hyperactivity disorder with serotonin 4 receptor gene polymorphisms in Han Chinese subjects

Neuroscience Letters 401 (2006) 6–9

Association of attention-deficit/hyperactivity disorder with serotonin 4 receptor gene polymorphisms in Han Chinese subjects Jun Li a , Yufeng Wang a,∗ , Rulun Zhou a , Bing Wang a , Haobo Zhang a , Li Yang a , Stephen V. Faraone b b

a Institute of Mental Health, Peking University (Peking University sixth hospital), Beijing 100083, People’s Republic of China Genetics Research Program and Department of Psychiatry and Behavioral Sciences, SUNY Upstate Medical University, Syracuse, NY, USA

Received 30 December 2005; received in revised form 20 February 2006; accepted 21 February 2006

Abstract Attention-deficit/hyperactivity disorder (ADHD) is an important public health problem. Although serotonin is believed to be an important neurotransmitter in the etiology of this disorder, it remains unclear which specific 5-HT receptors are involved in regulating the symptoms of ADHD. Previous studies have provided favorable evidence for the association of ADHD with both the serotonin transporter gene and serotonin 1B receptor gene. To further investigate the role of other genes of the serotonergic pathway in ADHD, the current study examined variants of the serotonin 4 receptor gene in a relatively large sample of ADHD nuclear families. The T allele of the 83097 C > T polymorphism of HTR4 showed a tendency of preferential transmission to probands with ADHD (χ2 = 2.699, P = 0.100). When haplotype TDT analysis of HTR4 was performed, we further found that the C/G haplotype of the 83097 C > T and 83198 A > G polymorphisms (χ2 = 8.783, P = 0.003) and the C/G/C haplotype of these and the −36 C > T polymorphism (χ2 = 5.762, P = 0.016) were under-transmitted to probands with ADHD. These results suggest that the HTR4 gene may play a role in the genetic predisposition to ADHD. © 2006 Published by Elsevier Ireland Ltd. Keywords: Serotonin; HTR4; Attention-deficit hyperactivity disorder (ADHD); Transmission disequilibrium test (TDT); Haplotype

Attention-deficit/hyperactivity disorder (ADHD) is an important public health problem affecting about 8–12% of children worldwide [13] with estimates from China ranging from 4 to 7.4% [18,29]. The predisposition to ADHD is likely determined by several factors, including genetic factors [12]. Family studies have suggested that ADHD aggregates in families [3,9], while twin and adoption studies corroborated the hypothesis that part of the observed familial aggregation of ADHD is due to genetic factors. Recent twin studies have estimated the heritability of ADHD at about 0.80 [11], indicating that genes play a substantial role in the etiology of this disorder. Over the last decade, there has been growing interest in the neurobiology of ADHD [7,8,10]. Studies have consistently provided evidence that monoamines play important roles in ADHD. The “hyperdopaminergic hypothesis” is a leading theory on the pathophysiology of ADHD, but one limitation of this theory is that it overlooks the interaction between the dopamine (DA)

∗

Corresponding author. Tel.: +86 10 82801969; fax: +86 10 62027314. E-mail address: [email protected] (Y. Wang).

0304-3940/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2006.02.053

and serotonin (5-HT) neurotransmitter systems [27]. A large body of experimental evidences suggested that 5-HT can modulate the activity of DA in human brain and alteration in 5-HT transmission can modify DA-mediated behaviors, such as hyperactivity and aggression [32]. Recently, many researchers have proposed that an imbalance between DA and 5-HT is responsible for the occurrence of several behaviors in ADHD [27]. Although 5-HT has been studied less thoroughly in the neurobiology of ADHD, mounting evidence from both human and animal studies has suggested a role of this neurotransmitter in the etiology of the disorder [15,31]. Thus, genes that control the brain 5-HT pathways seem to be good candidates for mediating genetic susceptibility to ADHD. A growing number of molecular genetic studies have been carried out to investigate the relationship between serotonergic gene polymorphisms and ADHD. The serotonin transporter promoter region (5-HTTLPR) [14,19,22,37] and serotonin receptor 1B gene (HTR1B) [16,26] have repeatedly been reported to be associated with ADHD. As for tryptophan hydroxylase gene (TPH), no association with ADHD was reported in Han Chinese population [20,34]; however, recently, two reports revealed

J. Li et al. / Neuroscience Letters 401 (2006) 6–9

positive association of polymorphisms in the gene of TPH2, a second form of TPH, and ADHD in Europe population [28,36]. 5-HT4 receptors are more abundant in limbic system and frontal cortex in brain. Several in vivo studies demonstrated that 5-HT4 receptors facilitate DA release [5]. By the use of antagonist and agonists of this receptor, lines of evidences indicated that 5-HT4 receptors contributed to cognitive processing [4]. 5HT4 gene (HTR4) is located on chromosome 5q32, spanning a distance of ∼1.3 Mb. The gene is composed of five exons and produces at least eight splice variants, including seven Cterminal splice variants (a–g) and one internal splice variant (h) [2]. The gene knock-out mice have been reported to display attenuated novelty-induced exploratory activity [6], suggesting that this gene may be related to certain characteristics of individuals with ADHD, who also show an abnormal response to novelty and abnormal exploratory activity. Ohtsuki et al. [24] once sequenced the exon region and the branch site of HTR4 and found eight variants, two of which, 83097 C > T and 83198 A > G, were associated with bipolar disorder and another two, 6 G > A and −36 T > C, were associated with schizophrenia, haplotype analysis revealed significant association with both bipolar disorder and schizophrenia [24]. Later, another report about Japans population found haplotype association of HTR4 and schizophrenia [33]; however, the haplotypes used in the two reports were different. The current study selected three of these exon variants, including 83097 C > T and 83198 A > G and −36 T > C to genotype in ADHD trios to investigate the role of this gene. 83097 C > T and 83198A > G were located near exon d and −36 T > C was near exon h. All of the above three polymorphism may contribute to the production of splice variants. The current study is approved by the Ethics Committee of the Peking University. Written informed consent was obtained from parents. Three hundred and twenty six nuclear families participated in the present study. ADHD probands were recruited from the ADHD outpatient clinic at the Child and Adolescent Psychiatric Division of the Sixth Hospital, Peking University in Beijing, PRC. All probands were interviewed by at least two different child psychiatrists with the aid of their biological parents and teachers, fulfilled DSM-IV diagnostic criteria for ADHD [1]. All the subjects involved are Han Chinese. Children were excluded if they showed evidence of bipolar affective disorder, childhood schizophrenia, autism, mental retardation or other neurological or complex medical illnesses. Among these probands, 275 were male and 51 were female. The average age was 11.0 ± 2.3 years and the average intelligence quotient (IQ) was 101.1 ± 13.5. One hundred and thirty-four (41.1%) probands met criteria for ADHD combined type (ADHD-C), 19 (5.8%) met criteria for ADHD hyperactive-impulsive type (ADHD-HI), and 173 (53.1%) met criteria for ADHD inattentive type (ADHD-I). Within the total sample, 247 (75.8%) probands met diagnostic criteria for other disorders: 152 (46.7%) had comorbid oppositional defiant disorder (ODD) and/or conduct disorder (CD), 51 (15.6%) had a tic disorder, and 112 (34.4%) had a learning disability (LD). Genomic DNA was extracted from blood using standard procedures. 83097 C > T, 83198 A > G and −36 T > C poly-

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Table 1 The number of alleles transmitted or not transmitted from heterozygous parents to ADHD offspring Variant

Number of trios

Allele

T

NT

McNemar (χ2 )

P

C 83097T

326

C T

162 194

194 162

2.699

0.100

A 83198G

326

A G

157 162

162 157

0.050

0.823

C −36T

326

C T

183 196

196 183

0.380

0.538

T = transmitted; NT = not transmitted.

morphisms were genotyped using the conditions described by Ohtsuki et al. [24]. Genotypes were read by two independent readers, and ambiguous or unidentifiable results were reamplified and rescored. Samples that continued to amplify poorly were eliminated from the study. The transmission disequilibrium test (TDT) and the extended transmission disequilibrium test (ETDT) [30] were performed to test for evidence of distorted transmission of alleles and haplotypes, respectively. We used Holm’s sequential Bonferroni method [17] to correct for multiple testing. The T allele of the 83097 C > T polymorphism showed a tendency to be preferentially transmitted to ADHD probands (χ2 = 2.699, P = 0.100), although this result was not statistically significant. No other alleles of the three polymorphisms were transmitted preferentially to ADHD probands. Because the 83097 C > T polymorphism is very near to the 83198 A > G polymorphism (see Table 1), haplotypes of these two loci were determined by the same PCR process. Haplotypes C/A, C/G and T/G were observed, while haplotype T/A was not observed. Haplotype C/G was significantly under-transmitted to ADHD probands (χ2 = 8.783, P = 0.003), and haplotype T/G showed a tendency to be preferentially transmitted to ADHD probands (χ2 = 2.692, P = 0.100) (see Table 2). When the −36 T > C polymorphism was also considered, the haplotype for the three polymorphisms could be determined accurately only if recomTable 2 The number of haplotypes transmitted or not transmitted from heterozygous parents to ADHD offspring Variants

Number Haplotype T of trios

NT

McNemar (χ2 )

P

83097 C > T/83198 A>G

326

83097 C > T/83198 A > G/-36 C > T

326

C/A

158

162

0.050

0.823

C/G T/G

28 194

55 163

8.783 2.692

0.003 0.100

C/A/T

110

126

1.085

0.298

C/A/C C/G/T C/G/C T/G/T T/G/C

82 26 5 188 111

72 41 16 152 115

0.649 3.358 5.762 3.812 0.071

0.420 0.067 0.016 0.051 0.790

T = transmitted; NT = not transmitted.

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J. Li et al. / Neuroscience Letters 401 (2006) 6–9

bination was ignored. Under this assumption, both the C/G/T (χ2 = 3.358, P = 0.067) and C/G/C (χ2 = 5762, P = 0.016) haplotypes were under-transmitted to ADHD probands, while the T/G/T haplotype (χ2 = 3.812, P = 0.051) was over-transmitted (see Table 2). These analyses required 12 statistical tests, leading to a Holm Bonferroni corrected significance level of .005. One result met this criterion: the test of the C/G haplotype of 83097 C > T/83198 A > G (Table 2, P = 0.003). This study is the first to examine HTR4 gene polymorphisms in a large number of ADHD trios and found statistically significant under-transmission of the C/G haplotype of 83097 C > T/83198 A > G to probands with ADHD, even after correcting for multiple testing. These results suggest that the HTR4 gene may be associated with ADHD. Antagonists of the serotonin 4 receptor have been shown to reduce the release of dopamine in the striatum [21,25,35] and agonists of this receptor regulate the locomotor effect of cocaine mediated by the mesolimbic dopamine pathway [23]. These findings collectively indicate that the serotonin 4 receptor is an important link for the interaction between dopamine and serotonin. These factors also strengthen the assertion that variation within the HTR4 gene does indeed directly influence the interaction between dopamine and serotonin in the etiology of ADHD. However, due to no single marker was found to be association with ADHD after correction, the association between HTR4 and ADHD must be treated cautiously. The results of this study must of course be replicated in an independent sample, and other polymorphisms and haplotypes within this gene should be evaluated for an association with the illness. In summary, the present study examined the evidence for association of ADHD with three variants in HTR4 gene, and found that this gene may be related to the illness. The current results are preliminary and a replication study in an independent sample would be valuable to support our results. Acknowledgements Contract grant sponsor: Ministry of Science and Technology, China (2004BA720A20); Contract grant sponsor: Project of Science and Technology, Beijing (Y0204003040831); Contract grant sponsor: Key Project for Clinical Faculty Foundation, Ministry of Health, China (2004-468); Contract grant sponsor: Peking University Center for Human Disease Genomics (200112).

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