Association Between the Brain-Derived Neurotrophic Factor (BDNF) Gene and Schizophrenia in the Chinese Population

Neuroscience Letters 397 (2006) 285–290

Association between the brain-derived neurotrophic factor (BDNF) gene and Schizophrenia in the Chinese population Qing-Ying Chen a,b, Qi Chen b,c, Guo-Yin Feng d, Chun-Ling Wan b,c,∗, Klaus Lindpaintner e, Li-Jun Wang f, Zheng-Xiong Chen g, Zhen-Song Gao g, Ji-Sheng Tang h, Xing-Wang Li b,c, Lin He b,c,∗ a Institute for Nutritional Sciences, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, 319 Yue Yang Road, Shanghai 200031, PR China b Bio-X Center, Shanghai Jiaotong University, PO Box 501, Hao Ran Building, 1954 Hua Shan Road, Shanghai 200030, PR China c Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, PR China d Shanghai Institute of Mental Health, 600 South Wan Ping Road, Shanghai 200030, PR China e Roche (China) Ltd, 1100 Long Dong Avenue, Pudong New Area, Shanghai 201203, PR China f Shenyang Tiexi Institute of Mental Health, Liaoning, China g Shantou Institute of Mental Health, Guangdong, China h Shandong Institute of Mental Health, Shandong, China Received 12 October 2005; received in revised form 29 November 2005; accepted 12 December 2005

Abstract Brain-derived neurotrophic factor (BDNF) belongs to a family of the neurotrophin which plays important roles in the development of the brain. BDNF has been suggested as a factor that increases the risk of schizophrenia. In this study, we genotyped three single nucleotide polymorphisms (SNPs) in the BDNF gene using a set sample of Han Chinese subjects consisting of 560 schizophrenes and 576 controls. No signiﬁcant differences were found for either the genotype or allele distribution of analyzed polymorphisms, nor was any gender-speciﬁc association found. Thus, our data suggest that the BDNF gene may not be an important factor in susceptibility to schizophrenia. © 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: BDNF; Case-control; Association; Schizophrenia; Chinese

Schizophrenia is a heterogenous disorder involving genetic, bio- abundant of the neurotrophins in the brain, BDNF is important logic and environmental factors. Although schizophrenia afflicts for guiding the neurons of CNS during their development and approximately 1% of the population throughout the world, the maintaining their survival in adulthood [48]. ultimate biological cause of the disorder remains elusive. Lines BDNF is found to be involved in the maintenance of long- of evidence suggest that neurodevelopmental abnormalities of term potentiation (LTP), a cellular mechanism of learning specific brain areas, including disturbances of neuron migration, and memory, and participates in modulating the synthesis, alteration in neural plasticity and changes in synaptic connec- metabolism and release of neurotransmitters, and therefore hav- tion, are important factors in the pathogenesis of schizophrenia ing a role in regulating synaptic plasticity [1,13,26,34,36]. [2,3,25]. Animal experiments have revealed that BDNF is broadly The brain-derived neurotrophic factor (BDNF), the gene distributed in the central nervous system and is enriched in encoded on human chromosome 11p13, is a member of the the hippocampal formation, cerebral cortex and limbic areas superfamily of the neurotrophin which plays a critical role in [12,21,32]. During forebrain development in the rat, BDNF promoting and modifying growth, differentiation, and survival mRNA expression has the highest levels in the hippocam- of neurons in the central nervous system (CNS). As the most pus and the lowest in the striatum [10,21,49]. Furthermore, BDNF mRNA is variously expressed in the subfields of the hippocampus, with low expression in pyramidal cells in ∗ Corresponding author. Tel.: +86 21 62822491; fax: +86 21 62822491. CA1, moderate expression in CA2 and high expression in E-mail address: [email protected] (L. He). CA3 [7].

The function and distribution of BDNF in the CNS raise the any history of psychiatric disorders. The study was approved possibility that this neurotrophin is relevant to schizophrenia and by local psychiatry research ethics committees and informed a number of anatomical and clinical studies have been done to consent was obtained from all subjects. assess the potential contribution of BDNF to the pathophysiol- We selected three SNPs—rs3750934, rs6265 from dbSNP ogy of the disorder. Takahashi et al. reported that higher BDNF (http://www.ncbi.nlm.nih.gov) and ro 000011924 from the levels were detected specifically in the anterior cingulate cor- Roche database, spanning around 1047 bp in the BDNF gene. tex and hippocampus of schizophrenic patients, when compared rs3750934 is in the intron near the coding sequence (CDS) of with controls [46]. In their postmortem brain study, Durany the BDNF gene. rs6265 is located in the CDS of the gene, result- et al. reported that BDNF concentrations were significantly ing in an amino acid substitution from valine to methionine. increased in the cerebral cortex and decreased significantly in the ro 000011924 is in the penultimate exon of the gene. Genomic hippocampus of schizophrenic patients [9]. Recently, two post- DNA was extracted from venous blood collected from subjects mortem studies have reported decreased levels of BDNF mRNA by a standard phenol extraction procedure. and protein in the prefrontal cortex of subjects with schizophre- All SNPs were genotyped by allele-specific PCR, in which nia [20,55]. In addition, BDNF levels were significantly reduced primers were designed to specifically amplify the reference in the serum of schizophrenic patients but not in their whole allele or its variant in separate PCR reactions [18]. The assay blood [33,50]. On the other hand, several other studies have used in this study combines kinetic (real-time) PCR with failed to detect altered serum BDNF levels in schizophrenic allele-specific amplification [17]. The primers sequences patients [23,41]. used for three SNPs were as follows: For rs3750934: 5- Lines of genetic association studies have been carried out ACAATCAGATGGGCCACAC/T-3 (allele-specific primer) in order to determine the possible correlation between polymor- and 5-GGCTTTCTTTCACCGGGATG-3 (common primer); phisms in the BDNF gene and schizophrenia. Proschel et al. [35] for rs6265: 5-CATCCAACAGCTCTTCTATCAC/T-3 (allele- identified a GT dinucleotide repeat in the human gene for BDNF specific primer) and 5-CTTGACATCATTGGCTGACAC-3 in 1992. In a French Caucasian population, Krebs found an (common primer); for ro 000011924: 5-CACAACTTAAA- excess of the 172–176 bp alleles of the GT repeat polymorphism AAGTCTGCATTA/G-3 (allele-specific primer) and 5- in patients with late onset, in neuroleptic-responding patients ACGGCAACAAACCACAACA-3 (common primer). For and in non-substance-abusing patients. However, some studies real-time PCR, two PCR reactions were performed for each have given negative results [37,54]. A single nucleotide sub- sample, with 10 ng genomic DNA, 0.05 ␮l ∆Z05 enzyme (Roche stitution (C270T) polymorphism, located in the 5-noncoding company), 0.2 ␮M allele-specific primer, 0.2 ␮M common region of the BDNF gene, was found to be associated with primer and 0.2 × SYBR® Green I (Molecular Probe, Inc.) in a schizophrenia in a Caucasian population [45]. Nanko et al. found total volume of 25 ␮l. To reduce well-to-well variability in PCR the frequency of the T allele of the C270T polymorphism to be reaction conditions, an automated dispenser (Hydra® microdis- significantly increased in Japanese patients compared with con- penser, Robbins Scientific) and digital multichannel pipettes trols [29]. However, some recent studies have failed to confirm (Thermo Labsystems) were used. Kinetic PCR reactions were these findings. Galderisi et al. and Szczepankiewicz found no performed on an ABIPRISM 7900 Sequence Detection System differences in allele and genotype distribution between patients (Applied Biosystems). After an initial 2 min incubation step at and controls [16,44]. In addition, Hong et al. reported a trend 50 ◦C to activate the AmpErase® uracil-N-glycosylase (UNG) (p = 0.055) between genetic predisposition and a nonsynony- and a step of 12 min at 95 ◦C to deactivate UNG and activate mous mutation rs6265 in 93 schizophrenic patients [22]. AmpliTaq Gold® enzyme, 50 cycles consisting of 15 s at 95 ◦C All these data make the BDNF gene a good candidate for and 30 s at annealing temperature were performed, followed by association study of schizophrenia. Given the importance of a final stage of dissociation to check the PCR product. Allele independent observation of association findings in genetically calling was manually performed as in previous research in our complex diseases such as schizophrenia, we set out to inves- laboratory [47]. tigate the role of BDNF in the etiology of schizophrenia in Allele frequencies in different groups of subjects were com- an independent sample of schizophrenic patients and controls pared using the CLUMP program (version 1.9) [39] with 10,000 from China. Three SNP polymorphisms—rs3750934, rs6265 stimulations. The p-values reported are two-tailed and signifi- and ro 000011924 (from Roche database) were genotyped in cance was accepted at p < 0.05. A p-value of 0.05 was considered 560 Chinese patients and 576 Chinese control individuals. significant in tests for Hardy–Weinberg equilibrium. The stan- All subjects were Han Chinese in origin. A total of 560 dardized measure of linkage disequilibrium (LD), denoted as D, unrelated schizophrenic patients (53.4% male) with a mean age was estimated with software 2LD [57]. Haploypte frequencies 37.3 ± 13.6 were recruited from the Liaoning, Guangdong and were estimated by EHPLUS, which performs model-free anal- Shandong provinces of China. Consensus diagnosis of each ysis and permutation tests of allelic association based on EH patient was made by two independent psychiatrists according [56]. The odds ratio and 95% confidence interval were calcu- to the DSM-IV (Diagnostic and Statistical Manual of Men- lated on the web http://202.120.7.14/analysis/myAnalysis.php tal Disorders-Fourth Edition) [14] criteria for schizophrenia. [40]. Five hundred and seventy-six unrelated healthy subjects (51.9% In our case-control analysis, 560 schizophrenics were geno- male) with a mean age 33.2 ± 10.5 from the same geographical typed and compared with a set of 576 controls. Table 1 gives the region were used as controls. All were interviewed to exclude allele and genotype frequencies of the three markers. Genotypic Q.-Y. Chen et al. / Neuroscience Letters 397 (2006) 285–290 287

Table 1 Statistical analysis for polymorphisms of the BDNF gene for all subjects in the sample

Marker Distancea Genotypeb HWE (χ2) p-Value Alleleb p-Value OR (95% CI) (bp) (2d.f.) (1d.f.) rs3750934 0 CC CT TT C T Patients 2 (0.3) 44 (7.9) 514 (91.8) 1.0014 0.8946 48 (4.3) 1072 (95.7) 0.6875 0.907 (0.609–1.350) Controls 3 (0.5) 48 (8.4) 523 (91.1) 2.5948 54 (4.7) 1094 (95.3) rs6265 279 AA AG GG A G Patients 144 (25.7) 259 (46.3) 157 (28.0) 3.1082 0.25752 547 (48.8) 573 (51.2) 0.5330 0.941 (0.798–1.109) Controls 127 (22.0) 291 (50.5) 158 (27.4) 0.1026 545 (47.3) 607 (52.7) ro 000011924 1047 AA AG GG A G Patients 440 (78.6) 115 (20.5) 5 (0.9) 0.7088 0.2448 995 (88.8) 125 (11.2) 0.3045 1.146 (0.888–1.479) Controls 443 (76.9) 121 (21.0) 12 (2.1) 1.185 1007 (87.4) 145 (12.6) a The distance from rs3750934. b Frequencies are shown in parenthesis (%).

Table 2 and 3-noncoding region of the BDNF gene. rs6265, located Estimates of linkage disequilibrium between the three markers in codon 66, leading to a valine (Val) to methionine (Met) rs3750934 rs6265 ro 000011924 substitution in the 5 pro-region of the human BDNF protein, rs3750934 – exhibits a capacity to affect intracellular trafficking and rs6265 0.464 – activity-dependent secretion of BDNF [5,11]. Previous studies ro 000011924 0.326 0.003 – suggested that the Met allele of rs6265 is linked with abnormal hippocampal activation and impaired episodic memory in For each pair of markers, the standard D is shown. humans [11,19]. Many association studies have been performed to inves- distributions of these three polymorphisms did not deviate sig- tigate the relationship between rs6265 and Alzheimer’s dis- nificantly from Hardy–Weinberg equilibrium. ease, bipolar disorder and Parkinson’s disease (PD). Some As shown in Table 1, no SNP showed significant difference studies reported positive association between the G allele in either genotype or allele frequencies between the 576 con- of the rs6265 and bipolar disorder [31,43]. However, controls and the 560 patients taken as a whole. In addition, when trary results have also been reported [24,28]. Coincidently, groups were divided according to gender, we failed to find any the overrepresentation of the G allele and GG genotype of significant differences in genotype or allele distribution (data the BDNF rs6265 polymorphism were found in Italian and not shown). Japanese Alzheimer’s disease (AD) patients [27,52]. In con- LD between each pair of all the SNPs is presented in Table 2. trast, investigations into Spanish, American, Finnish and Tai- Each pair of SNPs is in low LD (D < 0.5). We analyzed the wan Chinese subjects did not detect significant differences frequencies of the haplotypes of the three SNPs but only those of the allele or genotype frequencies between AD patients were common (at least 1% frequency in either case or control and controls [6,8,51,53]. Interestingly, a significantly high fre- groups) (Table 3). There was no difference in frequencies of quency of the GG genotype in female controls compared haplotypes constructed by the three SNPs between cases and with female patients was detected in Mainland Chinese sub- controls (global χ2 = 0.747, p = 0.976). jects [4]. Recently, Foltynie et al. demonstrated that Parkin- We studied three SNPs: rs3750934, rs6265 and son’s disease patients with the A allele of the BDNF rs6265 ro 000011924, respectively, in the intron, coding region polymorphism perform better at planning tasks than patients

Table 3 Estimated haplotype frequencies and association signiﬁcance Haplotypea rs3750934 rs6265 ro 000011924 Haplotype frequency (%) χ2 p-Value Odds ratio (95% CI) Case Control

1 C G A 34.86 (3.1) 35.46 (3.1) 0.001 0.898 1.007 (0.627–1.619) 2 C A A 11.14 (1.0) 12.54 (1.1) 0.052 0.835 0.912 (0.405–2.054) 3 T G A 485.48 (43.3) 487.34 (42.5) 0.186 0.659 1.037 (0.878–1.225) 4 T G G 71.49 (6.4) 79 (6.9) 0.227 0.619 0.923 (0.663–1.285) 5 T A A 463.52 (41.4) 468.67 (40.8) 0.074 0.792 1.023 (0.866–1.210) 6 T A G 51.51 (4.6) 59 (5.1) 0.357 0.549 0.890 (0.607–1.305) Global 0.747 0.976 a Haplotypes were omitted from analysis if the estimated haplotype probabilities were less than 1%. 288 Q.-Y. Chen et al. / Neuroscience Letters 397 (2006) 285–290 with the G alleles and the effect is most apparent in women psychiatrists and mental health workers for their help in the [15]. recruitment of schizophrenic patients. 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