Introduction
Attention deficit hyperactivity disorder (ADHD) is one of the most common neuropsychiatric and behavioral childhood disorders (1), affecting approximately 12% of school-aged children, with a higher prevalence in boys than girls (2). The disorder is characterized by reduced attention and hyperactivity (3–5); ADHD children are disorganized and have trouble fulfilling plans and completing tasks (6). The symptoms have been attributed, mainly, to lower norepinephrine and dopamine activities. Research has indicated a malfunction in dopamine-β-hydroxylase (DBH), which is responsible for maintaining the balance between dopamine and norepinephrine concentrations (2,7–11).
Recent data suggest that various polymorphisms of the dopamine-related genes are related to ADHD. Studies involving children from the US, Czech Republic, Finland, Brazil and India demonstrated a correlation between DBH gene polymorphisms and increased susceptibility to ADHD (1,10–14). Moreover, studies from the US, Taiwan, UK and China revealed strong associations between ADHD and polymorphisms of the dopamine transporter gene (DAT1) (12,15,16). Despite accumulating evidence, no studies have examined these relationships in Arab/Middle Eastern populations. Therefore, in this study the plasma levels of DBH enzyme activity were evaluated in ADHD children from Jordan. In addition, the association between polymorphisms of the DAT1 and DBH genes and ADHD was examined.
Materials and methods
Subjects
Fifty children with ADHD (2.5–14 years of age) were recruited from the King Abdullah University Hospital and the Princess Rahmah Teaching Hospital. The children were diagnosed with ADHD according to the Diagnostic and Statistical Manual of Mental Disorder-IV (DSM-IV) criteria by a pediatric neurologist. Fifty healthy children, matched for age and gender, were included in the study as controls. The control children attended the same hospitals for reasons other than neuropsychiatric disorders. Written informed consent was obtained from the participants’ guardians according to the Jordan University of Science and Technology Institutional Review Board.
DNA extraction
DNA was extracted from EDTA blood samples obtained from the participants using the Wizard DNA Extraction kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The concentration of the extracted DNA was measured using SmartSpect™3000 (Bio-Rad, Hertfordshire, UK). DNA samples were stored at −20°C until use.
Genotyping of the DAT1 polymorphism
The 40-bp variable number tandem repeat (VNTR) of the DAT1 3′-untranslated region was analyzed using polymerase chain reaction (PCR) as previously described (17). Detection of various repeats was performed using 12% polyacrylamide gel and proper DNA ladders.
Genotyping of the DBH polymorphism
The DBH dinucleotide repeat was performed using previously described primers and PCR conditions (18). The PCR products were digested with HaeIII at 37°C for 2 h. The digested PCR products were analyzed using 12% polyacrylamide gels, and the sizes of the dinucleotide repeat were measured using proper DNA ladders (19).
Determination of dopamine-β-hydroxylase activity
The activity of the DBH enzyme was measured using the patch adsorption method according to the protocol used by Nagatsu and Udenfriend (20). In brief, 50 μl of serum was added to 350 μl of distilled water, and the diluted serum was added to 600 μl of a reagent mixture [200 μl of sodium acetate buffer (1 mol/l, pH 5.0), 50 μl of sodium fumarate (0.2 mol/l), 50 μl of ascorbic acid (0.2 mol/l), 50 μl of catalase (1 mg/ml), 50 μl of tyramine (0.4 mol/l), 50 μl of pargyline (20 mmol/l) and 150 μl of N-ethylmalemide (0.2 mol/l)]. As a blank, 50 μl of serum was diluted to 400 μl by distilled water, boiled at 95°C for 5 min and then added to 600 μl of the reagent mixture described above. The reaction mixtures were incubated at 37°C for 60 min with continuous shaking. At the end of the incubation period, the reactions were stopped by adding 200 μl of trichloroacetic acid (3 molar), and the mixtures were centrifuged at 2,000 x g for 10 min. The supernatant was transferred to a new tube containing 200 μl of Dowex-50 (H+, 200–400 mesh), which was fully equilibrated with acetate buffer. The precipitants were then washed three times with distilled water coupled with centrifugation at 2,000 x g for 5 min each. After the last wash, 1 ml of NH4OH (4 molar) was added to elute the adsorbed amines. The mixtures were then centrifuged at 2,000 x g for 5 min, and the supernatants were transferred to new tubes. Following this, 100 μl of NaIO4 (200 g/l) was added to the tube to convert the octopamine in the supernatant to para-hydroxybenzaldehyde. Finally, 100 μl of Na2S2O5 (10 g/l) was added to reduce the excess periodate. Absorbance was then measured at 330 nm using microcuvets with a 1-cm light path. The activity of the enzyme was expressed in terms of μmol/min; the level of DBH enzyme.
Statistical analysis
Statistical evaluation of the results was carried out by comparing allele, genotype and predicted phenotype distributions using the χ2 test. If n<5, the Fisher’s exact test was used. The SPSS 15.0 statistical software package (SPSS Inc., Chicago, IL, USA) was used for all statistical evaluations. Comparisons that involved two groups were carried out using the Student’s t-test. A p-value of <0.05 was considered significant.
Results
The sample ratio of boys to girls was 4:1. The median age was 7.1±1.2 years for the ADHD subjects and 8±1.4 for the control group (p>0.05).
According to the distribution of the DAT1 polymorphism genotypes shown in Table I, 9/9 and 9/10 were most common in the controls (34 and 28%, respectively). However, the most common genotypes in the ADHD group were 9/10 and 10/10 repeats (34 and 32%, respectively). Additionally, Table II shows that the 9-repeat allele (58%) was common in the controls, while the 10-repeat allele was common in the ADHD group (50%, p<0.05).
As shown in Table III, the frequency of (GT)n DBH polymorphism A2/A3 and A4/A6 genotypes was higher in the ADHD children (58 and 40%, respectively), while the frequency of the A2/A4 genotype was higher in the controls (56%, p<0.05). In agreement with the genotype distribution, Table IV shows that the A3 and A6 allelic frequencies were higher in the ADHD group, while the frequency of the A4 allele was higher in the control group (p<0.001).
The result also revealed a significant decrease (p<0.01) in the plasma DBH activity in the ADHD (25.412±2.32 μmol/min) compared to the control (84.689±5.01 μmol/min) children (Table V). Moreover, the percentage of children with abnormally low activity was higher (p<0.01, Table V) in the ADHD vs. the control group.
Discussion
The present study revealed that the frequency of the (GT)n repeat 5’ in the DBH gene was higher in the ADHD group among Jordanian children. In addition, a significantly lower level of DBH activity was detected in ADHD children compared to the controls.
Previous studies have revealed that an imbalance between the dopaminergic and noradrenergic systems is implicated in the development of ADHD in children (10,16,21). The two systems are connected by the activity of the DBH enzyme, which is expressed in the central and peripheral nervous systems and catalyzes the conversion of dopamine to norepinephrine (22). Genetic variations in the DBH gene suggest that a lower enzyme activity increases the susceptibility of developing ADHD in children. Several studies have shown an increased TaqI A polymorphism in children with ADHD (12,14,23). In addition, several single-nucleotide polymorphisms in the DBH gene have been implicated in ADHD etiology (21). Moreover, the DBH (GT)n repeat polymorphism was found to contribute to the development of ADHD in children from the US (23). In the the present study, the DBH (GT)n polymorphism was associated with ADHD children, supporting the hypothesis that DBH plays a key role in the ADHD trait. Thus, similar to other populations, genetic variations in the DBH gene may play a role in the development of ADHD in Jordanian children.
Another gene that plays a key role in regulating the dopaminergic system is DAT1, which codes for a dopamine transporter expressed in pre-synaptic neurons (24). Previous studies have implicated excess dopamine clearance in the synapses of ADHD as part of the etiology of the disease (25). In addition, positron emission tomography imaging indicated dysregulation of DAT1 in the striatum of subjects with ADHD (26,27). The present results indicate that VNTR in the 3′ untranslated region of DAT1 is associated with ADHD with a high frequency of the 10R allele in affected children. In agreement with this result, several studies (18,28) have found that homozygosity for the 10R allele of the DAT1 gene was significantly greater in ADHD children from the US, Ireland and Czech Republic (12,29,30). In addition, a meta-analysis found a significant association between the 10R allele and ADHD development (31,32). Thus, similar to other populations, VNTR in the 3′ untranslated region of DAT1 may play a role in the etiology of ADHD in children from Jordan.
In support of the genetic analysis, DBH enzyme activity was significantly different in the ADHD group compared to the controls. The plasma DBH level can be used as a marker to reflect sympathetic noradrenergic activity, since the enzyme is released into the blood stream during synaptic transmission (33). Although plasma DBH enzyme activity varies among unrelated individuals (18), it is generally accepted that children with ADHD have decreased plasma and urine DBH activities (10,19,34).
As is the case in most association studies, reported negative and positive associations with a given genetic variant are common. In the examined polymorphisms, lack of association between ADHD and the DBH (GT)n repeat was reported in Indians (35). Similarly, the absence of an association between ADHD and VNTR of the DAT1 3′-untranslated region was observed in studies from Brazil and Norway (1,17,23,36–38). The discrepancy may be due to the complexity of ADHD, the differences in the genetic background of the studied populations, and the impact of environmental factors.
Previous studies have demonstrated that certain environmental factors, including maternal smoking or drinking habits during pregnancy, low birth weight, maternal age, stress and/or exposure to heavy metals such as zinc and mercury contribute to the development of ADHD (6,39). However, the contribution of environmental factors to the development of ADHD in Jordanian children was not examined in the present study; it will be investigated in future research.
Notably, DBH enzyme activity has been shown to change in individuals with certain diseases such as schizophrenia, Tourette syndrome, familial dysautonomia and orthostatic hypotension (40. Similarly, polymorphisms of DAT1 are associated with bipolar disorder, Parkinson disease and drug abuse (41,42). Therefore, future studies should examine common variants of the DBH and DAT1 genes in Jordanians.