Introduction
Attention-deficit/hyperactivity disorder (ADHD) is a common childhood-onset psychiatric disorder that features symptoms of age-inappropriate inattention and/or impulsivity and hyperactivity. ADHD affects 5–6 % of children (Polanczyk et al.
2007) and frequently persists into adulthood (Faraone et al. 2006) causing a prevalence of ADHD of between 2.5 and 4.9 % in the adult population (Simon et al.
2009). The heritability of ADHD is around 0.8 in both children (Faraone et al.
2005) and adults (Larsson et al.
2013). ADHD’s complex genetic etiology likely involves multiple genes of small to moderate effect (Akutagava-Martins et al.
2013).
The dopamine neurotransmission system has been an important focus of genetic research in ADHD, since it is the main site of action of stimulant drugs, the primary pharmacological treatment for the disorder (Cortese
2012; Faraone et al.
2014a). One of the most appealing and extensively studied candidate genes for ADHD is the dopamine transporter (
DAT1) gene (official name
SLC6A3) (Faraone et al.
2005; Franke et al.
2012). The dopamine transporter is a key determinant of synaptic dopamine levels by regulating the reuptake of dopamine from the extracellular space, thereby terminating its synaptic action (Madras et al.
2005). The association between
DAT1 and ADHD was suggested in linkage and association studies and is confirmed in meta-analyses (Franke et al.
2010; Gizer et al.
2009; Li et al.
2006) showing small but significant effects on the susceptibility to ADHD. Meta-analyses of genetic association studies have indicated that the 10-repeat allele of the 3′ untranslated region (UTR) variable number of tandem repeat (VNTR) is overrepresented in children with ADHD (Gizer et al.
2009). More recent studies suggested that the 10-repeat allele might increase ADHD risk in children particularly in the context of a haplotype with the 6-repeat allele of another VNTR in intron 8 of the gene (Asherson et al.
2007; Brookes et al.
2008). A recent study also found an association between this 10-6 haplotype and ADHD symptom measures in nonclinical adults (Tong et al.
2015), but association studies in clinical samples of adults with ADHD could not confirm this relationship (Brüggemann et al.
2007) and reported an association of the 9-6 haplotype with adult ADHD (Franke et al.
2008,
2010). Together, these findings suggest a role for
DAT1 in modulating the ADHD phenotype across the lifespan, with different associations depending on age and diagnostic status.
The specific mechanisms by which
DAT1 genetic variants affect the risk for ADHD are not well understood. Two imaging genetics studies showed that genetic variation of the
DAT1 gene is associated with altered striatal volume, which may contribute to ADHD susceptibility; the caudate nucleus, a sub-region of the striatum, was found to be smaller in children homozygous for the 10-repeat allele (10/10) than in carriers of the 9-repeat allele (Durston et al.
2005; Shook et al.
2011). Although both studies did not found an interaction between presence/absence of ADHD and genotype, Durston et al. (
2005) reported that the effect of
DAT1 genotype on caudate volume was only significant in the subgroup of patients with ADHD. Studies investigating the effect of the
DAT1 gene on prefrontal gray matter volume, cortical thickness, or white matter integrity found no association between 10-repeat allele carriers (10/10) and 9-repeat allele carriers (Durston et al.
2005; Hong et al.
2015; Shaw et al.
2007), suggesting that this gene primarily affects regions, where it is highly expressed (i.e., the striatum) (Ciliax et al.
1999; Durston et al.
2009).
The effect of the
DAT1 gene on striatal volumes may help explain smaller volumes of caudate nucleus and putamen typically found in children with ADHD (Ellison-Wright et al. 2008; Frodl and Skokauskas
2012; Nakao et al.
2011; Valera et al. 2007). It has been shown that volumetric differences in caudate nucleus and the putamen gradually disappear with age (Castellanos et al.
2002; Frodl and Skokauskas
2012; Greven et al.
2015; Maier et al.
2015; Nakao et al.
2011). The largest study to date by the ENIGMA ADHD Working Group containing 1713 participants with ADHD and 1529 controls show (among others) reduced accumbens, caudate nucleus, and putamen volume in ADHD. Case–control differences were most pronounced in childhood confirming a model of delayed brain growth and maturation (Hoogman et al., submitted). Nonetheless, there is evidence from studies of adults with persistent ADHD that differences in caudate nucleus volume (Almeida Montes et al.
2010; Onnink et al.
2014; Proal et al.
2011; Seidman et al.
2011; Shaw et al.
2014) and putamen volume (Seidman et al.
2011; Shaw et al.
2014) persist into adulthood.
To summarize, existing literature points to different alleles of the
DAT1 increasing susceptibility to categorically defined ADHD from childhood to adulthood, with a possible role of striatal volume in the pathway from gene to disease. The evidence for an influence of
DAT1 on striatal volume is based on relatively small-sampled studies [
N = 59 in Shook et al. (
2011) and
N = 72 in Durston et al. (
2005)]. Moreover, these studies examined only one variant of the
DAT1 gene (10/10 homozygotes versus 9-repeat carriers), not taking into account the potentially stronger effects of the two-VNTR haplotypes. Importantly, they were conducted in children only and could not test possible different effects of gene variation on striatal volume across the lifespan.
In the current study, we therefore set out to investigate the effects of the three different DAT1 risk variants on striatal brain volume (nucleus accumbens, caudate nucleus, putamen) and the potential interaction with diagnostic status and age. We defined the DAT1 10/10 genotype, the 10-6 haplotype, and the 9-6 haplotype as risk alleles, based on associations with ADHD in children (10/10 genotype and 10-6 haplotype) and in adults (9-6 haplotype), respectively. Participants were derived from three cohorts with cross-sectional MRI data, a childhood/adolescent sample (NeuroIMAGE, 301 patients with ADHD and 186 healthy controls) and two adult samples (IMpACT, 118 patients with ADHD and 111 healthy controls; BIG, 1718 healthy participants).
Discussion
In the current study, the effect of the dopamine transporter gene DAT1/SLC6A3 on striatal brain volume was investigated in children and adults with ADHD and healthy participants in three different cross-sectional cohorts. In the adult case–control cohort IMpACT, carriers of the 9-6 haplotype, the risk allele for adult ADHD, had larger striatal volume than participants not carrying this haplotype. This effect varied by diagnostic group, with the risk haplotype affecting striatal volumes only in patients with ADHD and not in the healthy participants from this cohort. Consistent with this, the effect was not found in the BIG cohort of adult healthy participants. It was also not observed in the case–control children/adolescents cohort from NeuroIMAGE. Through an interaction analysis within the IMpACT cohort, also the 10/10 genotype was shown to affect striatal volume in patients only when compared to carriers of 9R allele(s), which was a smaller effect than for the 9-6 haplotype (and probably was just the other side of the same coin).
The finding in the IMpACT cohort showing smaller striatal volume in adult ADHD patients homozygous for the 10R allele (10/10 carriers) compared to 9R carriers is consistent with previous studies performed in children (Durston et al.
2005; Shook et al.
2011). However, as 84 % of the 9R carriers consisted of 9-6 haplotype carriers, this effect might be driven by the subgroup of 9-6 haplotype carriers. Indeed, the regression coefficient of −0.64 (
p = .013,
N = 118) (supplementary Table 4) dropped to −0.074 (
p = .78,
N = 92) when the 9-6 haplotype carriers (
N = 26) were excluded from the analysis (data not shown). The diagnosis-specificity of
DAT1 only affecting striatal volume in the subgroup of patients with ADHD was also suggested in the previous study by Durston et al. (
2005). Larger striatal volume in adult carriers of the
DAT1 risk haplotype 9-6 for adult ADHD may represent compensatory mechanisms for the increased expression/activity of the dopamine transporter, which has been found in 9-repeat allele carriers (Faraone et al.
2014b). The increased levels of DAT in these individuals might lead to more efficient clearing of extracellular dopamine, yielding lower extracellular levels and reduced dopamine signaling (Faraone et al.
2014b). Importantly, a study by Spencer and coworkers showed that an ADHD diagnosis made an additional, independent contribution to DAT binding (Spencer et al.
2013). The diagnosis-specificity of our findings may thus reflect an interaction between genetic and environmental risk factors, where cumulative effects allow for a bigger impact of
DAT1 genotype on striatal volume in the patients. We emphasize, nonetheless, that replication of our findings is needed before firm conclusions can be drawn.
Our explorative 3-way interaction analysis in the cohorts combined (
N = 2434) investigating the effect
DAT1 9-6 haplotype, diagnostic status, and age suggests that carriership of the 9-6 haplotype predisposes to a slower age-related decay of striatal volume, which is specific for ADHD patients (Fig.
1). Importantly, age effects have shown a differential decay of
DAT1 expression for different genotypes (Shumay et al.
2011), which may be consistent with the compensation hypothesis mentioned above. Shumay et al. demonstrated that 9-repeat homozygotes showed the steepest decline of DAT availability with increasing age. Great care is needed in interpreting the age effects we observed, as this is a cross-sectional study. Interestingly, a recent study suggests that individuals can meet symptom criteria for ADHD as adults without having a history of childhood ADHD (Moffitt et al.
2015). Although this study by Moffitt et al. is in need of replication, our results may suggest that carriership of the
DAT1 9-6 haplotype might be a mechanism contributing to the emergence of new cases of ADHD during adulthood. However, to replicate our age-dependent effect and to explore this more fully, analysis of longitudinal MRI data is required.
The functional implications of larger striatal volume for the pathophysiology of adult ADHD remain to be investigated. As smaller caudate volume in male patients with ADHD has been associated with an increased number of hyperactivity/impulsivity symptoms (Onnink et al.
2014), larger striatum volume in a subgroup of ADHD patients may be linked to neurobiological processes that go along with the reported age-dependent decline in hyperactivity/impulsivity symptoms in people with ADHD (Biederman et al.
2000). Increased volume may also reflect compensatory ‘hypertrophy’ because of reduced dopamine neurotransmission (see above).
Our findings should be viewed in the light of certain strengths and limitations. A clear strength was the investigation of haplotypes of DAT1 in addition to the 3′UTR VNTR genotype variants in a large sample including patients with ADHD and healthy individuals at different ages. This case–control design maximized the variance in the phenotype and may have magnified gene effects. A strong limitation was the cross-sectional MRI study design, especially since the participants of this study were partly derived from different cohorts. Another limitation was the restricted availability of data at early childhood age and late adult age, which reflects insufficient focus of imaging research in our field on such age groups. The developmental trajectories our data propose need to be confirmed in additional studies, optimally from longitudinal studies including data across a wide age range collected using the same study protocol.
In summary, our cross-sectional findings showed that adult patients with ADHD carrying the DAT1 9-6 risk haplotype for adult ADHD had increased striatal volume. Furthermore, based on our exploratory analysis on age effects, we hypothesize that ADHD patients carrying the 9-6 haplotype follow a different trajectory of brain development over the lifespan than those ADHD patients not carrying this haplotype. These findings are in need of replication, preferably using longitudinal designs. Clarifying the nature of the involvement of DAT1 variants in brain development would provide a key step towards understanding part of ADHD’s pathophysiology. The present results demonstrate the importance of taking into account interindividual variability, as indexed by DAT1 haplotype, presence of an ADHD diagnosis, and age, when assessing striatal volume effects in ADHD.
Acknowledgments
We thank Angelien Heister, Marlies Naber and Remco Makkinje for help with genotyping, Janneke Dammers for assistance with recruitment and testing and Paul Gaalman for technical MRI assistance. We are grateful to all participants for their contribution.
NeuroIMAGE: This study used the sample from the NeuroIMAGE project. NeuroIMAGE was performed between 2009 and 2012, and is the follow-up study of the Dutch part of the International Multisite ADHD Genetics (IMAGE) project which is a multi-site, international effort. Funding support for the IMAGE project was provided by NIH Grants R01MH62873 and R01MH081803 to Dr. Faraone and the genotyping of samples was provided through the Genetic Association Information Network (GAIN). The dataset used for the analyses described in this manuscript were obtained from the database of Genotypes and Phenotypes (dbGaP) found at
http://www.ncbi.nlm.nih.gov/gap through dbGaP Accession Number #20726-2. This work was further supported by an NWO Large Investment Grant 1750102007010 and NWO Brain & Cognition an Integrative Approach Grant (433-09-242) (to Dr. Buitelaar), and grants from Radboud University Nijmegen Medical Center, University Medical Center Groningen and Accare, and VU University Amsterdam. The research leading to these results also received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under Grant Agreement Number 278948 (TACTICS) and number 602450 (IMAGEMEND). Dr. Franke is supported by a Vici grant from NWO (Grant number 016-130-669) and she and Dr. Buitelaar received funding from the National Institutes of Health (NIH) Consortium Grant U54 EB020403, supported by a cross-NIH alliance that funds Big Data to Knowledge Centers of Excellence. Dr. Faraone is supported by the K.G. Jebsen Centre for Research on Neuropsychiatric Disorders, University of Bergen, Bergen, Norway, the European Community’s Seventh Framework Programme (FP7/2007–2013) under Grant Agreement No 602805 and NIMH grants R13MH059126 and R01MH094469.
IMpACT: This study used the sample of the Dutch node of the International Multicentre persistent ADHD Collaboration (IMpACT). IMpACT unites major research centres working on the genetics of ADHD persistence across the lifespan and has participants in The Netherlands, Germany, Spain, Norway, the United Kingdom, the United States, Brazil and Sweden. Principal investigators of IMpACT are: Barbara Franke (chair), Andreas Reif (co-chair), Stephen V. Faraone, Jan Haavik, Bru Cormand, Antoni Ramos Quiroga, Philip Asherson, Klaus-Peter Lesch, Jonna Kuntsi, Claiton Bau, Jan Buitelaar, Stefan Johansson, Henrik Larsson, Alysa Doyle, and Eugenio Grevet. The Dutch IMpACT study is supported by grants from the Netherlands Organization for Scientific Research (NWO), i.e., the NWO Brain & Cognition Excellence Program (Grant 433-09-229) and a Vici grant to BF (Grant 016-130-669), and by grants from the Netherlands Brain Foundation (Grant 15F07[2]27) and BBMRI-NL (Grant CP2010-33). The research leading to these results also received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under Grant Agreements No 602805 (Aggressotype) and no 602450 (IMAGEMEND), and from the European Community’s Horizon 2020 Programme (H2020/2014–2020) under Grant Agreement No 643051 (MiND). In addition, the work was supported by a Grant for the ENIGMA Consortium (Grant number U54 EB020403) from the BD2K Initiative of a cross-NIH partnership.
BIG: This study used the BIG database which was established in Nijmegen in 2007. This resource is now part of Cognomics, a joint initiative by researchers of the Donders Centre for Cognitive Neuroimaging, the Human Genetics and Cognitive Neuroscience departments of the Radboud University Medical Centre and the Max Planck Institute for Psycholinguistics. The Cognomics Initiative is supported by the participating departments and centres and by external grants, i.e., the Biobanking and Biomolecular Resources Research Infrastructure (Netherlands) (BBMRI-NL), the Hersenstichting Nederland and the Netherlands Organisation for Scientific Research (NWO).