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Joint Analysis of the DRD5 Marker Concludes Association with Attention-Deficit/Hyperactivity Disorder Confined to the Predominantly Inattentive and Combined Subtypes
2004; Elsevier BV; Volume: 74; Issue: 2 Linguagem: Inglês
10.1086/381561
ISSN1537-6605
AutoresNaomi Lowe, Aiveen Kirley, Ziarih Hawi, Pak C. Sham, Harvey Wickham, Christopher J. Kratochvil, Shelley D. Smith, Saretta Y. Lee, Florence Lévy, Lindsey Kent, Fiona Middle, Luís Augusto Rohde, Tatiana Roman, Eda Tahir Turanlı, Y. Yazgan, Philip Asherson, Jonathan Mill, Anita Thapar, Antony Payton, Richard D. Todd, Timothy G. Stephens, Richard P. Ebstein, Iris Manor, Cathy L. Barr, Karen Wigg, Richard J. Sinke, Jan K. Buitelaar, Susan L. Smalley, Stan F. Nelson, Joseph Biederman, Stephen V. Faraone, Michael Gill,
Tópico(s)Autism Spectrum Disorder Research
ResumoAttention-deficit/hyperactivity disorder (ADHD) is a highly heritable, heterogeneous disorder of early onset, consisting of a triad of symptoms: inattention, hyperactivity, and impulsivity. The disorder has a significant genetic component, and theories of etiology include abnormalities in the dopaminergic system, with DRD4, DAT1, SNAP25, and DRD5 being implicated as major susceptibility genes. An initial report of association between ADHD and the common 148-bp allele of a microsatellite marker located 18.5 kb from the DRD5 gene has been followed by several studies showing nonsignificant trends toward association with the same allele. To establish the postulated association of the (CA)n repeat with ADHD, we collected genotypic information from 14 independent samples of probands and their parents, analyzed them individually and, in the absence of heterogeneity, analyzed them as a joint sample. The joint analysis showed association with the DRD5 locus (P=.00005; odds ratio 1.24; 95% confidence interval 1.12–1.38). This association appears to be confined to the predominantly inattentive and combined clinical subtypes. Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable, heterogeneous disorder of early onset, consisting of a triad of symptoms: inattention, hyperactivity, and impulsivity. The disorder has a significant genetic component, and theories of etiology include abnormalities in the dopaminergic system, with DRD4, DAT1, SNAP25, and DRD5 being implicated as major susceptibility genes. An initial report of association between ADHD and the common 148-bp allele of a microsatellite marker located 18.5 kb from the DRD5 gene has been followed by several studies showing nonsignificant trends toward association with the same allele. To establish the postulated association of the (CA)n repeat with ADHD, we collected genotypic information from 14 independent samples of probands and their parents, analyzed them individually and, in the absence of heterogeneity, analyzed them as a joint sample. The joint analysis showed association with the DRD5 locus (P=.00005; odds ratio 1.24; 95% confidence interval 1.12–1.38). This association appears to be confined to the predominantly inattentive and combined clinical subtypes. Attention-deficit/hyperactivity disorder (ADHD [MIM 143465]) is a common disorder of childhood onset that manifests itself as a combination of inattentive, hyperactive, and impulsive symptoms. It is known to affect 3%–9% of school-aged children worldwide (Faraone et al., Faraone et al., in pressFaraone SV, Sergeant J, Gillberg C, Biederman J. The worldwide prevalence of ADHD: is it an American condition? World Psychiatry (in press)Google Scholar) and may often persist into adulthood (Faraone et al. Faraone et al., 2000Faraone SV Biederman J Spencer T Wilens T Seidman LJ Mick E Doyle AE Attention deficit hyperactivity disorder in adults: an overview.Biol Psychiatry. 2000; 48: 9-20Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). The heritability of ADHD is estimated to be 70%–90% (Levy et al. Levy et al., 1997Levy F Hay DA McStephen M Wood C Waldman I Attention deficit hyperactivity disorder: a category or continuum? Genetic analysis of a large-scale twin study.J Am Acad Child Adolesc Psychiatry. 1997; 36: 737-744Abstract Full Text PDF PubMed Scopus (741) Google Scholar; Thapar et al. Thapar et al., 1999Thapar A Holmes J Poulton K Harrington R Genetic basis of attention deficit and hyperactivity.Br J Psychiatry. 1999; 174: 105-111Crossref PubMed Scopus (315) Google Scholar). Children with this disorder often develop severe problems with personal relationships and academic development. Compared with control subjects, ADHD probands exhibit lower grades, fail more courses, have worse performance on standardized tests, have fewer friends, and are rated less adequate in psychosocial adjustment (Mannuzza and Klein Mannuzza and Klein, 2000Mannuzza S Klein RG Long-term prognosis in attention-deficit/hyperactivity disorder.Child Adolesc Psychiatr Clin N Am. 2000; 9: 711-726PubMed Google Scholar). In addition, individuals with this disorder are at greater risk for substance abuse (Flory et al. Flory et al., 2003Flory K Milich R Lynam DR Leukefeld C Clayton R Relation between childhood disruptive behavior disorders and substance use and dependence symptoms in young adulthood: individuals with symptoms of attention-deficit/hyperactivity disorder and conduct disorder are uniquely at risk.Psychol Addict Behav. 2003; 17: 151-158Crossref PubMed Scopus (104) Google Scholar). Furthermore, individuals with ADHD are reported to have increased driving problems; as part of a case-control study, individuals with ADHD self-reported more traffic citations—including, speeding, vehicular crashes, and license suspensions—than their counterparts in the control group (Barkley et al. Barkley et al., 2002Barkley RA Murphy KR Dupaul GI Bush T Driving in young adults with attention deficit hyperactivity disorder: knowledge, performance, adverse outcomes, and the role of executive functioning.J Int Neuropsychol Soc. 2002; 8: 655-672Crossref PubMed Scopus (277) Google Scholar). The exact etiology of ADHD is unknown, but it is widely recognized to have a significant genetic component, as demonstrated by family (Biederman et al. Biederman et al., 1992Biederman J Faraone SV Keenan K Benjamin J Krifcher B Moore C Sprich-Buckminster S et al.Further evidence for family-genetic risk factors in attention deficit hyperactivity disorder: patterns of comorbidity in probands and relatives psychiatrically and pediatrically referred samples.Arch Gen Psychiatry. 1992; 49: 728-738Crossref PubMed Scopus (637) Google Scholar), twin (Silberg et al. Silberg et al., 1996Silberg J Rutter M Meyer J Maes H Hewitt J Simonoff E Pickles A Loeber R Eaves L Genetic and environmental influences on the covariation between hyperactivity and conduct disturbance in juvenile twins.J Child Psychol Psychiatry. 1996; 37: 803-816Crossref PubMed Scopus (219) Google Scholar), and adoption (Cadoret and Stewart Cadoret and Stewart, 1991Cadoret RJ Stewart MA An adoption study of attention deficit/hyperactivity/aggression and their relationship to adult antisocial personality.Compr Psychiatry. 1991; 32: 73-82Abstract Full Text PDF PubMed Scopus (105) Google Scholar) studies. Although both the serotonergic and noradrenergic systems have been implicated in ADHD, the dopaminergic system is by far the most extensively explored to date. Neuropharmacological studies have demonstrated that methylphenidate and dexamphetamine—stimulant medications that are effective in ∼70% of patients with ADHD (Spencer et al. Spencer et al., 1996Spencer T Biederman J Wilens T Harding M O'Donnell D Griffin S Pharmacotherapy of attention-deficit hyperactivity disorder across the life cycle.J Am Acad Child Adolesc Psychiatry. 1996; 35: 409-432Abstract Full Text PDF PubMed Scopus (673) Google Scholar)—block the reuptake of dopamine by the dopamine transporter DAT1 (Amara and Kuhar Amara and Kuhar, 1993Amara SG Kuhar MJ Neurotransmitter transporters: recent progress.Annu Rev Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (981) Google Scholar; Krause et al. Krause et al., 2000Krause KH Dresel SH Krause J Kung HF Tatsch K Increased striatal dopamine transporter in adult patients with attention deficit hyperactivity disorder: effects of methylphenidate as measured by single photon emission computed tomography.Neurosci Lett. 2000; 285: 107-110Crossref PubMed Scopus (428) Google Scholar). Neuroimaging studies have shown abnormalities in the frontal lobe and subcortical structures, regions that are known to be rich in dopaminergic neurotransmission and important in the control of attention and response to organization (Lou et al. Lou et al., 1990Lou HC Henriksen L Bruhn P Focal cerebral dysfunction in developmental learning disabilities.Lancet. 1990; 335: 8-11Abstract PubMed Scopus (123) Google Scholar; Zametkin et al. Zametkin et al., 1990Zametkin AJ Nordahl TE Gross M King AC Semple WE Rumsey J Hamburger S Cohen RM Cerebral glucose metabolism in adults with hyperactivity of childhood onset.N Engl J Med. 1990; 323: 1361-1366Crossref PubMed Scopus (757) Google Scholar; Rubia et al. Rubia et al., 1997Rubia K Overmeyer S Taylor E Bullmore E Brammer M Williams S Simmos A Andrew C Inhibitory control of hyperactive adolescents in FMRI.in: Toga AW Frackowiak RSJ Mazziotta JC Neuroimage: proceedings of the Third International Conference on Functional Mapping of the Human Brain (May 19–23, 1997, Copenhagen). Academic Press, New York1997Google Scholar). Multiple animal models have been produced, including the DAT1 knockout mouse, which expresses spontaneous hyperactivity and difficulty with learning tasks (Davids et al. Davids et al., 2003Davids E Zhang K Tarazi FI Baldessarini RJ Animal models of attention-deficit hyperactivity disorder.Brain Res Brain Res Rev. 2003; 42: 1-21Crossref PubMed Scopus (176) Google Scholar). In addition, molecular genetic studies have implicated susceptibility genes, including those for the dopamine D4 receptor (DRD4), the dopamine transporter (DAT1), dopamine β hydroxylase (DBH), synaptosomal associated protein 25 (SNAP25), and the dopamine D5 receptor (DRD5). To date, the most extensively examined genes of the dopaminergic system are DRD4 and DAT1 (Hawi et al. Hawi et al., 2003aHawi Z Kirley A Lowe N Fitzgerald M Gill M Recent genetic advances in ADHD and diagnostic and therapeutic prospects.Expert Rev Neurother. 2003a; 3: 453-464Crossref PubMed Scopus (10) Google Scholar). The primary focus of attention at DAT1 has been the 480-bp allele of a 40-bp repeat located in the 3′ UTR of the gene. In the case of DRD4, the majority of studies have reported on a 48-bp microsatellite located in exon 3 of the gene and translated to the third intracellular loop of the protein. Recently, meta-analyses have been performed on both of these markers, in an attempt to definitively confirm or refute association with ADHD (Faraone et al. Faraone et al., 2001Faraone SV Doyle AE Mick E Biederman J Meta-analysis of the association between the 7-repeat allele of the dopamine D(4) receptor gene and attention deficit hyperactivity disorder.Am J Psychiatry. 2001; 158: 1052-1057Crossref PubMed Scopus (613) Google Scholar; Maher et al. Maher et al., 2002Maher BS Marazita ML Ferrell RE Vanyukov MM Dopamine system genes and attention deficit hyperactivity disorder: a meta-analysis.Psychiatr Genet. 2002; 12: 207-215Crossref PubMed Scopus (188) Google Scholar). In 1999, Daly et al. reported a significant association between ADHD and the 148-bp allele of a microsatelite marker located 18.5 kb 5′ to the DRD5 gene on chromosome 4. Subsequent studies of this (CA)n repeat marker have shown nonsignificant trends toward association with the same allele (Barr et al. Barr et al., 2000Barr CL Wigg KG Feng Y Zai G Malone M Roberts W Schachar R Tannock R Kennedy JL Attention-deficit hyperactivity disorder and the gene for the dopamine D5 receptor.Mol Psychiatry. 2000; 5: 548-551Crossref PubMed Scopus (50) Google Scholar; Tahir et al. Tahir et al., 2000Tahir E Yazgan Y Cirakoglu B Ozbay F Waldman I Asherson PJ Association and linkage of DRD4 and DRD5 with attention deficit hyperactivity disorder (ADHD) in a sample of Turkish children.Mol Psychiatry. 2000; 5: 396-404Crossref PubMed Scopus (135) Google Scholar; Payton et al. Payton et al., 2001Payton A Holmes J Barrett JH Hever T Fitzpatrick H Trumper AL Harrington R McGuffin P O'Donovan M Owen M Ollier W Worthington J Thapar A Examining for association between candidate gene polymorphisms in the dopamine pathway and attention-deficit hyperactivity disorder: a family-based study.Am J Med Genet. 2001; 105: 464-470Crossref PubMed Scopus (107) Google Scholar). To avoid the premature conclusion of the presence or absence of an association with ADHD (and guided by the meta-analyses of DRD4 and DAT1), a joint analysis of DRD5 was proposed. Although there is no evidence to suggest that the D5 microsatellite is itself functional, the association reported by Daly et al. (Daly et al., 1999Daly G Hawi Z Fitzgerald M Gill M Mapping susceptibility loci in attention deficit hyperactivity disorder: preferential transmission of parental alleles at DAT1 DBH and DRD5 to affected children.Mol Psychiatry. 1999; 4: 192-196Crossref PubMed Scopus (332) Google Scholar) is too strong to be ignored. Therefore, we hypothesized that if the association with ADHD were true, the microsatellite may be in linkage disequilibrium (LD) with one or more functional variants. To this end, we invited all known groups with samples based on parent-proband trios to genotype their sample for the marker and present us with their data for analysis (table 1).Table 1Information about Individual GroupsGroup NumberPrimary InvestigatorNo. of CasesPublication StatusaU = unpublished data; P = previously published data; P + U = previously published data with the addition of new unpublished data.Origin of SampleInstrument of DiagnosisbKSADS = Kidi schedule for affective disorders and schizophrenia (Kaufman et al. 1997). ATBRS = Australian Twin Behavior Rating Scale (Hay et al. 2001). CAPA = Child and Adolescent Psychiatric Assessment (Angold et al. 1995). K-SADS-E = Schedule for Affective Disorders and Schizophrenia for School-Age Children–Epidemiologic Version (Orvaschel et al. 1987). MAGIC = Missouri Assessment of Genetics Interview for Children (Reich 2000). PICS-IV = Parent Interview for Child Symptoms (A. Schachar, A. Ichowicz, unpublished data). DISC = Diagnostic Interview Schedule for Children (Shaffer et al. 2000). K-SADS-PiL = Schedule for Affective Disorders and Schizophrenia for School-Age-Children Present and Lifetime version (Kaufman et al. 1997).1C. J. Kratochvil41UUnited StatesKSADS2F. Levy66UAustraliaATBRS3L. Kent69UUnited KingdomCAPA4S. V. Faraone82UUnited StatesK-SADS-E5L. A. Rohde85UBrazilK-SADS-E6E. Tahir100PcTahir et al. (2000).TurkeyKSADS7P. Asherson103UUnited KingdomCAPA8A. Thapar106PdPayton et al. (2001).United KingdomCAPA9R. D. Todd161UUnited StatesMAGIC10M. Gill168PeDaly et al. (1999). + UIrelandCAPA11R. P. Ebstein176UIsraelClinical interview12C. L. Barr178PfBarr et al. (2000). + UCanadaPICS-IV13R. J. Sinke207UHollandDISC14S. L. Smalley438PgKustanovich et al. (in press).United StatesK-SADS-PiLa U = unpublished data; P = previously published data; P + U = previously published data with the addition of new unpublished data.b KSADS = Kidi schedule for affective disorders and schizophrenia (Kaufman et al. Kaufman et al., 1997Kaufman J Birmaher B Brent D Rao U Flynn C Moreci P Williamson D Ryan N Schedule for affective disorders and schizophrenia for school-age children-present and life-time version (K-SADS-PL): initial reliability and validity data.J Am Acad Child Adolesc Psychiatry. 1997; 36: 980-988Abstract Full Text PDF PubMed Scopus (6594) Google Scholar). ATBRS = Australian Twin Behavior Rating Scale (Hay et al. Hay et al., 2001Hay D McStephen M Levy F The diagnostic genetics of ADHD symptoms and subtypes.in: Levy F Hay D Attention genes and ADHD. Hove, United Kingdom, Brunner-Routledge2001: 35-37Google Scholar). CAPA = Child and Adolescent Psychiatric Assessment (Angold et al. Angold et al., 1995Angold A Prendergast M Cox A Harrington R Simonoff E Rutter M The Child and Adolescent Psychiatric Assessment (CAPA).Psychol Med. 1995; 25: 739-753Crossref PubMed Scopus (434) Google Scholar). K-SADS-E = Schedule for Affective Disorders and Schizophrenia for School-Age Children–Epidemiologic Version (Orvaschel et al. Orvaschel and Puig-Antich, 1987Orvaschel H Puig-Antich J Schedule for Affective Disorder and Schizophrenia for School-Age Children-Epidemiological. 4th version. Nova University Center for Psychological Study, Fort Lauderdale1987Google Scholar). MAGIC = Missouri Assessment of Genetics Interview for Children (Reich Reich, 2000Reich W Diagnostic interview for children and adolescents (DICA).J Am Acad Child Adolesc Psychiatry. 2000; 39: 59-66Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). PICS-IV = Parent Interview for Child Symptoms (A. Schachar, A. Ichowicz, unpublished data). DISC = Diagnostic Interview Schedule for Children (Shaffer et al. Shaffer et al., 2000Shaffer D Fisher P Lucas CP Dulcan MK Schwab-Stone ME NIMH Diagnostic Interview Schedule for Children, version IV (NIMH DISC-IV): description differences from previous versions, and reliability of some common diagnoses.J Am Acad Child Adolesc Psychiatry. 2000; 39: 28-38Abstract Full Text Full Text PDF PubMed Scopus (2661) Google Scholar). K-SADS-PiL = Schedule for Affective Disorders and Schizophrenia for School-Age-Children Present and Lifetime version (Kaufman et al. Kaufman et al., 1997Kaufman J Birmaher B Brent D Rao U Flynn C Moreci P Williamson D Ryan N Schedule for affective disorders and schizophrenia for school-age children-present and life-time version (K-SADS-PL): initial reliability and validity data.J Am Acad Child Adolesc Psychiatry. 1997; 36: 980-988Abstract Full Text PDF PubMed Scopus (6594) Google Scholar).c Tahir et al. (Tahir et al., 2000Tahir E Yazgan Y Cirakoglu B Ozbay F Waldman I Asherson PJ Association and linkage of DRD4 and DRD5 with attention deficit hyperactivity disorder (ADHD) in a sample of Turkish children.Mol Psychiatry. 2000; 5: 396-404Crossref PubMed Scopus (135) Google Scholar).d Payton et al. (Payton et al., 2001Payton A Holmes J Barrett JH Hever T Fitzpatrick H Trumper AL Harrington R McGuffin P O'Donovan M Owen M Ollier W Worthington J Thapar A Examining for association between candidate gene polymorphisms in the dopamine pathway and attention-deficit hyperactivity disorder: a family-based study.Am J Med Genet. 2001; 105: 464-470Crossref PubMed Scopus (107) Google Scholar).e Daly et al. (Daly et al., 1999Daly G Hawi Z Fitzgerald M Gill M Mapping susceptibility loci in attention deficit hyperactivity disorder: preferential transmission of parental alleles at DAT1 DBH and DRD5 to affected children.Mol Psychiatry. 1999; 4: 192-196Crossref PubMed Scopus (332) Google Scholar).f Barr et al. (Barr et al., 2000Barr CL Wigg KG Feng Y Zai G Malone M Roberts W Schachar R Tannock R Kennedy JL Attention-deficit hyperactivity disorder and the gene for the dopamine D5 receptor.Mol Psychiatry. 2000; 5: 548-551Crossref PubMed Scopus (50) Google Scholar).g Kustanovich et al. (Kustanovich et al., in pressKustanovich V, Ishii J, Crawford L, Yang M, McGough JJ, McCracken JT, Smalley SL, Nelson SF. Transmission disequilibrium testing of dopamine-related candidate gene polymorphisms in ADHD: confirmation of association of ADHD with DRD4 and DRD5. Mol Psychiatry (in press)Google Scholar). Open table in a new tab In the event of an overall significant association between the variant and ADHD, we proposed that, owing to the unique opportunity presented by a data set of this magnitude, we would further examine the association in relation to a number of different subdivisions. Three times more males than females receive a diagnosis of ADHD (Anderson et al. Anderson et al., 1987Anderson JC Williams S McGee R Silva PA DSM-III disorders in preadolescent children: prevalence in a large sample from the general population.Arch Gen Psychiatry. 1987; 44: 69-76Crossref PubMed Scopus (1139) Google Scholar), and it has been reported by teachers that affected boys are more inattentive and more hyperactive/impulsive than affected girls (Hartung et al. Hartung et al., 2002Hartung CM Willcutt EG Lahey BB Pelham WE Loney J Stein MA Keenan K Sex differences in young children who meet criteria for attention deficit hyperactivity disorder.J Clin Child Adolesc Psychol. 2002; 31: 453-464Crossref PubMed Scopus (50) Google Scholar). Furthermore, Clarke et al. (Clarke et al., 2003Clarke AR Barry RJ McCarthy R Selikowitz M Clarke DC Croft RJ EEG activity in girls with attention-deficit/hyperactivity disorder.Clin Neurophysiol. 2003; 114: 319-328Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) have reported results indicating that girls with ADHD also exhibit abnormalities in their electroencephalograms; however, there is far less variance in their profiles than appears in boys. We therefore proposed analysis by the sex of the affected child. In addition, we planned to examine the sex of the transmitting parent, because the literature has suggested the possibility of imprinting at the DRD2 (Kirley et al. Kirley et al., 2002Kirley A Hawi Z Daly G McCarron M Mullins C Millar N Waldman I Fitzgerald M Gill M Dopaminergic system genes in ADHD: toward a biological hypothesis.Neuropsychopharmacology. 2002; 27: 607-619PubMed Google Scholar) and SNAP-25 loci (Brophy et al. Brophy et al., 2002Brophy K Hawi Z Kirley A Fitzgerald M Gill M Synaptosomal-associated protein 25 (SNAP-25) and attention deficit hyperactivity disorder (ADHD): evidence of linkage and association in the Irish population.Mol Psychiatry. 2002; 7: 913-917Crossref PubMed Scopus (135) Google Scholar) in conjunction with multiple known imprinted genes in various other areas of psychiatric genetics (Davies et al. Davies et al., 2001Davies W Isles AR Wilkinson LS Imprinted genes and mental dysfunction.Ann Med. 2001; 33: 428-436Crossref PubMed Scopus (44) Google Scholar). Finally, recent attention has focused on more homogeneous diagnostic symptoms and subtypes. Waldman et al. (Waldman et al., 1998Waldman ID Rowe DC Abramowitz A Kozel ST Mohr JH Sherman SL Cleveland HH Sanders ML Gard JM Stever C Association and linkage of the dopamine transporter gene and attention deficit hyperactivity disorder in children: owing to diagnostic subtype and severity.Am J Hum Genet. 1998; 63: 1767-1776Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar) reported that, in between-family association analyses, levels of predominantly hyperactive-impulsive symptoms were related to the number of DAT1 high-risk alleles but that levels of the inattentive symptoms were not, whereas within-family analysis showed association between DAT1 and the combined subtype. We therefore also proposed to analyze the joint sample in relation to diagnostic subtype. All children in the present study were given a diagnosis of one of the three clinical subtypes (predominantly inattentive, predominantly hyperactive/impulsive, or combined) of ADHD, according to criteria of the Diagnostic and Statistical Manual, fourth edition (DSM-IV) (American Psychiatric Association American Psychiatric Association, 1994American Psychiatric Association (1994) Diagnostic and statistical manual of mental disorders, 4th ed. Washington, DCGoogle Scholar). To receive a diagnosis of predominantly inattentive or predominantly hyperactive/impulsive ADHD, children must display a minimum of six of the nine symptoms from the inattentive or hyperactive/impulsive sections of the DSM-IV in a minimum of two settings (e.g., home and school). If a child has six or more symptoms in both sections, then he or she receives a diagnosis of combined-type ADHD. Inclusion criteria for the present study were the presence of a DSM-IV diagnosis of childhood-onset ADHD and the genotyping of one or both parents. Exclusion criteria were the presence of pervasive developmental disorders, fragile X syndrome, major neurological disorders, fetal alcohol syndrome, Tourette syndrome, psychosis, and a full-scale IQ score <70. Information on the sex of the children was provided along with the clinical subtype. Genotypic data for the microsatellite marker were collected from each group, along with at least three DNA samples, which were genotyped at the Neuropsychiatry Genetics Laboratory, Dublin, to ensure consistent allele calling between centers. The data received from each group consisted of genotypes from affected probands and either or both of their parents. To obtain as complete a sample as possible—and to avoid potential bias—we contacted all members of the ADHD collaborative network (Faraone et al. Faraone, 2003Faraone SV Report from the Fourth International Meeting of the Attention Deficit Hyperactivity Disorder Molecular Genetics Network.Am J Med Genet. 2003; 121B: 55-59Crossref PubMed Scopus (20) Google Scholar) and invited their participation. We also searched PubMed using the keywords "ADHD" and "DRD5." This revealed an earlier meta-analysis (Maher et al. Maher et al., 2002Maher BS Marazita ML Ferrell RE Vanyukov MM Dopamine system genes and attention deficit hyperactivity disorder: a meta-analysis.Psychiatr Genet. 2002; 12: 207-215Crossref PubMed Scopus (188) Google Scholar) that contained data from four published studies, which we have included, and from an additional unpublished sample, which we excluded because it did not meet our inclusion criteria. We are aware of five additional samples that have not yet been genotyped for this marker. This approach to sample collection should provide adequate power to confirm a gene of small effect and to minimize publication bias. A funnel plot (Egger et al. Egger et al., 1997Egger M Davey Smith G Schneider M Minder C Bias in meta-analysis detected by a simple graphical test.BMJ. 1997; 315: 629-634Crossref PubMed Scopus (28520) Google Scholar) was produced to examine for evidence of sample bias in the study. This was achieved by plotting the number of affected children for each sample against its equivalent odds ratio (OR), in conjunction with the OR for the combined sample (fig. 1). The larger samples should result in ORs that are closer to the true OR than the smaller samples. In addition, we regressed the normal SD of the OR against the precision of the OR, according to the methods used by Egger et al. (Egger et al., 1997Egger M Davey Smith G Schneider M Minder C Bias in meta-analysis detected by a simple graphical test.BMJ. 1997; 315: 629-634Crossref PubMed Scopus (28520) Google Scholar) through use of STATA 6.0 (StataCorp StataCorp, 1999StataCorp Reference manual for Stata, version 6.0. StataCorp, College Station, TX1999Google Scholar). This analysis works on the same basis as the funnel plot, in that the precision of the OR increases with sample size. Egger et al. (Egger et al., 1997Egger M Davey Smith G Schneider M Minder C Bias in meta-analysis detected by a simple graphical test.BMJ. 1997; 315: 629-634Crossref PubMed Scopus (28520) Google Scholar) showed that, in the absence of bias, the regression would run through the origin (fig. 2). This method produces an R2 (measure of the fit of the regression to the data) and a value for the intercept of the line, along with corresponding statistics.Figure 2Regression of the standardized effect size versus the precision of the OR. The intercept of the line will occur close to 0, implying no bias in the samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Initially, association analyses were performed on each individual group's sample, using the family-based extended transmission/disequilibrium test (ETDT) (Sham and Curtis Sham and Curtis, 1995Sham PC Curtis D An extended transmission disequilibrium test (TDT) for multi-allele marker loci.Ann Hum Genet. 1995; 59: 323-336Crossref PubMed Scopus (575) Google Scholar). This test was designed to avoid population stratification by using parental genotypes as internal controls and to disregard duos whose inclusion could potentially lead to bias (Curtis and Sham Curtis and Sham, 1995Curtis D Sham PC A note on the application of the transmission disequilibrium test when a parent is missing.Am J Hum Genet. 1995; 56: 811-812PubMed Google Scholar). It is adapted from the transmission/disequilibrium test (TDT) and is used to examine for linkage in the presence of association between multiallelic markers and disease phenotypes. The analysis involves the comparison of transmissions and nontransmissions of the risk allele from heterozygous parents to affected offspring. To test for heterogeneity among the samples, we used the transmission and nontransmission information of the 148-bp allele from the TDT analysis. Two different logistic regression models were compared. The first model assumed homogeneity (i.e., that the regression coefficients were the same across studies, so only one regression coefficient was estimated); the second model assumed heterogeneity (i.e., that all the regression coefficients were different, so that as many regression coefficients were estimated as there were studies). Both models produced a −2 log likelihood value, a measure that attempts to assess the suitability of the statistical method used for a given set of data. Using the formula (-2log likelihoodindividual)-(-2 log likelihoodcombined)=χ2, we tested for evidence of a significant difference between the two models, which would be indicative of heterogeneity among the samples. For the sensitivity analysis, each group's data were removed in turn from the combined total, and the remaining data were reanalyzed. This was performed to ensure that no individual group was biasing the combined result. The attributable fraction (Levin Levin, 1953Levin M The occurrence of lung cancer in man.Acta Union International Contra Cancrum. 1953; 9: 531-541PubMed Google Scholar) for the marker was calculated according to the formula f(RR-1)/[f(RR-1)+1], where f is the frequency of the 148-bp allele in the sample of nontransmitted alleles and RR is the relative risk. The value was calculated as RR=[a/(a+c)]/[b/(b+d)], where a is the number of transmissions of the risk allele, b is the number of nontransmissions of the risk allele, c is the number of transmissions of the nonrisk alleles, and d is the number of nontransmissions of the nonrisk alleles. To test the combi
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