Association Between HTR1A Gene Polymorphisms and Attention Deficit Hyperactivity Disorder in Korean Children

Abstract

Attention deficit hyperactivity disorder (ADHD) is a common disorder of the school age population. ADHD has been shown to be familial, and genetic studies estimate its heritability at 80%-90%. The aim of the present study was to investigate the association between the genetic type and alleles for the HTR1A gene in Korean children with ADHD. The sample consisted of 142 ADHD children and 139 control children. We diagnosed ADHD according to the Diagnostic and Statistical Manual of Mental Disorders-4th edition. ADHD symptoms were evaluated with Conners' Parent Rating Scales and Dupaul Parent ADHD Rating Scales. Blood samples were taken from the 281 subjects, DNA was extracted from blood lymphocytes, and polymerase chain reaction was performed for HTR1A polymorphism. Alleles and genotype frequencies were compared using the chi-square test. We compared the allele and genotype frequencies of HTR1A gene polymorphism in the ADHD and control groups. This study showed that there was a significant correlation among the frequencies of the rs10042486 (OR=1.55, 95% CI=1.02-2.30, p=0.041), rs1423691(OR=1.55, 95% CI=1.02-2.30, p=0.041),and rs878567(OR=1.60, 95% CI=1.06-2.43, p=0.027) alleles of HTR1A, but the final conclusions are not definite. Follow-up studies with larger patient or pure subgroups are expected. These results suggested that HTR1A might be related to ADHD symptoms.

Introduction

Attention deficit hyperactivity disorder (ADHD) is a common childhood neuropsychiatric disorder characterized by behavioral problems, such as attention deficit, hyperactivity, and impulsivity (American Psychiatric Association Committee on Nomenclature and Statistics, 1994). It has a prevalence of 2%-7.6% among children of school age in Korea (Cho and Shin, 1994; Kim et al., 1999). Family studies reported that ADHD showed a heredity as high as 80%-90% (Faraone and Doyle, 2001), and molecular genetic studies are actively carried out accordingly. Recent genetic studies on ADHD have usually been conducted on the dopamine-related genes and dopamine-related neurotransmitters, and there are a few previous studies conducted on the serotonin receptors and related neurotransmitters.

Serotonin is a neurotransmitter involved in various bodily functions, such as aggression, attention, appetite, locomotion, and the sleep-wake cycle, and the depletion of the serotonin functions is related to depression, anxiety, appetite loss, aggression, increased pain sensation, and ADHD symptoms (Lucki, 1998). Serotonin dysfunction is correlated with impulsivity, which is one of the common characteristics of ADHD. It was reported that injection of fluoxetine, a selective serotonin reuptake blocker, sedated hyperactivity in mice whose dopamine transporter gene was knocked out (Gainetdinov et al., 1999). The gene for the serotonin 1A receptor (HTR1A) is located at chromosome 5q11.2-13 which contains no intron (Kobilka et al., 1987). HTR1A is located at both the presynapse and the postsynapse. A study from China (Wu and Comings, 1999) discovered C-1019 polymorphism at the promoter site of the human HTR1A receptor. A study (Le Francois et al., 2008) reported that HTR1A expression was affected by C-1019G promoter polymorphism of the HTR1A gene at both the presynapse and the postsynapse. HTR1A gene polymorphism also affects the postnatal development of the hippocampus and cortex and adult neurogenesis later. HTR1A agonist was reported to increase striatum dopamine secretion (Kim et al., 1999). These results indicate that the 5-HT in the striatum affects dopamine. The inhibitory HTR1A receptors are concentrated in the prefrontal cortex, which plays an important role in the working memory, recognition, and adjusted behavior in ADHD, and the prefrontal cortex is linked with the raphe nucleus (Puig et al., 2004). Additionally, the HTR1A was reported to cause brain serotonin-dopamine imbalance and related symptoms. Recently, a Korean study (Shim et al., 2010) first reported the correlation between HTR1A rs6295 (C-1019G) and ADHD through a study conducted with 78 ADHD children and 107 normal children in Korea. However, there has been no other study reporting on a correlation between neurodevelopmental disorders, such as ADHD and HTR1A.

This study was conducted to verify the correlation between the genetic type and alleles of the HTR1A gene and ADHD.

Materials and Methods

Subjects

A questionnaire was conducted with about 16,000 elementary school students in a city whose population is about 500,000 from September 2008 and August 2010. An interview was performed randomly with the children whose Korean version of the Dupaul ADHD Rating Scales (K-ARS) (Kim et al., 2002) score was 19 or higher, and 142 ADHD children who consented to the genetic study were selected. For the control group, 139 children in the same area were selected by matching the sex and age of the subjects in the patient group. For both the patient and control groups, a clinical evaluation and the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV) diagnosis (American Psychiatric Association Committee on Nomenclature and Statistics, 1994) were performed by a child psychiatrist. The number of ADHD children was 142, including 101 boys (71.1%) and 41 girls (28.9%), and the mean age was 8.67±0.84. The number of the children in the control group was 139, including 93 boys (66.9%) and 46 girls (33.1%), and the mean age was 8.65±0.81. There was no significant difference in the sex and age between the two groups (Table 2). Subjects were excluded from the study if there was any evidence of conduct disorder, mood disorder, anxiety disorder, Tourette's disorder, pervasive development disorder, mental retardation (IQ<70), and neurological disorders, including epilepsy. None of the children who participated in the study has ever undergone drug treatment before the evaluation. Informed consent was obtained before study entry. The study was also approved by the Hospital Ethics Committee. None of the children was taking psychostimulants at the time of the study.

Table 2.

Epidemiological Characteristics Between the Attention Deficit Hyperactivity Disorder Group and the Control Group

Rating scale	ADHD group (N=142) Mean±SD	Control group (N=139) Mean±SD	F or χ²	p value
Age^a	8.67±0.84	8.65±0.81	0.05	0.827
Sex (N,%)^b
Female	41 (28.9%)	46 (33.1%)	0.52	0.262
Male	101 (71.1%)	93 (66.9%)

These data represent mean±SD, by independent t test^a, or N (%), by chi-square test^b, significant p-value <0.05.

Independent t test.

Chi-square test.

ADHD, attention deficit hyperactivity disorder.

On the day of visiting the hospital, the child psychiatrist performed a clinical interview as well as Kovac's Children's Depression Inventory (Kovacs, 1983), State Anxiety Inventory, Trait Anxiety Inventory (Cho and Choi, 1989), and Dupaul ADHD Rating Scales (K-ARS) (Kim et al., 2002), the computerized ADHD Diagnostic System (ADS) (Shin et al., 2000) as well as completing a questionnaire survey regarding the pregnancy, infancy, developmental history, and anamnesis of the children with their parents. Subjects were included from our sample if they had a score over two standard deviations from the norm on the tests for ADS (T-score>70). ADHD had a lot of comorbid disorders, such as depressive disorder and anxiety disorder. So we excluded children with a high score of depressive symptoms and anxiety symptoms. Subjects with high anxiety scores (a Spielberger trait/state anxiety scale score>47/49) on the Korean version of Spielberger trait-state anxiety scale for children were excluded, and subjects with high depression scores (Kovacs depression inventory score>29) on Kovacs depression inventory for children were also excluded. In addition, a professional clinical psychologist performed a comprehensive psychological test, including an intelligence test, on each subject.

DNA extraction and genotyping

DNA was extracted from leukocytes using a commercial DNA extraction kit, the Wizard Genomic DNA purification kit (Promega, Madison, WI). The HTR1A single nucleotide polymorphism (SNP) was genotyped by polymerase chain reaction according to the protocol originally described by a study (Serretti et al., 2007) and HTR1A rs10042486(T/C), rs1423691(T/C), rs6295(G/C), rs878567(T/C) were genotyped by Illumina, Inc. (San Diego, CA) through the use of their Integrated Bead Array System (Table 1). We supplied Illumina with barcoded DNA microtiter plates containing the DNA quantified with Pico Green to be at 100 ng/mL and Illumina delivered genotypes with quality scores calculated by proprietary Illumina algorithms. Genotyping methods for the Korean samples were previously reported (Shim et al., 2010).

Table 1.

Single Nucleotide Polymorphisms Considered in this Study

SNP ID	Chromosome	Location	Position (coordinate)	Distance	NCBI gene ID (Accession)	Alleles
HT1A					3350(NM000524.2)
rs10042486	5	Flanking 5	63297084	−3783		T/C
rs1423691	5	Flanking 3	63287417	−4616		T/C
rs6295	5	Flanking 5	63294320	−1019		C/G
rs878567	5	Flanking 3	63291746	−287		C/T

HT1A, Serotonon 1A receptor; SNP, single nucleotide polymorphism.

Statistical analysis

We performed independent t tests for age, chi-square tests for sex, and chi-square tests to compare the results of the control group and the ADHD group through the frequency of the genotypes and alleles. SPSS PC software (version 15.0) was used for the statistical analysis and the significance level was set to the p-value being less than 0.05. The calculation revealed that a sample size of 210 subjects is required to obtain a power that is 95% or higher in the chi-square test between the control group and the patients group. Our study was conducted with 281 subjects and the power was 97.41%. This indicates that the association of the HT1A receptor gene polymorphism and ADHD can be sufficiently accounted for by the results in this study. However, we performed the power program analysis for the chi-square test with 281 subjects and the result showed that the effect size was 0.46 (moderate level).

Results

Comparison of the frequency of the genotypes and alleles with genetic polymorphism between the control group and the attention deficit hyperactivity disorder group

The HT1A-rs10042486 and HT1A-rs1423691 genotypes of the 139 subjects in the control group and the 142 subjects in the ADHD group were T/T (69.57%; 62.86%), T/C (28.26%; 28.57%), and C/C (2.17%; 8.57%), and there was a significant difference in the frequency between the two groups (χ²=2.26, df=2, p=0.024) (Table 3). The HT1A-rs878567 genotypes of the 139 subjects in the control group and the 142 subjects in the ADHD group were C/C (68.84%; 60.71%), C/T (28.99%; 30.71%), T/T (2.17%; 8.57%), and there was a significant difference in the frequency between the two groups (χ²=2.29, df=2, p=0.022) (Table 3).

Table 3.

Multivariate Model for Genotype Distributions and Allele Frequencies in the ADHD Group and the Control Group

	Control		ADHD
Characteristics	N	%	N	%	OR	95% CI	χ²	P
HTR1A-rs10042486(T-3783C)
Genotype					4.48	(1.22-16.45)	2.26	0.024
TT	96	69.57	88	62.86
TC	39	28.26	40	28.57
CC	3	2.17	12	8.57
Allele					1.55	(1.02-2.36)	2.04	0.041
T	231	83.7	216	77.14
C	45	16.3	64	22.86
HTR1A-rs1423691(C-4616T)
Genotype					4.48	(1.22-16.45)	2.26	0.024
TT	96	69.57	88	62.86
TC	39	28.26	40	28.57
CC	3	2.17	12	8.57
Allele					1.55	(1.02-2.36)	2.04	0.041
T	231	83.7	216	77.14
C	45	16.3	64	22.86
HTR1A-rs6295(C-1019G)
Genotype					2.64	(0.89-7.85)	1.75	0.081
CC	88	63.77	82	58.57
CG	45	32.61	46	32.86
GG	5	3.62	12	8.57
Allele					1.36	(0.92-2.03)	1.52	0.128
C	221	80.07	210	75.0
G	55	19.93	70	25.0
HTR1A-rs878567(C-287T)
Genotype					4.59	1.25-16.88	2.29	0.022
CC	95	68.84	85	60.71
CT	40	28.99	43	30.71
TT	3	2.17	12	8.57
Allele					1.60	1.06-2.43	2.22	0.027
C	230	83.33	213	76.07
T	46	16.67	67	23.93

These data represent N (%) by chi-square test, significant p-value <0.05.

OR, odds ratio; CI, confidence interval.

Odds ratio of the genotypes and alleles with genetic polymorphism in the control group and the ADHD group

For the HTR1A rs10042486 and the HTR1A rs1423691 genotype, the odds ratio was significant at 4.48 (confidence interval: 1.22-16.46) and for the allele, the odds ratio was significant at 1.55 (confidence interval: 1.02-2.36). For the HT1A rs878567 genotype, the odds ratio was significant at 4.59 (confidence interval: 1.25-16.88) and for the allele, the odds ratio was significant at 1.60 (confidence interval: 1.06-2.43).

Discussion

This study is a case-controlled study in which the frequency of the genotypes and alleles were compared between the ADHD children and the control group in Korea. The correlation between the genotypes and alleles of four candidate HTR1A SNPs was investigated. This study showed that there was a significant correlation between the frequencies of the HTR1A, this result is reported for the first time domestically and internationally. In the study of Korean children (Shim et al., 2010), which is the only previous study about the correlation between ADHD and HTR1A, the correlation between the rs6295 genetic polymorphism of the HTR1A gene and ADHD was reported first. However, the correlation between ADHD and rs6295 genetic polymorphism was not found in this study. One study from Japan (Tsuchida et al., 2009) reported that the effect of amphetamine to repress hyperactivity was perfectly blocked by the 5-HT1A antagonist and that 5-HT1A mediated inhibition in the prefrontal lobe and corticostriate was partially related to the therapeutic effect of psychostimulants on ADHD.

This result can be understood to indicate that the failure of HTR1A regulation may cause changes in 5-HT and may be correlated with vulnerability toward various psychiatric diseases, including ADHD and depression. Moreover, the neurotransmitter systems of dopamine and serotonin are considerably correlated with each other (Kelland and Chiodo, 1996), because the 5-HT regulates the dopamine activity by means of various receptors, including HTR1A. In a previous study, HTR1A showed an important link with the relationship between dopamine and serotonin. We suggested that the same result of the HT1A-rs10042486 and HT1A-rs1423691 genotypes of the 139 subjects in the control group and the 142 subjects in the ADHD group was a coincidence of SNPs.

This study showed that the HTR1A SNP has a significant effect on the ADHD vulnerability and that the increased frequency of the C-allele gene is correlated with ADHD. The increased frequency indicates the increased risk of ADHD occurrence. This study also suggests that the failure to regulate HTR1A expression causes changes in the HTR1A expression and the structural development of the brain regions related with nerve activity, attention, and impulsivity. In this study, we assumed that the variation of the HTR1A gene affects these two neurotransmitters that are the cause of ADHD. The analysis in their study (Ribasés et al., 2009) of 19 SNP genes showed that they were related to serotonin neurotransmission, including HTR1A and that there was not a significant correlation between the HTR1A gene and ADHD. Hence, the correlation between the HTR1A gene and ADHD should be carefully handled and the result of our study should be verified in the future study with a large number of independent samples.

The limitations of this study are as follows: first, the number of subject children was small. The subjects of this study were 142 ADHD children and 139 children in the control group. Second, the results of this study may not be generalized for the cases of other racial or ethnic groups since the frequency of alleles can vary due to local or racial differences. The distribution of the allele frequency in the ADHD patient children and parents group in this study was also different from that of other countries. Third, only a few SNPs were investigated in this study among the many genes related with the various ADHD phenotypes. Although it is clear that not just one genetic factor causes the increased ADHD vulnerability, we did not consider the interaction with other risk factors.

Despite the methodological limitations described before, this study has several advantages. First, the patient group and the control group were matched so that there was no difference in the frequency of sex and age. The prevalence of ADHD is higher among males and in adolescence; thus, the sex and age characteristics can have a great effect. Considering this, our study was evaluated by matching the age and sex of the patient group and the control group with each other. Second, this study used population-based samples. Previous studies in Korea were hardly considered to represent the general population because the subjects were usually ADHD children who visited hospitals for their clinical symptoms. In this study, the subjects in the risk group were selected by the questionnaire survey from the whole population in a region and the patient and control samples were obtained by random contact. Thus, the subjects in this study may be more appropriate to the characteristics of general population than those of the study performed with the patients who visited hospitals. Third, this study might have compared relatively homogenous groups that had the characteristics of Koreans, different from the studies conducted in other countries with subjects from various ethnic groups and nations. Fourth, both the patient group and control group in this study underwent clinical evaluation and DSM-IV diagnosis by children psychiatrists, applying the inclusion and exclusion criteria strictly, and thus, the patient group was composed of pure ADHD diagnosed subjects.

We expect that different allele distribution results may be produced from future studies on the quantitative correlation of the ADHD performance in the pure ADHD group from which coexisting diseases are excluded, the patients group composed of only boys or girls, the subtype groups, such as hyperactivity dominant group and attention deficiency dominant group, and the drug response group.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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