Attention Deficit Hyperactivity Disorder is a childhood disease that affects all developmental stages of the individual, shows its effects throughout life, and shows significant functional impairment and high inheritance [12].
In this study, we aim to determine the role of candidate genes in the pathophysiology of ADHD by investigating the expressions of candidate genes that we think are involved in response to treatment. Most studies based on related candidate genes, meta-analyses, and genome-wide association studies show that dopaminergic, serotonergic and glutamatergic signaling, neuronal transmission, neuronal migration, and cell adhesion pathways play a role in the etiology of ADHD [13, 14]. Analyzes on ADHD sensitivity and MPH response in many meta-analyzes do not show consistent results [14]. Also, pharmacological studies on candidate genes could confirm SLC6A3 as a key molecular target in drugs containing methylphenidate and atomoxetine in ADHD [15].
In our study, it was observed that the expression level of the SLC6A3 (DAT) gene before treatment was doubled (p≤0.001) in patients compared to the control group. In addition, after methylphenidate and atomoxetine treatments, it was observed that the expression level of SLC6A3 decreased compared to the expression level of the control group (p≤0.05). This shows that these drugs act by regulating the expression of this gene.
Grünblatt et al. studied the expression levels of the SLC6A3 gene in 108 adult ADHD patients and 35 healthy controls. Accordingly, it has been observed that the expression level of the SLC6A3 gene in patients is higher than in healthy individuals [16]. Our research on pediatric patients is consistent with the results of this study. With positron emission tomography, the amount of DAT in the internal globus pallidus (output nucleus of the basal ganglia) was high and this situation caused a decrease in dopamine levels; as a result, it was concluded that neuronal circuits that are effective in initiating behavior are affected and thus impulsive behaviors emerge [17]. Since DNA methylation silences gene expression by preventing transcription factors from binding to DNA, they hypothesized that the excess of DAT in the impulsive group is due to methylation in the binding site of a suppressor transcription factor (regulatory protein) that suppresses DAT expression (suppression of suppression) [18].
SLC6A4 gene reuptakes serotonin from the synaptic gap on the presynaptic membrane. Therefore, SLC6A4 concentration in the membrane is one of the most important factors determining the amount of synaptic serotonin. Polymorphism in the promoter region of this gene affects the transcription rate of the transporter protein that performs the reuptake, hence the presynaptic SLC6A4, thus affecting the serotonergic system and mood [19].
While no difference was observed between the control and patient groups in the SLC6A4 gene expression level study on adults [16], in our study on children, the expression level of the SLC6A4 gene increased to the control group expression levels in patients. In addition, the expression level of the gene increased 3 times (p≤0.001) in patients after the use of methylphenidate, and the expression level doubled after the use of atomoxetine (p≤0.05).
In the expression study conducted by Sener et al. in children with autism spectrum, a significant difference was found in the patient group in terms of SLC6A4 gene expression compared to the healthy control, parallel to our study [20]. Detection of lower expression in the patient group suggests a deficiency in serotonin reuptake. Allelic variants in the serotonin transporter gene (SLC6A4), lower transcriptional efficiency, changes in serotonin concentration in various brain regions, and differences in SLC6A4 mRNA expression have been associated with the development of ADHD [21].
SLC1A2 plays an important role in preventing extracellular glutamate concentrations from reaching neurotoxic levels and recycling glutamate at synapses by transporting glutamate to astrocytes to convert it to glutamine [22].
SLC1A2 (EAAT2 or GLT1), which encodes glutamate transporter with high affinity especially in astroglial cells, is a brain-specific gene with a high degree of disorder. It is known that its expression changes in the glutamatergic system changes in the brain, especially in psychiatric disorders [23]. Decreases in the expression of this gene have been observed in many human and animal depression models [24]. The dysregulation of SLC1A2 causes amyotrophic lateral sclerosis, Alzheimer disease, and epilepsy, as well as psychiatric disorders such as depression and autism [25]. In this study, the SLC1A2 gene expression level was found to be significantly lower in patients than in the control group (p≤0.001). In addition, it was observed that the expression level of the SLC1A2 gene significantly increased after methylphenidate and atomoxetine treatments (p≤0.05).
The vesicular monoamine transporter type 2 gene (VMAT2) has a very important role in the storage and synaptic release of all monoamines, including serotonin (5-HT). VMAT2 level changes are associated with depression, bipolar disorder, and schizophrenia. In addition, studies show that changes in VMAT2 levels cause Tourette syndrome, alcohol addiction, ADHD symptoms in children, and cognitive consequences after traumatic brain injuries in adults [26]. In studies conducted on mice models with VMAT2 deficiency, dopamine intake and release into vesicles decreased more than 80%, pathophysiologically, dopaminergic adrenergic, cellular oxidative stress, alterations in alpha-synuclein accumulation and as behaviorally decreased in mobility, increased in depressive mood and sleep disturbances has been observed [26–28]. VMAT2 over-expression resulted in an increase in uptake of dopamine into vesicles by 100% and its release into vesicles by about 80% and it resulted in increased mobility, anxiety and decreased depressive behaviors. High DAT and low VMAT2 levels will theoretically result in cytosolic dopamine accumulation and minimal dopamine release. Low DAT and high VMAT2 levels would theoretically result in low cytosolic dopamine content and high extracellular dopamine [29].
Our study showed that the VMAT2 gene expression level of the control group consisting of healthy individuals was six times the expression levels of the patients (p≤0.001). In addition, it was determined that VMAT2 expression in patients increased up to the expression levels of healthy individuals after methylphenidate and atomoxetine treatments (p≤0.001).
ADRA2C plays a role in the regulation of norepinephrine release from sympathetic nerves in the central nervous system in the adrenergic system. Noradrenergic neurons play a role in modulating wakefulness, regulation of visual attention, learning, and memory.[30].
According to the study conducted by Cho et al. on Korean patients with ADHD, a connection was established between ADRA2C (GT) repeat polymorphism and ADHD [31]. Barr et al. they worked on the same repeat polymorphism. Although Barr et al. could not establish a link between polymorphism and ADHD, they stated that other stronger SNPs in the ADRA2C gene are linked to ADHD and should be investigated [32].
In the expression study that we conducted on children, significant results were obtained between the expression levels of patients and healthy individuals. The ADRA2C expression level in healthy individuals was twice that of the patients (p≤0.001), and as a result of MPH (p≤0.05) and ATX treatment, ADRA2C expression in patients increased above the level of normal individuals.
Monoamine oxidase A (MAOA) is involved in breaking down monoamine neurotransmitters such as dopamine, 5-hydroxytryptamine (5-HT, serotonin), and norepinephrine. [33]
MAOA enzyme level is known to affect human behavior and characteristics. Some research has shown that a genetic polymorphism with low MAOA activity has an abnormal emotional response to environmental and social cues [34]. Additionally, a family study reported that MAOA enzyme activity is highly correlated with impulsivity. MAOA enzyme activity is known to be associated with the EcoRV polymorphism of the MAOA gene [35].
It has been reported that MAOA polymorphisms are associated with the hyperactive/impulsive ADHD type and the development of borderline personality disorder [36].
Weder et al. have found a correlation between exposure to moderate traumatic conditions during childhood with the low MAOA gene expression and risk of aggressive behavioral problems [37]. In this study, the expression level of the MAOA gene in healthy individuals was more than six times that of the patients (p≤0.001). MAOA expression levels of the patients increased significantly after MPH or ATX treatments (p≤0.001). Expression level in patients approached that of the control group.
Catechol O-methyl transferase (COMT) plays a role in the inactivation of catecholamines, including dopamine [38]. COMT gene has been seen as a focal point in studies on psychiatric disorders; SNP scans were performed, expression analyzes and protein studies were performed [39–42]. In the study conducted by Chen et al., all three parameters were evaluated. This study was conducted among healthy and schizophrenic patients, male and female individuals, and people of white and African descent, and provides a wide range of statistical results. No significant difference was found in COMT expression for age and disease parameters based on mRNA studies. Although the Val158Met SNP and the SNP in the 3 'end region are important risk factors for schizophrenia, the presence of these SNPs does not have a significant effect on mRNA expression. Researchers cannot explain the differences in protein studies and enzyme activities with mRNA expression and think that the functional effect of COMT has more complex genetic bases [42]. In another ADHD study, a general decrease in the surface area of the total premotor cortex was observed in males [43]. In our study, the COMT expression level was found to be lower in the patient group compared to the control (p≤0.001). In the patient group treated with methylphenidate, there was a decrease in the COMT gene expression level after the treatment, but it was not statistically significant (p> 0.05). A statistical decrease in the expression level was observed in the patient group after atomoxetine treatment (p≤0.02).
GLYAT encodes the enzyme Glycine-N-acyltransferase, which is responsible for metabolizing some metabolites in cells. Drugs are primarily metabolized to acyl-CoA intermediates. The glycine-N-acyltransferase enzyme catalyzes the combination of mitochondrial Acyl-CoAs with glycine [44]. Studies on the GLYAT gene on drug metabolism have been carried out, but they have not been focused on individuals with ADHD. In this respect, the significant results for GLYAT have great importance in this study. The GLYAT gene expression levels of the patients were lower than half the expression levels of normal individuals. (P≤0.02) In addition, the use of MPH brought the GLYAT mRNA level of the patients to the expression levels of normal individuals (p≤0.01).
Glutamate is the main stimulating neurotransmitter in the brain and plays a role in a number of ADHD-related processes such as brain development, modulation of neuronal activity, bidirectional regulation of dopamine signaling, synaptic flexibility, memory formation, and learning [45]. GRM5 appears to be critical for inhibitory learning mechanisms because impaired receptor function causes inappropriate retention of deterrent memories that can lead to anxiety disorders [46]. Deletion in the CNV region of GRM5, one of the glutamate metabotropic receptor genes, has been associated with the presence of comorbid anxiety disorders [47].
In expression level studies conducted on patients with autism, it was observed that the GRM5 expression level was low in these patients [48]. Our study was conducted on ADHD and it was observed that the GRM5 expression level was lower in the patient group compared to the control group, as in children with autism, and also the use of MPH increased the GRM5 expression level significantly (p≤0.01).
DRD4 is one of the dopaminergic system genes and one of the dopamine receptors that is most associated with ADHD. In the study conducted by Grünnblatt et al., DRD4 and DRD5 gene expression levels were found to be higher compared to patients [12]. The study we conducted supported this study and it was observed that the expression level of the DRD4 gene was high in patients. But these results were statistically insignificant (p>0.05). It was also observed that MPH and ATX treatments did not significantly alter DRD4 gene expression levels.
Tryptophan hydroxylase 1 gene (TPH1) encodes a rate limiting enzyme in the biosynthesis of the monoamine neurotransmitter serotonin. Many studies have reported that TPH1 and TPH2 polymorphisms are associated with ADHD [49].
According to the study published by Taurines et al. (2011), no difference was found between TPH1 gene expression levels, while according to a study published by Grünnblatt et al., (2012), expression level of the TPH1 gene was higher in patient group than in control group [12, 16]. There are different results for many studies. As a result of our study, TPH1 gene expression levels in patients were found to be statistically significantly lower than healthy individuals (p≤0.01). In addition, it was observed that MPH and ATX treatments increased TPH1 expression levels (p≤0.01), closer to TPH1 gene expression levels of healthy individuals.
There may be many reasons why the expression levels of the genes mentioned above differ between children with ADHD and the control group. These reasons may be variants in the regulatory regions of genes, as well as epigenetic regulatory mechanisms such as DNA methylation, histone modifications, and micro-RNAs seen in CpG islets in the promoter regions of these genes [16]. Therefore, post-transcriptional regulators activate and inactivate the translation of mRNA in some cases [49]. Although methylations generally create a silencing effect by suppressing the transcription of the gene, methylation of a regulatory region can sometimes lead to an increase in the gene product [18, 50].
In this study, the expression levels of genes (SLC6A3, SLC6A4, SLC1A2, VMAT2, MAOA, COMT, GLYAT, GRM5, DRD4, TPH1), which are called candidate genes in the literature, differed between ADHD patients and the control group.
The SLC6A3 gene expression level was found to be higher in children with ADHD compared to the control, and this elevation was reduced by medical treatments. (p≤0.01)
Expression levels of SLC6A4, SLC1A2, VMAT2, MAOA, COMT, GLYAT, GRM5, TPH1 genes were found to be less in children with ADHD compared to the control, and this decrease was increased with medical treatments. (p≤0.01)
As a result, with this study, we reveal that these genes are potential molecular biomarkers that can be used to diagnose ADHD. Improvement was observed in children whose ADHD status was evaluated according to the post-treatment scale. Both the differences in expression between patients and control groups and the correction of these differences with treatment show that the studied genes are biomarkers in the diagnosis of ADHD and the monitoring of treatment. Future studies aim to further investigate these genes in a larger independent population, including different ADHD subgroups (inattentive, hyperactive, and compound type) that are unresponsive to treatment. We aim to obtain more specific sets of biomarkers to compare these not only with healthy controls, but also with other psychiatric disorders, and to distinguish between childhood, adolescent and adult forms of each sub-disorder and ADHD. In addition, miRNAs known as post-transcriptional regulators that target these candidate genes can be investigated and can help find therapeutic molecular agents that target factors that cause the suppression or degradation of the mRNA of these genes.