Pathogenic study on catechol-O-methyltransferase gene and catecholaminergic neurotransmitters with attention deficit hyperactivity disorder in Chinese children

Background: This study analyzed a correlation between the Val158Met polymorphisms of catechol-O-methyltransferase (COMT) gene and catecholaminergic neurotransmitters in ADHD children. Methods: All subjects were genotyped for the Val158Met polymorphisms of COMT gene and determined in the difference of dopamine and noradrenalin by a 1:1 paired case-control study. Results: The frequencies of A/A, G/A and G/G were 51.67%, 41.11% and 7.22% in the case group, 62.22%, 31.11% and 6.67% in the control group. There was a significant difference in the distribution of all genotypes of COMT gene between the two groups (OR=1.85, χ2=7.80, P<0.05). The serum concentrations of dopamine and noradrenalin were 1.42±0.34 ng/ml and 177.70±37.92 pg/ml in the case group, 1.94±0.42 ng/ml and 206.20±42.45 pg/ml in the control group. There were the significant differences in the levels of dopamine and noradrenalin between the two groups (dopamine: t=4.30, P<0.01. noradrenalin: t=2.24, P<0.05). Conclusions: Our study suggested that there was the positive association between the Val158Met polymorphisms of COMT gene and catecholaminergic neurotransmitters in ADHD children. The Val158Met polymorphisms of COMT gene were genotyped by the quantitative PCR amplification. The TaqMan Genotyping Master Mix was purchased from TaqMan R Drug Metabolism Genotyping Assay. The following primers were used in the Polymerase Chain Reaction (PCR): 5’-ACT GTG GCT ACT CAG CTG TG-3’ (forward) and 5’-CCT TTT TCC AGG TCT GAC AA-3’ (reverse). The Assays consisted of a 20x mix of unlabeled PCR primers and TaqMan R MGB probes (FAM tm and VIC R dye-labeled). These assays were designed for the allele discrimination of the specific Single Nucleotide Polymorphisms (SNPs). In each of reaction tubes, we added a 10 ng DNA,2 TaqMan R MGB probes (FAM tm and VIC R dye-labeled) in a 25 µl reaction system. The TaqMan R MGB probes tested for A (methionine) or G (valine) alleles symbolizing FAM tm or VIC R dye-labeled respectively. The peak of FAM tm index implicated G/G homozygethe peak of VIC R index implicated A/A homozygeand the peaks of FAM tm and VIC R indexes implicated G/A heterozyge. Researchers involved in COMT genotyping were blind to the neuropsychological results, and researchers involved in neuropsychological assessments were blind to the COMT genotyping results. COMT genotype was coded as a categorical variable (met/met, met/val and val/val) for further analysis.

inattention, hyperactivity and impulsivity in ADHD children. Furthermore, Catechol-O-methyltransferase (COMT) enzyme is specifically responsible for the inactivation of catecholaminergic neurotransmitters in the prefrontal cortex [1,2]. Since COMT enzyme is involved in the degradation of catecholaminergic neurotransmitters, it is possible that COMT enzyme catalyzes the inactivation of catecholaminergic neurotransmitters by a transfer of a methyl group to catechol compounds. So COMT enzyme is an indispensable regulator in the catecholamine metabolism of ADHD children.
The Val158Met polymorphisms of COMT gene, coding for COMT enzyme, have attracted great interest as a candidate gene for ADHD children. Because the Val158Met polymorphisms of COMT gene adjust the activity of COMT enzyme, the high activity of valine variant degrades dopamine three to four times more quickly than the low activity of methionine variant in ADHD children [3]. Therefore, the Val158Met polymorphisms of COMT gene should be extremely worthwhile for further study in ADHD children.
Recently, there were seldom reports on the correlation between the Val158Met polymorphisms of COMT gene, catecholaminergic neurotransmitters and clinical phenotypes of ADHD children [4]. Against this background, we analyzed our extended samples to manifest the regulatory mechanism of the Val158Met polymorphisms of COMT gene and catecholaminergic neurotransmitters in ADHD children.

Study subjects
The subjects in this study, a total of 180 paired ADHD and non-ADHD children aged Val158Met polymorphisms of COMT gene The Val158Met polymorphisms of COMT gene were genotyped by the quantitative PCR amplification. The TaqMan Genotyping Master Mix was purchased from TaqMan R Drug Metabolism Genotyping Assay. The following primers were used in the Polymerase Chain Reaction (PCR): 5'-ACT GTG GCT ACT CAG CTG TG-3' (forward) and 5'-CCT TTT TCC AGG TCT GAC AA-3' (reverse). The Assays consisted of a 20x mix of unlabeled PCR primers and TaqMan R MGB probes (FAM tm and VIC R dyelabeled). These assays were designed for the allele discrimination of the specific Single Nucleotide Polymorphisms (SNPs). In each of reaction tubes, we added a 10 ng DNA,2 TaqMan R MGB probes (FAM tm and VIC R dye-labeled) in a 25 µl reaction system. The TaqMan R MGB probes tested for A (methionine) or G (valine) alleles symbolizing FAM tm or VIC R dye-labeled respectively. The peak of FAM tm index implicated G/G homozyge the peak of VIC R index implicated A/A homozyge and the peaks of FAM tm and VIC R indexes implicated G/A heterozyge. Researchers involved in COMT genotyping were blind to the neuropsychological results, and researchers involved in neuropsychological assessments were blind to the COMT genotyping results. COMT genotype was coded as a categorical variable (met/met, met/val and val/val) for further analysis.

Serum concentrations of catecholaminergic neurotransmitters
The serum concentrations of dopamine and noradrenalin were determined by the Enzyme-linked Immunosorbent Assay (ELISA). The dopamine and noradrenalin kits were purchased from Labor Diagnostika Nord GmbH and Co. KG. In each of the reaction tubes, we added a 25 µl enzyme solution, 100 µl standard, 100 µl control and 100 µl sample incubated at room temperature on a shaker set at 400-500 rotation per minute (RPM) for 30 minutes, added a 50 µl antiadrenergic serum incubated at room temperature on a shaker set at 400-500 RPM for 30 minutes, added a 100 µl enzyme addition incubated at room temperature on a shaker set at 400-500 RPM for 30 minutes, added a 100 µl assay and extraction buffer incubated at room temperature on a shaker set at 600-900 RPM for 20-30 minutes, and added a 100 µl stop solution. The reaction was monitored at 450 nm with the amount of antibody bound to dopamine and noradrenalin concentrations of the solid phase being inversely proportional to that of the sample. Researchers involved in dopamine and noradrenalin assessments were blind to neuropsychological results, and researchers involved in neuropsychological assessments were blind to the dopamine and noradrenalin results.

Statistical analysis
The database was established by Visual FoxPro (VFP 6.0), and analyzed by Statistical Analysis System (SAS 8.1) in this study. The data for the case and control groups underwent 1:1 paired χ 2 -test and t-test to examine the association between the Val158Met polymorphisms of COMT gene and catecholaminergic neurotransmitters, and conducted Odds Ratio (OR) to analyze the probability of all the alleles and genotypes in ADHD children.

Allele distribution of COMT gene
The alleles of COMT gene were A (methionine) and G (valine) in ADHD children. The frequencies of A and G were 72.22% and 27.78% in the case group, and 77.78% and 22.22% in the control group. The distribution of COMT gene was under Hardy-Weinberg equilibrium. There was no significant difference between the two groups (OR = 0.74, χ 2 = 2.96, P > 0.05), thus no distortion in the transmission of the variants of COMT gene to ADHD children as demonstrated in Table 1.  noradrenalin: t = 2.24, P < 0.05)as analyzed in Table 3.  Table 4.

Discussion
The etiology and pathogenesis of ADHD children have been poorly understood recently. The evidences from some family, twin and adoption studies suggested that the genetic factors played an important role in the etiology of ADHD children. As a result, there has been growing interest in the molecular genetic basis for ADHD children [6]. Meanwhile, recent studies showed that catecholaminergic neurotransmitters might be involved in the regulatory mechanism of ADHD children.
The findings from some structural and functional cerebral studies supported the involvement of catecholaminergic neurotransmitters in ADHD children.
As expected, it was biologically valuable to hypothesize an association between the Val158Met polymorphisms of COMT gene and clinical phenotypes in ADHD children [7][8][9]. According to this hypothesis, G (valine) variant of COMT gene was associated with the faster depletion of catecholaminergic neurotransmitters from synapses in the prefrontal cortex [10][11][12][13]. COMT gene [14][15][16][17][18]. Hence, these findings raised the possibility that the Val158Met polymorphisms of COMT gene might be necessary in the development of certain ADHD symptoms [19][20][21][22][23]. Our study found a significant association between G/A variations of COMT gene and clinical phenotypes of ADHD children.
However, some studies have also found no correlation between the Val158Met polymorphisms of COMT gene and clinical phenotypes of ADHD children [24][25][26]. The conclusions of these studies might be influenced by the following factors [27][28][29]: First, many studies were obtained from a small number of selected subjects which were not reasonable to consider these results of the correlation studies. Second,

Declaration of conflict of interest
We declared that we had no conflicts of interest in this study.

Ethics approval and consent to participate
Informed written consent was obtained from all participants and/or their parents.
The study protocol was approved by the Ethics Committee of Maternal and Child Health Hospital of Hubei Province. Every sample of 2-ml venous blood was drawn from each subject in both groups at the time of recruitment. Pathogenesis and study routine of ADHD children Figure 1 Pathogenesis and study routine of ADHD children