Introduction
Attention-deficit hyperactivity disorder (ADHD), characterized by persistent symptoms of inattention, hyperactivity and impulsivity, is one of the most common psychiatric and behavioural disorders in children and adolescents, with more than 5% of the paediatric population affected worldwide, and ADHD often persists into adulthood with a prevalence of 2.5–4.9% in adults (Thomas et al.
2015; Polanczyk et al.
2014). ADHD has been shown to have a high genetic component with around 80% heritability (Faraone and Larsson
2018). A recent genome-wide association study of over 20,000 ADHD patients has identified 12 independent loci to be genome-wide significantly associated with ADHD (Demontis et al.
2019). Nevertheless, genetic variations such as variable number tandem repeats (VNTRs) cannot be captured on such arrays, and therefore, conventional gene association studies may provide further information. One of these VNTRs (rs28363170), located on the dopamine transporter gene (
SLC6A3 /
DAT1) in the 3′ untranslated region (UTR), with the 10- and 9-repeat alleles that are most common (Doucette-Stamm et al.
1995), were found to be associated with ADHD. In particular, the 10-repeat allele was described in several meta-analyses to associate with child and adolescent ADHD, though with a rather low effect size and in most cases with high heterogeneity between studies due to clinical phenotyping, age and ethnicity (Gizer et al.
2009; Yang et al.
2007). Nevertheless, further studies kept looking into the association between
DAT1 gene and ADHD, also due to significant linkage findings with the chromosomal location 5p13 containing the gene and ADHD (Friedel et al.
2007). Since it is known that there are further variants (3–13-repeats) on the
DAT1 3′-UTR VNTR, expressing in various ethnicities in different frequencies (Mazei-Robinson and Blakely
2006), some studies looked also into associations with the 9- or the 11-repeat alleles. However, no association has been detected in child and adolescent ADHD both in European and Asian ethnicity (Li et al.
2006), whereas in adult ADHD a trend for association was found for 9-repeat allele carriers following a recent meta-analysis (Bonvicini et al.
2016).
The dopamine transporter is a key player in the dopaminergic system, regulating the synaptic dopamine homeostasis and its signalling. Since psychostimulants, such as amphetamine and methylphenidate, provide an effective treatment for ADHD (e.g., Faraone and Buitelaar
2010), are known to have high affinity to the transporter and inhibiting the transporter (Markowitz and Patrick
2008; Han and Gu
2006), the dopamine transporter has become one of the risk candidates for ADHD research. Dopamine controls numerous functions including attention, mood, cognition, reward and movement (Iversen and Iversen
2007). And altered dopamine homeostasis and particularly dopamine transporter is not exclusive for ADHD, but has been implicated in several disorders, including paediatric Bipolar Disorder (rs40184) (Mick et al.
2008), Major Depressive Disorder (9-repeat) (Lopez-Leon et al.
2008), Posttraumatic Stress Disorder (9-repeat) (Li et al.
2016), Tourette Syndrome (9-repeat associated with increased tics) (Tarnok et al.
2007), Obsessive–Compulsive Disorder (9-repeat) (Taylor
2013), Alzheimer’s disease (9-repeat) (Feher et al.
2014) and Alcoholism (9-repeat associated with alcohol withdrawal seizure and delirium tremens) (Du et al.
2011), while no association was found in Schizophrenia (Gamma et al.
2005)(see also meta-analysis in
http://www.szgene.org) and Parkinson’s disease (Geissler et al.
2017) [for detailed review on dopamine transporter and various CNS disorders see (McHugh and Buckley
2015)]. However most studies demonstrated either conflicting findings or having only a single finding lacking replications or having no significant findings. In a meta-analysis of a collection of naturalistic studies ADHD children without 10/10-repeat genotypes responded better to methylphenidate; however, this effect was not found in clinically monitored studies (Soleimani et al.
2018). On the other hand, Parkinson’s disease carriers of the 9-repeat allele required lower levopoda doses, as well as were at risk of hallucination/psychosis following treatment (Politi et al.
2018). Similarly, in methamphetamine substance use, 9-repeat was shown to be a strong risk factor for a worse prognosis of methamphetamine psychosis (Ujike et al.
2003).
Investigation regarding the functional consequence of the aforementioned variants has shown some mixed results. In vitro, using various cell culture models and reporter gene designs, showed either the 9- or the 10-repeat to increase
DAT1 gene expression (Fuke et al.
2001; VanNess et al.
2005; Mill et al.
2005; Greenwood and Kelsoe
2003; Inoue-Murayama et al.
2002; Hill et al.
2010; Miller and Madras
2002). Ex vivo, gene expression in post-mortem brain as well as in periphery also showed some inconsistent results [3 monkey substantia nigra (SN) (Miller and Madras
2002], 20 post-mortem cerebellum and temporal lobe and 18 volunteers lymphocytes (Mill et al.
2002), 7 post-mortem midbrain (Brookes et al.
2007), post-mortem of 30 Alzheimer’s disease SN and polar region of the frontal lobe (Pinsonneault et al.
2011), post-mortem of the ventral midbrain from 18 controls and 18 cocaine users (Zhou et al.
2014). To our knowledge, no data are available linking between central nervous system and blood
DAT1 expression and
DAT1 genotypes. However, Wiers et al. (
2018) reported correlations in a small post-mortem study investigating
DAT1 mRNA expression in SN of 3 adult ADHD and 13 controls and DAT protein expression in the Caudate. Indeed, they found a significant positive correlation between mRNA and protein expression in the two brain regions (Wiers et al.
2018). Furthermore, DNA methylation of the
DAT1 cluster A in blood was significantly correlated with the DNA methylation of the
DAT1 cluster A in SN (Wiers et al.
2018). In a recent meta-analysis of a collection of positron emission tomography (PET) a highly significant evidence for the 9-repeat allele was shown to be associated with increased dopamine-transporter activity in human adults, that was significant in healthy adults and only marginally significant in adult ADHD patients (Faraone et al.
2014). In the single-photon emission computed tomography (SPECT) analysis, although containing small sample size, similar results were obtained for healthy adults, while for affected adults (ADHD, Parkinson’s disease, Schizophrenia, and alcoholism) the opposite results were observed (Faraone et al.
2014). Interestingly, in adult age span (20–75 years of age) 10-repeat homozygotes showed reduced striatal activity during working memory task that reduced with age, that led to the hypothesis of earlier manifestation of cognitive impairment in 10-repeat homozygotes (Sambataro et al.
2015). Yet, most studies focused on the functional effects of the most frequent repeats, the 9- and 10-repeats, while few studies looked whether the other variants have any functional effect. In a recent study, the binding of HESR1 and two transcription factors, known to bind at the VNTR-site of the
DAT1 gene, was found to be inhibited depending on the length of the VNTR variant, with 11-repeats having low
DAT1 expression compared to the short variants up to 6-repeats (Kanno and Ishiura
2011). Moreover, the non-coding RNA, miR-491 was found to inhibit
DAT1 expression in a dose-dependent manner, in which higher repeat number (11-repeats) inhibited the expression compared to low number of repeats (up to 1-repeat) (Jia et al.
2016).
Therefore, in the current case–control and family study, we assessed whether the association between DAT 3′-UTR VNTR 10-repeat carriers or long-allele (10-repeats and higher) carriers conveys a risk for ADHD. In addition, we conducted a systematic review of the literature followed by an updated meta-analysis, including all current available associations with DAT1 3′-UTR VNTR for case–control and family studies in child and adolescent as well as adult ADHD in various ethnicities. To address the possible confounding effects by age of patients, and their ethnicity, a stratified meta-analysis for each variable was conducted.
Discussion
The current meta-analysis, could confirm previous findings showing weak association between the 10-repeat allele of the
DAT1 3′-UTR VNTR gene and child and adolescent ADHD, that reached significance only in the European population; however, this was accompanied by high heterogeneity that was in some cases due to literature bias but in other cases due to heterogeneity in clinical phenotyping, age or ethnicity (Li et al.
2006; Gizer et al.
2009). On the other hand, we were not able to confirm a significant association between the 9-repeat allele and adult ADHD as previously reported (Bonvicini et al.
2016), and only a nominal significant association was observed for the European adult ADHD with 9-repeat as risk allele (see Supplementary Table S2).
Interestingly, assessing the functional approach, in which the long-allele was suggested to result in decreased dopamine transporter expression (Faraone et al.
2014; Kanno and Ishiura
2011; Jia et al.
2016), seem to show significant association with ADHD. Indeed, the meta-analysis including the overall ADHD studies, as well as the child and adolescent ADHD, the Caucasian child and adolescent ADHD, the European child and adolescent ADHD, and nominally the Asian child and adolescent ADHD resulted in significant association with Long-allele as risk allele. In the other ethnical groups the associations did not reach significance, however all seem to show some tendency toward the long-allele as risk allele, however further studies should be conducted to confirm this hypothesis.
The current study included the largest sample size available (total
n = 40,681 consisting of 14,821 cases) for a powerful meta-analysis of the
DAT1 3′-UTR VNTR gene variants, and used when needed the random-effects model that incorporate heterogeneity among trials (Borenstein et al.
2010). Indeed, following quality assessment of all included studies according to traditional epidemiological considerations (Liu et al.
2015) indicated that some studies did not reach high quality scores due to sample size, diagnostic criteria, recruitment strategies and quality control of the genetic analysis. This was reflected in significant heterogeneity, that in some cases, was also confirmed with significant publication bias assessed with Begg’s test (Begg and Mazumdar
1994) and Egger’s regression test (Egger et al.
1997). In these few cases we corrected the results using trim and fill correction (Duval and Tweedie
2000), that kept their significance even after correction.
To summarize the current study, we could show further evidence of the DAT1 3′-UTR VNTR variants to play a role in ADHD, in particularly in child and adolescents with the Long-allele as risk allele. As previously hypothesized, this could indeed be due to the possible functional effect of the variants length in controlling the expression of the DAT1 gene. However, as still high study heterogeneity was observed, with some studies not reaching high quality, further analysis is necessary to establish a robust conclusion.
Acknowledgements
The authors thank the families, patients, and control volunteers who participated in this research. The authors would like to acknowledge Dr. Ian Gizer providing data from his meta-analysis from 2009, Dr. Susan Shur-Fen Gau, Dr. Chi-Yung Shang, Dr. Philip Asherson, Dr. Martine Hoogman, Dr. Marten Onnink, Dr. Mara Helena Hutz, Dr. Carlos S Cruz Fuentes, Dr. Gabriela Ariadna Martinez Levy, Dr. Kate Langley, Dr. Kanchan Mukhopadhyay, Mrs. Julia Morgan, Dr. Zsofia Nemoda, Dr. Jenny Ortega-Rojas, Dr. Ayusi Garcia Carmen and Dr. Clara Isabel Gomez Sanchez providing failing data of their publication and samples, Dr. Jie Li for translation of publications in Chinese into English, Ms. Rea Müller, Ms. Miryame Hofmann and Mrs. Susanne Kunert-Dümpelmann for their laboratory technical support. The authors acknowledge the Swiss National Science Foundation (Grant number SW 320030-130237) and the postdoctoral grant support for Anna Maria Werling by the University of Zurich, “Filling the Gap program”.
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