Association study of the adrenergic receptors and childhood-onset mood disorders in Hungarian families.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:227 –233 (2006) Association Study of the Adrenergic Receptors and Childhood-Onset Mood Disorders in Hungarian Families I. Burcescu,1 K. Wigg,1 L. Gomez,1 N. King,2 Á. Vetró,3 E. Kiss,3 K. Kapornai,3 J. Gádoros,4 J.L. Kennedy,2 M. Kovacs,5 C.L. Barr1,6* and The International Consortium for Childhood-Onset Mood Disorders 1 Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada Neurogenetics Section, Centre for Addiction and Mental Health, Toronto, Ontario, Canada 3 Department for Child and Adolescent Psychiatry, Szeged University, Szeged, Hungary 4 Vadaskert Hospital, Budapest, Hungary 5 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 6 The Hospital for Sick Children, Toronto, Ontario, Canada 2 The adrenergic system has been implicated in the etiology of depression based on a number of lines of evidence, particularly, the mechanism of some classes of antidepressants which increase the synaptic levels of norepinephrine. Further, several genome scans for mood disorders, both unipolar and bipolar, have indicated linkage to the chromosomal regions of 5q23–q33.3, 8p12– p11.2, 4p16, and 10q24–q26, the location of the adrenergic receptors a1B (ADRA1B), b3 (ADRB3), a2C (ADRA2C), a2A (ADRA2A), and b1 (ADRB1). In this manuscript, we report on the relationship of the adrenergic receptors and depression using a family based association approach and 189 families (223 affected children) with childhoodonset mood disorder (COMD) collected in Hungary. We found no significant evidence for an association with any of the 24 markers, in total, tested across these genes using single marker analysis or haplotypes of markers across these genes. The results in the present sample indicate that these nine genes are unlikely to be major susceptibility genes contributing to COMD. ß 2006 Wiley-Liss, Inc. KEY WORDS: adrenergic receptors; child; depression; genetics; mood disorders; gene INTRODUCTION The adrenergic system has long been suggested to be involved in mood disorders with the main line of evidence stemming from the mechanisms of action of antidepressant treatments that increase the synaptic levels of norepinephrine Members are listed in the acknowledgements. Grant sponsor: National Institute of Mental Health Program Project grant; Grant number: MH 56193; Grant sponsor: National Alliance for Research on Schizophrenia and Depression. *Correspondence to: C.L. Barr, 399 Bathurst St., MP14-302, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada. E-mail: CBarr@uhnres.utoronto.ca Received 12 May 2005; Accepted 22 November 2005 DOI 10.1002/ajmg.b.30292 ß 2006 Wiley-Liss, Inc. [Brunello et al., 2003]. Further, the adrenergic system regulates key biological processes that are often dysregulated in depression, sleep, learning, memory, attention, arousal, and adaptation to stress [Ressler and Nemeroff, 2000]. While a number of the antidepressants affect multiple monoamines, a specific contribution of the adrenergic system can be deduced from the efficacy of noradrenergic specific agents such as Reboxetine, a selective noradrenaline reuptake inhibitor [Wong et al., 2000]. After the initial increase in synaptic monoamines through monoamine transporter blockade, or receptor blockade, the exact mechanism by which antidepressants exert their effect is not clear. The synaptic levels of the monoamines increase rapidly, however, the therapeutic response is not seen for days or weeks after the beginning of treatment, indicating a more complicated mechanism than the simple increase in monoamine levels. Several other lines of evidence suggest a dysregulation of the adrenergic system in depression. These include alterations in platelet a2-adrenergic receptor binding, upregulation of badrenoreceptors, decreased cAMP response to b-adrenergic agonists, and blunted response in growth hormone secretion to the a2-agonist clonidine [Leonard, 1997; Schatzberg, 1998]. However, these findings have not always been consistent across studies leading to speculation of biologically subtypes [Charney, 1998]. The most convincing evidence for biological subtypes related to the adrenergic system comes from monoamine depletion studies. Depletion of noradrenaline results in relapse in individuals with a history of depression [Berman et al., 1999]. There are also indications that relapse in response to catecholamine depletion is specific to individuals that respond to noradrenergic antidepressants, but not to individuals that respond to serotonergic agents [Delgado and Moreno, 2000]. Further, monoamine depletion does not appear to affect individuals without a previous depressive episode, or without a family history of depression, indicating that this differential response is based on an underlying biological vulnerability with different biological mechanisms [Delgado and Moreno, 2000]. The location of six of the adrenergic receptors in regions identified as linked to mood disorders, also points to these particular genes as potential risk factors for mood disorders. Four adrenergic receptors are located in chromosomal regions identified in a genome scan of families with recurrent (2 episodes), early onset (onset age 25) major depressive disorders [Zubenko et al., 2003]. These are the adrenergic receptors; a1B (ADRA1B) located on 5q23–q33.3, b3 (ADRB3) located on 8p12–p11.2, and a2A (ADRA2A) and b1 (ADRB1) both located on 10q24–q26. The location of the adrenergic receptors a1A (ADRA1A, formerly ADRA1C) on 8p21.2 [Cichon 228 Burcescu et al. TABLE I. Markers Genotyped by Taqman Assays on-Demand Gene Marker ADRA1A rs1383914 rs544215 rs527340 rs2055197 rs3739215 rs1019920 rs2030373 rs10515805 rs11743363 rs13171967 rs11953285 rs11739589 rs1886844 rs946188 rs835878 rs734290 rs638019 rs1168965 rs13019188 rs11957757 ADRA1B ADRA1D ADRA2A ADRA2B ADRB2 DNA variant C!T A!G C!T A!T C!G A!C A!C A!G C!G G!T A!C G!T G!T A!G A!G C!T A!G C!G A!G A!G Flanking sequence TGCTCCTGGCCCGCAGTTACCTACA[C/T]TTTGAGCTGCCCCACCGAAGGCTCC TCCATGAGAGCCACCATACCTGTTA[A/G]TGTGGCAGCCAGTGTGGTGGGGTAG CTGCAGCGCCCATTGCACGGGTGCA[C/T]GATCACATGTATGTATATTAGTAAA TCTGTGCAAAAACCATGTTTTTTCC[A/T]TCTTCGACATTCTCATGCCTGCTTC GAACGAAGGGGATTTTGTTTTGCAG[C/G]GTGATTTTCATGTCTATGAATGTTC TCCCCTCCCTGACCTCTGCCTTACT[A/C]AGGAGCCTGGTCACATTCTGTTCTC CACAATAAAAAGTGAGAGGTTCTTG[A/C]CACCTTGATGCTATCTTGTTTCTCT CTTCAAGGCTGCACCTGGCTTCAAA[A/G]AGGAAAAATGAACTACAGGCATCTG TCCGATAACTTTATGGGGACCTCTT[C/G]GGTACTGTCAGTCTTTACACACAGG AGAGGGGGCAGAGATGATGACCCTT[G/T]TAGCTCCTCCCTGCAAGTCAGCCCC CCTCATCCTCTTCACCACCTTCTTC[A/C]TCTTGGTAGCAGTGGTACTGGTGGT GGGTGGCCCACTGGAGTCCTCACTT[G/T]GTGAGACCCCAGTGTCAGGCTCCAT TTTCTGATGGAAGAGGTTTGAGTGT[G/T]TTTATAAGCTTTGAGAAAAGATTCA AACGGCCCTAACCTTGGAGCTCAGT[A/G]GCAGCCTTCCCCAGGAGAGGCTGGG AACATTAATATGGTAATTTATGCTC[A/G]ACCAACCTGAAGTCTGCTGTGAGCT AGCCCAGAACCTGCTGTAGACTCAC[C/T]GTGTGAAATGTCTGCTCCAAGCCCT TAGAAAACACCTGGCTTAATTTTCC[A/G]GGCTGGGGCAGGATGGGTTAGCACG GCATCTTCAAGAGAACAAGATCGGA[C/G]ACCAAGAGACAAATCCCAATGG GCTGTGTTTAACCCTTGGCTCCCCA[A/G]CAATTGACTGGGGAATCCAAGCAGT ATATTTGTACAAATCTAGGTTTTTT[A/G]TACACTTCCATATGATTGCTTAAAT et al., 2001] and a2C (ADRA2C) located on 4p16 [Blackwood et al., 1996; Ewald et al., 2002] were identified in independent genome scans for bipolar disorder. As well as supported by the genome scan of recurrent, early-onset depression [Zubenko et al., 2003], the 10q24–q26 region was also identified in independent genome scan studies of bipolar disorder [Cichon et al., 2001; Kelsoe et al., 2001; Ewald et al., 2002]. Given substantial evidence of the role of the adrenergic system in depression, several of the receptors genes have been tested in linkage and association studies of mood disorders. Early studies used a linkage approach and large, multigenerational families. No evidence for linkage was found for the ADRA2C [Wang et al., 1992; Byerley et al., 1994] or ADRA2A receptor genes [Wang et al., 1992]. However, linkage studies, particularly those using a limited number of multigenerational families, have low power to detect genes of minor effect and therefore their results cannot be conclusive. Using an association study, three of the adrenergic receptors, ADRA1C (ADRA1A), ADRA2A, and ADRA2C, were tested for association to bipolar I disorder in a sample of 120 German families and no evidence for association was found [Abou Jamra et al., 2004]. The ADRA2A gene was also investigated in a case-control (103 cases and 114 controls) study of mood disorder patients that found no evidence for an association [Ohara et al., 1998]. The ADRB1 gene was investigated as a candidate for mood disorders using a functional polymorphism, Gly389Arg, in 259 patients and 206 controls; however, no evidence for association was observed [Zill et al., 2003]. These association studies used only one polymorphism in each of the genes and therefore these studies would have limited power to detect an association. Based on strong biological evidence implicating the adrenergic system in depression, as well as the location of six of the receptors as positional candidates based on genome scans, we TABLE II. Markers Genotyped by Taqman Assays-by-Design Gene Marker DNA variant ADRA1B rs3729604 G!A ADRA2A rs553668 A!G rs1800544 G!C rs2429511 G!A rs18011252 A!G rs2183378 G!A rs1042718 C!A rs1042717 G!A rs1042719 G!C rs4994 T!C rs4998 C!G ADRB1 ADRB2 ADRB3 Primers F: CTTGTCCACCGTCATCTCCAT R: CCGCACTCCTTGTCATCGTT F: CAGGGCTGCCCTTAGCATT R: TAATTCCCCTTCCATTCCCAACT F: GTGCCCGTTGCGTTCTG R: TGGGAGTTGGCCATGCA F: CCAATCCCTTGGCTATTTTGAGACT R: GATTAGAGGCATGAGCCACTGT F: CCGCCCGCCTCGTT R: CGCTGTCCACTGCTGAGA F: TTGTTAAGCACATTTTCCTAGCTTATGTT R: TGCAGAATTTCTGTCTGGCAATTT F: CCTTCTTGCCCATTCAGATGCA R: GCATAGCAGTTGATGGCTTCCT F: ACTGGCCTGTGCTGATCTG R: TGGGCGGCCCCAAAG F: GCCTTCCAGGAGCTTCTGT R: ACTCTGCTCCCCTGTGTTG F: GTTGGTCATGGTCTGGAGTCT R: GCAACCTGCTGGTCATCGT F: TGAACTTCACTCCTCCCTCAGT R: GGGTGCCCCTACCAAAGC Probes VIC: CTCTCCTTGGgTGGAAG FAM: TCTCCTTGGaTGGAAG VIC: CTCTCTCTCTTTTTaAAGAAA FAM: TCTCTCTTTTTgAAGAAA VIC: CCGTCGGCCCgGAG FAM: CCGTCGGCCCcGAG VIC: CTTCTGGACCCaCAAGG FAM: TCTGGACCCgCAAGG VIC: CCAGCGAAaGCCCCGA FAM: CAGCGAAgGCCCCGA VIC: CAGAGGAACgGGCACA FAM: CAGAGGAACaGGCACA VIC: CTGGTACcGGGCCAC FAM: CTGGTACaGGGCCAC VIC: CATGGGCCTgGCAGT FAM: TCATGGGCCTaGCAGT VIC: CCTATGGgAATGGCT FAM: CTATGGcAATGGCT VIC: CATCGCCtGGACTC FAM: ATCGCCcGGACTC VIC: CACGGCAcCTGGACA FAM: ACGGCAgCTGGACA Association of Adrenergic Receptor Genes and Childhood-Onset Mood Disorders 229 TABLE III. Categorical Analysis of the Markers Genotyped for the Adrenergic Genes Gene Location 50 rs1383914 Intron rs544215 Intron rs527340 Intron rs2055197 Exon 30 rs1048101 Arg347Cys rs3739215 50 rs1019920 Exon rs3729604 Intron rs2030373 Intron rs10515805 Intron rs11743363 Intron rs13171967 Intron rs11953285 30 rs11739589 ADRA1A ADRA1B ADRA1D 3 0 rs946188 Intron rs835878 50 rs1886844 5 0 rs1800544 3 UTR rs553668 50 rs1168965 3 ADRB1 0 30 UTR 5 0 Exon ADRB2 ADRB3 rs638019 50 0 ADRA2C rs734290 Intron ADRA2A ADRA2B Marker rs13019188 rs11269124 rs2429511 30 rs1801252 Gly49Ser rs2183378 30 rs11957757 Exon rs1042719 Exon rs1042718 Exon rs1042717 Exon rs4994 Arg64Trp rs4998 30 UTR Allele Allele frequency T C G A C T T A T C G C A C G A C A G A G C G T A C G T C T A G A G G T G A C G G A G A G C Ins Del G A A G G A G A G C C A G A T C C G 0.561 0.439 0.485 0.515 0.690 0.310 0.552 0.448 0.541 0.459 0.545 0.455 0.663 0.337 0.841 0.159 0.763 0.237 0.815 0.185 0.827 0.173 0.766 0.234 0.872 0.128 0.752 0.248 0.742 0.258 0.751 0.249 0.575 0.425 0.689 0.311 0.747 0.253 0.742 0.258 0.827 0.173 0.631 0.369 0.670 0.330 0.865 0.135 0.525 0.475 0.858 0.142 0.862 0.138 0.604 0.396 0.657 0.343 0.811 0.189 0.772 0.228 0.919 0.081 0.923 0.077 Transmission 94 83 86 86 75 82 88 89 88 87 88 87 75 94 40 58 56 76 49 67 45 63 61 71 40 39 59 75 71 57 57 69 75 76 71 65 74 55 68 58 49 45 71 70 78 72 40 38 87 95 44 39 44 42 74 98 68 80 49 66 57 64 30 19 28 19 Non-transmission 83 94 86 86 82 75 89 88 87 88 87 88 94 75 58 40 76 56 67 49 63 45 71 61 39 40 75 59 57 75 69 57 76 75 65 71 55 74 58 68 45 49 70 71 72 78 38 40 95 87 39 44 42 44 98 74 80 68 66 49 64 57 19 30 19 28 w2 P-value 0.684 0.408 0.000 1.000 0.312 0.577 0.006 0.938 0.006 0.938 0.006 0.938 2.136 0.144 3.306 0.069 3.030 0.082 2.793 0.095 3.000 0.083 0.758 0.384 0.013 0.909 1.910 0.167 1.531 0.216 1.143 0.285 0.007 0.933 0.265 0.607 2.798 0.094 0.794 0.373 0.170 0.680 0.240 0.624 0.007 0.933 0.051 0.821 0.352 0.553 0.301 0.583 0.047 0.828 3.349 0.067 0.973 0.324 2.513 0.113 0.405 0.525 2.469 0.116 1.723 0.189 230 Burcescu et al. investigated these genes as risk factors for childhood-onset mood disorder (COMD). We used a sample of 189 nuclear families (189 probands and 34 affected siblings) identified through a proband with onset of depression by 14 years of age. As well as the six genes specifically identified in the genome scans of mood disorders listed above, we investigated the genes for the adrenergic receptors a1D (ADRA1D) on 20p13, a2B (ADRA2B) on 2p13–q13, and b2 (ADRB2) on 5q32. METHODS Subjects This genetics study was conducted as part of a multidisciplinary Program Project of research on multiple risk factors in COMD. The families have been previously described in detail [Adams et al., 2005]. Our sample consisted of 189 families with 223 affected children recruited from 21 mental health facilities across Hungary. The probands met DSM-IV criteria for either unipolar major depression or bipolar disorder, with the onset of the first episode by age 14. The Psychiatric Interview Schedule for Children and AdolescentsDiagnostic Version (ISCA-D), an extension and modification of the ISCA [Sherrill and Kovacs, 2000] was used for the diagnoses. Proband and parent informants were separately interviewed on two occasions, approximately 1 month apart, by trained clinicians. Consensus diagnosis of two independent child psychiatrists was used as the final diagnosis. Genotyping DNA was extracted from blood using a high salt method [Miller et al., 1988]. To genotype the Cys492Arg polymorphism of ADRA1A, (rs1048101) we amplified a 501 bp fragment using a reaction mixture of 100 ng of genomic DNA, the primers ADRA1A-Pst1 (50 atg ctc cag cca aga gtt ca 30 ) and ADRA1APst2 (50 tcc aag aag agc tgg cct tc 30 ), 1.5 mM MgCl2, and 1 unit of Taq polymerase [Shibata et al., 1996]. The polymerase chain reaction (PCR) reaction consisted of an initial denaturation stage of 4 min at 948C followed by 35 cycles of denaturing at 948C for 30 sec, annealing at 588C for 30 sec, and an extension of 728C for 30 sec. A final extension step of 728C was added for 10 min after the last cycle. To detect the polymorphism, 10 ml of the PCR product was digested with 8 U of PstI restriction enzyme (New England Biolabs, Beverly MA) for approximately 2 hr at 378C. The alleles were separated on a 2% agarose gel. Allele 1 (C, Cys492) was not cut with the restriction enzyme and was seen as a 501 bp band. Allele 2 (T, Arg492) was cut into two bands of 442 and 58 bp. The ADRA2C insertion/deletion polymorphism was genotyped using the following primers: ADRA2C-indelF: cgg tct tca acc agg att tc and ADRA2C-indelR: tct ctc tgc caa gct cct g. The alleles were separated on a 3% agarose gel. The insertion and deletion alleles yield fragments of 249 bp and 228 bp, respectively. The remaining polymorphisms were genotyped using the TaqMan1 50 nuclease assay with primers and probes available commercially (Table I, Applied Biosystems, Foster, CA, Assay by Demand) or designed specifically for this study (Table II, Applied Biosystems, Assay by Design). The reference sequence for Assays by Demand or the primers and probes for Assay by Design are listed in the Tables I and II, respectively. Five microliters of PCR reactions contained 30 ng of genomic DNA, 5 mmol of TaqMan1 Universal PCR Master Mix (Applied Biosystems), and 0.125 ml of allelic discrimination mix which contained 18 mM of each primer and 4 mM of each probe. The thermal cycling conditions for the Assays-on-Demand1 obtained from Applied Biosystems were 958C for 10 min, then 50 cycles of 928C for 15 sec and 1 min at the annealing temperature of 608C. Two negative controls were included within each 96-well plate. Plates were read on the ABI 7900HT Sequence Detection System using the allelic discrimination end-point analysis mode of the software package version 2.0 (Applied Biosystems). Statistical Analysis The extended TDT (ETDT) program [Sham and Curtis, 1995] was used to test for biased transmission of alleles for single markers and the transmission of haplotypes were analyzed using the TRANSMIT program [Clayton, 1999]. The robust estimator option was used for the TRANSMIT analysis, and only haplotypes with frequencies greater than 0.10 were used. The degree of linkage disequilibrium (LD) between marker alleles in this study was evaluated using Haploview v3.11 (http://www.broad.mit.edu/mpg/haploview) [Barrett et al., 2005]. RESULTS We genotyped in total 24 markers across the adrenergic receptor genes. We targeted polymorphisms that had been confirmed with known allele frequencies at the time we began the study. The adrenergic receptors are encoded by relatively small genes with either one or two exons. We chose markers that covered the coding region as well as the 50 and 30 regions wherever possible. We designed assays to test a number of unconfirmed polymorphisms to improve our coverage of the genes when necessary. Of these unconfirmed polymorphisms tested, two were not polymorphic in our sample. We examined data from the HapMap project (http://www.hapmap.org/) to determine the coverage of the genes by the markers we selected. Several of the genes, ADRA2A, ADRA2B, ADRA2C, ADRB1, and ADRB2, are contained within a single LD block. For the remaining genes, we genotyped at least one marker in TABLE IV. Haplotype Analysis of the ADRA1B Gene Haplotype Haplotype frequency Observed Expected Var (O–E) Chisq (1 df) ADRA1B-rs1019920, rs3729604, rs2030373 AGC 0.648 287.94 288.12 52.948 CGC 0.114 48.043 49.222 21.969 CAA 0.143 66.984 61.647 25.799 Global chisquared test, on 6 degrees of freedom ¼ 5.854, P ¼ 0.440 Chi-squared test on 3 degrees of freedom ¼ 1.846, P ¼ 0.605 ADRA1B-rs11743363, rs13171967, rs11953285 CGA 0.668 288.4 288.74 45.388 GTA 0.137 58.565 57.197 24.571 Global chisquared test, on 7 degrees of freedom ¼ 6.026, P ¼ 0.537 Chi-squared test on 2 degrees of freedom ¼ 0.093, P ¼ 0.955 P-value 0.001 0.063 1.104 0.980 0.802 0.401 0.003 0.076 0.959 0.480 Association of Adrenergic Receptor Genes and Childhood-Onset Mood Disorders 231 TABLE V. Haplotype Analysis of the ADRB2 Gene Haplotype Haplotype frequency Observed Var (O–E) Chisq (1 df) P-value 39.901 42.895 17.688 6.082 1.305 0.706 0.014 0.253 0.401 Expected ADRB2-rs11957757, rs1042719, rs1042718, rs1042717 GGCG 0.351 125.24 140.82 AGCG 0.304 129.76 122.27 GCCG 0.118 49.835 46.3 Global chisquared test, on 9 degrees of freedom ¼ 16.344, P ¼ 0.060. Chisquared test on 3 degrees of freedom ¼ 6.278, P ¼ 0.099. TABLE VI. Linkage Disequilibrium Between Markers ADRA1A rs1383914 rs1383914 rs544215 rs527340 rs2055197 rs1048101 rs3739215 0.07 0.00 0.01 0.00 0.01 rs544215 0.29 0.48 0.00 0.00 0.00 rs527340 0.09 0.98 0.00 0.00 0.00 rs2055197 0.09 0.05 0.01 rs1048101 0.08 0.03 0.02 0.91 0.79 0.77 rs3739215 0.10 0.04 0.02 0.89 1.00 0.98 ADRA1B rs1019920 rs1019920 rs3729604 rs2030373 rs10515805 rs11743363 rs13171967 rs11953285 rs11739589 0.35 0.50 0.05 0.04 0.07 0.01 0.01 rs3729604 0.96 0.54 0.03 0.03 0.04 0.01 0.00 rs2030373 0.90 0.93 0.01 0.01 0.01 0.01 0.00 rs10515805 0.34 0.82 0.12 rs11743363 0.31 0.94 0.13 1.00 0.92 0.55 0.00 0.00 ADRA1D rs1886844 rs1886844 rs946188 rs835878 rs734290 0.04 0.03 0.00 rs946188 0.63 0.24 0.09 rs835878 0.28 0.98 rs734290 0.16 0.79 0.43 0.11 ADRA2A rs638019 rs638019 rs1800544 rs553668 0.93 0.91 rs1800544 0.97 rs553668 0.97 0.98 0.92 ADRA2B rs1168965 rs1168965 rs13019188 rs13019188 0.99 0.83 ADRB1 rs2429511 rs2429511 rs1801252 rs2183378 0.15 0.14 rs1801252 1.00 rs2183378 0.98 0.99 0.94 ADRB2 rs11957757 rs11957757 rs1042719 rs1042718 rs1042717 0.03 0.00 0.00 rs1042719 0.27 0.37 0.46 rs1042718 0.08 0.91 rs1042717 0.01 0.92 0.99 0.81 ADRB3 rs4994 rs4994 rs4998 rs4998 0.98 0.91 D0 values are shown in the upper half of the table, r2 values in the lower half of the table. 0.61 0.00 0.00 rs13171967 0.33 0.84 0.12 0.86 0.95 0.00 0.00 rs11953285 0.38 0.54 0.45 0.05 0.05 0.05 0.05 rs11739589 0.09 0.08 0.01 0.01 0.02 0.00 0.33 232 Burcescu et al. each LD block and additional markers flanking the blocks. For the ADRA2C gene, only one polymorphism was genotyped, an insertion/deletion located in the 30 untranslated region. Currently there is only one other confirmed polymorphism for this gene with a minor allele frequency of 0.09, and thus too low to be sufficiently informative for an association study. ADRA2C is very small (2,826 bp) and the coding region is contained in a single exon. For three genes, ADRA1A, ADRB1, and ADRB3, we genotyped nonsynonymous SNPs that had minor allele frequencies sufficiently polymorphic to be informative in the analyses. For the ADRA1A gene, we genotyped a polymorphism (rs1048101) that results in a nonsynonymous amino acid change (cysteine to arginine) at codon 492 [Shibata et al., 1996]. This amino acid variation is unlikely to cause a change in phenotype, since no differences in receptor function were observed between the two protein variants [Shibata et al., 1996]. The results of the TDT analysis for the markers used in this study are shown in Table III. We did not observe significant evidence for association for any of the markers. We did observe trends for biased transmission (w2 > 3) for three of the markers located in the ADRA1B gene and one marker in the ADRB2 gene, however, given the number of tests involved in this study, these are likely to be the result of chance. To investigate these results further, an analysis of the haplotypes was performed. For the ADRA1B gene, the haplotype analysis was performed for the markers that were in strong linkage disequilibrium, the first block contained markers rs1019920, rs3729604, and rs2030373. The second block contained rs11743363, rs13171967, and rs11953285 (Table IV). The analysis did not provide significant evidence for association and therefore did not provide additional support for the trends identified for the three markers with the single point analysis. For the ADRB2 gene, the GGCG haplotype (see Table V) for the four markers that were genotyped showed significant evidence of association (w2 ¼ 6.082, 1d.f., P ¼ 0.014), however, the global analysis was not significant (w2 ¼ 16.344, 9 d.f. P ¼ 0.06). Our analysis of haplotypes for the other six genes did not provide significant evidence for association (data not shown). The linkage disequilibrium between the markers was estimated using Haploview, these results are shown in Table VI. DISCUSSION Despite strong biological evidence supporting the adrenergic system in depression, and the evidence from genome scans supporting the location of six of the genes as contributing to mood disorders, we found no significant evidence for association with 24 markers for the 9 adrenergic receptors in this sample of Hungarian families. This is the first association study to investigate five of the adrenergic receptor genes and mood disorders and this sample is the largest to be used in an association study for the ADRA1A, ADRA2A, ADRA2C, and ADRB1 genes that have been previously tested for association. Therefore, this is the most comprehensive test of the adrenergic receptors and mood disorders to date. Power is always an issue in association studies. The previous results from the genome scans for bipolar disorder and earlyonset recurrent depression indicate that if there are susceptibility genes in these regions, they must have a major effect to be identified using a linkage approach. An association study should have much more power than a linkage approach if the marker that is chosen is in LD with the risk allele [Risch and Merikangas, 1996]. Of course there is no way to determine this until the risk alleles are identified. However, our use of multiple markers across eight of the nine genes, and the use of haplotypes, maximized the possibility that we would detect LD with any potential risk alleles and subsequently increased the power of our search. Power is also affected by the mode of inheritance and the mechanism by which genes contribute to the susceptibility. Risk genes with more complex modes of inheritance that involve gene by gene or gene–environment interactions would likely have low power to be detected in this sample, and cannot be ruled out at this time. The trend for association for some of these genes may reflect true association with a complex mode of inheritance and we will pursue these findings as the sample size increases. Genetic alteration in the adrenergic receptors could contribute to depression by increasing presynaptic a2-autoreceptor activity resulting in decreased release of norepinephrine or reduced sensitivity of the postsynaptic receptors. Our failure to find significant association with the receptor genes suggest that genetic changes in the receptors themselves may not be the contributing factor. We cannot rule out other mechanisms influencing the adrenergic system such as a decrease of release, or production of norephinephrine, increased catabolism or intracellular response such as changes in second messengers. Genes contributing to these mechanisms are also likely candidates and the focus of our continued research. ACKNOWLEDGMENTS Members of the International Consortium for ChildhoodOnset Mood Disorders: István Benák, Viola Kothencné Osváth, Edit Dombovári, Emı́lia Kaczvinszky, and László Mayer; Department of Child and Adolescent Psychiatry, Szeged University Medical Faculty, Szeged. Júlia Gádoros, Ildikó Baji, Zsuzsa Tamás, Márta Besnyö; Vadaskert Hospital, Budapest. Judit Székely; Department of Pediatrics, Semmelweis University I., Budapest. Heads of the Units and Departments where the patients were collected and collaborators: Rózsa Hasuly, M.D.; Outpatient Unit of Child Psychiatry, Szent Rókus Hospital, Budapest. Ilona Riegler, M.D., Márta Fohn, M.D.; Outpatient Unit of Child Psychiatry, Heim Pál Hospital, Budapest. Katalin Benkö, M.D.; Outpatient Unit of Child Psychiatry, Szeged. Mária Mojzes, M.D.; Outpatient Unit of Child Psychiatry, Baja. Róza Oláh, M.D.; Department of Child and Adolescent Psychiatry, Kenézy Gyula Hospital, Debrecen. Mária Károlyfalvi, M.D., Gyöngyi Farkas, M.D., Zsuzsa Bánk, M.D.; Outpatient Unit of Child Psychiatry, Debrecen. Ferenc Dicsö, M.D., Dénes Kövendy, M.D.; Outpatient Unit of Child Psychiatry, Jósa András Hospital, Nyı́regyháza. Mária Gyurcsó, M.D.; Outpatient Unit of Child Psychiatry, Petz Aladár Hospital, Györ. Zsuzsanna Fekete, M.D., Mariann Vados, M.D.; Outpatient Unit of Child Psychiatry, Szent György Hospital, Székesfehérvár. Zsuzsa Takács, M.D.; Outpatient Unit of Child Psychiatry, Szekszárd Hospital, Szekszárd. Eszter Gyenge, M.D., Mária Palaczky, M.D., Ágnes Horváth, M.D., Outpatient Department of Child Psychiatry, Pécs. Ilona Mógor, M.D., Péter Steiner, M.D.; Outpatient Unit of Child Psychiatry, Csolnoky Ferenc Hospital, Veszprém. Enikö Juhász; Outpatient Unit of Child Psychiatry, Szolnok Hospital, Szolnok. Mária Révhelyi, M.D., Department of Child and Adolescent Psychiatry, Petz Aladár Hospital, Györ. Éva Gyulai, M.D.; Baja Hospital Outpatient Unit of Child Psychiatry, Baja. Katalin Bense, M.D., Edina Farkas, M.D.; Outpatient Unit of Child Psychiatry, Kecskemét Hospital, Kecskemét. Sörfözö Zsuzsanna, M.D.; Outpatient Unit of Child Psychiatry, Kaposi Mór Hospital, Kaposvár. Magdolna Gácser; Department for Child and Adolescent Psychiatry, Kálmán Pándy Hospital, Gyula. Emöke Endreffy Ph.D.; Szeged University Pediatric Department. Interviewers: Judit Tömöri, Tünde Horváth M., Eszter Szamosi, Laura Német. Regional research office members: Edit Sitkei, Noémi Takács, Erika Kolonics. Central research office members: Szilvia Szabó Kelemenné, Zoltán Széll, Márta Dobó, and Éva Lehoczky. Association of Adrenergic Receptor Genes and Childhood-Onset Mood Disorders REFERENCES Abou Jamra R, Schumacher J, Golla A, Richter C, Otte AC, Schulze TG, Ohlraun S, Maier W, Rietschel M, Cichon S, Propping P, Nothen MM. 2004. Family-based association studies of alpha-adrenergic receptor genes in chromosomal regions with linkage to bipolar affective disorder. Am J Med Genet (Neuropsychiatr Genet) Part B 126B:79–81. Adams JH, Wigg KG, King N, Burcescu I, Vetro A, Kiss E, Baji I, George CJ, Kennedy JL, Kovacs M, Barr CL. 2005. Association study of neurotrophic tyrosine kinase receptor type 2 (NTRK2) and childhood-onset mood disorders. Am J Med Genet (Neuropsychiatr Genet) Part B 132B:90–95. Barrett JC, Fry B, Maller J, Daly MJ. 2005. Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics 21(2):263–265. Epub 2004 August 5. Berman RM, Narasimhan M, Miller HL, Anand A, Cappiello A, Oren DA, Heninger GR, Charney DS. 1999. Transient depressive relapse induced by catecholamine depletion: Potential phenotypic vulnerability marker? Arch Gen Psychiatry 56(5):395–403. Blackwood DH, He L, Morris SW, McLean A, Whitton C, Thomson M, Walker MT, Woodburn K, Sharp CM, Wright AF, Shibasaki Y, St. Clair DM, Porteous DJ, Muir WJ. 1996. A locus for bipolar affective disorder on chromosome 4p. Nat Genet 12(4):427–430. Brunello N, Blier P, Judd LL, Mendlewicz J, Nelson CJ, Souery D, Zohar J, Racagni G. 2003. Noradrenaline in mood and anxiety disorders: Basic and clinical studies. Int Clin Psychopharmacol 18(4):191–202. Byerley W, Hoff M, Holik J, Coon H. 1994. A linkage study with D5 dopamine and alpha 2C-adrenergic receptor genes in six multiplex bipolar pedigrees. Psychiatr Genet 4(3):121–124. Charney DS. 1998. Monoamine dysfunction and the pathophysiology and treatment of depression. J Clin Psychiatry 59(suppl 14):11–14. Cichon S, Schumacher J, Muller DJ, Hurter M, Windemuth C, Strauch K, Hemmer S, Schulze TG, Schmidt-Wolf G, Albus M, BorrmannHassenbach M, Franzek E, Lanczik M, Fritze J, Kreiner R, Reuner U, Weigelt B, Minges J, Lichtermann D, Lerer B, Kanyas K, Baur MP, Wienker TF, Maier W, Rietschel M, Propping P, Nothen MM. 2001. A genome screen for genes predisposing to bipolar affective disorder detects a new susceptibility locus on 8q. Hum Mol Genet 10(25):2933–2944. Clayton D. 1999. A generalization of the transmission/disequilibrium test for uncertain-haplotype transmission. Am J Hum Genet 65(4):1170–1177. Delgado PL, Moreno FA. 2000. Role of norepinephrine in depression. J Clin Psychiatry 61(suppl 1):5–12. Ewald H, Flint T, Kruse TA, Mors O. 2002. A genome-wide scan shows significant linkage between bipolar disorder and chromosome 12q24.3 and suggestive linkage to chromosomes 1p22-21, 4p16, 6q14-22, 10q26 and 16p13.3. Mol Psychiatry 7(7):734–744. Kelsoe JR, Spence MA, Loetscher E, Foguet M, Sadovnick AD, Remick RA, Flodman P, Khristich J, Mroczkowski-Parker Z, Brown JL, Masser D, 233 Ungerleider S, Rapaport MH, Wishart WL, Luebbert H. 2001. A genome survey indicates a possible susceptibility locus for bipolar disorder on chromosome 22. Proc Natl Acad Sci USA 98(2):585–590. Epub 2001 January 9. Leonard BE. 1997. Noradrenaline in basic models of depression. Eur Neuropsychopharmacol 7(suppl 1):S11–S16; discussion S71-77. Miller SA, Dykes DD, Polesky HF. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16(3):1215. Ohara K, Nagai M, Tani K, Tsukamoto T, Suzuki Y. 1998. Polymorphism in the promoter region of the alpha 2A adrenergic receptor gene and mood disorders. Neuroreport 9(7):1291–1294. Ressler KJ, Nemeroff CB. 2000. Role of serotonergic and noradrenergic systems in the pathophysiology of depression and anxiety disorders. Depress Anxiety 12(suppl 1):2–19. Risch N, Merikangas K. 1996. The future of genetic studies of complex human diseases. Science 273(5281):1516–1517. Schatzberg AF. 1998. Noradrenergic versus serotonergic antidepressants: Predictors of treatment response. J Clin Psychiatry 59(suppl 14):15– 18. Sham PC, Curtis D. 1995. An extended transmission/disequilibrium test (TDT) for multi-allele marker loci. Ann Hum Genet 59(Pt 3):323– 336. Sherrill JT, Kovacs M. 2000. Interview schedule for children and adolescents (ISCA). J Am Acad Child Adolesc Psychiatry 39(1):67–75. Shibata K, Hirasawa A, Moriyama N, Kawabe K, Ogawa S, Tsujimoto G. 1996. Alpha 1a-adrenoceptor polymorphism: Pharmacological characterization and association with benign prostatic hypertrophy. Br J Pharmacol 118(6):1403–1408. Wang Z, Crowe RR, Tanna VL, Winokur G. 1992. Alpha 2 adrenergic receptor subtypes in depression: A candidate gene study. J Affect Disord 25(3):191–196. Wong EH, Sonders MS, Amara SG, Tinholt PM, Piercey MF, Hoffmann WP, Hyslop DK, Franklin S, Porsolt RD, Bonsignori A, Carfagna N, McArthur RA. 2000. Reboxetine: A pharmacologically potent, selective, and specific norepinephrine reuptake inhibitor. Biol Psychiatry 47(9):818–829. Zill P, Baghai TC, Engel R, Zwanzger P, Schule C, Minov C, Behrens S, Bottlender R, Jager M, Rupprecht R, Moller HJ, Ackenheil M, Bondy B. 2003. Beta-1-adrenergic receptor gene in major depression: Influence on antidepressant treatment response. Am J Med Genet (Neuropsychiatr Genet) Part B 120B:85–89. Zubenko GS, Maher B, Hughes HB, IIId, Zubenko WN, Stiffler JS, Kaplan BB, Marazita ML. 2003. Genome-wide linkage survey for genetic loci that influence the development of depressive disorders in families with recurrent, early-onset, major depression. Am J Med Genet (Neuropsychiatr Genet) Part B 123B:1–18.