. Biochem. Behav. 58, 763–766.
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Kreek, M.J. (1996). Neurochem. Res. 21, 1469–1488. Lesch, K.P., Bengel, D., Heils, A., Sabol, S.Z., Greenberg, B.D., Petri, S., Benjamin, J., Muller, C.R., Hamer, D.H. and Murphy, D.L. (1996). Science 274(5292), 1527–1531. Madras, B.K., Miller, G.M. and Fischman, A.J. (2002). Behav. Brain. Res. 130, 57–63. Malison, R.T., McDougle, C.J., van Dyck, C.H., Scahill, L., Baldwin, R.M., Seibyl, P., Price, L.H., Leckman, J.F. and Innis, R.B. (1995). Am. J. Psychiatry 152, 1359–1361. Martinez, D., Gelernter, J., Abi-Dargham, A., van Dyck, C.H., Kegeles, L., Innis, R.B. and Laruelle, M. (2001). Neuropsychopharmacology 24(5), 553–560. McBurnett, K., Lahey, B.B., Rathouz, P.J. and Loeber, R. (2000). Arch. Gen. Psychiatry 57, 38–43. Miller, G.M., Bendor, J., Tiefenbacher, S., Yang, D., Novak, M. and Madras, B.K. (2004). Mol. Psychiatry 9, 99–108. Miller, G.M., de la Garza, R. 2nd, Novak, M.A. and Madras, B.K. (2001). Mol. Psychiatry 6, 50–58. Miller, G.M. and Madras, B.K. (2002). Mol. Psychiatry 7, 44–55. Nakamura, M., Ueno, S., Sano, A. and Tanabe, H. (2000). Mol. Psychiatry 5(1), 32–38. Pajer, K., Gardner, W., Rubin, R.T., Perel, J. and Neal, S. (2001). Arch. Gen. Psychiatry 58, 297–302. Rubie, C., Schmidt, F., Knapp, M., Sprandel, J., Wiegand, C., Meyer, J., Jungkunz, G., Riederer, P. and Stober, G. (2001). Neurosci. Lett. 297(2), 125–128. Trefilov, A., Berard, J., Krawczak, M. and Schmidtke, J. (2000). Behav. Genet. 30(4), 295–301. Trefilov, A., Krawczak, M., Berard, J. and Schmidtke, J. (1999). Electrophoresis 20(8), 1771–1777. Vandenbergh, D.J., Persico, A.M., Hawkins, A.L., Griffin, C.A., Li, X. and Jabs, E.W. (1992). Genomics 14, 1104–1106. van der Weyden, L., Adams, D.J. and Bradley, A. (2002). Physiol. Genomics 11(3), 133–164. van Dyck, C.H., Quinlan, D.M., Cretella, L.M., Staley, J.K., Malison, R.T., Baldwin, R.M., Seibyl, J.P. and Innis, R.B. (2002). Am. J. Psychiatry 159(2), 309–312. Veenstra-VanderWeele, J., Anderson, G.M. and Cook, E.H. Jr. (2000). Eur. J. Pharmacol. 27, 410(2-3), 165–181. Waldman, I.D., Rowe, D.C., Abramowitz, A., Kozel, S.T., Mohr, J.H. and Sherman, S.L. (1998). Am. J. Hum. Genet. 63, 1767–1776. Wand, G.S., McCaul, M., Yang, X., Reynolds, J., Gotjen, D. and Lee, S. (2002). Neuropsychopharmacology 26, 106–114.
SIMILARITIES OF NON-HUMAN PRIMATES TO HUMANS
Bennett, A.J., Lesch, K.P., Heils, A., Long, J.C., Lorenz, J.G., Shoaf, S.E., Champoux, M., Suomi, S.J., Linnoila, M.V. and Higley, J.D. (2002). Mol. Psychiatry 7(1), 118–122. Benton, D. (1985). Pharmacol. Biochem. Behav. 23, 871–876. Bond, C., LaForge, K.S., Tian, M., Melia, D., Zhang, S. and Borg, L. (1998). Proc. Natl. Acad. Sci. USA. 95, 9608–9613. Cheon, K.A., Ryu, Y.H., Kim, Y.K., Namkoong, K., Kim, C.H. and Lee, J.D. (2003). Eur. J. Nucl. Med. Mol. Imaging 30, 306–311. Cheon, K.A., Ryu, Y.H., Namkoong, K., Kim, C.H., Kim, J.J. and Lee, J.D. (2004). Psychiatry Res. 130, 85–95. Comings, D.E., Gade-Andavolu, R., Gonzalez, N., Wu, S., Muhleman, D., Blake, H., Chiu, F., Wang, E., Farwell, K., Darakjy, S., Baker, R., Dietz, G., Saucier, G. and MacMurray, J.P. (2000a) Clin. Genet. 58(1), 31–40. Comings, D.E., Gade-Andavolu, R., Gonzalez, N., Wu, S., Muhleman, D., Blake, H., Mann, M.B., Dietz, G., Saucier, G. and MacMurray, J.P. (2000b). Clin. Genet. 58(5), 375–385. Dougherty, D.D., Bonab, A.A., Spencer, T.J., Rauch, S.L., Madras, B.K. and Fischman, A.J. (1999). Lancet 354, 2132–2133. Dresel, S., Krause, J., Krause, K.H., LaFougere, C., Brinkbaumer, K., Kung, H.F., Hahn, K. and Tatsch, K. (2000). Eur. J. Nucl. Med. 27(10), 1518–1524. Frost, J.J., Rosier, A.J., Reich, S.G., Smith, J.S., Ehlers, M.D. and Snyder, S.H. (1993). Ann. Neurol. 34, 423–431. Grosch, S., Niederberger, E., Lotsch, J., Skarke, C. and Geisslinger, G. (2001). Br. J. Clin. Pharmacol. 52, 711–714. Gwynn, G.J. and Domino, E.F. (1984). J. Pharmacol. Exp. Ther. 231, 306–311. Heinz, A., Goldman, D., Jones, D.W., Palmour, R., Hommer, D., Gorey, J.G., Lee, K.S., Linnoila, M. and Weinberger, D.R. (2000). Neuropsychopharmacology 22(2), 133–139. Hoehe, M.R., Kopke, K., Wendel, B., Rohde, K., Flachmeier, C., Kidd, K.K., Berrettini, W.H. and Church, G.M. (2000). Hum. Mol. Genet. 9(19), 2895–2908. Jacobsen, L.K., Staley, J.K., Zoghbi, S.S., Seibyl, J.P., Kosten, T.R., Innis, R.B. and Gelernter, J. (2000). Am. J. Psychiatry 157(10), 1700–1703. Kalin, N.H., Larson, C., Shelton, S.E. and Davidson, R.J. (1998). Behav. Neurosci. 112, 286–292. Kalin, N.H. (1999). J. Clin. Psychiatry 60, Suppl 15, 29-32. Krause, K.H., Dresel, S.H., Krause, J., la Fougere, C. and Ackenheil, M. (2003). Neurosci. Biobehav. Rev. 27, 605–613.