Genes and attention deficit hyperactivity disorder - Springer Link

Genes and Attention Deficit Hyperactivity Disorder James Swanson, PhD*, Michael Posner, PhD, John Fusella, PhD, Michael Wasdell, MA, Tobias Sommer, MS, and Jin Fan, PhD

Address * Child Development Center, University of California at Irvine, 19722 MacArthur Boulevard, Irvine, CA 92612, USA. E-mail:jmswanso@uci,edu Current Psychiatry Reports 2001, 3:92–100 Current Science Inc. ISSN 1523-3812 Copyright © 2001 by Current Science Inc.

The initial molecular genetic studies of attention deficit hyperactivity disorder (ADHD) evaluated two candidate genes (DAT and DRD4) suggested by dopamine theories of this common disorder and its treatment with stimulant medication. The initial reports of weak associations with ADHD have been replicated by many (but not all) investigators, as is expected for genes with small effects. This literature is reviewed, along with emerging literature generated by active research groups investigating additional genes that might contribute to the genetic basis of this complex disorder.

Introduction Attention deficit hyperactivity disorder (ADHD) is the most prevalent psychiatric disorder of childhood, and for over 50 years a specific pharmacologic intervention (low doses of stimulant drugs such as amphetamine or methylphenidate) has been used as a treatment for this condition [1]. Extensive research and clinical experience has provided some validity for the diagnosis of ADHD and its treatment with stimulant medication [2], but controversies exist. Defining the biologic bases of ADHD has been a major goal of investigators [3], but the lack of clear and convincing evidence to support this goal has been emphasized by vocal critics [4]. Wender [5] developed a seminal hypothesis of a possible biologic basis, emphasizing dysfunction in catecholaminergic neurotransmitters (dopamine and norepinephrine) and suggesting a possible genetic origin. Levy [6] refined this neurotransmitter hypothesis by developing a dopamine deficit theory of ADHD. In the mid 1990s, several brain imaging studies that compared groups of ADHD and control children documented anatomic abnormalities in specific brain regions, including some (eg, reduced size of right frontal lobe and

caudate nucleus) where dopamine receptors are dense [7]. These brain regions are crucial to components of attention as defined by the neuroanatomic network theory of attention [8]. In the late 1990s, the mechanism of action of clinical doses of methylphenidate (the primary stimulant used in pharmacologic treatment) was clearly defined by positron emission tomography imaging to be a blockade of the dopamine transporter [9]. These emerging developments in the 1990s provided converging support for dopamine theories of ADHD [5,6]. This empiric and theoretic work set the stage for the initial molecular genetic gene studies of ADHD. Wh en t he molecular gen et ic studies of ADHD were initiated in the mid 1990s, it was already established that ADHD had a genetic component, based on behavioral genetic studies that documented high familiality and heritability. These behavioral genetic methods (eg, from adoption [10], family [11], and twin [12] studies) evaluate the phenotypic relationships of ADHD probands to parents and siblings who share well-defined genetic overlap, but they are not designed to implicate specific genes or biologic mechanisms. Molecular genetic methods utilize genotypic information from DNA as well as phenotypic information to investigate specific genes that may contribute to a disorder or a behavior. Two common molecular genetic approaches are the genome scan approach and the candidate gene approach [13]. The genome scan approach is used when there is no a priori hypothesis, and it considers the location of all genes based on the use of a set of genetic markers spread across the human genome. In groups of affected siblings, higher than expected allele sharing (> 0.5) of a marker suggest “linkage” with a disease gene. This identifies the approximate chromosomal region but not a specific gene, so “position cloning ” is then necessary to locate the disease gene [13]. Also, the power of this method is low, so large sample sizes are required [14]. For example, this approach was adopted in the area of autism, because “the lack of candidate genes meant that there were poor leads for any association study and hence the choice of an affected sibpair design was the obvious way to go” [15]. However, the results [16] suggest that many genes of small effects

Genes and Attention Deficit Hyperactivity Disorder • Swanson et al.

probably accumulate as the genetic cause of autism. Risch et al. [17] estimated that the number of genes was large (> 15) and concluded that positional cloning of susceptibility loci by linkage analysis may be a formidable task, and that other approaches may be necessary. A candidate gene approach can be used when there is an a priori hypothesis about a gene (or genes) that may be associated with a disorder. However, there is some bias against the use of this approach in psychiatric genetic studies because of the risk of false positive findings [18], and the assumption that many unimagined genes will be involved in complex disorders [13]. Despite this caution, in the initial studies of the molecular basis of ADHD, the candidate gene approach was used. This approach has been remarkably successful [19,20•], probably because the selection of candidate genes was based on sound neuroscience theory. However, this initial step is certainly not sufficient to unravel the genetic bases of a complex disorder such as ADHD. In this review, we take a historic approach to summarize the initial findings, present the current literature, and describe the next stage of investigations that are now in progress.

Candidate Gene Methods Candidate gene approaches are based on evaluation of the relationship between genotype (eg, the alleles of a specific gene) and phenotype (eg, the presence of symptoms of ADHD). One way this can be accomplished is in a population-based (case-control) design that compares the variation in genotype (alleles frequencies of the candidate gene) in groups of subjects defined by variation in phenotype (presence or absence of the disorder). If a candidate gene plays a causative role in the manifestation of the disorder in question, then the frequency distribution of the alleles should differ for the groups defined by the presence or absence of the disease. In general, a “risk allele” is defined by an allele that is higher in the group with the disease. However, if the groups also differ on other factors that may produce variation in allele frequency, such as ethnicity, then the relationship between the genotype and phenotype may be artifactual [18]. To avoid this, a family-based design may be used that compares the transmission of alleles from parents to affected children rather than allele frequencies across groups of cases and controls. Under the null hypothesis of no association, the genotype should be independent of the phenotype, so the transmission of alleles of a gene from parents to affected probands should conform to Mendel’s law of segregation and be around 0.5. On the other hand, if the probability of transmission of a risk allele is significantly higher than expected (ie, > 0.5), then the gene is either associated with the disorder, or if it is not, then it is in linkage disequilibrium with a nearby gene that is associated with the disorder.

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Selection of Candidate Genes The dopamine theories of ADHD [5,6] and of attentional networks [8] influenced the selection of two candidate genes. These theories emerged from developments in a relatively new area called cognitive neuroscience, which draws on cognitive science as well as basic neurosciences [21]. The cognitive neuroscience approach suggests the schematic of a dopamine synapse shown in Figure 1 [21], which integrates brain anatomy, neurotransmitter pathways, and attentional networks. This shows the termination of dopamine neurons (originating from the substantia nigra or ventral tegmental areas in the midbrain) as they synapse with dopamine receptive neurons (located in the caudate nucleus or cingulate gyrus). The density of two types of dopamine receptors (D2 and D4) differ in the two brain regions shown [22••], with a greater than two to one density of the D4 receptor in the prefrontal cortex than in the caudate nucleus. These brain regions are integral parts of the circuitry for component processes of attention (alerting, orienting, and executive control) according to Posner and Raichle’s [8] neuroanatomic network theory. Neuronal firing produces release of dopamine into the synapse, and Figure 1 shows two fates of released dopamine. The dopamine molecule may attach to a dopamine receptor located in the postsynaptic membrane, which would propagate the neuronal firing by a cascade of events. Or, the dopamine molecule may attach to a transporter protein that removes the dopamine molecule from the synapse by reuptake back into the presynaptic membrane, which is one of the processes that regulates synaptic levels of dopamine and is the primary site of action of methylphenidate, which blocks this reuptake process [9]. The dopamine transporter density is more than an order of magnitude higher in the caudate nucleus than the prefrontal cortex [22••], which is the reverse pattern of relative density of the D4 receptors, so the regulation of levels of synaptic dopamine by the reuptake process should differ dramatically in these two brain regions [21]. These factors led to selection of candidate genes. For example, the dopamine deficit hypothesis of ADHD [5,6] led to investigations of the dopamine transporter (DAT) gene, because it may influence individual differences in the process that regulates synaptic dopamine (especially for D2 synapses in the caudate nucleus). This may be a mechanism by which genetic variation produces a functional dopamine deficit in some individuals who, as a result, manifest symptoms of ADHD. The dopamine deficit theory also suggested investigations of dopamine receptor genes, because they may influence individual differences in dopamine levels required for the activation of dopamine receptive neurons (especially for D4 synapes in the frontal cortex). This also may be a mechanism by which genetic variation produces a functional dopamine deficit. A cognitive theory of attention [8,23] was used to focus on the D4 receptor, which is selectively expressed in the mesolimbic (rather than the nigrostriatal) dopamine

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Child and Adolescent Disorders Figure 1. The nigrostriatal dopamine pathway (arrows showing connections from substantia nigra to caudate nucleus) and the mesolimbic dopamine pathway (arrows showing connections from ventral tegmentum to frontal cortex are shown. The density of receptor types (D2 and D4 synapses shown) differ in these two pathways, and the regulation of synaptic dopamine by re-uptake may differ, due to differences in density of dopamine transporters (shown as the primary the site of action of methylphenidate, which blocks the re-uptake process).

pathway of the brain (Fig. 1) and is localized in brain regions related to the components of attention called executive control and alerting (Fig. 2).

The Initial Candidate Gene Investigations The initial molecular genetic studies of ADHD were conducted by groups at the University of Chicago and the University of California, Irvine/University of Toronto. Both groups were led by experts in psychiatric genetics (Ed Cook at the University of Chicago and James Kennedy at the University of Toronto) and clinical experts in the area of ADHD (Mark Stein at the University of Chicago and James Swanson at the University of California, Irvine). These groups capitalized on recent developments in the cognitive neurosciences to select candidate genes. Two candidate genes (Fig. 3) were selected for the initial investigations of specific genes that may be associated with ADHD: 1) the DAT gene located on chromosome 5p15.3 [24], and 2) the dopamine receptor D4 (DRD4) gene on chromosome 11p15.5 [25]. The polymorphism of interest in both of these candidate genes is based on alleles defined by a variable number of tandem repeats (VNTR). As outlined above [23], hypotheses based on developments in the cognitive neuroscience speculate how genetic variations might contribute to neuronal activity in dopamine pathways: alleles of the DAT gene may regulate the efficiency of the reuptake process in the nigrostriatal dopamine pathways and alleles of the DRD4 gene may affect the sensitivity of D4 receptors to levels of synaptic dopamine. The VNTR in the DAT gene [26] is a 40-base pair (bp) repeat sequence in the 3’ untranslated region of the gene.

Because this VNTR is not in a coding region of the DAT gene, it does not affect the protein sequence of the dopamine transporter, but it may affect the amount of protein (and thus the density of dopamine transporters) expressed in brain regions. In the human population, the primary alleles have nine or 10 repeats of the VNTR (denoted as DAT.9 and DAT.10). The allele frequencies vary across ethnic groups, but in some populations the reported allele frequencies are about 0.23 for the DAT.9 allele and 0.76 for the DAT.10 allele [19]. Because a primary mechanism of action of methylphenidate is the inhibition of reuptake of dopamine [9]), the DAT gene is a logical candidate based on the site-of-action strategy. In the initial molecular genetic study of DAT and ADHD, a family-based control association study, Cook et al. [24] investigated parent-tochild transmission rates of the DAT alleles, and reported that an increased prevalence (0.85) and parent-to-proband transmission rate (0.60) of the 10-repeat allele in children with ADHD. The VNTR in the DRD4 gene [27] is a 48-bp repeat sequence in exon III of the gene. Because this VNTR is in a coding region of the DRD4 gene, it does affect the protein structure of the D4 receptor. The primary alleles have from two to 10 repeats (denoted as alleles D4.2 to D4.10), which produce structural differences in the D4 receptor across individuals in the size of the third intracellular loop involved in G-protein coupling and mediation of postsynaptic effects. In humans the allele frequencies of DRD4 vary across ethnic groups [27], but in some populations [19] the frequencies of the most prevalent alleles have been reported to be about 0.10 (D4.2 allele), 0.67 (D4.4 allele), and 0.12 (D4.7 allele), with about 0.11 for all other


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Figure 2. Two parallel segregated circuits (networks) involved in component processes of attention. These networks are defined by cortical-striatal-thalamic-cortical loops. In the Posner and Raichle neuroanatomic network theory of attention, the neural circuitry underlying "executive control" includes the anterior cingulate gyrus and nucleus accumbens, and the neural circuitry underlying "alerting" involves the right prefrontal cortex and caudate nucleus.

alleles. In the initial molecular genetic study of the DRD4 gene and ADHD, a population based-association study, LaHoste et al. [25] reported and increased allele frequency (0.28) of the 7-repeat allele in children with ADHD as compared with an ethnically matched control group (0.12). This finding was suspect because the case-control design did not protect against the feared artifact of population stratification (despite the care taken for ethnic matching), so the same investigators used a family-based association design to correct this problem. Swanson et al. [28] reported a replication of an increased frequency of the 7-repeat allele in an ADHD sample (0.27) compared with a control group defined by the nontransmitted parental alleles (0.17), which provide perfect ethnic matching. The haplotype relative risk test revealed a statistically significant effect that extended the results of the populationbased association study.

Replications of the Initial Candidate Gene Studies Usually, an initial positive finding that emerges from a candidate gene study is not replicated [18]. However, in several subsequent studies of the DAT and DRD4 genes and their association with ADHD, replications of the initial results have been reported. For example, following the initial study by Cook et al. [24], Gill et al. [29] provided an independent replication of an increased prevalence and transmission rate of the 10-repeat allele of the DAT gene. And, following the study by Swanson et al. [28], Smalley et al. [30] provided an independent replication of an increased prevalence and transmission rate of the 7-repeat allele of the DRD4 gene. An early review [19] of three studies of the DAT gene and five studies of the DRD4 gene summarized the early literature,

pointing out all three DAT studies were positive and four of the five DRD4 studies were positive. These replications suggest that the initial findings were not statistical artifacts. However, the relative risk values for the DAT and the DRD4 genes are relatively low (about 1.5). Based on this, it should be expected that many studies would not duplicate the initial findings [31]. This pattern has emerged in recent studies. Recently, a formal meta-analysis [32] was conducted of the growing body of literature of the DRD4-ADHD association, which included seven case-control studies (four positive) and 14 family-based studies (nine positive). The conclusion of the meta-analysis was that a statistically significant association between ADHD and the 7-repeat allele of DRD4 existed, with a relative risk of 1.9 (P < 0.00000008) for seven case-control studies and 1.4 (P < 0.02) for 14 family-based studies. Others have reviewed this literature and suggested that it may reflect “a major achievement in psychiatric genetics: an association finding which has been observed in an overwhelming majority of attempts at replication” [20•]. The replication of the DAT-ADHD association is received less attention, and an exhaustive review of the literature has not been reported. Two groups that have reported positive results for the DRD4-ADHD association have reported negative results for the DAT-ADHD association. Swanson et al. [19] conducted a family-based association study with 80 trios and reported a trend toward more transmission of the 9-repeat than the 10-repeat allele, which opposite the pattern reported in the initial studies [24,29]. Holmes et al. [33] conducted a family-based study of 137 probands as well as a population-based comparison with 295 control subjects, and reported negative findings, due to a slight tendencies opposite the predictions from the initial studies: lower frequency of the 10-repeat allele in the ADHD group (72.6%

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Child and Adolescent Disorders

Figure 3. A schematic representation of the 22 pairs of autosomes and one pair of sex chromosomes with bands from chemical staining (the human karyotype). The dopamine transporter (DAT) gene is shown on the short arm of chromosome 5 and the dopamine receptor type 4 (DRD4) gene is shown on the short arm of chromosome 11.

vs 73.6%) and a slight tendency for nontransmission of the 10-repeat allele (40 transmitted, 45 not transmitted). However, Barr et al. [34•] conducted a family-based study of 333 probands with ADHD and reported a trend (P < 0.06) toward increased transmission of the 10-repeat as predicted by prior studies (58 transmitted vs 42 not transmitted). Furthermore, when a haplotype analysis was performed using two additional markers in the DAT gene, the most common haplotype (which included the 10-repeat allele) showed significantly (P < 0.0x) biased transmission, which confirmed an association of ADHD with the DAT gene. Cook [35] summarized the result of 11 family-based studies (including all those mentioned above) in a meta analysis and concluded that the association between the DAT gene and ADHD was highly significant (P < 0.000132).

Nonreplications of the DRD4-Attention Deficit Hyperactivity Disorder Association Because nonreplications are expected, some of the studies that did not find evidence of an association between DRD4 and ADHD are discussed. McCracken et al. [36] expanded the sample size reported by Smalley et al. [30] from 220 to 371 probands, and the data from the additional 151 trios rendered the prior significant finding nonsignificant (P < 0.297). Sunohara et al. [37] reported on an extension of the sample reported by Swanson et al. [28], and in an

additional 52 trios found no evidence for biased transmission of the 7-repeat allele. These studies suggest that a sample from recruitment late in a series of patients may produce a different pattern of association than a sample from recruitment early in the series, perhaps due to subtle differences in the phenotype. Nonreplication has been reported in samples from different ethnic groups. For example, in a family-based study with an Irish sample, Hawi et al. [38] failed to replicate the DRD4-ADHD association, and two reports of family-based studies in Israel [39,40] failed to replicate association of the DRD4 gene with ADHD. These studies suggest that the association of DRD4 and ADHD may differ in different ethnic groups. Also, partial replication (or nonreplication) has been reported in two recent large studies in the United Kingdom, based on population-based and family based designs. Holmes et al. [33] and Mill et al. [41] reported an increased prevalence of the 7-repeat allele in ADHD groups compared with control groups, but family-based tests showed no increase in the transmission rate from parent to proband. Both reports pointed out that this pattern may occur if a gene-environment interactions is operating, such that offspring (with or without a 7-repeat allele) of parents with a 7-repeat allele were to be affected by some nongenetic factors (eg, consumption of alcohol or tobacco during pregnancy, exposure to stress in the family, and so on).


What Is the Risk Allele? The early studies suggested that the 10-repeat allele was the risk allele of the DAT gene and that the 7-repeat allele was the risk allele of the DRD4 gene. However, these speculations provide just a starting point for specifying the underlying biologic mechanisms involved in the expression of the disorder. The statistical associations of genotype with phenotype that are provided by the candidate gene studies do not provide evidence about the biologic consequences of genetic variation, and allelic differences may confer risk or protection from disease related factors. One way to approach the issue of risk is to compare the cases with and without a putative risk allele on performance measures (quantitative traits). An initial study of this type was performed to evaluate the consequences of having a 7-repeat allele in a group of children with ADHD [42]. The prior studies [25,28] suggested that about half of the ADHD cases would have the 7-present genotype and about would have the 7-absent genotype. Based on the risk implied by an increased prevalence of the 7-repea t allele in ADHD groups, a prediction was made for more severe and pervasive impairment in subgroups of individuals with (7-present) than without (7-absent) this putative risk allele. The hypothesis of greater impairment in the 7-present genotype was tested using a quantitative trait from reaction time tests administered in a laboratory setting [42]. Performance (speed and variability of reaction time) on three neuropsychologic tasks (the Stroop task, the Generate-Read task, and the Stop task) was evaluated. Based on the notion that these tasks place high demands on the attentional networks proposed by Posner and Raichle [8], slower and more variable reaction times were predicted for the 7-present group than for the 7-absent group of ADHD children. At the beh avioral level (eg, ratings of ADHD symptoms), there were no significant differences between the two groups. Surprisingly, at the cognitive level (eg, speed and variability of response) there were differences, but they were opposite the predictions: those ADHD subjects with a 7-repeat allele of the DRD4 gene showed better performance (faster and less variable responding on all three tasks) than those without a 7-repeat allele. The 7-present subgroup was not significantly different in performance on these tasks when compared to a control group of nonaffected classmates. One hypothesis suggested by these results is that the 7-absent allele may produce a partial syndrome with manifestations at the behavioral level of analysis (subjective impressions of symptom severity judged by parents and teachers) but not at the cognitive level of analysis (objective performance measures on tasks requiring attention). This is also consistent with principles of evolutionary biology that have been applied to account for psychiatric disorder [43,44]. For example, both Leckman et al. [43]

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and Pani [44] have speculated that ancient environments shaped (by positive Darwinian selection) current genotypes that are not well-suited to modern environments. Thus, disorders such as ADHD may be due to an environmental mismatch of formerly adaptive genotypes with the demands of modern society. A recent evaluation of ethnic variation in the prevalence of the DRD4 7-repeat allele, Chen et al. [45] speculated about the role this allele might have played in migration. This study was based on the notion that the 7-repeat allele was associated with the personality trait of novelty seeking [46], which may have been beneficial in the distant past for exploration and migration. They reported a high correlation (r = 0.85) between the 7-repeat allele frequency and miles of migration estimated for ethnic groups around the world, which may have been established by progressive founder effects as the human population expanded out of Africa. In the context of the environmental mismatch hypothesis, in the present environment of societies that require focused attention and socialization (and are not conducive to migration), the formerly adaptive personality trait of high novelty seeking may produce impairment. However, this speculation should be put into context of the literature on the DRD4 gene and novelty seeking, because a critical part of the hypothesis (ie, the molecular basis of novelty seeking) has not been firmly established [47].

Haplotype Analysis The function of the 7-repeat alleles may vary depending on particular combination with other alleles in the same chromosomal region (ie, a haplotype). Barr et al. [48] investigated this in an analysis of haplotypes based on three additional polymorphic sites in the DRD4 gene: a 120-bp repeat 1.2 kilobases upstream to the transcription start site, a single nucleotide polymorphism defined by a C to T change at –521-bp, and a C to G change at –616-bp. In this study, the most common DRD4 haplotype, defined by the combination of the 7-repeat allele of the 48-bp polymorphis m, t he 2-re pe at allele of t he 120- bp polymorphism, the T allele at –521-bp, and the C allele at –616-bp (the 7-2-T-C haplotype), had a biased transmission to ADHD probands. However, McCracken et al. [36] evaluated the haplotype based on the 48-bp 7-repeat and the 120-bp 2-repeat, and reported a biased transmission (66 transmitted vs 51 nontransmitted) in the opposite direction for the 7-2 haplotype that contained the 7-repeat allele.

Sequence Analysis The usual analysis of DNA to determine polymorphism in the DRD4 and DAT genes specifies the length of the 48-bp VNTR (ie, two to 10 repeats). However, several lines of research indicate that the length alone is not sufficient to define alleles of this gene. For example, it is has been

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established that 48-bp repeat is imperfect [49••,50], because not all 48-bp sequences are the same. The end 48-bp repeat sequences typically differ by three amino acids, and the middle 48-bp sequences are even more variable [50]. If these sequence differences affect behavior, then it is possible (or likely) that specifying just the length polymorphism defined by the 48-bp VNTR will not be sufficient to capture important features of the DRD4 gene that may be associated with the manifestations of symptoms of ADHD (ie, the phenotype). For example, the longer length may be more likely to harbor an abnormal 48-bp sequence, and it (rather than length) may have an effect on the phenotype. Moyzis et al. [51] have initiated a project to sequence the DRD4 gene in individuals with and without a diagnosis of ADHD, but this project has not yet determined the significance of the 7-repeat allelic variation.

Other Genes Associated with Attention Deficit Hyperactivity Disorder Because the heritability of ADHD is high (h2 > 0.75) and the relative risk of the initial candidate genes is so low (< 1.5), it is assumed that other genes will be discovered that are associated with ADHD. The continued search for catecholaminergic genes has generated several recent positive reports in the literature. In a recent issue of Molecular Psychiatry [52], candidate gene studies of ADHD were reported for the DRD2, DRD3, and DRD5 receptor genes, as well as for genes related to the enzymes monoamine oxidase and catechol-O-methyltransferase (COMT). These initial findings are not discussed here, because most are not expected to be replicated in future studies. For example, the plausible theory about COMT (an enzyme important in the metabolism of dopamine) led to the hypothesis that genetic variation in this enzyme might play a role in the manifestation of ADHD symptoms. This hypothesis was supported in an initial candidate gene study [53], but this finding was not replicated, either by the same group in another sample [54] or by another group [55]. This reinforces the advice of Crowe [18] to require multiple replications before accepting the findings of candidate gene studies. Studies of noncatecholaminergic genes have also been conducted based on animal models of ADHD such as the Coloboma mouse model of ADHD [56], which is related to a mutation in the SNAP-25 gene that produces a mouse with hyperactivity that is responsive to amphetamine. Barr et al. [57] reported a significant association with ADHD. Another line of research is based on the serotonin hypothesis of ADHD [58], which led Quist et al. [59] to investigate and report an association of the 5HIAA gene and ADHD.

Next Steps The initial molecular genetic studies of ADHD used the candidate gene approach rather than the genome scan

approach. This favored strategy is to use a set of markers to identify chromosome regions likely to be involved in a disorder, and then to follow-up with intensive search for genes in those implicated chromosomal regions. So far, no report of a genome scan has been published, but in the near future the reports of findings from multiple genome scans are expected. The use of pooled samples may be essential to obtain a sample size large enough for this task. An ADHD Molecular Genetics Network [60] has been formed for this purpose, which has spurred the development of operational definitions for a common assessment of cases across multiple sites and methods [61]. These represent concrete steps in the direction of large-scale collaborations to elucidate the complex molecular genetic bases of ADHD. There are many pitfalls for a genome scan investigation of ADHD. If ADHD is a complex genetic disorder, then multiple genes of small effects combine to produce the phenotype. Thus, many different combinations of genes could produce the same ADHD phenotype. Terwilliger and Weiss [62] point out that such a many to one mapping of genotype to phenotype would make it virtually impossible to identify a clear genetic cause as with simple Mendelian (single gene) disorders, where the mapping of genotype to phenotype is one to one. In fact, they suggest that these methods are inappropriate for the study of complex disorders. For complex disorders, traditional association studies may not take advantage of the optimal design. Allison [63] outline the benefits of a quantitative trait approach, which has considerable increased statistical power compared with other designs based on the qualitative definition of phenotype. However, the increased power is derived from selection of extremes of an underlying distribution, from the well end to the ill end. This approach may not be applicable for most measures of ADHD symptom severity, because typical measures evaluate only in the ill end of the distribution. For example, on the SNAP rating scale, which evaluates symptom presence on a four-point scale (not at all, just a little, quite a bit, and very much), those individuals without psychopathology all have the same score (reflecting no psychopathology). A new rating scale of Strengths and Weaknesses of ADHD-symptoms and Normal-behaviors has been developed [64,65] to allow for measurement across the entire range of behavior (from psychopathology to superiority). Also, a new Attention Network Task has been developed [66], based on laboratory assessment of reaction time to simple stimuli (eg, arrows), with manipulations within a short series of trials to vary temporal cues (alerting), directional cues (orienting), and conflicting flankers (executive control). This task provides reliable estimates of ability to perform these cognitive processes, and the variation across individuals for two of the processes (alerting and executive control) has a genetic component (ie, it is heritable). The application of the quantitative trait approach is predicted [63] to increase the power of the statistical tests based on a quantitative transmission disequilibrium test (Q-TDT).


Conclusions Even though the candidate gene approach has been very successful in the ADHD area [19,20,32,35], many of the initial finding with genes of small effects are not expected to be duplicated in all studies. The expected pattern of replication and nonreplication is present in the emerging literature in this relatively new field of molecular genetics of childhood psychiatric disorder. Meta analyses have confirmed the statistical association of ADHD with the DRD4 gene [32] and the DAT gene [35]. The next steps are underway to identify additional genes associated with ADHD as well as to perform finegrained analysis of the DRD4 and DAT genes. This research should advance our knowledge about the molecular genetic bases of this complex disorder.

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