An investigation of the neurotrophic factor genes GDNF NGF and NT3 in susceptibility to ADHD.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:375 –378 (2007) Brief Research Communication An Investigation of the Neurotrophic Factor Genes GDNF, NGF, and NT3 in Susceptibility to ADHD Zahoor Syed,1 Frank Dudbridge,2 and Lindsey Kent3* 1 Addenbrookes Hospital, Cambridge, United Kingdom MRC Biostatistics Unit, Cambridge, United Kingdom 3 Developmental Psychiatry, University of Cambridge, Cambridge, United Kingdom 2 Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable, neurodevelopmental disorder with onset in early childhood. Genes involved in neuronal development and growth are, thus, important etiological candidates and neurotrophic factors have been hypothesized to play a role in the pathogenesis of ADHD. Glial derived neurotrophic factor (GDNF), nerve growth factor (NGF (beta subunit)), and neurotrophic factor 3 (NT3) are members of the neurotrophin family and are involved in the survival, differentiation, and maintenance of neuronal cells. We have examined 10 coding and intronic single nucleotide polymorphisms (SNPs) across GDNF, NGF, and NT3 in a family-based association sample of 120 DSM-IV ADHD probands and their biological parents, as well as a case-control analysis with 120 sex-matched controls. Borderline significant overtransmission of the C allele of a non-synonymous C/T SNP (rs6330) in NGF which codes an alanine/valine change was found in the family-based sample (Chi-square ¼ 3.69, odds ratio (OR) ¼ 1.65, P ¼ 0.05). Although this SNP is located in the 50 pro-NGF sequence and not the mature NGF protein, it may affect intracellular processing and secretion of NGF. ß 2006 Wiley-Liss, Inc. KEY WORDS: attention deficit hyperactivity disorder; association study; neurotrophic factor; polymorphism Please cite this article as follows: Syed Z, Dudbridge F, Kent L. 2007. An Investigation of the Neurotrophic Factor Genes GDNF, NGF, and NT3 in Susceptibility to ADHD. Am J Med Genet Part B 144B:375–378. Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder that affects 2–5% of school-aged children with more boys diagnosed than girls [Swanson et al., 1998]. It is characterized by marked and pervasive inattention, overactivity, and impulsiveness and causes significant social, educational, and psychological problems. Quantitative genetic research over the last decade, from family, twin, and adoption studies has firmly established that ADHD has a significant Grant sponsor: The Wellcome Trust. *Correspondence to: Lindsey Kent, Ph.D., Developmental Psychiatry, University of Cambridge, Douglas House, 18b Trumpington Road, Cambridge CB2 2AH, UK. E-mail: email@example.com Received 28 June 2006; Accepted 21 September 2006 DOI 10.1002/ajmg.b.30459 ß 2006 Wiley-Liss, Inc. genetic contribution [Thapar et al., 1999]. Given that ADHD is a neurodevelopmental disorder, genes involved in neuronal development and growth represent an important set of candidates for involvement in the pathogenesis. The neurotrophins are a family of polypeptide growth factors which are essential for the proliferation, differentiation, survival, and death of neuronal, and non-neuronal cells [see review Chao et al., 2006]. A role has been suggested for brain-derived neurotrophic factor (BDNF) in ADHD susceptibility [Lanktree et al., 2004; Kent et al., 2005], raising the possibility that other neurotrophic factors may also be involved in the pathogenesis of this condition. Of particular interest is glial derived neurotrophic factor (GDNF), the gene for which is located at 5p13. Nominal evidence for linkage to chromosome 5p13 has been demonstrated in all four published genome-wide linkage scans of ADHD [Bakker et al., 2003; Ogdie et al., 2003, 2004; ArcosBurgos et al., 2004; Hebebrand et al., 2006]. GDNF plays a role in the development, maintenance, and survival of dopaminergic midbrain neurons [Granholm et al., 2000] and has been shown, in culture to enhance neuronal dopamine release [Bourque and Trudeau, 2000]. Michelato et al.  provided some evidence of association with a GDNF repeat polymorphism and schizophrenia although others have not found this to be the case [Lee et al., 2001a]. The gene for neurotrophin-3 (NT3) is located at 12p13, a region implicated in a genome-wide scan of quantitative ADHD traits [Fisher et al., 2002] and although there has been some evidence to implicate genetic variation within neurotrophic factor 3 (NT3) in the pathogenesis of schizophrenia [e.g., Hattori et al., 2002], most later studies have failed to replicate this association [see review Lin and Tsai, 2004]. NT3 mRNA is an abundant neurotrophin in the developing brain which is highly expressed in the hippocampi of newborn and in immature neocortical regions of the fetus [Maisonpierre et al., 1991]. Finally, nerve growth factor (NGF) exerts within the CNS, a trophic and functional role on basal forebrain cholinergic neurones, which are involved in attentional systems [see review by Sarter et al., 2006]. The NGF complex consists of three types of subunits: alpha, beta, and gamma, but it is the beta subunit which is solely responsible for nerve growth stimulating activity of NGF. The gene for beta NGF (NGFB) is located at 1p13.1. For this study, 120 ADHD probands and their parents were recruited from two child psychiatry clinics in the UK, following approval from the appropriate research ethics committees. Of these, 72 were full trios with DNA available from both parents. After complete description of the study to the subjects, written informed consent was obtained. Parents were interviewed by trained psychiatrists or psychologists employing the Child and Adolescent Psychiatric Assessment (CAPA) [Angold et al., 1995]. In addition, Conners Teacher Rating Scale (CTRS) [Conners, 1998] were completed on all children to confirm that 0.47 0.53 0.41 0.59 0.78 0.22 0.47 0.53 0.67 0.33 0.60 0.40 0.82 0.18 0.64 0.36 0.52 0.74 0.26 0.48 0.45 0.55 0.44 0.56 0.73 0.27 0.49 0.51 0.73 0.27 0.52 0.48 0.79 0.11 0.54 0.46 0.55 0.73 0.27 0.45 0.48 0.52 0.39 0.61 0.75 0.25 0.49 0.51 0.69 0.31 0.56 0.44 0.86 0.14 0.57 0.43 0.53 0.47 0.72 T C G A G T C T T C T A A C A G rs6330 rs11111 rs12514589 rs3749692 rs1862574 0.28 Probands N ¼ 120 Parents N ¼ 192 Controls N ¼ 120 T C G C rs7132127 rs910330 rs6332 rs7958038 NTF3 NGFB GDNF symptoms met the criterion of pervasiveness. Established cutoff points for possible and likely ADHD caseness on the CTRS were adhered to, that is, a T score above 55 was required. All 120 probands were white and born in the UK (mean age ¼ 11.1 years; range 5–16 years; SD ¼ 3.1 years). The sample was predominantly male (91%). All probands fulfilled DSM-IV diagnostic criteria for ADHD. Of these, 97 (81%) had ADHD combined type, 11 (9%) had the inattentive subtype, and 12 (10%) had the hyperactive impulsive subtype. Children with an IQ below 70, autistic spectrum disorder, or significant medical conditions such as epilepsy were excluded. Twenty-nine children (24%) had comorbid oppositional defiant disorder and 15 children (13%) fulfilled criteria for comorbid conduct disorder. In addition to the family-based sample, a control group of 120 ethnically and sex-matched adolescents from schools and colleges, local to the ADHD clinics were also employed for a case-control analysis. Control subjects were psychiatrically screened with the Strengths and Difficulties Questionnaire [Goodman, 2001] and their mean age was 15.5 years (range 13–17 years). Recruitment of these controls was approved by the same research ethics committees as before and they consented specifically to be used as control samples for an ADHD genetic study. For this study, all cases, controls, and only parents which contributed to full trios were genotyped, that is, where only one parent for a proband was available they were not genotyped. High molecular weight genomic DNA was extracted from either whole blood or cheek swab according to routine procedures. Single nucleotide polymorphisms (SNPs) for the three genes were chosen from the ABI assay on demand or assay by design service (https://www2.appliedbiosystems.com) and all genotyping was performed by Geneservice (www.geneservice.co.uk), blind to affection status. Known coding SNPs with a minor allele frequency greater than 0.1 were chosen where possible, otherwise intronic SNPs were genotyped. Additionally, 20 Geneservice control samples were typed in duplicate with 100% concordance and 98.5% of all genotyping was successful. The transmission disequilibrium test (TDT) [Spielman et al., 1993] was used to test for the presence of non-random transmission of alleles to ADHD probands at each polymorphism, indicative of allelic association and case-control data were analyzed using Pearson Chi-squared test. Parental, proband, and control allele frequencies are shown in Table I and were not significantly different from frequencies reported in Caucasian/CEPH populations from several databases including Hapmap (www.hapmap.org) and summarized on https://www2.appliedbiosystems.com.. Genotype frequencies demonstrated no significant departure from Hardy Weinberg equilibrium. There was no significant difference in allele or genotype frequencies between cases and controls for each polymorphism. TDT analysis demonstrated borderline significant overtransmission of the C allele of rs6330, a non-synonymous SNP in NGF (odds ratio ¼ 1.65, Chi-square ¼ 3.7, P ¼ 0.05) (Table II). This study provides some preliminary evidence that another neurodevelopmentally relevant gene, in addition to BDNF, may be involved in the pathogenesis of ADHD. This common, non-synonymous C/T SNP in NGF produces an alanine-tovaline substitution at amino acid position 35 and although located in the 50 pro-NGF sequence and not the mature NGF protein, it may affect intracellular processing and secretion of NGF as has been demonstrated for the Val66Met allele of propeptide BDNF [Suter et al., 1991; Chen et al., 2004]. Additionally, proneurotrophins are more effective in inducing p75 neurotrophin receptor-dependent apoptosis, than mature NGF, whilst the mature forms of neurotrophins selectively rs4930767 Syed et al. TABLE I. Parent, Proband, and Control Allele Frequencies for GDNF, NGFB, and NTF3 SNPs 376 ADHD and Neurotrophic Factors 377 TABLE II. TDT Analyses on 72 Trios Gene SNP GDNF NGFB NTF3 Overtransmitted allele T:U Chi-square P-value C A A T C T G T C T 15:10 31:27 24:18 31:25 38:23 29:20 34:30 31:26 33:29 36:27 1.0 0.28 0.86 0.64 3.69 1.65 0.25 0.44 0.26 1.29 n.s. n.s. n.s. n.s. 0.05 n.s. n.s. n.s. n.s. n.s. rs1862574 rs3749692 rs12514589 rs11111 rs6330 rs910330 rs6332 rs7958038 rs7132127 rs4930767 T, transmitted allele; U, untransmitted allele. activate the Trk family of receptors to promote survival [Lee et al., 2001b]. Cell death, mediated by proneurotrophins and the p75 receptor may be important for correct targeting and pruning of neuronal populations during development [see review, Chao et al., 2006]. Genetic variation within the pro domain of NGF may therefore be of biological relevance in susceptibility to a developmental disorder such as ADHD. However, these results should be viewed with caution. 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