Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University, Nashville, TN, USA

Correspondence to: M K Hahn, Vanderbilt University, Department of Pharmacology and Center for Molecular Neuroscience, 418 Preston Research Building, Nashville, TN 37232-6420, USA. Tel: (615) 936-2578 Fax: (615) 936-0212 E-mail: maureen.k.hahn@vanderbilt.edu

The norepinephrine, dopamine and serotonin transporters (NET, DAT and SERT, respectively), limit cellular signaling by recapturing released neurotransmitter, and serve as targets for antidepressants and drugs of abuse, emphasizing the integral role these molecules play in neurotransmission and pathology. This has compelled researchers to search for polymorphisms in monoamine (MA) transporter genes. Studies support linkage and association of MA transporter genetic variation in psychiatric and other complex disorders. Understanding the contribution of MA transporter polymorphisms to human behavior, disease susceptibility and response to pharmacotherapies will involve further progress in linkage and association that will be aided by both definition of highly selective phenotypes and utilization of a large number of polymorphic markers. The relationship of polymorphisms to alterations in transport capacity, likely a complex interaction, involving genetic background, disease state, and medication, will elucidate the means by which MA transporter genetic variability contributes to our individuality.

The Pharmacogenomics Journal (2002) 2, 217-235. doi:10.1038/sj.tpj.6500106

SNP; polymorphism; transporter; norepinephrine; dopamine; serotonin

INTRODUCTION

The monoamine (MA) neurotransmitters, norepinephrine (NE), dopamine (DA) and serotonin (5-HT), play important roles in mood, cognition, learning, motor activity, reward, sleep, appetite, and cardiovascular functions. Noradrenergic pathways subserve arousal, mood, attention and the response to stress. It has long been hypothesized that depression involves the noradrenergic system as evidenced by the mood altering effects of compounds that impact catecholamine levels, and by studies of noradrenergic metabolite levels and receptor binding sites in depression.^1,2,3,4 NE is also the major neurotransmitter in postganglionic sympathetic synapses, and NE uptake sites and activity are compromised in cardiomyopathy, heart failure, hypertension and ischemia.^5,6,7,8,9,10 Dopamine (DA) systems are important mediators of motor function, mood, reward, particularly the reinforcing properties of drugs of abuse, and cognition.^11,12 DA systems are thought to play a role in many disorders including attention deficit/hyperactivity disorder (ADHD), schizophrenia, drug abuse, Parkinson's disease and Tourette's syndrome.¹³ Serotonin plays a role in mood, aggression, response to alcohol, appetite, sleep, cognition, and sexual and motor activity and likely contributes to multiple mental illnesses related to these biological processes.^14,15,16,17 The availability of extracellular NE, DA and 5-HT is limited by presynaptically localized transporters, NET, DAT and SERT, respectively.The MA transporters retrieve released neurotransmitter, thus limiting the spread and duration of synaptic excitability and allowing the neurotransmitter to be repackaged into synaptic vesicles inside the terminal (Figure 1). NET and SERT are the targets for several classes of antidepressants, including the tricyclic agents and the selective serotonin reuptake inhibitors (SSRIs; Table 1) and, with DAT, are the targets for the psychostimulants, amphetamine and cocaine.¹⁸ DAT is also a more selective target for neurotoxin substrates including MPP+ and 6-hydroxydopamine. Tissue localization of MA transporters, each demonstrating expression largely restricted to its own neurotransmitter system, coupled with their existence as single-copy genes, suggests a potentially large impact of MA transporter polymorphisms due to a limited opportunity for compensation by other gene products. This is supported by transgenic mouse models in which genetic disruption of the MA transporters results in alterations in a number of behavioral and biochemical measures.^19,20,21 The importance of monoamine transporters to the regulation of neurotransmitter signaling, to disease processes and as the site of action for psychoactive compounds implores us to uncover the genetic variability that directs their expression and activity and contributes to individual differences in disease susceptibility. This review describes the human MA gene structure and polymorphic features that have been identified, including amino acid substitutions and polymorphic sites in promoter regions and other noncoding elements. We review the progress made in functional analyses to elucidate the effect of polymorphisms on expression level and other transport properties, including drug response that suggest potential pharmacogenomic applications. We review the linkage and association studies attempting to establish relationships between this genetic diversity and the presence and severity of complex disorders, which will be strengthened in the future by both defining an increased number of informative markers in the MA transporter genes as well as refining phenotypes to comprise individuals whose characteristics are influenced by transporter polymorphisms.

GENE SUBFAMILY OF MA TRANSPORTERS

NET, DAT and SERT are members of a Na⁺- and Cl^--coupled cotransporter gene family, designated SLC6A, which also includes GABA, glycine, proline and taurine transporters. Transporters in this family have a putative 12 transmembrane domain (TMD)-containing structure with intracellularly oriented amino and carboxy termini. Amino acid identity in the gene family is highest among the MA transporter subfamily, and is highest in the TMDs and boundaries, particularly TMD 1 and 2 and 4-8 and lowest in the amino and carboxy terminal regions and extracellular loops.²² The large second extracellular loop contains consensus sites for glycosylation that have been shown to play a role in transporter trafficking and stability.^23,24 There are also potential sites for serine and threonine and tyrosine kinase phosphorylation in the amino- and carboxy-termini and cytoplasmic loops and evidence supports an important role for protein kinases and phosphatases in the acute modulation of transporter trafficking and activity.^{25,26,27,28,29,30}

The genes for human NET, DAT and SERT (hNET, hDAT and hSERT) are single copy genes localized to chromosomes 16, 5 and 17, respectively, and share exon/intron structural organization and high sequence identity^{31,32,33,34,35,36,37,38,39,40,41,42,43} presumably arising from gene duplication events (see Hahn and Blakely for more detailed discussion).⁴⁴ Alternative splicing, which could provide a substrate for potential effects of genetic variability, with polymorphic sequences driving the selection of one splice pathway over others, has been identified in the hNET and 5-HTT* genes. Recent identification of a novel exon in hNET, exon 16, revealed that this exon is used in two splice variants that skip exon 15 and differ in usage of 3' splice acceptor sites and stop codons of exon 16, yielding proteins with 3 (C-t var1) or 18 (C-t var2) additional amino acids (Figure 2a).⁴⁵ The C-t var2, demonstrates an increase in K_m and V_max for norepinephrine transport in transfected cells whereas C-t var1 is inactive and appears to be retained intracellularly.⁴⁶ Removal of the terminal amino acid in the exon 15-containing hNET variant, results in both a loss of interaction with a PDZ-protein, PICK1, and decreased surface expression of hNET in transfected cells (Bauman and Blakely, submitted).⁴⁷ Therefore, splice variants lacking exon 15 lose this PDZ-binding site and may explain, in part, the observed trafficking effects. To date, however, no confirmation of native expression of protein splice variants has been obtained. An alternative splice site in the 5' noncoding region of the 5-HTT gene, was found and splice variants, with the insertion or deletion of the newly identified exon 1B, were expressed in equal proportions in JAR cells and human dorsal raphe.⁴⁸ No alternative splicing of hDAT has been documented.

INFLUENCE OF POLYMORPHISMS ON TRANSPORTER FUNCTION: PHARMACOGENOMIC ASPECTS

Polymorphisms in monoamine transporter genes could influence transporter function in a variety of ways. Single nucleotide polymorphisms (SNPs) that substitute one amino acid for another (nonsynonymous) could alter expression levels by producing an unstable protein conformation, resulting in retention in the endoplasmic reticulum via quality control mechanisms, or decreasing the time resident on the plasma membrane surface. In addition to global deleterious effects on transporter structure, the replacement of an amino acid that specifically participates in substrate binding or translocation could alter transport kinetics and extracellular neurotransmitter levels and synaptic signaling patterns. Such amino acid substitutions could yield transporters with either diminished or enhanced function. Variants located at positions of antagonist interaction could modify binding properties and influence the response to drugs, ie, an altered sensitivity to medication (SSRIs) or to environmental substances (drugs of abuse). Indeed, polymorphisms in receptors and ion channels alter intrinsic activity as well as the clinical response to medications.^49,50 Furthermore, recent work reveals highly dynamic regulation of expression and trafficking of transporters, with both substrates and antagonists influencing the retention of transporter molecules at the cell surface.^25,51,52 Signal transduction events, such as activation of PKC, also downregulate transporter binding sites through a redistribution of transporters expressed on the plasma membrane surface to intracellular compartments.^28,53,54 Transporter polymorphisms could impact this ability to redistribute in response to drugs and signal transduction mechanisms.

Polymorphisms that do not alter amino acid sequence of transporters, including synonymous SNPs, noncoding SNPs and variable number of tandem repeat (VNTR) regions are generally present at higher allele frequencies than nonsynonymous cSNPs but are without obvious effect on protein function. Such polymorphisms, through a modulation of transcriptional or translational efficiency, may still alter transporter expression level, an effect potentially as consequential as amino acid mutations. The density of transporter at a terminal would be predicted to impact neurotransmitter tone as well as the response to drug. Indeed, PET scan analysis of cocaine users demonstrated that greater than 60% of DATs need to be occupied in order to elicit the reinforcing 'high' of cocaine.⁵⁵ Drugs of abuse may also influence transporter number and result in further phenomena including sensitization and motor and cognitive performance deficits. For example, PET scan analysis of methamphetamine abusers reveals diminished DAT binding sites in the caudate and putamen that correlate with detriments on tests of motor and cognitive function⁵⁶ and persistence of psychosis.⁵⁷ (This may reflect damaged terminals but there is some support for selective down-regulation of DAT with other DA neuron markers unaffected).⁵⁸ Thus, altered transporter density could be a risk factor for both susceptibility to drug abuse as well as detrimental consequences in response to loss of transporter. Furthermore, the level of expression of transporter may influence a balance of transporter activity between the cell body and terminal regions. Whereas transporter present on the terminal contributes to clearance of neurotransmitter and termination of synaptic signaling, transporter present on the soma of neurons clear neurotransmitter that could otherwise stimulate autoreceptors to inhibit neuronal firing. These opposing actions may exist in a finely tuned balance dictated by the level of transporter expression. Indeed, the delay of action of antidepressant treatment is hypothesized to be dictated by the time it takes for somatodendritic autoreceptors to become desensitized following chronic presence of transporter antagonist and elevated extracellular levels of neurotransmitter.⁵⁹ The relationship of noncoding polymorphisms, exhibiting common allelic frequencies, to both transporter expression level and altered drug response will be key to understanding the role of transporters in complex disease.

Such common polymorphisms may contribute to the heterogeneity of response to pharmacotherapy. For example, major depression can be treated successfully with agents that selectively target either SERT or NET, and treatment efficacy is approximately 60% regardless of class of compound employed.⁶⁰ Given the complexities of psychiatric disorders and the interactions of biogenic amine pathways, it is not surprising that therapeutic effects can be achieved with distinct classes of drugs that target the monoamine transporters with differing levels of selectivity and that varied responses among individuals to the same compound are observed. One proposed model for the treatment of major depression suggests that drugs targeting serotonergic neurotransmission might be employed to alleviate symptoms of anxiety and distress, whereas selective norepinephrine reuptake inhibitors would influence motivation and pleasure.⁶¹ However, no predictors to direct selection of a drug that would ensure treatment response have been identified conclusively and a large proportion of patients with depression remain treatment-resistant following an initial monotherapy. Genetic variability in transporter genes may correlate with positive treatment outcome to a particular antidepressant. Indeed, examining the polymorphic sites in NET, SERT and DAT as a whole may yield higher-order patterns on which to overlay customized therapeutic strategies of one or more compounds.

hNET POLYMORPHISMS

A Taq1 restriction fragment length polymorphism (RFLP) was the first genetic variation identified in hNET.⁶² A lack of linkage with bipolar disorder or major depression was reported⁶³ and this polymorphism has not been examined further. Stöber and coworkers were the first to identify SNPs in the coding region (cSNPs) of the hNET gene (Table 2; Figure 2b).⁶⁴ They identified 13 SNPs; five nonsynonymous, three synonymous and five in introns. The nonsynonymous cSNPs were all observed at frequencies of less than 0.02 and were not associated with bipolar disorder, schizophrenia or Tourette's syndrome (TS).^64,65 A lack of association for the more common synonymous G155A polymorphism in exon 10 (frequency of A allele = 0.35) with major depression or with suicidal ideation has also been reported.⁶⁶ The functional impact of the five identified nonsynonymous cSNPs assayed in in vitro heterologous expression systems demonstrated Gly478Ser, a variant identified in a control population, to express a 4-fold increase in the K_m value for NE uptake.⁶⁷ A recent study examined the prevalence of SNPs in subjects in the top and bottom 2.5 percentile of blood pressure measurements and identified seven nonsynonymous, three synonymous and three noncoding SNPs in the hNET gene (Table 2; Figure 2b).⁶⁸ Our laboratory has studied the effects of the nonsynonymous SNPs on hNET expression and transport through transfection into heterologous cell culture systems.⁶⁹ These studies reveal both loss-of-function as well as gain-of-function hNET alleles that now must be explored in the context of individual patient phenotypes.

A457P and Orthostatic Intolerance

Our laboratory has attempted to refine the candidate gene approach further, narrowing the phenotype definition to better encompass those populations likely to specifically harbor transporter mutations. In so doing, we have focused on autonomic disturbances in which NET dysfunction has been hypothesized. One such syndrome, Orthostatic Intolerance (OI), is a disorder characterized by an increase in standing heart rate of at least 30 bpm that is not accompanied by hypotension.⁷⁰ A proband with OI was identified who demonstrated standing-induced increased NE spillover and decreased clearance, and decreased intraneuronal metabolism of NE as measured by decreased dihydroxyphenylglycol (DHPG) to NE ratios.⁷¹ DHPG is the metabolite formed by monoamine oxidase in the cytoplasm of cells and therefore is considered a correlative measure for NE that has been taken up by NET following release.⁷² In addition, the proband demonstrated a blunted response to tyramine, a compound that causes an increase in plasma NE following uptake through NET (Figure 3).⁷¹ Direct sequencing of the hNET gene revealed the proband to be a heterozygote for a G to C substitution at nucleotide 237 in exon 10 that results in a proline substitution for alanine at amino acid position 457 located in transmembrane domain 9 (Table 2; Figure 2b).⁷³ Although this is a rare variant that has not been found in other kindreds, analysis of our proband's family revealed a significant correlation between presence of the A457P polymorphism and elevation in standing-induced heart rate and plasma NE and decreased plasma DHPG to NE ratio.⁷³

Transient transfection of A457P into heterologous expression systems revealed a protein that is devoid of transport activity (Figure 3).⁷³ In addition, A457P is greatly diminished in both the mature, fully glycosylated form of the transporter and its surface expression (Hahn et al, in preparation). Furthermore, A457P exerts a dominant-negative effect on hNET uptake activity (Hahn et al, in preparation). This supports evidence that transporters form multimeric complexes^74,75 and suggests that individuals heterozygous for A457P, or other transporter polymorphisms, may be affected to a greater extent than predicted for harboring one mutant allele. This is the first demonstration of a coding mutation in a monoamine transporter gene with consequences for both in vitro transporter activity and neurotransmitter biochemistry and behavioral phenotype in the affected proband and family. Elevated standing heart rate may be an endophenotype indicative of hNET dysfunction that may successfully identify subgroups of subjects with functional hNET polymorphisms within populations bearing broader psychiatric diagnoses.⁷⁶

hDAT POLYMORPHISMS

A variable number of tandem repeats (VNTR) of a 40-bp element, containing 9-, 10- or more rare 3-,5-,7-,8- and 11-repeat units, was identified in the 3' untranslated region of hDAT (Figure 4a).³⁶ The 10-repeat allele was most common, present at a frequency of 0.70, followed by the 9-repeat allele at 0.24. However, numerous studies have made it evident that great heterogeneity exists in allele frequencies across different ethnic groups.^77,78 Indeed, some populations of North/Central or South American ancestry demonstrated solely the 10-repeat allele whereas in some African and middle Eastern populations, the 10-repeat allele was as low as 0.35-0.50.⁷⁹ A population with Siberian ancestry revealed further heterogeneity that included both a relatively high frequency of the 7-repeat allele and the presence of a 13-repeat allele.⁸⁰ This variability is evident even among groups often considered as one population in association studies, such as Caucasian, which may in fact be composed of groups with differing ancestry and allele frequencies for the hDAT VNTR. This can result in positive results in case-control association studies due to ethnic differences, or stratification, between the control and affected groups. A Taq1 RFLP and a VNTR in intron 8, the latter consisting of five or six copies of a 30-bp repetitive element, have also been reported.^38,81,82

A large-scale SNP discovery study that examined 106 genes encoding proteins with functions relevant to cardiovascular and neurologic disorders was the first to identify SNPs, six synonymous and one nonsynonymous, in the hDAT gene (Table 3; Figure 4b).⁸³ Vandenbergh and coworkers recently identified 12 variants, including two novel nonsynonymous cSNPs (Table 3; Figure 4b).³⁸ Another group identified ten SNPs, including two novel nonsynonymous cSNPs, in controls and bipolar disorder patients (Table 3; Figure 4b).⁸⁴ Other synonymous or noncoding SNPs have also been reported (Table 3).^85,86,87 A subset of these SNPs used in linkage analysis are listed in Table 3.

hDAT Polymorphisms: Linkage and Association studies

Substance abuse/dependence: A significant increase in the 9-repeat allele of the 3' VNTR was observed in a cocaine-dependent group with paranoia, both for the total group and white patient subgroup.⁸⁸ Another study found no association of drug use with the 9- or 10-repeat alleles or with the Taq1 polymorphism, although types of drugs used by subjects were not specified.⁸⁹ Given that the action of cocaine at DAT to increase extracellular levels of DA is critical to reinforcing properties of this drug, it is clear that additional studies are needed to explore the contribution of the hDAT gene to cocaine abuse and dependence.

A series of studies have attempted to establish a relationship between the hDAT 3' VNTR and alcoholism. One group demonstrated an increased frequency of the 9- vs 10-repeat allele in German alcoholics experiencing withdrawal seizures and/or delirium compared to controls or alcoholics negative for these symptoms.^90,91 These researchers have also recently demonstrated an increase in the 9-repeat allele in epilepsy, suggesting that the association of the 9-repeat allele may be with seizure susceptibility rather than alcohol withdrawal per se.⁹² Other studies failed to establish a role for the 3' VNTR in alcoholism.^93,94 In an association study using a Japanese population, where the 7-, 9- and 10-repeat alleles had frequencies in controls of 0.013, 0.06 and 0.90, respectively, alcoholics with a mutation in the aldehyde dehydrogenase-2 gene had a 2.5-fold greater frequency of the 7-repeat allele.⁹⁵ This suggests that different allelic variants may contribute to a disease when in the context of a different genetic background. Examination of the single nucleotide coding variants identified by Vandenbergh and coworkers demonstrated no differences in allele frequencies in alcohol-dependent patients vs controls.³⁸ One group has found association of the A allele of the 3' UTR G2319A polymorphism and the 10-repeat of the hDAT 3' VNTR in Japanese alcoholics.⁸⁵ Overall, the link of the 9- or 7-repeat allele of DAT 3' VNTR with alcoholism is not yet strong, and although the potential link of the 9-repeat allele to seizure susceptibility is intriguing, it calls for further replication.

Affective disorders: Association studies of hDAT polymorphisms and both bipolar disorder and major depressive disorder (MDD) have met with negative results.^96,97 However, family-based studies of linkage, that provide a means for removing population stratification due to ethnic or socioeconomic influences, have yielded intriguing results. The transmission disequilibrium test (TDT) is an example of this type of analysis, which determines if there is preferential allele transmission to the affected offspring by comparing parental alleles transmitted vs not transmitted. Using this approach, the 10-repeat allele of the 3' VNTR and the longer alleles of the Taq1 polymorphism were found to be in linkage disequilibrium (LD) with bipolar disorder.⁹⁸ Other studies failed to replicate this linkage.^99,100 Using haplotypes generated from markers in the 3' region of the hDAT gene, significant LD with bipolar disorder was attained.⁸⁷ Thus, the 3' end of the hDAT gene appears to contain informative markers potentially explaining the conflicting results obtained when using the 3' VNTR allelic variation as the sole marker for analysis. In summary, the data are equivocal regarding a role for the hDAT 3' VNTR in affective disorder, but evidence of linkage of other sequences in the 3' region of the hDAT gene to bipolar disorder appears to be emerging. This highlights the power of using a dense SNP map in haplotype studies of disorders thought to be linked to hDAT but for which positive evidence has been elusive. Indeed, this approach is also yielding positive results in other studies including those of ADHD in which there has been conflicting evidence regarding involvement of the 3' VNTR (see below).

Attention deficit/hyperactivity disorder: The proposed role of DA in ADHD, the fact that DAT is a target for psychostimulant drugs used to treat ADHD, and the hyperactive phenotype of DAT knockout mice all suggest that hDAT polymorphisms may be identified in ADHD that contribute to this disorder.¹⁰¹ Recent imaging studies have reported increases of 15-70% in hDAT binding sites in the striatum in adult ADHD.^102,103,104 In two of these studies, elevated hDAT binding sites were decreased to less than control by methylphenidate treatment.^103,104 Cook and coworkers used a haplotype-based haplotype relative risk (HHRR) approach to study 56 families, composed of ADHD affected offspring with one or two parents, to demonstrate that the 10-repeat allele was preferentially transmitted to the child.¹⁰⁵ Other within-family designs have demonstrated similar linkage of the 10-repeat allele.^{106,107,108,109} However, other within-family studies as well as case-control association studies have failed to replicate these positive findings.^101,110,111 Interestingly, one of the latter studies involved a group of ADHD-Combined Type patients with a demonstrated clinical response to methylphenidate, and it is possible that patient selection affected the outcome.¹¹⁰ Indeed, a case-control study of African-American children with ADHD that assessed methylphenidate response found a large excess of 10/10 genotypes among the treatment nonresponders.¹¹² The 10/10 genotype may influence treatment response through directing different levels of hDAT expression that set the tone for the response to drug. For example, similar to the effects of cocaine, occupancy of a minimum percentage of transporters may be necessary to elicit the neurochemical response to provoke a behavioral effect. Additionally, the decrease in hDAT expression observed following methylphenidate may provide treatment efficacy by setting a new level of dopaminergic tone and this response may be dependent on the initial density of transporters. However, the relationship of the 3' VNTR genotype to protein expression is far from understood and is discussed further below. This also highlights the importance of phenotype definition, as it may be fruitful to select a particular phenotype in some cases but not in others. The decision in one instance to study solely treatment responders may have eliminated the gene effect whereas the comparison of methylphenidate responders to nonresponders revealed a significant contribution of the hDAT 3' VNTR.

Vandenbergh and coworkers have assessed the relationship of the SNPs identified by their laboratory to ADHD.¹¹³ Using the TDT, they demonstrated a three-fold greater transmission to the affected offspring of the 10-repeat allele of the 3' VNTR (previously shown in these same patients by Cook and coworkers) and an approximately two-fold greater transmission of C2026, located in the 3' UTR (Table 3).^105,113 There was no transmission disequilibrium of Val55Ala or Val382Ala in the ADHD families. An analysis of several markers, the 3' VNTR, an exon 9 SNP, A1343G, and an intron 9 SNP, G1553A, found no preferential transmission of any of the alleles individually.¹¹³ However, a haplotype analysis revealed evidence of linkage; haplotypes containing the 10-repeat allele and the A allele of the intron 9 polymorphism but differing in the presence of the A or G allele of the exon 9 polymorphism were preferentially transmitted or not transmitted, respectively (Table 3).¹¹³ These data may explain the apparent conflicting results generated from studies examining a link between the 10-repeat allele and ADHD. The 10-repeat allele may not be a marker or the sole marker for ADHD but may signify the presence of another nearby marker that is in linkage disequilibrium. The grouping of mixed haplotypes in an analysis of solely the 10-repeat allele could contribute to the lack of significance observed in other studies. As more markers are identified to construct haplotypes and perform haplotype analyses, the region of the hDAT gene linked to ADHD and other diseases will become more refined. Evidence of both linkage and association of the 10-repeat allele of the hDAT 3' VNTR and 3' region haplotypes with ADHD, in combination with reports of altered hDAT expression observed in ADHD, makes this a promising link between genetic variation, protein expression and phenotype.

Parkinson's disease: In a case-control association study of French PD patients and controls, there were no differences in the allele or genotype frequencies of the hDAT 3' VNTR and an analysis of variance revealed no effect of the 9- vs 10-repeat allele on age of onset of PD.¹¹⁴ A study of Australian Caucasian PD patients found an increase in the rare 11-repeat allele in PD patients compared to controls, generating an odds ratio of 10, or a 10-fold increased risk, associated with the 11-repeat allele.¹¹⁵ The latter study exhibits a large effect, albeit for a rare allele of the hDAT 3' VNTR, suggesting contribution to only a minority of cases. The A1343G SNP was examined for association with PD and the 1343G allele was found to be significantly higher in controls vs PD patients.⁸⁶ Given the highly selective pathology of dopaminergic neurons in Parkinson's disease, more work is clearly needed in this area.

hDAT 3' VNTR Genotype and hDAT Expression

It is not known how the hDAT 3' VNTR polymorphism, which is located in the 3' UTR and thus does not change amino acid sequence, influences hDAT protein expression. The 3' untranslated region of genes may serve a variety of functions, including sorting of mRNA to distinct compartments in the cell, regulating rate of translation and signaling stabilization or destabilization of mRNA to influence turnover rate.^116,117,118 Recently, the 10-repeat allele of the hDAT 3' VNTR was reported to act as a transcriptional enhancer in both transfected cell lines as well as substantia nigra slice preparations.¹¹⁹ Determining the ability of the number or sequence of repeats in the 3' VNTR to differentially enhance transcription of hDAT will provide a functional consequence to the allelic variation. Studies performed in vitro or in situ may elucidate the role of the 3' VNTR without the interference of complex, differing genetic backgrounds, presence of disease, and environmental factors to contribute to the regulation of hDAT expression levels.

Several studies have examined the relationship of genotype of the hDAT 3' VNTR to DAT binding sites in the brain. A single positron emission computerized tomography (SPECT) analysis, using [³H]-CIT as ligand, demonstrated a small 13% decrease in binding to the transporter in the striatum in the 10/10 genotype group.¹²⁰ However, a study of 31 abstinent alcoholics and 23 controls demonstrated a 22% increase in -CIT binding in the putamen in those subjects with a 10/10 genotype (with no genotype difference between controls and alcoholics).⁹⁴ The latter findings are consistent with the association of the 10-repeat allele with ADHD and an increase in striatal hDAT binding sites in ADHD that has been reported.^105,109 A SPECT study in controls and schizophrenics revealed no effect of genotype or diagnosis on [¹²³I]-CIT binding or DA availability following a single amphetamine injection.¹²¹ The latter work suggests that the hDAT 3' VNTR genotype does not give rise to a functional change in transport activity of hDAT in the striatum. It is worth noting that a study of alcoholics admitted to detoxification treatment demonstrated decreased striatal [¹²⁵I]-CIT binding compared to controls, that increased to control levels following 4 weeks of abstinence, highlighting the potential of a disease state to confound results.¹²² In summary, further studies, particularly ones using unaffected individuals, are required to understand the potential relationship of the hDAT 3' VNTR genotype to hDAT expression levels and function in brain. Furthermore, comparison of haplotypes, particularly those suggested to be linked to disease, will need to be incorporated into these analyses. Such studies will facilitate our understanding of the apparent complex interaction of genotype, disease symptomatology and drug exposure to impact hDAT expression levels and activity.

hSERT POLYMORPHISMS

One of the first polymorphic features to be observed in the 5-HTT gene was a Pst1 RFLP of two alleles in the 3' untranslated (UTR) region of the 5-HTT gene that has remained largely unexamined.¹²³ Two repeat region polymorphisms have been studied more extensively. Heils and coworkers identified a repeat element with a 44-bp deletion polymorphism at position -1212 to -1255 in the 5' region of the 5-HTT gene (Figure 5a).¹²⁴ The polymorphism, termed the 5-HTTLPR, demonstrated two alleles, long (l) and short (s), with frequencies in Caucasians of 0.39 and 0.61, respectively. Several studies expanded on these observations to reveal large variation in allele frequencies of the 5-HTTLPR among a variety of ethnic populations.^125,126,127 Rare 15-, 19-, 20-, and 22-repeat alleles, often limited to a particular ethnic ancestry, have also been identified.^{125,126,128,129} Novel alleles of s and l that differ in sequence composition of the individual repeat units have also been found.¹³⁰ Expression of promoter constructs containing the l or s allele in cell culture demonstrated that the l allele confers three-fold greater basal transcriptional activity.^124,131,132 Lymphoblast cell lines generated from individuals demonstrated that the ll genotypes had greater SERT mRNA levels, [¹²⁵I]RTI-55 membrane binding and 5-HT uptake.^131,132 Enhanced 5-HT uptake associated with the 5-HTTLPR ll genotype has also been observed in platelets.¹³³ Furthermore, it was recently shown that the ll genotype exhibits greater SERT expression and transport activity in cultured pulmonary artery smooth muscle cells (PA-SMC).¹³⁴ Interestingly, increased 5-HT uptake in the PA-SMC in the lung is implicated in hyperplasia of these cells that contributes to primary pulmonary hypertension (PPH), and patients with PPH have demonstrated an increased ll genotype.¹³⁴ However, caution should be taken in attempting to model brain SERT activity on that of platelets or other peripheral tissues as conflicting results have been observed when correlation between genotype and brain SERT binding site availability has been examined. For example, a recent SPECT study of 16 unaffected individuals revealed no 5-HTTLPR genotype-associated differences in [1²³I]-CIT radioligand binding in several brain regions¹³⁵ (see further discussion below).

A VNTR has been identified in intron 3 (formerly designated intron 2 prior to the identification of a novel upstream intron) of the 5-HTT gene (Figure 5a).¹³⁶ The intron 3 VNTR is composed of 17-bp repeats, the most prevalent alleles being the 10- and 12-repeat.¹³⁶ Examination of different ethnic groups revealed wide variation in frequencies; the 10-repeat allele ranged from a frequency of 0.02 in the Japanese to 0.47 in European Americans.^126,127 The 10-repeat allele was present at 0.26 in African Americans. This is worth noting, as this difference in frequencies between the European and African Americans is greater than the size of effects commonly observed in association studies, in which these two ethnically diverse populations are often combined. A rare 9-repeat allele with a frequency of 0.01 and an 11-repeat allele with a frequency of 0.13 in a Mbuti population have also been identified.^126,127

Despite its location outside of the coding or promoter regions of the 5-HTT gene, expression in transgenic embryos and embryonic stem cells suggests that the intron 3 VNTR polymorphism acts as a strong regulator of transcription.^137,138 The 12-repeat demonstrated greater transcriptional activation which could be due to an increase in the number of transcriptional elements present in a the larger 12-repeat construct, or the size difference that potentially allows a more favorable conformation of DNA for binding transcriptional factors. These findings support that intron 3 of the 5-HTT gene plays a role in transcriptional regulation and the polymorphic region may direct different levels of expression. As a variant with potential effects on transcriptional activity, the intron 3 VNTR has promise for association with clinical conditions.

A novel 381-bp region of DNA, located between the 5-HTTLPR and the transcriptional start site, was recently identified in genomic clones (Figure 5a).^139,140 Transfection of promoter constructs containing this novel sequence into RN46A immortalized raphe cells and JAR cells enhances transcriptional expression compared to constructs lacking this region.¹³⁹ The difficulty in cloning this site suggests that it may be unstable in vivo and hence be polymorphic as well as demonstrate mosaicism in an individual, with deletion of the sequence in some cells but not others. One group found by PCR amplification that the deletion occurred in approximately 50% of normal individuals examined.¹⁴¹ However, two other groups, including our laboratory, have found that all subjects examined contain the full sequence.^139,140 It remains an open question as to whether this region represents a deletion carried by individuals that could contribute to genetic variability or an artifact of isolation and manipulation of genomic DNA or PCR amplification. Additionally, in judging relevance of this observation, it will be important to determine the extent to which this rearrangement occurs in tissues that express SERT, such as the dorsal raphe nucleus.

A small number of SNPs were identified in the course of cloning of the 5-HTT cDNA and gene^48,142,143 (Table 4). A larger survey of the occurrence of SNPs in genes that encode proteins with functions relevant to cardiovascular and neurologic disorders yielded four 5-HTT SNPs; two nonsynonymous, one synonymous and one noncoding (Table 4; Figure 5b).⁸³ Utilizing denaturing high performance liquid chromatography (DHPLC) and direct sequencing of DNA from 450 individuals in the DNA Polymorphism Discovery Resource, additional low frequency 5-HTT SNPs were identified, seven of which were novel amino acid variants (Table 4; Figure 5b).¹⁴⁴ The impact of these amino acid variants on expression or transport function remains unexplored.

5-HTT Polymorphisms: Linkage and Association Studies

Anxiety/personality traits: In addition to identifying the 5-HTTLPR, Lesch and coworkers also demonstrated the first association of this polymorphism with a phenotype. S-containing genotypes were associated with higher scores on the NEO personality inventory (NEO-PI-R) on the neuroticism scale, which is based on anxiety and depressive symptoms, and estimates harm avoidance, based on anxiety symptoms.¹³² Many studies have attempted to replicate this finding, using different populations and a variety of personality inventories. Several studies have failed to associate neuroticism with the 5-HTTLPR.^{145,146,147,148,149,150} One group has replicated the association of the s allele with increases in scores of harm avoidance on the TPQ and of anxiety and depression subscales of neuroticism on the NEO-PI-R.¹⁵¹ Recently, Lesch and colleagues returned to this issue and replicated their findings in a new population, both alone and as a combined group with the population from the initial study.¹⁵² Further refinement of the qualities that define personality traits may generate reproducibility and stronger association.

Affective disorders: Efforts to identify an association of the 5-HTT intron 3 polymorphism with affective disorder have yielded mixed results. One group has found an increase of the rare 9-repeat allele in unipolar depression in Scottish populations, supporting its contribution to affective disorder in only a small subset of patients.^153,154 Furthermore, others have failed to replicate this finding in bipolar depression or MDD.^155,156,157 An association of the 12-repeat allele with depression has also been reported.^128,158 A meta-analysis, that included the results of the studies described above, found no involvement of the intron 3 VNTR in unipolar or bipolar depression.¹⁵⁹ These conflicting results suggest further replication is necessary to support an involvement of the 5-HTT intron 3 polymorphism in affective disorder.

Two family-based studies found no linkage of the 5-HTTLPR and bipolar disorder.^160,161 However, one family-based study of bipolar probands and their parents used TDT to find a preferential transmission of the s allele.¹⁶² There is also some suggestion in case-control studies of association of the s allele and ss genotype with MDD, unipolar or bipolar depression.^159,163,164 Additionally, significant association of the s allele with seasonal affective disorder (SAD) has been reported.¹⁶⁵ However, the controls and patients in that study differed in gender and race. Furthermore, an equal number of reports support a lack of association of the 5-HTTLPR with depressive disorders.^{97,128,156,166,167} Overall, there is not strong support for the contribution of polymorphisms of the 5-HTT gene to affective disorder, and given that some of these studies employed large sample sizes or reported a high level of power to detect an effect, defining subtypes of affective disorder that suggest hSERT involvement may be an important step for future success.

The efficacy of SSRIs as antidepressant agents and their primary site of action as blockers of SERT activity indicate that polymorphisms in the 5-HTT gene could influence treatment response. Indeed, there are some intriguing recent reports of the impact of 5-HTTLPR genotype on antidepressant response. MDD patients with psychotic features treated with the SSRI, fluvoxamine, did not respond as well, as measured by scores on the Hamilton Depression Rating Scale (HDRS), if they were of the ss genotype.¹⁶⁸ Furthermore, this difference in response was eliminated when fluvoxamine treatment was supplemented with pindolol. Pindolol is hypothesized to hasten the ameliorative actions of SSRIs by antagonizing the 5-HT1A autoreceptors to alleviate the initial inhibitory effects of SSRIs on 5-HT neuronal firing. This group extended these findings to a group of MDD patients without psychotic features in which paroxetine-treated ll and ls genotypes improved significantly on HDRS scores at 2 and 4 weeks compared to ss genotypes.¹⁶⁹ Furthermore, the association of the s-containing genotypes with poorer treatment response and pindolol augmentation appears to be independent of diagnostic subtype or the presence of delusional features.¹⁷⁰ These results were supported by another group showing that older depressed patients with the ll genotype responded more rapidly to the SSRI, paroxetine, compared to those patients with s-containing genotypes. Interestingly, there was no difference in the response of different genotype groups to nortriptyline, an uptake inhibitor acting more selectively at NET.¹⁷¹ One group has examined 5-HTTLPR genotype in bipolar patients experiencing manic episodes during, and believed to be provoked by, antidepressant treatment.¹⁷² Highly significant results were obtained for association of the s allele and ss genotype with patients who had experienced such episodes. In summary, although there does not appear to be strong evidence for the 5-HTTLPR in affective disorder, the 5-HTTLPR genotype may have predictive power for SSRI treatment response. This presents an opportunity for a pharmacogenomic approach to the treatment of affective disorder whereby selection of therapeutics can be matched to genetic background.

Suicide: Findings of altered 5-HT metabolites and SERT binding sites in suicide suggest 5-HTT polymorphisms could influence susceptibility.¹⁴ Association of the s allele, l allele or no association with suicide have all been reported.^173,174,175 Another study failed to associate the 5-HTTLPR with suicide and, despite decreased [³H]cyanoimipramine binding to SERT in the prefrontal cortex in depressed patients and suicide victims, there was no effect of genotype on binding sites.¹⁷⁶ Therefore, this latter work supports neither an association of the 5-HTTLPR with suicide nor reports of lower uptake sites of s-containing genotypes described in cell lines and platelets. A lack of association of the intron 3 VNTR with suicide attempt was also reported.¹⁷⁷ Taken together, these findings fail to support a strong association of 5-HTT gene polymorphisms with suicide. Interestingly, research that focuses on suicide with elements of impulsive behavior has revealed an association with the 5-HTTLPR (see below), emphasizing once again the importance of refining phenotype definition.

Substance abuse and dependence: Examining the link of the 5-HTTLPR to alcoholism, several groups have found an excess of the s allele and/or ss genotype in alcohol-dependent subjects, particularly in a subgroup, such as withdrawal severity.^{178,179,180,181} However, one study associated the presence of the ll genotype with alcohol dependence.¹⁸² No association of the 5-HTTLPR or intron 3 VNTR with alcohol dependence was found in a large European American population.¹²⁶ Overall, there is some support for association of the s allele of the 5-HTTLPR with alcohol dependence, and continued success in this area may depend on the designation of subgroups, such as symptom severity or personality traits.

Two reports have examined the 5-HTTLPR as well as SERT binding sites in the brain in alcohol-dependent subjects and cocaine users. In these reports, there were no significant genotype differences between the controls and any of the drug use groups, but there were interesting findings regarding the relationship between genotype and binding site availability. In one report, the sl genotypes were accompanied by less post-mortem [¹²⁵I]-CIT binding and SERT mRNA in the dorsal raphe in a group combining cocaine users and controls compared to ll genotypes, but there were too few ss genotypes to make conclusions about this group.¹⁸³ However, [¹²⁵I]-CIT binding was increased in alcoholics with s-containing genotypes compared to cocaine user and control groups.¹⁸³ In the second study, examining controls only, the ll genotype demonstrated two-fold greater [¹²³I]-CIT binding in the dorsal brainstem than ss-carrier controls.¹⁸⁴ The increased binding in the controls with the ll genotype supports the data from cell culture and platelet studies that this genotype directs higher levels of expression. However, alcohol-dependent subjects with the ll genotype demonstrated less [¹²³I]-CIT binding than ll controls. This apparent interaction of genotype with alcoholism on SERT binding sites could be due to a different array of genetic contributions in alcoholics, or the effects of alcohol and many other environmental factors associated with chronic alcohol use. Therefore, studies correlating genotype with binding sites that use clinical populations as part of the sample should be interpreted with caution. This emphasizes that more comprehensive studies in groups free from psychiatric diagnoses and exposure to medications and drugs of abuse are required to understand the relationship between genotype and expression levels.

Impulsivity: 5-HT systems are hypothesized to mediate aggressive, impulsive behavior that may be present across a broad spectrum of psychiatric syndromes.¹⁴ There have been efforts to establish an association of 5-HTT gene polymorphisms with clinical conditions that involve elements of impulsivity, such as alcohol dependence and suicide by violent means. The Type 2 subgroup of alcoholics are defined by early onset, with dissocial and impulsive-aggressive behavior and also display alterations in aspects of serotonergic system function and high novelty seeking and low harm avoidance scores on the TPQ personality assessment.¹⁸⁵ One study reported a trend for an excess of ss genotypes in the dissocial alcoholic group, as well as a trend for higher novelty-seeking scores and lower harm avoidance scores in the dissocial alcoholics.¹⁸⁶ The increased frequency of the s allele was replicated in impulsive, violent, early-onset alcoholics compared to late-onset alcoholics.¹⁸⁷ However, this finding was not replicated in German alcohol-dependent patients, divided into high and low impulsivity groups, compared to controls.¹⁸⁸ An association has also been made between the 5-HTTLPR and suicidal behavior with elements of violence. A compelling study reported that 91% of suicides, mostly committed by violent means, but only 67% of controls, had an s-containing genotype.¹⁸⁹ Another group replicated this finding.¹⁹⁰ Taken together, the data suggest an association of the s allele of the 5-HTTLPR with impulsive violent behavior that may be alternately expressed as violence comorbid with alcohol dependence or suicide attempts. These findings advocate extracting a common phenotype from a cluster of characteristics within complex disorders that may more accurately reflect the traits to which the gene is contributing.

Autism: Case-control studies have found a lack of association of the 5-HTTLPR with autism.¹⁹¹ Cook and coworkers were the first to identify linkage of the s allele of the 5-HTTLPR gene polymorphism in autism.¹⁹² However, other family-based TDT studies, including subgroup analyses, support preferential transmission of the l allele.^193,194,195 Two of these reports agree in finding no preferential transmission of the intron 3 VNTR.^192,193 A genome-wide linkage analysis of 152 sib pairs revealed significant LOD scores for a marker on chromosome 17 located within intron 3 of the 5-HTT gene.¹⁹⁶ Furthermore, there appeared to be a parent-of-origin effect at this marker demonstrating increased paternal transmission. Genomic imprinting has been observed in the inheritance of a wide array of diseases, some of which implicate 5-HT systems, such as bipolar affective disorder.¹⁹⁷ To date there has been no evidence for genomic imprinting of the 5-HTT gene but these results suggest using genetic models that can evaluate parent-of-origin effects.

OCD: Analyses of the 5-HTTLPR and obsessive-compulsive disorder (OCD) have yielded evidence of both linkage disequilibrium of the l allele and association of the ll genotype with OCD.^198,199 However, other studies have failed to find a relationship.^200,201 Additionally, one group has sequenced 22 OCD patients and found no polymorphisms.²⁰² That OCD is uniquely treated by SERT-preferring antidepressants, suggests pharmacogenomic approaches to treatment of this disorder may prove useful and advocates further examination of 5-HTT gene variation in OCD.

CONCLUSIONS

The cloning of the MA transporter genes over the last 10 years has provided gene structure information that can be used to begin to understand transporter expression and function. There is much yet to learn about the role of the 5' promoter region, 3' region and introns in MA transporter gene expression, and the role of amino acids sequence in transporter structure and function, in order to understand how polymorphisms in any of these regions might influence behavior and personality and predisposition to illness. Additionally, it remains to be determined to what extent alternative splicing of hNET, hSERT, and potentially hDAT occurs in human brain, whether splice variants alter transporter expression and function in vivo, and if there are polymorphisms that favor one splice pattern over another.

Our expanding knowledge of complex disorders has allowed some hypotheses of what diseases are predicted to exhibit genetic variability in MA transporters. Armed with these tools, researchers have met with some success in identifying genetic variants in the MA transporter genes, their association with disease, and their potential impact on transporter function. The link between the hDAT 3' VNTR 10-repeat allele and ADHD serves as an instructive example of how promising inroads are being established while many questions remain. The selection of hDAT as a candidate gene was strongly hypothesis-driven, given the importance of DA systems in motor activity and attentional processes and evidence of altered hDAT binding sites in the brains of ADHD patients. However, the function of the 10- vs 9-repeat of the 3' VNTR with hDAT expression even in unaffected individuals is unknown. It will be of great interest to not only establish an association of a polymorphism with a trait but to understand the mechanism by which a polymorphism exerts its effects. Further work needs to explore the role of the 9 vs 10-repeat allele, either alone or in combination with other polymorphisms, on hDAT expression. Haplotype analyses reveal interactions of several markers in the 3' region of hDAT to contribute to disease. This demands that we not abandon the candidate gene approach without utilizing dense coverage of the gene of interest to ensure that sufficient relevant markers have been examined. Furthermore, a polymorphism that appears to have no functional consequences could alter an aspect of hDAT function not yet assayed, such as transcriptional activation under conditions of stress. The ability to assign function to polymorphic sites is complicated by technical difficulties encountered in measuring transporter expression and regulation in the living human brain, including access, available assay tools, and the widespread, heterogeneous, and relatively low levels of MA transporter expression. Use of cell culture models remains a highly attractive alternative or adjunct to such studies. Indeed, our interrogation of the A457P mutation in heterologous cell culture systems reveals aberrations in transporter protein processing, cell surface expression and transport which are not at present readily measurable in the individuals harboring this mutation. Finding an association of a polymorphism with disease is clearly the first of many steps towards understanding how genetic differences shape us.

Constructing hypotheses of disease etiology and designating candidate genes enhances the power of genetic studies but proving association of complex diseases influenced by many genes that contribute small individual effects requires large sample size and even then may prove elusive. It may be possible to combat this limitation by applying phenotype definitions that more narrowly define the disorder and more accurately model the underlying genetic composition.⁷⁶ Our laboratory has successfully used such an approach. Designating alterations in DHPG levels and tyramine responsiveness as indicative of hNET dysfunction, the identification of the nonfunctional polymorphism, A457P, was achieved. This provides a rational basis for assessing these biochemical measures in other potential subjects for hNET polymorphism discovery. The information obtained by identifying just one individual with a nonfunctional hNET is invaluable. Elevated standing heart rate and metabolite levels may constitute an endophenotype with great predictive power, particularly for those polymorphisms highly disruptive to hNET function. For example, ADHD has been studied primarily for hDAT polymorphisms, but the role of NE in cognition and attention through actions in the prefrontal cortex also suggests an involvement of this neurotransmitter system in ADHD.²⁰³ Selecting a subset of ADHD patents with elevated standing heart rate may greatly increase the chances of finding an association of hNET with this disorder.

What will also aid in the identification of MA transporter polymorphisms in disease is the rapid evolution of technology including microarray chips, DHPLC, and microsphere-based addressing and sorting of SNPs. These techniques allow increased throughput for both SNP discovery and genotyping of identified SNPs. Advantages include that SNPs and larger length variants can be identified without sequencing the entire region (DHPLC) and the researcher need only examine nucleotide positions identified as variable by the analysis software (microarray chips). In addition, multiplexing opportunities are feasible with all of these techniques. Higher throughput methodologies will aid in assaying larger sample sizes and the increasing number of markers that are being identified for use in more complex haplotype analyses, both vital to revealing significant effects. We are clearly in the early stages of this undertaking, but utilizing highly selective phenotype definitions in combination with multiple informative markers should enhance our ability to clarify how genetic variation in MA transporters contributes risk to complex human disease.

DUALITY OF INTEREST

None declared.

*We refer to serotonin transporter cDNA and protein with the acronym SERT and the cognate gene with the acronym 5-HTT, the latter proposed by Lesch and co-workers.

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Figure 1 Schematic representation of transporter function at a synaptic terminal. hNET, hDAT and hSERT are localized to the presynaptic plasma membrane in NE, DA and 5-HT neurons, respectively. Uptake of neurotransmitter from the extracellular space into the cytosol is achieved via a translocation process that is coupled to a concentration-dependent ion transport and can occur in the reverse direction. Neurotransmitter retrieved into the cytosol can be metabolized by monoamine oxidases or repackaged into synaptic vesicles for exocytosis.

Figure 2 (a) Exon/intron structure of the hNET gene showing the corresponding regions of the protein encoded by each exon. Untranslated regions of exons are shown in grey and protein transmembrane domains (TMD) are black boxes. The three protein variants generated by alternative splicing of the hNET gene are depicted. (Scale bar = 100 bp of exon sequence. Introns are not drawn to scale.) (b) Amino acid variants identified in hNET. hNET is depicted as a 12-TMD spanning protein with intracellular N- and C-termini. The approximate location of the variant residues is shown. The number refers to the amino acid position in the protein which is preceded by the single-letter code for the amino acid commonly found at that position and is followed by the single letter code for the variant.

Figure 3 Schematic representation of wild-type (wt) hNET (a) and the mutant A457P (b) function at a noradrenergic synapse. (a) Normal function of wt hNET. hNET takes up released NE that once in the cytoplasm can be repackaged into exocytotic vesicles or metabolized by monoamine oxidase (MAO) to dihydroxyphenylglycol (DHPG). Some NE escapes the reuptake process and appears in plasma (when sympathetic nerve terminals are the source of the NE). DHPG can diffuse out of the neuron and also be measured in plasma. Tyramine is a compound that is a substrate for hNET and once inside the cell also displaces NE from vesicles, thereby increasing cytoplasmic NE level and resulting in the exit of NE from the neuron via a reverse transport mechanism. (b) The failure of A457P to transport NE generates greater than normal spillover and diminished clearance of NE from the plasma. The lack of uptake diminishes the flux of NE through the MAO degradation pathway and, following release of NE, plasma DHPG levels do not increase as compared to normal. Additionally, tyramine is not able to induce release of NE through reverse transport. Thus, whereas A457P enhances basal and stimulated plasma NE levels, there is a blunted response to tyramine. Note that individuals are heterozygous for A457P and yet to be determined are ratios of wt and A457P surface expression in vivo and the role of transporter multimers to foster functional interactions.

Figure 4 (a) Exon/intron structure of the hDAT gene depicting the 3' VNTR located within the 3' untranslated region in exon 15. Untranslated regions of exons are shown in grey. (Scale bar = 100 bp of exon sequence. Introns are not drawn to scale.) (b) Amino acid variants identified in hDAT. hDAT is depicted as a 12-transmembrane domain spanning protein with intracellular N- and C-termini. The approximate location of the variant residues is shown. The number refers to the amino acid position in the protein which is preceded by the single-letter code for the amino acid commonly found at that position and is followed by the single letter code for the variant.

Figure 5 (a) Exon/intron structure of the 5-HTT/hSERT gene depicting several structural features. Untranslated regions of exons (boxes) are shown in grey. Exon 1B is an alternatively spliced noncoding exon. Sites localized to the 5' region of the gene are the 5-HTTLPR (black) and the 5-HTT del (grey). The intron 3 VNTR is shown in black in intron 3. (Scale bar = 100 bp of exon sequence. Introns are not drawn to scale.) (b) Amino acid variants identified in hSERT. hSERT is depicted as a 12-transmembrane domain spanning protein with intracellular N- and C-termini. The approximate location of the variant residues is shown. The number refers to the amino acid position in the protein which is preceded by the single-letter code for the amino acid commonly found at that position and is followed by the single letter code for the variant.

Table 1 Selected antagonists of monamine transporters

Table 2 Single nucleotide polymorphisms in hNET gene

Table 3 Single nucleotide polymorphisms in hDAT gene

Table 4 Single nucleotide polymorphisms in the 5-HTT gene