Association study of the brain-derived neurotropic factor (BDNF) gene in attention deficit hyperactivity disorder.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:976 –981 (2007) Association Study of the Brain-Derived Neurotropic Factor (BDNF) Gene in Attention Deficit Hyperactivity Disorder Jonghun Lee,1,2 Nancy Laurin,1 Jennifer Crosbie,3 Abel Ickowicz,3 Tejaswee Pathare,3 Molly Malone,3 Rosemary Tannock,3 James L. Kennedy,4 Russell Schachar,3 and Cathy L. Barr1,3* 1 Cell and Molecular Biology Division, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada Department of Psychiatry, Catholic University of Daegu, Daegu, South Korea 3 Brain and Behaviour Programme, The Hospital for Sick Children, Toronto, Ontario, Canada 4 Department of Psychiatry, Neurogenetics Section, University of Toronto, Toronto, Ontario, Canada 2 Attention deficit hyperactivity disorder (ADHD) is a prevalent neurodevelopmental childhood psychiatric disorder. Brain-derived neurotropic factor (BDNF) has been suggested to play a role in the pathogenesis of ADHD and two family-based association studies demonstrated an association of BDNF polymorphisms with ADHD. The aim of the current study was to investigate the BDNF gene for association with ADHD in a large sample of families from Toronto. The transmission of three polymorphisms of the BDNF gene (rs6265, rs11030104, and rs2049046) was examined in 266 nuclear families ascertained through a proband with ADHD (315 affected children) using the transmission/disequilibrium test (TDT). In addition, we conducted quantitative analysis to assess the relationship between these marker alleles and the symptom dimensions of ADHD (inattention and hyperactivity/impulsivity) and cognitive measures of working memory. None of the individual marker alleles showed significant evidence of association with ADHD, dimensional symptom scores, or working memory ability in our sample of ADHD families. There was no significant evidence for biased transmission of individual haplotypes with frequency >10% (global x2 for these three haplotypes: x2 ¼ 6.349, df ¼ 3, P ¼ 0.096). One uncommon haplotype (A-G-G; frequency 2.2%) showed a significant association with ADHD in the categorical (x2 ¼ 5.293, df ¼ 1, P ¼ 0.021) and quantitative analyses (parents’ rated inattention: Z ¼ 2.504, P ¼ 0.012; and hyperactivity/impulsivity: Z ¼ 2.651, P ¼ 0.008). These results should be interpreted cautiously, however, because of the low haplotype frequency. In light of the evidence for an involvement of BDNF in ADHD, further analysis of the BDNF gene in ADHD is warranted. ß 2007 Wiley-Liss, Inc. Grant sponsor: Hospital for Sick Children Psychiatric Endowment Fund; Grant sponsor: Canadian Institutes of Health Research; Grant numbers: MT14336, MOP14336. *Correspondence to: Cathy L. Barr, The Toronto Western Hospital, 399 Bathurst St., Room MP14-302, Toronto, ON, Canada. M5T 2S8. E-mail: CBarr@uhnres.utoronto.ca Received 11 May 2006; Accepted 16 August 2006 DOI 10.1002/ajmg.b.30437 ß 2007 Wiley-Liss, Inc. KEY WORDS: attention deficit hyperactivity disorder (ADHD); genetics; brainderived neurotropic factor; BDNF; transmission/disequilibrium test Please cite this article as follows: Lee J, Laurin N, Crosbie J, Ickowicz A, Pathare T, Malone M, Tannock R, Kennedy JL, Schachar R, Barr CL. 2007. Association Study of the Brain-Derived Neurotropic Factor (BDNF) Gene in Attention Deficit Hyperactivity Disorder. Am J Med Genet Part B 144B:976–981. INTRODUCTION Attention deficit hyperactivity disorder (ADHD) is a common childhood-onset psychiatric condition characterized by marked and pervasive inattention, hyperactivity and impulsiveness. Family, twin, and adoption studies provided compelling evidence that genetic influences play an important role in mediating susceptibility to ADHD [Faraone et al., 2005]. ADHD is a neurodevelopmental disorder. Neuroanatomical studies have suggested that the regions subserving complex motor and cognitive process are implicated in the pathophysiology of ADHD [Casey et al., 1997; Faraone and Biederman, 1998; Lou et al., 1998; Rubia et al., 2000]; therefore genes related to neuronal development and differentiation may represent an important set of candidates for involvement in the pathogenesis of ADHD. Brain-derived neurotropic factor (BDNF) is recognized as a critical regulator in the survival and differentiation of central neurons during childhood development and in adulthood, and is also implicated in the synaptic plasticity of brain functions [Ernfors et al., 1994; Linnarsson et al., 2000; Schinder and Poo, 2000; Huang and Reichardt, 2001]. BDNF was suggested to play an important role in the pathophysiology of several psychiatric diseases, including mood disorders [Duman, 2002; Neves-Pereira et al., 2002; Sklar et al., 2002; Geller et al., 2004; Hashimoto et al., 2004; Strauss et al., 2005], symptoms of which are often observed in individuals with ADHD. Deficits in executive functions are a main feature of the cognitive difficulties in children with ADHD. Executive functions are defined as neurocognitive processes that maintain an appropriate problem-solving set to attain a later goal and include processes such as response inhibition, vigilance, working memory, and planning [Willcutt et al., 2005]. Several lines of evidence suggest that the gene coding for BDNF plays a role in the pathogenesis of ADHD [Tsai, 2003]. Mice in which the BDNF gene were inactivated postnatally demonstrated significantly more locomotor activity when stressed, for example, after transfer to a new cage than normal mice and, in contrast to normal mice, BDNF mutant mice are still agitated after a period of habituation [Rios et al., 2001]. Animal studies indicate that BDNF is critical for 0.655 0.200 0.655 0.200 0.819 0.053 37 39 42 38 38 42 0.777 0.080 0.490 0.484 39 37 38 42 42 38 0.413 0.671 33 40 28 23 24 26 0.627 0.237 0.536 0.464 40 33 23 28 26 24 0.534 rs6265 rs11030104 w2 (1 df) T P-value w2 (1 df) NT T P-value w2 (1 df) 0.388 100 109 80 71 73 79 109 100 71 80 79 73 0.464 0.536 0.235 0.765 0.217 0.783 T A G A A G Diagnostic Assessment and Subjects Subject assessment and diagnostic criteria for inclusion in this study are described in detail elsewhere [Barr et al., 1999; Quist et al., 2000; Laurin et al., 2005]. Briefly, probands and affected siblings between 7 and 16 years old were referred to the Child Development and Neuropsychiatry Clinics at the Hospital for Sick Children, Toronto. Diagnosis was based on information from semi-structured interviews of parents and teachers: Parent Interview for Child Symptoms (PICS-IV) [Ickowicz et al., 2006] and Teacher Telephone Interview (TTI-IV) [Tannock et al., 2002]. Children who met DSM-IV criteria for ADHD were included in the study. All children were free of medication for 24 hr before assessment. This protocol was approved by the Hospital for Sick Children’s Research Ethics Board and informed written consent or assent was obtained for rs2049046 MATERIALS AND METHODS NT working memory [Kiprianova et al., 1999; Mizuno et al., 2000]. Several drugs acting on the catecholamine-mediated regulation of executive control functions, including amphetamine, tricyclic antidepressants, and serotonin reuptake inhibitors, lead to elevation of central BDNF mRNA levels in animal studies [Nibuya et al., 1995; Meredith et al., 2002]. Three recent studies have assessed the role of the BDNF gene in ADHD. Kent et al.  found evidence of preferential transmission of the valine allele of the Val66Met polymorphism (rs6265) in 341 ADHD families from the UK and Ireland while Xu et al.  reported evidence of association with the C270T polymorphism, but not with the Val66Met variant, in ADHD samples from the UK and Taiwan. In contrast, Friedel et al.  did not find an association between ADHD and the same BDNF polymorphisms (Val66Met, C270T) in a small sample of 88 ADHD cases. The gene encoding BDNF is located at chromosome 11p14.1 [Hanson et al., 1992] and codes for a precursor peptide (proBDNF), which is proteolytically cleaved to form the mature protein [Mowla et al., 2001]. The gene displays an important structural complexity with seven alternatively used noncoding exons and one coding exon. The multiple promoters, in addition to alternative splicing and several polyadenylation sites, result in various transcripts with different levels of expression in different tissues and different brain regions [Liu et al., 2005]. Antisense transcripts are also produced at the BDNF locus and could potentially be part of the BDNF gene regulation mechanism [Liu et al., 2005]. The aim of the current research was to test for an association between BDNF markers and ADHD in an independent sample of families having a child proband with ADHD. We examined three single nucleotide polymorphisms (SNPs), including the Val66Met (rs6265) polymorphism previously studied [Friedel et al., 2005; Kent et al., 2005; Xu et al., 2005]. We performed a transmission/disequilibrium test (TDT) analysis to test for evidence of biased transmission of alleles and haplotypes in 266 nuclear families selected through a proband with ADHD. We also conducted quantitative analysis to assess the relationship between these marker alleles or haplotypes and the symptom dimensions of ADHD (inattention and hyperactivity/impulsivity) and a measure of working memory. T 0.273 0.241 0.863 Frequency 1.000 0.988 0.987 Allele rs11030104 rs6265 rs6265 Polymorphism D2 Paternal transmissions D0 All transmissions rs2049046 rs2049046 rs11030104 Marker 2 TABLE II. Allele Frequencies and TDT Analysis of BDNF SNPs Marker 1 Maternal transmissions TABLE I. Linkage Disequilibrium Estimates (D and D ) Between BDNF Markers NT 2 P-value BDNF and ADHD 0 977 978 Lee et al. TABLE III. BDNF Haplotype Frequencies and TRANSMIT Analysis* Transmission Haplotypea TAG AAG AGA AGG TAA AAA Frequency Observedb Expectedc Var(O-E) 0.469 0.294 0.213 0.022 0.001 0.001 272.00 159.00 112.00 6.00 — — 261.39 163.64 111.94 11.48 — — 64.85 59.70 46.97 5.68 — — w2 (1 df) 1.735 0.360 7.54E-5 5.293 — — P-value 0.188 0.548 0.993 0.021 — 2 *Global w on 3 df for haplotypes with frequencies >10% ¼ 6.349, P ¼ 0.096. a Test statistic representing the observed number of transmissions. b Expected value of the test statistic under the null hypothesis of no association. c Haplotypes are composed, in order, of rs2049046, rs11030104, and rs6265. all participants. The study sample comprised 266 nuclear families recruited in the Toronto area, including 49 affected siblings of the probands, for a total of 315 affected children (80.8% boys). For the quantitative analysis, we used the symptom scores obtained for both ADHD dimensions (inattention and hyperactivity/impulsivity) from the PICS-IV and TTI-IV semistructured interviews. Verbal short-term and working TABLE IV. FBAT Analysis of BDNF Allele Transmission in Relation to ADHD Symptom Scores and Short-Term and Working Memory Measures Polymorphism Allele Informative families Parent rated inattention rs2049046 T A rs11030104 G A rs6265 A G Parent rated hyperactivity/impulsivity rs2049046 T A rs11030104 G A rs6265 A G Teacher rated inattention rs2049046 T A rs11030104 G A rs6265 A G Teacher rated hyperactivity/impulsivity rs2049046 T A rs11030104 G A rs6265 A G Digit span forward rs2049046 T A rs11030104 G A rs6265 A G Digit span backward rs2049046 T A rs11030104 G A rs6265 A G a b 132 101 101 130 101 101 128 98 98 128 98 98 100 76 72 100 76 72 Sa E(S)b Var(S) Z P-value 642.300 759.100 318.600 787.600 325.900 735.200 636.358 765.042 355.592 750.608 334.767 726.333 1250.314 0.168 0.168 1.258 1.258 0.307 0.307 0.867 631.800 732.200 335.800 765.800 346.200 719.600 613.850 750.150 341.650 759.950 322.800 743.000 1302.868 0.497 0.497 0.196 0.196 0.802 0.802 0.619 583.800 692.400 299.200 699.200 323.600 659.000 582.017 694.183 306.383 692.017 306.433 676.167 1128.936 0.053 0.053 0.261 0.261 0.619 0.619 0.958 471.500 579.000 263.000 558.000 274.500 518.000 480.375 570.125 257.375 563.625 251.250 541.250 1007.797 0.280 0.280 0.215 0.215 0.893 0.893 0.780 113.600 191.200 58.200 139.800 64.300 128.700 122.767 182.033 65.583 132.417 63.683 129.317 341.597 133.600 207.200 54.200 149.800 53.300 147.700 151.433 189.367 57.917 146.083 57.517 143.483 Test statistic. Expected value of the test statistic under the null hypothesis of no association. 864.053 833.628 894.522 852.225 755.206 770.209 682.984 677.375 226.255 209.845 440.264 256.571 241.711 0.496 0.496 0.491 0.491 0.043 0.043 0.850 0.850 0.232 0.232 0.271 0.271 0.208 0.759 0.845 0.423 0.794 0.536 0.830 0.372 0.620 0.624 0.966 0.395 0.817 0.786 BDNF and ADHD 979 TABLE V. FBAT Analysis of BDNF Haplotype Transmission in Relation to ADHD Symptom Scores and Short-Term and Working Memory Measures Haplotypesa Sb E(S)c Var(S) Z P-value 783.000 576.300 332.900 12.650 771.596 545.300 351.504 35.675 1247.449 1038.162 733.625 84.534 0.323 0.962 0.687 2.504 0.747 0.336 0.492 0.012 815.000 531.400 353.200 9.700 790.225 540.400 336.575 38.150 1351.116 1147.848 728.022 115.137 0.674 0.266 0.616 2.651 0.500 0.791 0.538 0.008 730.500 529.900 328.600 17.700 718.392 534.400 323.208 29.750 987.161 1059.743 634.222 70.133 0.385 0.138 0.214 1.439 0.700 0.890 0.830 0.150 569.750 400.250 277.500 10.750 570.187 398.750 260.812 26.375 957.788 843.396 587.084 69.078 0.014 0.052 0.689 1.880 0.989 0.959 0.491 0.060 145.800 129.600 55.600 — 151.867 114.600 59.033 — 340.832 282.217 167.472 — 0.329 0.893 0.265 — 0.742 0.372 0.791 — 161.800 188.600 63.600 — 175.533 171.100 64.367 — 440.382 397.267 193.339 — 0.654 0.878 0.055 — 0.513 0.380 0.956 — Informative families Parent rated inattention TAG 116 AAG 119 AGA 84 AGG 12 Parent rated hyperactivity/impulsivity TAG 114 AAG 117 AGA 84 AGG 12 Teacher rated inattention TAG 112 AAG 117 AGA 81 AGG 11 Teacher rated hyperactivity/impulsivity TAG 112 AAG 117 AGA 81 AGG 11 Digits span forward TAG 90 AAG 87 AGA 65 AGG 9 Digits span backward TAG 90 AAG 87 AGA 65 AGG 9 a Haplotypes are composed, in order, of rs2049046, rs11030104, and rs6265. Test statistic. c Expected value of the test statistic under the null hypothesis of no association. b memory were assessed using the digit span subtest of the WISC-III [Wechsler, 1991; Kaplan et al., 1999]. This test provides two subscale scores (digits span forward and digits span backward) which index the ability to store and manipulate auditory-verbal information, respectively, as well as an overall memory score. Isolation of DNA and Genotyping of Markers DNA was extracted from blood lymphocytes using a standard high salt extraction method [Miller et al., 1988]. A total of three SNPs (rs6265, chr. 11:27, 636, 492; rs11030104, chr. 11:27, 641, 093; and rs2049046, chr. 11:27, 680, 351) were genotyped using the ABI 7900-HT Sequence Detection Systems1 (Applied Biosystems, Foster City, CA) and TaqMan 50 nuclease assays for allelic discrimination [Livak et al., 1995]. The Val66Met (rs6265) polymorphism is located in the coding exon and the two other SNPs (rs11030104 and rs2049046) are located in intronic sequences. Primer and probe sequences are available on request. The PCR reactions (5 ml) contained 30 ng of genomic DNA, 10 mM of TaqMan Universal PCR Master Mix1 (Applied Biosystems) and 0.1 ml of allelic discrimination mix (Applied Biosystems). The thermal cycling conditions were 958C for 10 min and 40–50 cycles of 958C for 15 sec and the annealing temperature (58–598C) for 1 min. Statistical Analysis We examined the allelic transmission of the three individual markers using the TDT statistic calculated with the extended TDT (ETDT) program [Sham and Curtis, 1995]. Given previous findings of preferential paternal transmission bias with the Val66Met variant in ADHD [Kent et al., 2005], we assessed paternal and maternal transmission separately for all markers. Evidence for linkage disequilibrium (LD) between the three makers was examined by calculating the D0 and D2 coefficients using the Haploview program [Barrett et al., 2005]. For the analysis of the haplotypes, we employed the TRANSMIT program version 2.5, using the robust estimator of variance option [Clayton, 1999]. Quantitative trait TDT analyses, examining the transmission of individual alleles or haplotypes in relation to symptom scores of inattention and hyperactivity/ impulsivity and to short-term memory and working memory measures, were carried out using the FBAT program version 1.5.5, with the additive model of inheritance [Laird et al., 2000; Horvath et al., 2001]. We used population-based mean scores for the tests as an offset value to mean center the trait. The P values reported in this study were not corrected for multiple tests. We did not observe any significant departure from Hardy–Weinberg equilibrium for the genotype frequencies. RESULTS According to the HapMap data (build 21) and genetic studies on this gene, BDNF is enclosed in a unique haplotype block [Barrett et al., 2005; Liu et al., 2005; Strauss et al., 2005]. We thus selected three SNPs, as they tag the major haplotypes of this block, to perform an association study. Pairwise LD coefficients D0 and D2 to assess the strength of LD support the high correlation between the three markers in our sample of families (Table I). 980 Lee et al. As shown in Table II, when considering ADHD as a categorical trait, we did not observe significant evidence of biased transmission of these three individual markers, including the Val66Met allele implicated previously in ADHD. Moreover, we did not observe preferential transmission of the paternal Val66Met allele after separate analysis of paternal and maternal transmissions, as was suggested in the study by Kent et al. The transmission of the haplotypes consisting of the three polymorphisms was tested using the TRANSMIT program (Table III). No significant evidence for biased transmission of any of the three haplotypes with frequency >10% was found (global w2 for haplotypes with frequencies >10%: w2 ¼ 6.349, df ¼ 3, P ¼ 0.096). We then analyzed the markers using a quantitative approach for ADHD inattentive and hyperactive-impulsive symptom dimensions as well as for short-term and working memory measures. We found no significant evidence for a relationship between individual alleles or common haplotypes (with frequency >10%) and the two ADHD symptom dimensions or short-term and working memory scores (Tables III and V). Of note, one uncommon haplotype, A-G-G (frequency 2.2%), demonstrated a significant association with ADHD in categorical (undertransmission w2 ¼ 5.293, df ¼ 1, P ¼ 0.021) and quantitative analyses (parents’ rated inattention: Z ¼ 2.504, P ¼ 0.012; and hyperactivity/impulsivity: Z ¼ 2.651, P ¼ 0.008) (Tables III and V). However, the number of transmission for this haplotype was considered too low to provide a definitive result (i.e., quantitative analysis was based on 12 informative families). DISCUSSION Genetic variations in the BDNF gene have recently been found to be associated with ADHD [Kent et al., 2005; Xu et al., 2005]. To further evaluate the association of variation in BDNF with ADHD, we performed a genetic study using three SNPs in a relatively large family-based sample from Toronto. Using TDT and quantitative analysis, we found no evidence for association between ADHD and the individual alleles or the haplotypes with frequency >10%. Significant undertransmission of the haplotype A-G-G was found in our sample, however, this haplotype was present at a low frequency (2.2%). Our results regarding BDNF markers differ from previous family-based association studies. Those studies found evidence for preferential transmission of the valine (G) allele of the Val66Met polymorphism (rs6265) with a strong paternal effect in a sample from the UK and Ireland [Kent et al., 2005] or with the C270T polymorphism in a combined sample from the UK and Taiwan [Xu et al., 2005]. Although we did not directly assess the C270T polymorphism, this variant is embedded in the haplotype block tagged by the markers genotyped for this study. Another study did not find an association between the Val66Met (rs6265) or C270T polymorphisms and ADHD in 88 patients with ADHD [Friedel et al., 2005]. The reasons for the discrepancies between studies are unclear. The present study was carried out using approximately the same number of affected children as the Kent et al.’s  study (315 children vs. 341 children, respectively). The Xu et al.  study was composed of two samples of ADHD probands from the UK (180 children) and Taiwan (212 children). Therefore, our study would have been adequately powered to detect an association assuming that there are no betweensample differences in effect size or LD structure in the gene. The ethnic composition, however, was different among the study samples. In our sample, the majority of the families were of European Caucasian descent, while 10% of families were of ‘‘other’’ or ‘‘mixed’’ background, including Chinese, African, Indian, and Native-Americans. The sample of Kent et al.  was described as consisting of Caucasians with all individuals born in the UK or Ireland and Xu et al.’s  was a combination of European-Caucasian and Taiwanese. These ethnic differences could have had an effect on the results since variable allelic frequencies and genotypes of the BDNF gene polymorphism (Val66Met) have been reported in different ethnic groups [Shimizu et al., 2004]. The allele frequency of this polymorphism between our sample and the Kent et al.  sample was not strikingly different (80% Val, 20% Met vs. 87% Val, 13% Met, respectively), however, an important difference is present with regards to the same allele frequencies of the Taiwanese sample in the Xu et al.’s  report (47% Val, 53% Met). Another aspect potentially explaining discrepancies in the different association studies is a variable distribution of the assessed phenotype in each sample. A higher proportion of Kent et al.  sample (81%) and Xu et al.  (UK 100%, Taiwan 78%) were diagnosed with DSM-IV ADHD combined subtype compared to our sample (62%). Conversely, more children were diagnosed with predominantly inattentive subtype in our sample (24%) than in Kent et al.  sample (9%). It is possible that the clinical heterogeneity between samples may have masked a real association, but it is currently unclear how clinical heterogeneity may relate to genetic risk for this gene. BDNF has been implicated in learning and memory functions in animal studies [Kiprianova et al., 1999; Mizuno et al., 2000]. Children with ADHD often perform poorly on tasks of working memory, with deficits reported both in spatial and verbal components [Hervey et al., 2004; Martinussen et al., 2005]. Based on these findings, we also tested the association between BDNF and verbal working memory in this sample of families with an ADHD proband. These analyses did not yield significant results suggesting that BDNF is not playing a major role in the verbal WM performance of those children, as measured by the digit span subtest. It is difficult to make firm conclusions about the relationship between BDNF and ADHD given the contrasting results between studies. In the present study, we found little evidence that BDNF is a major genetic susceptibility factor contributing to ADHD. However, in light of the evidence for an involvement of BDNF in ADHD in other samples, and the evidence for biased transmission of an uncommon haplotype in our sample, further study of the BDNF gene in ADHD is warranted. ACKNOWLEDGMENTS This work was supported by Postdoctoral Fellowships from the Hospital for Sick Children Research Training Centre (N.L.) and the Canadian Institutes of Health Research (N.L.) and by grants from The Hospital for Sick Children Psychiatric Endowment Fund (C.L.B), and the Canadian Institutes of Health Research MT14336 and MOP14336 (C.L.B). REFERENCES Barr CL, Wigg K, Malone M, Schachar R, Tannock R, Roberts W, Kennedy JL. 1999. 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