Dopamine Receptors and the Pharmacogenetics of Side-Effects of Stimulant Treatment for Attention-Deficit/Hyperactivity Disorder

To The Editor:

T here is considerable interest in the pharmacogenetics of responses to stimulant treatment for attention-deficit/hyperactivity disorder (ADHD) (Froehlich et al. 2010). By contrast, little attention has been paid to individual differences in side effects. Dopamine function in the prefrontal cortex (PFC) is a strong candidate system for understanding these (Arnsten, 2009). D1 receptors are the most abundant dopamine (DA) receptor subtype, and mediate most of the cellular effects of DA in this region (Lewis and Gonzalez-Burgos 2006). They are thought to be involved in the regulation of sustained firing of dorsolateral PFC neurons during the delay phase of delayed-response tasks that require working memory (Sawaguchi and Goldman-Rakic 1994; Seamans and Yang 2004; Lewis and Gonzalez-Burgos 2006). Sawaguchi and Goldman-Rakic (1994) showed that D1 dopamine receptors in the dorsolateral prefrontal cortex of monkeys participated in the maintenance of internalized visuospatial representations and/or in the control of eye movements governed by internal cues. They suggested that the D1 family of dopamine receptors may have a critical role in PFC-mediated working memory and cognitive deficits.

Variations in D1 polymorphism rs4532 are associated with ADHD (Bobb et al. 2005) and predict side effects to medication for schizophrenia (Potkin et al. 2003; Lai et al. 2011), although in the latter studies the effects may relate to excess dopamine at the D1 receptor. In both cases, there are good reasons to suggest that D1 receptor genetics may predict side-effects from stimulant medication for ADHD.

We tested the relationship of stimulant side effects to common variations in DA receptor genes using 90 Caucasian children (79.2% males) who met formal criteria for American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, 4th ed. (DSM-IV) diagnosis of ADHD (American Psychiatric Association 1994); were 4–14 years of age (mean=8.85 years; SD=2.32); had no major neurological/physical illness; had an intelligence quotient (IQ) >70; were using stimulant medication; and were not using another psychoactive medication (full demographic and diagnostic data and medication use available from Dr. Levy). Ethics approval and fully informed consent was from the University of New South Wales.

Participants gave blood (52.5%) or saliva (or both for a smaller reliability check sample) via Oragene saliva collection kits (http://www.dnagenotek.com/). DNA extraction rates were >95% for both methods. Samples were genotyped for four dopamine receptor single nucleotide polymorphisms (SNPs): DRD1 rs4532, DRD2 rs6277, DRD3 rs6280; DRD4 rs7932167, and COMT rs4680. All were in Hardy–Weinberg equilibrium. Genotypes were determined using iPLEX Gold™ primer extension followed by mass spectrometry analysis on the Sequenom MassARRAY system (Sequenom, San Diego, CA) by the Australian Genome Research Facility (AGRF: http://www.agrf.org.au/). Age, weight sex, and IQ were noted, and the side effects rated as absent, mild, moderate, and severe, using a modified Barkley side-effect scale (Barkley et al. 1990). Medication usage was as follows: Primary medication Ritalin (immediate release [IR]) n=43, dosage mean=22.03 SD=11.7; Ritalin (modified release) n=14, dosage mean=26.43 SD=6.33; Concerta n=12, dosage mean=29.25 SD=7.79; dexamphetamine n=19, dosage mean=8.37 SD=4.47; dexamphetamine (long acting [Dex LA]) n=2, dosage mean=10 SD=0; secondary medication Ritalin (IR) n=5, dosage mean=14.0 SD=10.25; Catapres n=9, dosage mean=25.05 SD=10.83. Secondary medications were distributed as follows: 4 of the 43 children receiving primary Ritalin IR were also receiving Catapres at night; 6 of the 14 children receiving Ritalin (modified release) were receiving a secondary medication, that is, 3 were also receiving Catapres at night, and 3 were receiving an additional Ritalin IR during the day. Two of the 19 children receiving dexamphetamine were also receiving Ritalin IR during the day; none of those on Dex LA were receiving an secondary medication; 2 of the 12 on Concerta were receiving Catapres at night.

Medication doses were standardized to a standard Ritalin (IR) dose of 10 mg by the following formula: Dexamphetamine 5mg=Ritalin IR 10mg; Ritalin (modified release) 10mg=Ritalin IR 5mg b.i.d.; Concerta 18mg=Ritalin IR 5mg t.i.d.

We reduced the 16 side effects items to factors using principal components analysis. Both parallel analysis and Velicer's minimum average partial (MAP) test indicated that there were three nonrandom factors; therefore, we generated a three factor solution using Varimax rotation with Kaiser normalization. Factor loadings are available from Dr. Levy. The three factors, nausea, withdrawal (social), and irritability, accounted for only 48.2% of variance, six items failed to load, and one (emotional) cross-loaded on two factors. The factors aligned well, however, with our clinical and theoretical expectations. We first checked if level of side effects correlated with any demographic or dosage variables that might confound results. None of the side effects measures correlated with level of stimulant dose indexed as a raw dosage, standardized across medication variants, or after dividing by age or weight. Using other potential confounders, there were no significant correlations for nausea; however, both appearing rigid and “Zombie”-like and irritability correlated positively with age (r=0.23 and 0.27, p<0.05 respectively). The relationship of the polymorphisms to side effects was tested using multivariate analyses of covariance (MANCOVAs) in which side effect levels were the dependent variables, and allelic status was the independent variable (coded 0=minor; 1=heterozygote; 2=major homozygote; National Center for Biotechnology Information [NCBI] dbSNP build 79/137). Age, dose/weight, and IQ were all used as covariates.

Mean severities of nausea, withdrawal and irritability side effects split by children's status on each allele type are shown in Figure 1. A significant effect of allele status was found for rs4532, the DRD1 receptor polymorphism using Bonferroni correction, F(6,134)=5.73, p<0.001. Univariate follow-up tests showed that the SNP effect was significant for withdrawal, F(2,68)=15.99, p<0.001, with a nominally significant trend for nausea, F(2,68)=3.95, p=0.02. As can be seen in Figure 1, the minor homozygote CC was associated with more severe side effects than either the heterozygote CT or the major homozygote TT.

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FIG. 1.

Mean side-effects severity for nausea, withdrawal, and irritability for minor, heterozygous, and major alleles of DRD1, COMT, DRD2, DRD3, and DRD4 single nucleotide polymorphisms. Error bars show the standard error of the mean.

As methylphenidate and dexamphetamine have distinct pharmacological effects, we re-analyzed the data for the methylphenidate group alone (there were insufficient numbers to look at dexamphetamine alone). We also removed those receiving any secondary medication. The significant effect of the D1 receptor minor allele remained significant for withdrawal, F(2,49)=8.271, p<0.001, and borderline for nausea, F(2,49)=2.689, p=0.08.

To our knowledge, the present data are the first to demonstrate that D1 allelic status (rs4532) predicts side-effects from stimulant medication for ADHD. Previous research has shown that the minor allele of rs4532 is more common in ADHD probands (Bobb et al., 2005), and predicts response and side effects to drug treatment in schizophrenia, albeit associated with the DA antagonistic action of antipsychotics rather than the agonistic action of stimulants (Potkin et al., 2003; Lai et al., 2011). SNP rs4532 is located in the 5’ untranslated region (UTR) of exon one of the DRD1 gene, and whereas its functional relevance is unclear, it may affect DRD1 expression (Huang et al., 2008). For example, Potkin et al. (2003) found that treatment and brain regional metabolic responses to clozapine were significantly associated with rs4532 genotypes.

This research supports the value of research into the pharmacogenetics of individual differences in medication side-effects. On the basis of these initial data, we hypothesize that dopamine D1 receptor systems has value in predicting side effects to stimulant medication for ADHD. Further research with larger numbers and dose ranges should provide validation of these initial findings.

Disclosures

No competing financial interests exist.