Previous studies have suggested that genetic variations in the brain-derived neurotrophic factor (BDNF) gene may be associated with several neuropsychiatric diseases including bipolar disorder. The present study examined a microsatellite polymorphism located approximately 1.0 kb upstream of the translation initiation site of the BDNF gene for novel sequence variations, association with bipolar disorder, and effects on transcriptional activity. Detailed sequencing analysis revealed that this polymorphism is not a simple dinucleotide repeat, but it is highly polymorphic with a complex structure containing three types of dinucleotide repeats, insertion/deletion, and nucleotide substitutions that gives rise to a total of 23 novel allelic variants. We obtained evidence supporting the association between this polymorphic region (designated as BDNF-linked complex polymorphic region (BDNF-LCPR)) and bipolar disorder. One of the major alleles (‘A1’ allele) was significantly more common in patients than in controls (odds ratio 2.8, 95% confidential interval 1.5–5.3, P=0.001). Furthermore, a luciferase reporter gene assay in rat primary cultured neurons suggests that this risk allele (A1) has a lower-transcription activity, compared to the other alleles. Our results suggest that the BDNF-LCPR is a functional variation that confers susceptibility to bipolar disorder and affects transcriptional activity of the BDNF gene.
Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophic factor family and promotes the development, regeneration, survival and maintenance of function of neurons.1 It modulates synaptic plasticity and neurotransmitter release across multiple neurotransmitter systems, as well as the intracellular signal-transduction pathway.2 BDNF has been implicated in the pathogenesis of mood disorders and in the mechanism of action of therapeutic agents such as mood stabilizers and antidepressants.3 BDNF protein was reduced in postmortem brains of patients with bipolar disorder, compared to controls.4 Chronic electroconvulsive seizure and antidepressant drug treatments increase mRNA of BDNF and its receptor trkB.5 Lithium may also exert its neuroprotective effect through enhancing expression of BDNF and trkB.6
The BDNF gene is, therefore, an attractive candidate gene which may give susceptibility to bipolar disorder.7 In accordance with this, at least three previous studies reported a significant association between the Val66Met polymorphism (NCBI dbSNP rs6265) of the BDNF gene and bipolar disorder in Caucasian populations.8, 9, 10 In these studies, the Val66 allele was consistently found to have a risk-increasing effect on the development of bipolar disorder. However, this association was not replicated in other Caucasian11, 12, 13 or Asian populations including ours.14, 15, 16
Another polymorphism of the BDNF gene that has been well studied as to the possible association with neuropsychiatric diseases is the ‘GT repeat’ located approximately 1.0 kb upstream of the translation initiation site of the gene.17 With respect to the possible effect on mood disorders, a significant linkage disequilibrium with bipolar disorder8 and a significant association with childhood onset mood disorder18 have been reported in Caucasian populations, although one study failed to find such an association with bipolar disorder.13 Furthermore, there is some evidence suggesting that this polymorphism plays a role in the pathogenesis of schizophrenia.13, 19, 20 However, there is no study that examined whether this polymorphism has functional effects. Since micro- and minisatellite polymorphisms even located in intron have been shown to play a role in the expression of many genes,21 it might be intriguing to examine whether this microsatellite polymorphism of the BDNF gene is associated with transcriptional activity in an allele-dependent manner.
The aim of the present study was to examine this microsatellite polymorphism (designated here as BDNF-linked complex polymorphic region (BDNF-LCPR) due to its complex structure) for novel sequence variations, association with bipolar disorder, and effects on transcriptional activity.
Materials and methods
Subjects were 153 patients with bipolar disorder (71 males) and the same number of controls (71 males), matched for age, sex, ethnicity, and geographical area. These subjects, who were recruited from Showa University Hospital and Shiga University of Medical Science Hospital, Japan, were previously genotyped for the Val66Met polymorphism of the BDNF gene, yielding a result of no significant association.16 Mean age (standard deviation (s.d.)) in the patients was 47.8 (s.d. 15.3) years and that in the controls 47.1 (11.0). All the patients and controls were biologically unrelated Japanese. Consensus diagnosis of bipolar disorder was made for each patient by at least two experienced psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders, 4th ed. (DSM-IV),22 based on unstructured interviews and medical records. Among the patients, 94 individuals (61%) were diagnosed with bipolar I and the remaining 59 with bipolar II disorder. Patients who had one or more comorbid axis I disorders were excluded. The mean age of onset and number of episodes were 37.8 (s.d. 15.2) years and 3.9 (1.4) times, respectively. Thirty-four patients (22.2%) had at least one episode with psychotic features. Sixty-seven patients (43.8%) had a family history of major psychiatric illness (mood disorders or schizophrenia spectrum disorders) within their second-degree relatives. The controls were screened with a semi-structured interview and those individuals who had current or past contact to psychiatric services were excluded. In addition, those individuals who had a family history of major psychiatric illness or those who had a current or past history of regular use of psychotropic medication, including hypnotics, were excluded from the control group. After description of the study, written informed consent for the participation of the study was obtained from every subject. The study protocol was approved by ethics committee of each institution.
Venous blood was drawn and genomic DNA was extracted according to standard procedures. To determine accurate DNA sequences for the BDNF-LCPR, we cloned this polymorphic region and performed direct sequencing. An approximately 400 base-pair (bp) DNA fragment encompassing the polymorphic region was amplified by polymerase chain reaction (PCR) with primers of HindIII-tagged BDNF-LCPR-F1 and HindIII-tagged BDNF-LCPR-R1 (see Table 1 and Figure 1a). The purified PCR products were ligated into the HindIII site of the pBluescriptII SK (+) vector (Toyobo, Tokyo, Japan). The vector was transformed into Escherichia coli, DH5α and incubated. For sequencing, PCR amplification was performed with primers of IndexTermGTTGTAAAACGACGGCCAGTG (Universal primer) and IndexTermGGAAACAGCTATGACCATG (Reverse primer). At least four clones were examined for each individual. Direct sequencing was performed with the CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA).
Cloning and sequencing analysis described above suggested that the polymorphic region is not a simple dinucleotide repeat, but this polymorphism has a very complex structure. In addition, because of the stuttering effect in the PCR amplification, cloning and sequencing could not always determine the genotype of each individual. We then performed pyrosequencing that was able to differentiate true alleles from artifacts due to stuttering. The polymorphic region was amplified by PCR with primers of BDNF-LCPR-F2 and B-BDNF-LCPR-R2 for forward direction and B-BDNF-LCPR-F2 and BDNF-LCPR-R2 for reverse direction (Table 1). These primers were designed with the Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Pyrosequencing was performed with PSQ96MA System and PSQ96 SNP Reagent Kit (Pyrosequencing, AB, Uppsala, Sweden). Sequencing primers of both directions (forward: BDNF-LCPR-F3; reverse: BDNF-LCPR-R3, Table 1) were designed with the software supplied by Pyrosequencing, AB, Uppsala, Sweden (http://www.Pyrosequencing.com). On the basis of the sequences observed by cloning and direct sequence, described above, ‘the sequence to analyze’ was assumed to be 5′-IndexTermCGCGCACA[CCGCGCGCG]CACACACACACACACACACACACACACACACAGAGAGAGAACAT-3′ and dispensation order was set as 5′-IndexTermTCGCGCACAGCGCGCGCGTCACACACACACACACACACACACACA CACACATGAGAGAGAT-3′ for sequencing in the forward direction. For sequencing in the reverse direction, ‘the sequence to analyze’ and the dispensation order was set as 5′-IndexTermCTCTCTGTGTGTGTGTGTGTGTGTG[TGTGTGTGTGTGTGTG]CGCGCGCGCGTGTGCGCGCGCTCTGAGTT-3′ and 5′-IndexTermGCTCTCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTACGCGCGC GCGTGTGCGCGCGCTCTC-3′, respectively. Since the target sequence was rather long (∼70 bp), we added single-stranded binding protein (SSB, Sigma-Aldrich, St Louis, MO, USA) to avoid wearing down of signal for sequencing.
DNA sequences of two chromosomes of each individual were determined by referring to results of both direct sequencing of cloned fragments and pyrosequencing. For ambiguous genotypic data, we repeated experiments and determined genotype for every subject. Genotypes were read blind to affection status.
Association analysis with bipolar disorder
The presence of Hardy–Weinberg equilibrium in genotype distribution was examined by using the χ2-test for goodness of fit. Allele frequencies of the BDNF-LCPR were compared between patients and controls by using the χ2-test for independence. Then linkage disequilibrium and haplotype-based association analysis for the BDNF-LCPR and the Val66Met polymorphisms were carried out. These statistical analyses were performed by using the SPSS v11 (SPSS Japan Inc., Tokyo, Japan) and the COCAPHASE v2.403 program (http://www.hgmp.mrc.ac.uk/~fdudbrid/software/unphased/). All P-values reported are two-tailed.
Luciferase reporter gene assay in primary cultured neurons
Primary cultures were prepared from the cortex of postnatal 2 days old rats (SLC, Shizuoka, Japan) as described previously.23 To generate plasmids for the luciferase gene reporter assay (Figure 2a), the BDNF-LCPR was amplified by PCR with primers of SmaI-tagged BDNF-LCPR-F1 and SmaI-tagged BDNF-LCPR-R1 (Table1 and Figure 1). The PCR products were inserted into the SmaI site upstream of the SV40 promoter in the pGL3-Promoter vector (Promega, Tokyo, Japan). The four major alleles were subject to the assay. Plasmid constructs were transfected at 5 days in vitro. Cells on 24-well plates were co-transfected with 800 ng of pGL3-Promoter firefly luciferase vectors that included major alleles of the BDNF-LCPR and 25 ng of phRL-TK renilla luciferase vector (Promega, Tokyo, Japan) as an internal control by using Lipofectamine 2000 reagent (Invitrogen, Tokyo, Japan). Empty pGL3-Promoter vector was transfected simultaneously.
At 24 h after transfection, luciferase activity was measured by using Dual-Luciferase Reporter Assay System (Promega, Tokyo, Japan) and a Lumat LB9507 luminometer (Berthold Technologies, Bad Wildbad, Germany), as described previously.24 Firefly and renilla luciferase activities were quantified sequentially as relative light units (RLU) by addition of their respective substrates. The ratio of firefly RLU to renilla RLU of each sample was automatically computed. Then the activity of each construct was expressed as the relative value compared to that of empty pGL3-Promoter vector (relative luciferase expression, RLE). Primary cultured cells were prepared three times and transfection was performed triplicate for each cell culture. Comparisons in RLE were carried out by analysis of variance (ANOVA) or t-test.
Detection of novel variants
The structure of the BDNF gene25, 26 and DNA sequence of the cloned fragment according to the University of California, Santa Cruz (UCSC) genome database are illustrated in Figure 1a. We detected a total of 23 allelic variants in the BDNF-LCPR (registered to the DDBJ/EMBL/GenBank database, accession numbers AB212736 to AB212758). Sequences and allele frequencies in patients with bipolar disorder and controls are shown in Table 2. Allelic variants of the BDNF-LCPR consisted of three components of dinucleotide repeat of (CA)del/2(CG)del/4/5, (CA)9–15, and (GA)2/3, which were combined in succession (Figure 1b). In addition, there were four exceptional rare variants that contained a single nucleotide substitution (variants 2 and 4) or insertion of two nucleotides of cg (variants 1 and 3) immediately 5′ side of the repeats. The ‘GT repeat’ due to the original report17 was CA, but not GT, repeat when the sequence was read in the forward direction of the BDNF gene. There were four major alleles of Del-12-3 (allele 1; A1), 4-12-3 (A2), 5-12-2 (A3), and 5-13-3 (A4). To perform statistical analyses, the remaining rare alleles were combined and considered to be ‘allele 5 (A5)’. Supplementary figures S1 and S2 show images of direct sequencing of the major alleles, which were cloned and an example of pyrosequencing depicted in ‘pyrogram’.
Association analysis with bipolar disorder
Genotype and allele distributions in patients and controls are shown in Table 3. The genotype distributions were in Hardy–Weinberg equilibrium (for the patients: χ2=4.5, df=13, P=0.98; for the controls: χ2=8.2, df=13, P=0.83). The overall allele frequencies differed significantly between patients and controls (χ2=13.4, df=4, P=0.0093). The global-P-value estimated by the permutation test of 10 000 simulations, correcting for multiple testing, yielded a similar result (P=0.010). The A1 allele was clearly more common in patients than in controls (11.8 vs 4.6%, odds ratio (OR) 2.8, 95% confidential interval (CI) 1.5–5.3, χ2=10.5, df=1, P=0.001). When the three components of the BDNF-LCPR, that is, (CA)del/2 (CG)del/4/5, (CA)9–15, and (GA)2/3, were examined separately, only the first component showed a significant association with bipolar disorder (Table 4). Deletion of the first component, as seen in the A1 allele, was significantly more common in patients than in controls (14.6 vs 8.3%, OR 1.9, 95% CI 1.1–3.2, χ2=5.9, df=1, P=0.015).
Then we examined linkage disequilibrium and haplotype-based association for the BDNF-LCPR and the Val66Met polymorphism. As reported previously,16 there was no significant association between the Val66Met polymorphism and bipolar disorder in the current sample; the frequencies of the Val66 allele were 0.60 and 0.62 in patients and comparison groups, respectively (χ2=0.25, df=1, P=0.62). Results of haplotype-based analysis for these two polymorphisms are shown in Table 5. There was a very tight linkage disequilibrium between the BDNF-LCPR and Val66Met polymorphism (D′=0.91 for patients and D′=0.90 for cotrols; χ2=512, df=28, P=1.6 × 10−90 in total subjects). The Val66 allele was linked to the A1, A2, or A3 allele, while the Met66 allele was to the A4 allele. The haplotype-based association analysis yielded a significant result (global P=0.0069) estimated by the permutation test, correcting for multiple testing. Since the A1 allele was completely linked to the Val66 allele, the most significant individual P-value of 0.001 was obtained when the A1-Val66 was assumed to be the risk. When pairwise linkage disequilibrium across three components of the BDNF-LCPR and Val66Met was examined individually, there was a tight linkage disequilibrium between each component of the BDNF-LCPR and the Val66Met, while linkage disequilibrium within the three components were much weaker (Supplementary Table S1). The deletion of the first component of the BDNF-LCPR was completely linked to the Val66 allele; the (CA)del(CG)del allele was completely linked to the Val 66 allele, while the Val66 allele was linked to any of the (CA)del/2(CG)del/4/5 alleles.
Luciferase reporter gene assay in primary cultured neurons
Figure 2b shows observed RLEs for the major four alleles of the BDNF-LCPR, compared to RLE without insertion of such alleles (empty pGL3-Promoter vector). RLE decreased due to insertion of the polymorphic region for all the alleles compared to the empty pGL3-Promoter vector, suggesting that the BDNF-LCPR and its flanking region may have a silencer-like effect on transcriptional activity. When RLE was compared among the four alleles, there was a significant difference (F=5.9, df=3, 32, P=0.003, ANOVA). RLE for the A1 allele was the smallest among the four alleles. When RLE for the A1 allele was compared to that for the remaining three alleles combined, the difference was significant (t=−3.4, df=34, P=0.002), providing evidence suggesting that the A1 allele is associated with lower transcriptional activity.
The present study demonstrated that a microsatellite polymorphism of the BDNF gene originally reported as a ‘GT repeat’17 is not a simple dinucleotide repeat, but a very complex structure of polymorphism, containing three types of dinucleotide repeats, insertion/deletion, and nucleotide substitutions, which is consistent in part with a recent report. 27 We therefore designated this region as BDNF-linked complex polymorphic region (BDNF-LCPR). The nucleotide sequences were determined by combination of pyrosequencing together with direct sequencing after cloning. Thus sequencing errors are unlikely. As a result, a total of 23 novel allelic variants were detected, although only five alleles had been identified in the original report.17 We obtained evidence suggesting an association between the BDNF-LCPR and bipolar disorder. This is in accordance with a previous study8 that reported a significant association between this polymorphism and bipolar disorder. However, detected alleles and their distribution considerably differ between this previous study8 and the current study since the former genotyped the polymorphism by fragment-size analysis. We detected multiple alleles for each fragment size; for example, the A2 and A3 alleles had the same fragment size (407 bp, see Table 2). Therefore, fragment size analysis is not enough to perform an association study on the BDNF-LCPR.
Of note, the microsatellite corresponding to the BDNF-LCPR and its flanking region are conserved in rodents at similar location relative to the translation initiation site of the BDNF gene (1065 bp upstream in humans, 921 bp in rats, and 963 bp in mice). The nucleotide sequences flanking the microsatellite were highly homologous between humans and rodents (rat: 68% and mouse 66%, according to our calculation based on sequences from GenBank accession number AABR03134358.1 for rat and AY057907 for mouse). We then examined whether the BDNF-LCPR is associated with transcriptional activity in an allele-dependent manner, using luciferase reporter gene assay on primary cultured neurons from the rat brain cortex. The results provided evidence that the A1 allele is associated with lower transcriptional activity, compared to the other major alleles. This is interesting because the A1 allele, which is 12 or 16 bp shorter than the other major alleles (see Table 2), were found to be increased in patients with bipolar disorder, compared to controls. These results suggest that the A1 allele plays a role in giving susceptibility to bipolar disorder by reducing transcriptional activity of the BDNF gene. Since the A1 allele has deletion of the first component of the BDNF-LCPR, and this deletion was significantly more common in patients than in controls, it is possible that such deletion might be responsible for altering transcriptional activity and conferring the susceptibility. Our result is in line with a recent finding that BDNF protein was reduced in postmortem brains of patients with bipolar disorder, compared to controls.4
In previous studies8, 9, 10 that reported a positive association between the Val66met polymorphism of the BDNF gene and bipolar disorder, the Val66 allele was consistently found to be the risk allele. However, other studies11, 12, 13, 14, 15, 16 failed to find such an association. The Val66Met polymorphism has been found to have functional effects. The Met66 allele was associated with poorer episodic memory, abnormal hippocampal activation, and lower hippocampal n-acetyl aspartate in humans and that the Met66 allele showed lower depolarization-induced secretion and failed to localize to secretory granules or synapses in neurons.28 The relationship between the Met66 allele and poorer episodic memory has been further demonstrated.29 Since impairment in verbal episodic memory is one of the most consistently reported cognitive problems in individuals with bipolar disorder,30, 31 it is not feasible that the Val66 allele, but not the Met66 one, has consistently been reported to be the risk allele for bipolar disorder.8, 9, 10 In our linkage disequilibrium analysis between the BDNF-LCPR and the Val66Met polymorphisms, the A1 allele was completely linked to the Val66 allele, which may explain, at least in part, the inconsistent results in the previous studies. That is, the A1 allele might be a responsible allele; however, its linkage to the Val66 allele have made the Val66 allele over-represented in some samples but not in the other samples, since the Val66 allele is linked to not only the A1 allele but also A2, A3, and A5 alleles. To demonstrate this hypothesis, the association between the BDNF-LCPR and bipolar disorder should be reevaluated based on the current findings. In addition, studies examining the possible association of the BDNF-LCPR with brain structure and functions are warranted.
Several studies have performed an association study between the ‘GT repeat’ and schizophrenia, which have also yielded conflicting results.13, 19, 20, 32, 33, 34, 35 To resolve the inconsistent findings, further studies based on the current information are required.
In conclusion, we demonstrated that a microsatellite of the BDNF gene, which was originally reported as a ‘GT repeat’17 is not a simple dinucleotide repeat, but has a complex structure of polymorphism. Association analysis and luciferase reporter gene assay suggest that the BDNF-LCPR is a functional polymorphism that confers susceptibility to bipolar disorder and affects transcriptional activity.
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We thank Shizuno T and Okada K for their technical assistance in laboratory. This study was supported by the Health and Labor Sciences Research Grants (Research on Psychiatric and Neurological Diseases and Mental Health), the Japan Health Sciences Foundation (Research on Health Sciences focusing on Drug innovation) and Mitsubishi Pharma Research Foundation (HK).
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Cite this article
Okada, T., Hashimoto, R., Numakawa, T. et al. A complex polymorphic region in the brain-derived neurotrophic factor (BDNF) gene confers susceptibility to bipolar disorder and affects transcriptional activity. Mol Psychiatry 11, 695–703 (2006) doi:10.1038/sj.mp.4001822
- association study
- brain-derived neurotrophic factor (BDNF)
- bipolar disorder
- transcriptional activity
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