Investigation of the serotonin 2C receptor gene in attention deficit hyperactivity disorder in UK samples

BMC Research Notes BioMed Central Short Report Open Access Investigation of the serotonin 2C receptor gene in attention deficit hyperactivity disorder in UK samples Xiaohui Xu*1, Keeley Brookes2, Bo Sun3, Nicholas Ilott1 and Philip Asherson1 Address: 1MRC Social, Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, King's College London, UK, 2University of St Andrews, Bute Medical School, Fife, UK and 3School of Medicine, King's College London, UK Email: Xiaohui Xu* - ; Keeley Brookes - ; Bo Sun - ; Nicholas Ilott - ; Philip Asherson - * Corresponding author Published: 5 May 2009 BMC Research Notes 2009, 2:71 doi:10.1186/1756-0500-2-71 Received: 13 October 2008 Accepted: 5 May 2009 This article is available from: http://www.biomedcentral.com/1756-0500/2/71 © 2009 Xu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Attention deficit hyperactivity disorder (ADHD) is a common, childhood-onset neurodevelopmental disorder that is more frequent in males than females. Several genes on the X chromosome have been studied as candidate risk factors for ADHD including the 5-HT2C receptor (HTR2C) gene. Association between polymorphisms in HTR2C and ADHD were reported in a recent study. Findings: In this study we investigated the association between ADHD and two polymorphisms C-759T (rs3813929) and G-697C (rs518147) in the promoter region of the HTR2C gene using a sample of 180 UK ADHD probands and their parents. We have shown that the -697G allele was significantly over-transmitted to affected ADHD probands (P = 0.017). No association was detected between the C-759T polymorphism and ADHD. Haplotype analysis of the two markers revealed no significantly increased transmission of any haplotype to ADHD. Conclusion: The findings provide evidence that the G-allele of the G-697C HTR2C polymorphism may be involved in the development of ADHD. The results replicate one of the findings published recently. Findings Attention deficit hyperactivity disorder (ADHD) is a childhood onset, neurodevelopmental disorder characterized by inattention, hyperactivity and impulsivity. Molecular genetic and pharmacological studies suggest the involvement of the dopaminergic, serotonergic and noradrenergic neurotransmitter systems in the pathogenesis of ADHD. Polymorphic variants in several genes involved in regulation of the dopamine, and related neurotransmitter pathways are reported to be associated with ADHD [1-3]. Serotonin is a neurotransmitter in human brain, and involved in a variety of functions including learning, aggression and cognitive processes [4,5]. The serotonin receptors have been classified into at least seven families (5-HT1-7). The 5-HT2C receptor (HTR2C) gene is located on human chromosome Xq24. The previous studies showed that the 5-HT2C receptor is a key contributor to control of central dopamine functions [6,7] and may play an important role in the aetiology of mental disorders, including ADHD. ADHD is much more frequent in males Page 1 of 4 (page number not for citation purposes) BMC Research Notes 2009, 2:71 than females [8,9]. Genes coding to the X chromosome have been suspected as candidates for ADHD [10-14]. Yuan et al. [15] have suggested that the single nucleotide substitution polymorphisms in the upstream region of the 5-HT2C receptor could be involved in the promoter activity, as the -759C and -697G allele had less promoter activity than the -759T and -697C allele. One study has investigated association between HTR2C and ADHD in the Han Chinese population [16]. Two polymorphisms of HTR2C were investigated in 488 ADHD families in the study: C-759T (rs3813929) and G-697C (rs518147). The results showed that the -759C allele, the -697G allele, and haplotype -759C/-697G were significantly over-transmitted to affected probands, while haplotypes -759C/-697C and -759T/-697C were under-transmitted to ADHD probands. Li et al. [16] also found that the -697G allele and haplotype -759C/-697G were significantly over-transmitted to ADHD combined type (ADHD-C) probands, and haplotype -759T/-697C was under-transmitted to ADHD-C probands. Another study conducted by Brookes et al. [17] analysed 51 genes in a European collaborative sample of 776 DSM-IV ADHD combined type cases collected by the International Multi-centre ADHD Genetics (IMAGE) project and found association signals in DRD4, DAT1 and 16 other genes. Twenty-three single-nucleotide polymorphisms (SNPs) in HTR2C gene were investigated in this study, however, no association was found in any of HTR2C SNPs including C-759T and G-697C (P = 0.891 and P = 0.613, respectively). To provide further clarification of the reported association, in this study we examined the two associated polymorphisms previously reported in the Han Chinese population. Methods Sample DNA was collected from 180 DSM-IV ADHD combined subtype probands, from both parents for 116 of the ADHD probands and from the mother alone for 64 of the probands. Cases were recruited from child behaviour clinics in South-East England and referred for assessment if they were thought by experienced clinicians to have a diagnosis of the combined subtype of ADHD under DSMIV criteria, with no significant Axis I co-morbidity apart from oppositional defiant disorder (ODD) and conduct disorder (CD) and IQ greater than 70. Only those individuals fulfilling the recruitment criteria after completion of research assessments were included in the study. 96% of the sample was male. The age range was 5–15 years at the time of assessment (mean 10.41, SD 2.34). Parents were interviewed with a modified version of the Child and Adolescent Psychiatric Assessment (CAPA) [18]. Information on ADHD symptoms at school was obtained using http://www.biomedcentral.com/1756-0500/2/71 the long form of the Conner's questionnaire [19]. The subjects gave their written informed consent, and this study was approved by the Ethical Committee of King's College London (Reference number: G9814668). Genotyping The C-759T (rs3813929) and G-697C (rs518147) polymorphisms were genotyped using the method of PCR and enzyme digestion. Genomic DNA was amplified by using forward primers 5'-ATCTCCACCATGGGTCTCGC-3' (spanning nucleotide positions -885 to -866) and reverse primers 5'-CAATCTAGCCGCTCCAAAGG-3' (spanning nucleotide positions -653 to -634). The reaction was performed according to the protocol described in the previous study [15]. After denaturation at 95°C for 5 min, samples were amplified using 35 cycles of 96°C for 45 sec; 59°C for 30 sec; and 72°C for 30 sec, followed by a final step at 72°C for 10 min. The PCR products were incubated overnight at 37°C with AciI (New England Biolabs) and the digested PCR product (...truncated)