The Molecular Genetics of ADHD: A View From the IMAGE Project

By Stephen V. Faraone, Ph.D., and Philip Asherson, MRCPsych, Ph.D.
Psychiatric Times August 2005 Vol. XXII Issue 9

Although attention-deficit/hyperactivity disorder is frequently misunderstood as caused by normal childhood energy, boring classrooms, or overstressed parents and teachers, several decades of research show ADHD to be a valid disorder with a neurobiological basis (Faraone, in press). Genetic studies have played a leading role in clarifying the biological basis of the disorder. Family studies have documented familial transmission; adoption studies show this transmission occurs through biological, not adoptive relationships; and twin studies show that ADHD is highly heritable such that genes account for about 75% of the disorder's variability in the population (Faraone et al., in press). With a prevalence of 8% to 12% (Faraone et al., 2003), it is among the most common of psychiatric disorders. Given this strong evidence from epidemiologic studies, molecular genetic studies have begun the search for genes that increase susceptibility to ADHD. Two general approaches have been used. Genome scan linkage studies scan the entire genome in search of regions that might harbor susceptibility genes. They do not require a prior hypothesis about which genes cause the disorder. In contrast, candidate gene studies nominate specific genes based on a biological theory about their putative role. They use the method of association to test these prior hypotheses. To date, there have been three research groups performing genome linkage scans of ADHD (Arcos-Burgos et al., 2004; Bakker et al., 2003; Fisher et al., 2002; Ogdie et al., 2004, 2003). Table 1 summarizes their main results and gives their highest LOD scores (a statistic that measures the evidence for linkage). With the exception of chromosome 17p11, genomic regions implicated by these studies do not overlap. Because three of the implicated regions (17p11, 16p13 and 15q15) have also been implicated in studies of autism, these findings may suggest a shared genetic susceptibility for these disorders. For ADHD, the modest replication across studies completed so far suggests that genes of moderately large effect are unlikely to exist. For this reason, further studies are needed to increase the power of available linkage data. Difficulties in replication may also be due to genetic heterogeneity (i.e., there are different forms of ADHD influenced by different genes). In contrast to the small number of linkage studies, numerous candidate gene studies have used association to determine if biologically relevant genes influence the susceptibility to ADHD. Much of this work has focused on genes in catecholaminergic systems because the drugs that effectively treat ADHD are either dopamine reuptake inhibitors (e.g., stimulants) or norepinephrine reuptake inhibitors (e.g., tricyclic antidepressants, atomoxetine [Strattera]).

Compared with dopaminergic and noradrenergic systems, serotonergic systems have received relatively little attention in ADHD research. This is because measures of serotonin metabolism are minimally related to the clinical efficacy of the medicines that treat ADHD. Nevertheless, molecular genetic studies have examined serotonergic genes due to the well-known role of serotonin in impulsivity, one of the core symptoms of ADHD (Brunner and Hen, 1997). Another biological pathway to ADHD was discovered through study of coloboma mice, which have a hemizygous deletion of chromosome 2q (Wilson, 2000). This deletion region includes the gene encoding SNAP-25, a neuron-specific protein implicated in exocytotic neurotransmitter release. Coloboma mice show spontaneous hyperactivity, delays in achieving complex neonatal motor abilities and learning deficiencies. These problems are not seen if the mice are given a functioning SNAP25 gene through a transgenic procedure. Treatment with mixed amphetamine salts (Adderall) but not methylphenidate (Concerta, Metadate, Ritalin) reverses the hyperactivity, which is consistent with the mechanism of action of these medications. Methylphenidate treats ADHD by blocking the dopamine transporter. Mixed amphetamine salts block the dopamine transporter but also facilitates the non-vesicular release of dopamine through reverse transport, which would be expected to reverse the deficits in exocytotic neurotransmitter release caused by the coloboma mutation. To test genes associated with these biological hypotheses, candidate gene studies have used casecontrol or family-based designs. Case-control designs compare allele frequencies between patients with ADHD and non-ADHD controls. Alleles that confer risk for ADHD should be more common among patients with ADHD. The family-based design compares the alleles that parents transmit to children with ADHD to alleles parents do not transmit. If an allele increases the risk for ADHD, it should be more common among the transmitted alleles than the nontransmitted alleles. From both study designs, it is possible to derive an odds ratio (OR) that assesses the magnitude of the association between ADHD and the putative risk alleles. An OR of 1.0 indicates no association; >1.0 indicates the allele increases risk for ADHD; and <1.0 indicates the allele decreases the risk for ADHD. Faraone et al. (in press) reviewed the ADHD candidate gene literature and examined pooled ORs for candidate genes that had been examined in at least three case-control or family-based association studies. Table 2 shows seven genes that provide statistically significant evidence of association with ADHD. Other genes have been studied, showing either negative (catechol Omethyltransferase, norepinephrine transporter) or unclear (D2 and D3 dopamine receptors; tyrosine hydroxylase; noradrenergic receptors ADRA2A, 2C and 1C; monoamine oxidase A; serotonin 2A receptor; the A4 and A7 acetylcholine receptor subunits, and the 2A glutamate) results. The ORs for the positive associations range from 1.13 to 1.45 (Table 2). These small ORs are consistent with the idea that many genes of small effect mediate the genetic vulnerability to ADHD. Moreover, they suggest one explanation for the frequent failure to replicate initial reports of association: Many individual studies are underpowered to find significant associations if the effects are modest (Lohmueller et al., 2003). These small and sometimes inconsistent

effects emphasize the need for future molecular genetic studies to implement strategies that will provide enough statistical power to detect small effects. To address this project, we and colleagues from multiple sites in Europe conceived the International Multi-site ADHD Genetics (IMAGE) project. The main aim of IMAGE is to generate a clinical and genetic resource of 1,400 sibling pairs and their biological parents. The sibling pair design will enable the use of linkage analysis to identify chromosomal regions containing genes of moderate-to-large effect and association strategies to identify genes of small effect. The IMAGE group uses a novel approach by including ADHD probands and all available siblings in one dataset. In order to increase confidence in the diagnosis and decrease potential genetic and etiological heterogeneity, probands are recruited from ADHD treatment centers and selected for DSM-IV combined subtype. All siblings of probands are included in the study, whether or not they have ADHD, and continuous rating scale measures of symptoms will be used to map genes. The use of quantitative measures to map genes for common disorders is known as quantitative trait locus (QTL) analysis and reflects the view that genetic influences on ADHD are continuously distributed throughout the population (Asherson and IMAGE Consortium, 2004). This means that genes that increase risk for ADHD are also expected to influence individual differences in ADHD symptoms throughout the entire population. Similar approaches have been used for other common traits such as blood pressure (Harrap et al., 2002), cholesterol level (Lin, 2003) and dyslexia (Cardon et al., 1994). The use of quantitative trait measures for ADHD genetic research is based on findings from numerous population-based twin studies that show high heritability for parent and teacher ratings of ADHD symptoms (Thapar et al., 1999). We know, for example, that correlations for parentand teacher-rated ADHD symptoms are 70% to 75% for identical (monozygotic [MZ]) twins (who share 100% of their genes) and 30% to 35% for nonidentical (dizygotic [DZ]) twins (who share 50% of their genes). In order to examine the genetic correlation between ADHD diagnosis and continuous rating scale measures, a method called DF analysis estimates group heritability from the differential regression of identical and nonidentical co-twin trait scores to the population trait mean where twin probands are selected for extreme scores (DeFries and Fulker, 1988). In one study of 6,000 preschool twins, the group heritability for twins where probands were selected for extreme scores ranged from 0.83 to 0.93 (Price et al., 2001). Using the IMAGE dataset, we estimated the familial correlation between combined-type ADHD probands and continuous measures of ADHD symptoms among siblings. As expected, both inattentive and hyperactive-impulsive symptoms show familial associations to combined-type ADHD of around 0.2 to 0.3, which is similar to sibling correlations for DZ twins estimated from population samples (Table 3). There are several potential advantages to our study design. The collection of DNA from a large sample of ADHD probands allows us to perform powerful tests of association that can identify

small genetic effects, such as those seen for candidate genes listed in Table 2. At the same time, we can investigate whether alleles associated with ADHD correlate with quantitative ratings in siblings and we can increase the power of our analysis by combining within- and between-pair tests of association. We previously tested the utility of this approach by investigating whether some of the genes associated with ADHD showed QTL associations in a population twin sample with parent ratings of ADHD behavior (Mill et al., 2005). We found significant correlations with previously identified risk alleles from SLC6A3 and DRD5 and a protective allele from SNAP-25. Our analyses found positive correlations using within-pair tests of association that are independent of genetic stratification and are robust to potential stratification effects generated by parent raters who may scale similar behaviors differently. In other words, within-pair differences for behavioral scores are more reliable than across-pair comparisons. This was reflected in the strength of the genetic associations we observed. The QTL linkage also takes advantage of the reliable nature of within-pair differences by looking for correlations between the similarity between siblings and the number of parental chromosomes they share at a particular genetic locus. The IMAGE dataset contains a subset of affected sibling pairs that can be used for more traditional tests of association and linkage using diagnostic status alone. In our initial set of 608 ADHD combined-type probands, we found a sibling concordance rate for combined-subtype ADHD of 22.1% for males and 5.2% for females. This gives rise to similar sibling risk ratios for the combined subtype for males (9.2%) and females (11.5%) when expressed as a ratio against recent prevalence rates in a European population (2.4% males, 0.4% females). As we discover more genes, the goal of ADHD genetic research will shift from gene discovery toward gene functionality. Quantitative genetic findings have shifted perception of ADHD toward that of a quantitative trait sharing etiological influences with other developmental, behavioral and cognitive traits. Molecular genetics has confirmed a priori hypotheses of dopamine system dysregulation and promises to identify additional genes in the coming decade. Combining genetic, environmental and neurobiological research has the potential to delineate causal links between ADHD and the developmental course of the disorder, including persistence of ADHD symptoms into adulthood and comorbidity with associated psychiatric disorders/traits. Acknowledgements The IMAGE project is a multi-site, international effort supported by National Institutes of Health grant R01MH62873 to Dr. Faraone. IMAGE Project principal investigators are Philip Asherson, MRCPsych, Ph.D.; Tobias Banaschewski, Ph.D.; Jan Buitelaar, M.D., Ph.D.; Richard P. Ebstein, Ph.D.; Stephen V. Faraone, Ph.D.; Michael Gill, M.D., MRCPsych; Ana Miranda, Ph.D.; Fernando Mulas, Dr.D.; Robert D. Oades, Ph.D.; Herbert Roeyers, Ph.D.; Aribert Rothenberger, M.D.; Joseph Sergeant, Ph.D.; Edmund Sonuga-Barke, Ph.D.; Eric Taylor, M.D., Ph.D.; and Hans-Christoph Steinhausen, M.D., Ph.D. Dr. Faraone is professor of psychiatry and behavioral sciences at the State University of New York Upstate Medical University in Syracuse. He is also director of medical genetics research

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