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Association study of PDE4B with panic disorder in the Japanese population
Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 545–549
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Association study of PDE4B with panic disorder in the Japanese population Takeshi Otowa a,b,⁎, Yoshiya Kawamura a, Nagisa Sugaya c, Eiji Yoshida d, Takafumi Shimada a, Xiaoxi Liu e, Mamoru Tochigi a, Tadashi Umekage a, Taku Miyagawa e, Nao Nishida e, Hisanobu Kaiya d,f, Yuji Okazaki g, Katsushi Tokunaga e, Tsukasa Sasaki h a
Department of Neuropsychiatry, Graduate School of Medicine, the University of Tokyo, Tokyo 113-8655, Japan Department of Psychiatry, Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA Faculty of Human Science, Waseda University, Saitama 359-1192, Japan d Outpatient Clinic for Anxiety Disorders, Akasaka Mental Clinic, Tokyo 107-0052, Japan e Department of Human Genetics, Graduate School of Medicine, the University of Tokyo, Tokyo 113-8655, Japan f Resarch Center for Panic Disorder, Nagoya Mental Clinic, Aichi 453-0015, Japan g Department of Neurology, Tokyo Metropolitan Matsuzawa Hospital, Tokyo 156-0057, Japan h Office for Mental Health Support and Graduate School of Education, University of Tokyo, Tokyo 113-8625, Japan b c
a r t i c l e
i n f o
Article history: Received 4 November 2010 Received in revised form 13 December 2010 Accepted 15 December 2010 Available online 22 December 2010 Keywords: Association Japanese population Panic disorder Phosphodiesterase 4B (PDE4B) Single nucleotide polymorphism
a b s t r a c t Background: Panic disorder (PD) is a severe and chronic psychiatric disorder with genetic components underlying in its etiology. The Phosphodiesterase 4B (PDE4B) gene has been reported to be associated with several psychiatric disorders. Several studies indicated that PDE4B may be involved in the regulation of anxiety and depression. Therefore, we investigate the association of PDE4B with PD in the Japanese population. Methods: We genotyped 14 single nucleotide polymorphisms (SNPs) of PDE4B in 231 PD cases (85 males and 146 females) and 407 controls (162 males and 245 females). Differences in the genotype, allele and haplotype frequencies between the two groups were compared. Results: We found a significant association between PDE4B and PD in the haplotype analysis (haplotype C-T-T-A, permutation P = 0.031, OR = 1.81, 95% CI = 1.30–2.51). Sex-specific analyses demonstrated that PDE4B was associated with PD in females in the allele/genotype and haplotype analyses (rs10454453, allele P = 0.042, genotype P = 0.0034; haplotype C-T-T-A, permutation P = 0.028). Conclusion: Our results suggest that PDE4B may play a role in the pathophysiology of PD in the Japanese population. Replication studies using larger samples will be needed for more reliable conclusions. © 2010 Elsevier Inc. All rights reserved.
1. Introduction PD is an anxiety disorder characterized by panic attacks and anticipatory anxiety, with a life-time prevalence of 1–3% and a female: male ratio of 2:1 (Eaton et al., 1994). PD frequently takes a chronic course, with many remissions and relapses, occasionally complicated by comorbidity with other psychiatric disorders, such as agoraphobia and major depression (Weissman et al., 1997). It is generally accepted that panic disorder has genetic as well as environmental causes. A 2.6to 20-fold relative risk in the first-degree relatives of probands with Abbreviations: PD, panic disorder; cAMP, cyclic adenosine monophosphate; PDE4B, phosphodiesterase 4B; DISC1, Disrupted in Schizophrenia 1; SNP, single nucleotide polymorphism; MINI, Mini-International Neuropsychiatric Interview; SSRI, serotonin selective reuptake inhibitor; CREB, cAMP response element binding protein; LD, linkage disequilibrium; TDT, transmission disequilibrium test; SD, standard deviation; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author. Department of Psychiatry, Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, 800 East Leigh Street, Richmond, VA 23298-0126, USA. Tel.: + 1 804 922 2606; fax: + 1 804 828 1471. E-mail address: [email protected] (T. Otowa). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.12.013
PD compared to the general population suggests a familial component in this disorder (Crowe et al., 1983; Goldstein et al., 1997). Twin studies show that about 40% of the liability towards PD consists of heritable factors (Kendler et al., 1993; Hettema et al., 2001). Thus far, however, the etiology of PD is currently unknown. The phosphodiesterases regulate intracellular concentrations of cAMP, a second messenger implicated in learning, memory and mood (Davis et al., 1995; O'Donnell and Frith, 1999). PDE4B is encoded by a gene of approximately 580 kb located on chromosome 1p31.2, consisting of 17 exons. Millar et al. (2000) reported that PDE4B was disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with a chronic psychiatric disorder. PDE4B binds DISC1 that is a candidate susceptibility factor for psychiatric disorders including schizophrenia, schizoaffective disorder, bipolar disorder and depression (Millar et al., 2005). Recently, case–control association studies reported associations between PDE4B and schizophrenia (Pickard et al., 2007; Fatemi et al., 2008; Numata et al., 2009a; Kähler et al., 2010), bipolar disorder (Kähler et al., 2010) and depression (Numata et al., 2009b). The expression of PDE4B isoforms in postmortem brain tissue from patients with schizophrenia
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or bipolar disorder has been shown to differ compared with controls (Fatemi et al., 2008). Several studies indicated that PDE4B may be involved in the regulation of anxiety and depression. PDE4 inhibitor rolipram produced antidepressant-like (O'Donnell and Frith, 1999) and anxiolytic-like behaviors in animals (Silvestre et al., 1999; Li et al., 2009). PDE4B is expressed in the amygdala, hypothalamus and frontal cortex (Cherry and Davis, 1999), which are key regions in the mediation of anxiety and stress responses (Charney and Deutch, 1996). It has been shown that cAMP signaling regulates anxiety-like behavior (Pandey et al., 2005). PDE4B may play a role in this process as a critical controller of this signaling pathway (Li et al., 2009). Chronic treatment with antidepressants decreases the expression of PDE4B in the hippocampus and increases neurogenesis in mice (Dlaboga et al., 2006; Malberg et al., 2000). Furthermore, benzodiazepine anxiolytic diazepam inhibits the expression of PDE4B (Cherry et al., 2001). In the light of the findings mentioned above, we investigated the association between PDE4B and PD in the Japanese population. In the present study, we investigated 14 SNPs of PDE4B in Japanese 231 PD cases and 407 controls. 2. Materials and methods 2.1. Subjects All cases and control subjects were ethnically Japanese and were recruited in the vicinity of Tokyo, Japan. Subjects comprised 231 unrelated Japanese with PD recruited from a clinic for anxiety (85 males and 146 females; age = 37.3 ± 8.8 years (mean ± SD)) and 407 unrelated healthy volunteers (162 males and 245 females; age = 39.7 ± 10.6 years) served as controls. The diagnosis of PD was confirmed according to DSM-IV criteria and by using the MINI (Sheehan et al., 1998) and clinical records. The controls received a short interview by one of the authors and filled out questionnaires to exclude history of major psychiatric illness. At the time of blood collection for the present study, most of the patients were treated with antidepressants such as SSRIs. The objective of the present study was clearly explained and written informed consent was obtained from all subjects. The study was approved by the Ethical Committee of the Graduate School of Medicine, the University of Tokyo. 2.2. Genotyping Genomic DNA was extracted from leukocytes by using the standard phenol-chloroform method. We selected 10 tagging SNPs (rs4077429, rs2840677, rs1937456, rs1937443, rs1354061, rs6588186, rs10454453, rs6588190, rs502958 and rs472952) with minor allele frequencies of 10% more in the Japanese population (HapMap database: www.hapmap.org), using HaploView 4.2 program (Barrett et al., 2005). The LD blocks were defined by the Gabriel method (Gabriel et al., 2002). We further added four SNPs (rs1317611, rs2503166, rs1040716 and rs2180335) investigated in
the previous studies (Pickard et al., 2007; Fatemi et al., 2008; Numata et al., 2009a, 2009b). The location of the SNPs assayed and their relationship to the exon/intron boundaries of the gene are shown in Fig. 1. Genotyping was performed using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol with ABI PRISM 7900 SDS2 Software (Applied Biosystems, Foster City, CA). 2.3. Statistical analysis Hardy–Weinberg equilibrium (HWE) was checked using chisquare analysis. Comparisons of groups between cases and controls with respect to allele and genotype frequencies were undertaken using chi-square tests. Genotype data were analyzed using UNPHASED ver. 3.1.4 (Dudbridge, 2008) to derive P-values for single-marker and global and individual haplotype tests. To maximize the genetic information extracted from the genotyped SNPs, two-, three- and four-marker sliding-window haplotype analyses were conducted for global haplotype tests. The expectation-maximization (EM) algorithm was used to estimate haplotype frequencies and the log-likelihood ratio test under a log-linear model was conducted. Permutation tests were performed using 10,000 iterations (random permutation). It adjusts the global P-value of the particular haplotype block tested within any window size or P-value of the individual haplotype tested within the particular haplotype block. ORs with 95% CIs were calculated relative to all other haplotypes pooled. Power calculation was performed using Genetic Power Calculator (Purcell et al., 2003). Significance for the result was set at P b 0.05. 3. Results The genotype and allele frequencies of the 14 SNPs are summarized in Table 1. Genotype frequency of SNP8 (rs2503166) in cases deviates from HWE (P = 0.028). Genotype frequencies of all SNPs in controls were within HWE. Significant differences were found in the allele and genotype frequencies of SNP9 (rs10454453) between cases and controls (Table 1). However, these associations did not reach statistical significance after Bonferroni correction. No significant difference was observed in the genotype or allele frequency of the other SNPs between cases and controls. When the cases with agoraphobia were studied, no significant difference was observed (Table 1). Considering the higher prevalence of PD in females and sexdependent findings in the previous association studies, sex-specific analyses would be necessary (Maron et al., 2010). When the data were subdivided on the basis of sex, significant associations of SNP9 (rs10454453) with PD were found in females after Bonferroni correction (allele P = 0.042, genotype P = 0.0034, corrected; Supplementary Table 1). In the haplotype analyses, nominally significant haplotype associations with PD were observed (Table 2). However, only the four-SNP haplotype (SNPs 9–12) remained significant after correction for multiple testing (global permutation P = 0.014). Individual
Fig. 1. The genomic structure of PDE4B and the locations of 14 SNPs selected for the association analysis.
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Table 1 Genotype distributions and allele frequencies of the PDE4B gene. SNP ID
M/ma
Phenotype
N
MAFb
P-value
Genotypec
P-value
Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls
231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407
0.310 0.359 0.343 0.292 0.278 0.275 0.455 0.480 0.500 0.429 0.439 0.469 0.485 0.470 0.439 0.338 0.374 0.385 0.435 0.409 0.391 0.390 0.394 0.360 0.437 0.419 0.366 0.329 0.308 0.310 0.435 0.409 0.404 0.217 0.242 0.259 0.186 0.207 0.226 0.204 0.222 0.217
0.225 0.674
105/109/17 38/51/10 177/181/49 120/87/24 51/41/7 218/154/35 67/118/46 22/59/18 105/197/105 72/120/39 27/57/15 116/200/91 59/120/52 23/59/17 131/195/81 99/108/24 37/50/12 152/197/58 73/115/43 33/51/15 142/212/53 94/94/43 41/38/20 176/169/62 78/104/49 34/47/18 160/196/51 105/100/26 49/39/11 195/172/40 73/115/43 34/49/16 144/197/66 140/82/9 54/42/3 227/149/31 154/68/9 61/35/3 249/132/26 148/72/11 60/34/5 255/127/25
0.175 0.448
SNP1
rs4077429
A/G
SNP2
rs2840677
A/T
SNP3
rs1937456
G/A
SNP4
rs1317611
G/C
SNP5
rs1937443
C/G
SNP6
rs1354061
C/T
SNP7
rs6588186
C/T
SNP8
rs2503166
C/T
SNP9
rs10454453
A/C
SNP10
rs6588190
C/T
SNP11
rs502958
A/T
SNP12
rs1040716
A/T
SNP13
rs2180335
C/T
SNP14
rs472952
C/T
0.516 0.942 0.118 0.610 0.160 0.449 0.111 0.429 0.095 0.780 0.121 0.634 0.292 0.373 0.0124e 0.167 0.474 0.967 0.282 0.900 0.087 0.627 0.094 0.565 0.557 0.884
0.747 0.759 0.231 0.118 0.252 0.209 0.207 0.102 0.231 0.847 0.158 0.848 0.529 0.480 0.0135e 0.303 0.773 0.849 0.556 0.978 0.142 0.199 0.248 0.412 0.762 0.794
Statistical analysis was performed using chi-square test. a M; major allele, and m: minor allele. b MAF: minor allele frequency. c Described as major homo/hetero/minor homo. d Cases with agoraphobia were compared with controls. e Bold numbers represent nominally significant P-value.
Table 2 Haplotype analysis of the PDE4B gene. SNP ID
Global haplotype P-valuea,b 2SNP
3SNP
4SNP
SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 SNP8 SNP9 SNP10 SNP11 SNP12 SNP13 SNP14
0.514 0.482 0.484 0.139 0.319 0.251 0.388 0.023 0.014 0.146 0.090 0.350 0.011
0.594 0.592 0.314 0.432 0.530 0.600 0.03 0.01 0.027 0.005 0.281 0.073
0.615 0.334 0.612 0.607 0.249 0.125 0.05 0.040 0.001c 0.008 0.095
a
P-values were calculated by log-likelihood ratio test. Bold numbers represent nominally significant P-value. c Haplotype showed significance after correction for multiple testing (permutation P = 0.014). b
haplotype analysis of the four-SNP haplotype (SNPs 9–12) is shown in Table 3. Significant difference was observed in frequency of the haplotype C-T-T-A between cases and controls (P = 3.48 × 10− 4, OR = 1.81, 95% CI = 1.30–2.51). This haplotype remained positive after correction for multiple testing by permutation (permutation P = 0.031). In the sex-specific analyses, the haplotype C-T-T-A showed a significant association with PD in females after correction for multiple testing (permutation P = 0.028; Supplementary Table 2). Table 3 Haplotype analysis among PD cases and controls. SNPs 9–12 haplotype A-C-A-A C-T-T-A C-T-T-T A-C-A-T C-C-A-A a b
Frequencya Cases
Controls
Chi-square
P-valueb
0.428 0.207 0.088 0.065 0.060
0.443 0.125 0.111 0.087 0.050
0.38 12.79 0.91 2.49 1.47
0.54 3.48E-04 0.34 0.11 0.22
Haplotypes whose frequencies were estimated N 5% were described. Bold number represents significant P-value (permutation P = 0.031).
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4. Discussion In the present study, we investigated the association between PDE4B and PD in the Japanese population. In the single SNP analyses, no association was found with PD. In the haplotype analyses, the fourSNP haplotype (SNPs 9–12) showed an association with PD (global permutation P = 0.014). The individual haplotype C-T-T-A showed an association with PD (permutation P = 0.031). Sex-specific analyses showed that the SNP rs10454453 and the haplotype C-T-T-A were associated with PD in females (rs10454453, allele P = 0.042, genotype P = 0.0034 corrected; haplotype C-T-T-A, permutation P = 0.028). PDE4B has been proposed as a candidate gene in psychiatric disorders including schizophrenia, bipolar disorder and depression. Our results demonstrated that PDE4B was associated with PD. However, the SNP and haplotype showing association with PD are different from those in the previous studies. Pickard et al. (2007) reported that haplotypes within intron 3 were associated with schizophrenia in females. The SNPs and haplotypes in introns 7 and 8 were reported to be associated with schizophrenia (Fatami et al., 2008; Numata et al., 2009a) and depression (Numata et al., 2009b). Kähler et al. (2010) reported that SNPs, flanking the start-site of the isoform PDE4B3, were associated with schizophrenia and bipolar disorder. Our results suggested that the haplotype in introns 3–7 was associated with PD. The sex-specific analyses in our study showed that the SNP in intron 3 and haplotype were associated with PD in females, while no significant association was observed in males. Considering the high female/male ratio of PD, the gender effect such as hormonal influences on transcriptional regulation of PDE4B might be postulated in its pathophysiology. However, the gender effect was not consistent in the previous studies. The inconsistency in the results of the gender effect and positive association regions among studies may be derived from sample size, different phenotypes and ethnic groups investigated. Since risk SNPs or haplotypes were reported to be different among various populations (Panguluri et al., 2004), it is possible that different SNPs are associated with psychiatric disorders among various populations. Therefore, further association studies of PDE4B with PD using other populations would be warranted. PDE4B has been shown to encode a number of distinct isoforms such as the long isoforms PDE4B1 (Bolger et al., 1993) and PDE4B3 (Huston et al., 1997) and the short isoform PDE4B2 (Bolger et al.,
1993). The altered expression of PDE4B in psychiatric disorders was reported in several studies. Reduced expression of isoforms PDE4B2 and PDE4B4 was found in the cerebellar tissue from schizophrenia patients. A decrease in isoform PDE4B3 expression in cerebellum has been shown in postmortem tissue from patients with bipolar disorder compared with controls (Fatemi et al., 2008). Numata et al. (2009b) reported that higher expression of the PDE4B mRNA in the leukocytes in the drug-naïve depression patients was observed compared with controls and the expression in patients decreased after antidepressant treatment. However, how these isoforms are regulated, their distinct biological function, and their relationship to disease state are still unclear. In our study, the haplotype showing significant association with PD located at the 5′-flanking region of the splice site of PDE4B2. The haplotype C-T-T-A is significantly more prevalent in PD compared with controls (20.7% in cases vs. 12.5% in controls). Investigation of changes in the expression of PDE4B after antidepressant treatment using PD patients would be important to clarify the involvement of PDE4B in PD. Several lines of studies suggest that PDE4 should be considered a prime target for therapeutic treatment in psychiatric disorders including depression and impaired cognition (Zhang, 2009; Rose et al., 2005). The selective inhibitor of PDE4 rolipram has been shown to produce antidepressant- and anxiolytic-like and memory-enhancing effects in animals (Li et al., 2009; Blockland et al., 2005). Chronic treatment with antidepressants such as SSRIs decreased the expression of PDE4B in the mouse hippocampus (Dlaboga et al., 2006). Inhibition of PDE4B with rolipram increased levels of cAMP and phosphorylation of CREB, resulting in neurogenesis in mice (Li et al., 2009). Dysfunction of fear-relevant memory has been proposed as a risk factor for the development of PD (Berksun, 1999). Therefore, it would be speculated that inhibition of PDE4B might improve fearrelevant memory and decrease anxiety (Fig. 2). Although there are no selective inhibitors of individual PDE4 subtypes, PDE4 inhibitors may be effective in treating psychiatric disorders including anxiety disorders. A number of limitations merit consideration. First, sample size might not be adequate. In the present sample size, the statistical power was calculated as 0.85 at the level of α = 0.05 (minor allele frequency = 0.25, odds ratio = 1.7). We might miss loci with statistically smaller effects than OR = 1.7. Second, the effect of
Fig. 2. Scheme of the role of PDE4B in the action of anxiolytic drugs. cAMP is synthesized from ATP by adenylyl cyclase (AC) which is activated by G protein-coupled receptors (GPCRs). The enzyme PDE4 catalyzes cAMP into AMP. Administration of PDE4 inhibitors (such as rolipram) and antidepressants (SSRIs) reduces the function of PDE4B, which increases intracellular concentration of cAMP and phospholylation of CREB in the hippocampus and prefrontal cortex. The activation of cAMP/CREB signaling increases neurogenesis in the hippocampus and produces antidepressant and anxiolytic effects. ATP, adenosine triphosphate.
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population stratification must be taken into account. However, all subjects were recruited in the central area of Japan. The effect of population stratification may be small because population from the main islands in Japan has been reported to be homogenous (Yamaguchi-Kabata et al., 2008). Third, gender and age are not matched between cases and controls. Though no significant difference of gender ratio was observed between the two groups, significant differences of age were observed. The differences of the condition between samples may affect results. Fourth, since family history of PD was explored only in a limited number of patients, TDT of trio samples was not performed in our study. Replication of our findings in investigations of triads or families using TDT would be important. Finally, although controls were checked to exclude history of major psychiatric illness, family history of PD was not screened among controls. Considering high relative risk among first-degree relatives of PD patients, this may have decreased sensitivity. However, given the relatively low prevalence of PD (b2–3%), it was less likely that inclusion of controls with family history of PD has major effect on statistical power. 5. Conclusions Our results suggest that PDE4B may play a role in the pathophysiology of PD in the Japanese population. Replication studies using larger samples will be needed for more reliable conclusions. Our findings could help explain in part the complex pathogenesis of PD and suggest new candidate for therapeutic target for PD. Supplementary materials related to this article can be found online at doi:10.1016/j.pnpbp.2010.12.013. Acknowledgements This work was supported by research grant from the Japan Ministry of Education, Culture, Sports, Science and Technology (No. 17019029). TO was supported by research fellowship from the Japan Society for the Promotion of Science (No. 21-8373). References Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263–5. Berksun OE. Panic disorder and memory: does panic disorder result from memory dysfunction? Eur Psychiatry 1999;14:54–6. Blockland A, Schreiber R, Prickaerts J. Improving memory: a role for phosphodiesterases. Curr Pharm Des 2005;11:3329–34. Bolger G, Michaeli T, Martins T, St John T, Steiner B, Rodgers L, et al. A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs. Mol Cell Biol 1993;13:6558–71. Charney DS, Deutch A. A functional neuroanatomy of anxiety and fear: implications for the pathophysiology and treatment of anxiety disorders. Crit Rev Neurobiol 1996;10:419–46. Cherry JA, Davis RL. Cyclic AMP phosphodiesterases are localized in regions of the mouse brain associated with reinforcement, movement, and affect. J Comp Neurol 1999;407:287–301. Cherry JA, Thompson BE, Pho V. Diazepam and rolipram differentially inhibit cyclic AMP-specific phosphodiesterases PDE4A1 and PDE4B3 in the mouse. Biochim Biophys Acta 2001;1518:27–35. Crowe RR, Noyes R, Pauls DL, Slymen D. A family study of panic disorder. Arch Gen Psychiatry 1983;40:1065–9. Davis RL, Cherry J, Dauwalder B, Han PL, Skoulakis E. The cyclic AMP system and Drosophila learning. Mol Cell Biochem 1995;149–150:271–8. Dlaboga D, Hajjhussein H, O'Donnell JM. Regulation of phosphodiesterase-4 (PDE4) expression in mouse brain by repeated antidepressant treatment: comparison with rolipram. Brain Res 2006;1096:104–12.
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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Association study of PDE4B with panic disorder in the Japanese population Takeshi Otowa a,b,⁎, Yoshiya Kawamura a, Nagisa Sugaya c, Eiji Yoshida d, Takafumi Shimada a, Xiaoxi Liu e, Mamoru Tochigi a, Tadashi Umekage a, Taku Miyagawa e, Nao Nishida e, Hisanobu Kaiya d,f, Yuji Okazaki g, Katsushi Tokunaga e, Tsukasa Sasaki h a
Department of Neuropsychiatry, Graduate School of Medicine, the University of Tokyo, Tokyo 113-8655, Japan Department of Psychiatry, Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA Faculty of Human Science, Waseda University, Saitama 359-1192, Japan d Outpatient Clinic for Anxiety Disorders, Akasaka Mental Clinic, Tokyo 107-0052, Japan e Department of Human Genetics, Graduate School of Medicine, the University of Tokyo, Tokyo 113-8655, Japan f Resarch Center for Panic Disorder, Nagoya Mental Clinic, Aichi 453-0015, Japan g Department of Neurology, Tokyo Metropolitan Matsuzawa Hospital, Tokyo 156-0057, Japan h Office for Mental Health Support and Graduate School of Education, University of Tokyo, Tokyo 113-8625, Japan b c
a r t i c l e
i n f o
Article history: Received 4 November 2010 Received in revised form 13 December 2010 Accepted 15 December 2010 Available online 22 December 2010 Keywords: Association Japanese population Panic disorder Phosphodiesterase 4B (PDE4B) Single nucleotide polymorphism
a b s t r a c t Background: Panic disorder (PD) is a severe and chronic psychiatric disorder with genetic components underlying in its etiology. The Phosphodiesterase 4B (PDE4B) gene has been reported to be associated with several psychiatric disorders. Several studies indicated that PDE4B may be involved in the regulation of anxiety and depression. Therefore, we investigate the association of PDE4B with PD in the Japanese population. Methods: We genotyped 14 single nucleotide polymorphisms (SNPs) of PDE4B in 231 PD cases (85 males and 146 females) and 407 controls (162 males and 245 females). Differences in the genotype, allele and haplotype frequencies between the two groups were compared. Results: We found a significant association between PDE4B and PD in the haplotype analysis (haplotype C-T-T-A, permutation P = 0.031, OR = 1.81, 95% CI = 1.30–2.51). Sex-specific analyses demonstrated that PDE4B was associated with PD in females in the allele/genotype and haplotype analyses (rs10454453, allele P = 0.042, genotype P = 0.0034; haplotype C-T-T-A, permutation P = 0.028). Conclusion: Our results suggest that PDE4B may play a role in the pathophysiology of PD in the Japanese population. Replication studies using larger samples will be needed for more reliable conclusions. © 2010 Elsevier Inc. All rights reserved.
1. Introduction PD is an anxiety disorder characterized by panic attacks and anticipatory anxiety, with a life-time prevalence of 1–3% and a female: male ratio of 2:1 (Eaton et al., 1994). PD frequently takes a chronic course, with many remissions and relapses, occasionally complicated by comorbidity with other psychiatric disorders, such as agoraphobia and major depression (Weissman et al., 1997). It is generally accepted that panic disorder has genetic as well as environmental causes. A 2.6to 20-fold relative risk in the first-degree relatives of probands with Abbreviations: PD, panic disorder; cAMP, cyclic adenosine monophosphate; PDE4B, phosphodiesterase 4B; DISC1, Disrupted in Schizophrenia 1; SNP, single nucleotide polymorphism; MINI, Mini-International Neuropsychiatric Interview; SSRI, serotonin selective reuptake inhibitor; CREB, cAMP response element binding protein; LD, linkage disequilibrium; TDT, transmission disequilibrium test; SD, standard deviation; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author. Department of Psychiatry, Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, 800 East Leigh Street, Richmond, VA 23298-0126, USA. Tel.: + 1 804 922 2606; fax: + 1 804 828 1471. E-mail address: [email protected] (T. Otowa). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.12.013
PD compared to the general population suggests a familial component in this disorder (Crowe et al., 1983; Goldstein et al., 1997). Twin studies show that about 40% of the liability towards PD consists of heritable factors (Kendler et al., 1993; Hettema et al., 2001). Thus far, however, the etiology of PD is currently unknown. The phosphodiesterases regulate intracellular concentrations of cAMP, a second messenger implicated in learning, memory and mood (Davis et al., 1995; O'Donnell and Frith, 1999). PDE4B is encoded by a gene of approximately 580 kb located on chromosome 1p31.2, consisting of 17 exons. Millar et al. (2000) reported that PDE4B was disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with a chronic psychiatric disorder. PDE4B binds DISC1 that is a candidate susceptibility factor for psychiatric disorders including schizophrenia, schizoaffective disorder, bipolar disorder and depression (Millar et al., 2005). Recently, case–control association studies reported associations between PDE4B and schizophrenia (Pickard et al., 2007; Fatemi et al., 2008; Numata et al., 2009a; Kähler et al., 2010), bipolar disorder (Kähler et al., 2010) and depression (Numata et al., 2009b). The expression of PDE4B isoforms in postmortem brain tissue from patients with schizophrenia
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or bipolar disorder has been shown to differ compared with controls (Fatemi et al., 2008). Several studies indicated that PDE4B may be involved in the regulation of anxiety and depression. PDE4 inhibitor rolipram produced antidepressant-like (O'Donnell and Frith, 1999) and anxiolytic-like behaviors in animals (Silvestre et al., 1999; Li et al., 2009). PDE4B is expressed in the amygdala, hypothalamus and frontal cortex (Cherry and Davis, 1999), which are key regions in the mediation of anxiety and stress responses (Charney and Deutch, 1996). It has been shown that cAMP signaling regulates anxiety-like behavior (Pandey et al., 2005). PDE4B may play a role in this process as a critical controller of this signaling pathway (Li et al., 2009). Chronic treatment with antidepressants decreases the expression of PDE4B in the hippocampus and increases neurogenesis in mice (Dlaboga et al., 2006; Malberg et al., 2000). Furthermore, benzodiazepine anxiolytic diazepam inhibits the expression of PDE4B (Cherry et al., 2001). In the light of the findings mentioned above, we investigated the association between PDE4B and PD in the Japanese population. In the present study, we investigated 14 SNPs of PDE4B in Japanese 231 PD cases and 407 controls. 2. Materials and methods 2.1. Subjects All cases and control subjects were ethnically Japanese and were recruited in the vicinity of Tokyo, Japan. Subjects comprised 231 unrelated Japanese with PD recruited from a clinic for anxiety (85 males and 146 females; age = 37.3 ± 8.8 years (mean ± SD)) and 407 unrelated healthy volunteers (162 males and 245 females; age = 39.7 ± 10.6 years) served as controls. The diagnosis of PD was confirmed according to DSM-IV criteria and by using the MINI (Sheehan et al., 1998) and clinical records. The controls received a short interview by one of the authors and filled out questionnaires to exclude history of major psychiatric illness. At the time of blood collection for the present study, most of the patients were treated with antidepressants such as SSRIs. The objective of the present study was clearly explained and written informed consent was obtained from all subjects. The study was approved by the Ethical Committee of the Graduate School of Medicine, the University of Tokyo. 2.2. Genotyping Genomic DNA was extracted from leukocytes by using the standard phenol-chloroform method. We selected 10 tagging SNPs (rs4077429, rs2840677, rs1937456, rs1937443, rs1354061, rs6588186, rs10454453, rs6588190, rs502958 and rs472952) with minor allele frequencies of 10% more in the Japanese population (HapMap database: www.hapmap.org), using HaploView 4.2 program (Barrett et al., 2005). The LD blocks were defined by the Gabriel method (Gabriel et al., 2002). We further added four SNPs (rs1317611, rs2503166, rs1040716 and rs2180335) investigated in
the previous studies (Pickard et al., 2007; Fatemi et al., 2008; Numata et al., 2009a, 2009b). The location of the SNPs assayed and their relationship to the exon/intron boundaries of the gene are shown in Fig. 1. Genotyping was performed using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol with ABI PRISM 7900 SDS2 Software (Applied Biosystems, Foster City, CA). 2.3. Statistical analysis Hardy–Weinberg equilibrium (HWE) was checked using chisquare analysis. Comparisons of groups between cases and controls with respect to allele and genotype frequencies were undertaken using chi-square tests. Genotype data were analyzed using UNPHASED ver. 3.1.4 (Dudbridge, 2008) to derive P-values for single-marker and global and individual haplotype tests. To maximize the genetic information extracted from the genotyped SNPs, two-, three- and four-marker sliding-window haplotype analyses were conducted for global haplotype tests. The expectation-maximization (EM) algorithm was used to estimate haplotype frequencies and the log-likelihood ratio test under a log-linear model was conducted. Permutation tests were performed using 10,000 iterations (random permutation). It adjusts the global P-value of the particular haplotype block tested within any window size or P-value of the individual haplotype tested within the particular haplotype block. ORs with 95% CIs were calculated relative to all other haplotypes pooled. Power calculation was performed using Genetic Power Calculator (Purcell et al., 2003). Significance for the result was set at P b 0.05. 3. Results The genotype and allele frequencies of the 14 SNPs are summarized in Table 1. Genotype frequency of SNP8 (rs2503166) in cases deviates from HWE (P = 0.028). Genotype frequencies of all SNPs in controls were within HWE. Significant differences were found in the allele and genotype frequencies of SNP9 (rs10454453) between cases and controls (Table 1). However, these associations did not reach statistical significance after Bonferroni correction. No significant difference was observed in the genotype or allele frequency of the other SNPs between cases and controls. When the cases with agoraphobia were studied, no significant difference was observed (Table 1). Considering the higher prevalence of PD in females and sexdependent findings in the previous association studies, sex-specific analyses would be necessary (Maron et al., 2010). When the data were subdivided on the basis of sex, significant associations of SNP9 (rs10454453) with PD were found in females after Bonferroni correction (allele P = 0.042, genotype P = 0.0034, corrected; Supplementary Table 1). In the haplotype analyses, nominally significant haplotype associations with PD were observed (Table 2). However, only the four-SNP haplotype (SNPs 9–12) remained significant after correction for multiple testing (global permutation P = 0.014). Individual
Fig. 1. The genomic structure of PDE4B and the locations of 14 SNPs selected for the association analysis.
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Table 1 Genotype distributions and allele frequencies of the PDE4B gene. SNP ID
M/ma
Phenotype
N
MAFb
P-value
Genotypec
P-value
Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls Cases Agoraphobiad Controls
231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407 231 99 407
0.310 0.359 0.343 0.292 0.278 0.275 0.455 0.480 0.500 0.429 0.439 0.469 0.485 0.470 0.439 0.338 0.374 0.385 0.435 0.409 0.391 0.390 0.394 0.360 0.437 0.419 0.366 0.329 0.308 0.310 0.435 0.409 0.404 0.217 0.242 0.259 0.186 0.207 0.226 0.204 0.222 0.217
0.225 0.674
105/109/17 38/51/10 177/181/49 120/87/24 51/41/7 218/154/35 67/118/46 22/59/18 105/197/105 72/120/39 27/57/15 116/200/91 59/120/52 23/59/17 131/195/81 99/108/24 37/50/12 152/197/58 73/115/43 33/51/15 142/212/53 94/94/43 41/38/20 176/169/62 78/104/49 34/47/18 160/196/51 105/100/26 49/39/11 195/172/40 73/115/43 34/49/16 144/197/66 140/82/9 54/42/3 227/149/31 154/68/9 61/35/3 249/132/26 148/72/11 60/34/5 255/127/25
0.175 0.448
SNP1
rs4077429
A/G
SNP2
rs2840677
A/T
SNP3
rs1937456
G/A
SNP4
rs1317611
G/C
SNP5
rs1937443
C/G
SNP6
rs1354061
C/T
SNP7
rs6588186
C/T
SNP8
rs2503166
C/T
SNP9
rs10454453
A/C
SNP10
rs6588190
C/T
SNP11
rs502958
A/T
SNP12
rs1040716
A/T
SNP13
rs2180335
C/T
SNP14
rs472952
C/T
0.516 0.942 0.118 0.610 0.160 0.449 0.111 0.429 0.095 0.780 0.121 0.634 0.292 0.373 0.0124e 0.167 0.474 0.967 0.282 0.900 0.087 0.627 0.094 0.565 0.557 0.884
0.747 0.759 0.231 0.118 0.252 0.209 0.207 0.102 0.231 0.847 0.158 0.848 0.529 0.480 0.0135e 0.303 0.773 0.849 0.556 0.978 0.142 0.199 0.248 0.412 0.762 0.794
Statistical analysis was performed using chi-square test. a M; major allele, and m: minor allele. b MAF: minor allele frequency. c Described as major homo/hetero/minor homo. d Cases with agoraphobia were compared with controls. e Bold numbers represent nominally significant P-value.
Table 2 Haplotype analysis of the PDE4B gene. SNP ID
Global haplotype P-valuea,b 2SNP
3SNP
4SNP
SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 SNP8 SNP9 SNP10 SNP11 SNP12 SNP13 SNP14
0.514 0.482 0.484 0.139 0.319 0.251 0.388 0.023 0.014 0.146 0.090 0.350 0.011
0.594 0.592 0.314 0.432 0.530 0.600 0.03 0.01 0.027 0.005 0.281 0.073
0.615 0.334 0.612 0.607 0.249 0.125 0.05 0.040 0.001c 0.008 0.095
a
P-values were calculated by log-likelihood ratio test. Bold numbers represent nominally significant P-value. c Haplotype showed significance after correction for multiple testing (permutation P = 0.014). b
haplotype analysis of the four-SNP haplotype (SNPs 9–12) is shown in Table 3. Significant difference was observed in frequency of the haplotype C-T-T-A between cases and controls (P = 3.48 × 10− 4, OR = 1.81, 95% CI = 1.30–2.51). This haplotype remained positive after correction for multiple testing by permutation (permutation P = 0.031). In the sex-specific analyses, the haplotype C-T-T-A showed a significant association with PD in females after correction for multiple testing (permutation P = 0.028; Supplementary Table 2). Table 3 Haplotype analysis among PD cases and controls. SNPs 9–12 haplotype A-C-A-A C-T-T-A C-T-T-T A-C-A-T C-C-A-A a b
Frequencya Cases
Controls
Chi-square
P-valueb
0.428 0.207 0.088 0.065 0.060
0.443 0.125 0.111 0.087 0.050
0.38 12.79 0.91 2.49 1.47
0.54 3.48E-04 0.34 0.11 0.22
Haplotypes whose frequencies were estimated N 5% were described. Bold number represents significant P-value (permutation P = 0.031).
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4. Discussion In the present study, we investigated the association between PDE4B and PD in the Japanese population. In the single SNP analyses, no association was found with PD. In the haplotype analyses, the fourSNP haplotype (SNPs 9–12) showed an association with PD (global permutation P = 0.014). The individual haplotype C-T-T-A showed an association with PD (permutation P = 0.031). Sex-specific analyses showed that the SNP rs10454453 and the haplotype C-T-T-A were associated with PD in females (rs10454453, allele P = 0.042, genotype P = 0.0034 corrected; haplotype C-T-T-A, permutation P = 0.028). PDE4B has been proposed as a candidate gene in psychiatric disorders including schizophrenia, bipolar disorder and depression. Our results demonstrated that PDE4B was associated with PD. However, the SNP and haplotype showing association with PD are different from those in the previous studies. Pickard et al. (2007) reported that haplotypes within intron 3 were associated with schizophrenia in females. The SNPs and haplotypes in introns 7 and 8 were reported to be associated with schizophrenia (Fatami et al., 2008; Numata et al., 2009a) and depression (Numata et al., 2009b). Kähler et al. (2010) reported that SNPs, flanking the start-site of the isoform PDE4B3, were associated with schizophrenia and bipolar disorder. Our results suggested that the haplotype in introns 3–7 was associated with PD. The sex-specific analyses in our study showed that the SNP in intron 3 and haplotype were associated with PD in females, while no significant association was observed in males. Considering the high female/male ratio of PD, the gender effect such as hormonal influences on transcriptional regulation of PDE4B might be postulated in its pathophysiology. However, the gender effect was not consistent in the previous studies. The inconsistency in the results of the gender effect and positive association regions among studies may be derived from sample size, different phenotypes and ethnic groups investigated. Since risk SNPs or haplotypes were reported to be different among various populations (Panguluri et al., 2004), it is possible that different SNPs are associated with psychiatric disorders among various populations. Therefore, further association studies of PDE4B with PD using other populations would be warranted. PDE4B has been shown to encode a number of distinct isoforms such as the long isoforms PDE4B1 (Bolger et al., 1993) and PDE4B3 (Huston et al., 1997) and the short isoform PDE4B2 (Bolger et al.,
1993). The altered expression of PDE4B in psychiatric disorders was reported in several studies. Reduced expression of isoforms PDE4B2 and PDE4B4 was found in the cerebellar tissue from schizophrenia patients. A decrease in isoform PDE4B3 expression in cerebellum has been shown in postmortem tissue from patients with bipolar disorder compared with controls (Fatemi et al., 2008). Numata et al. (2009b) reported that higher expression of the PDE4B mRNA in the leukocytes in the drug-naïve depression patients was observed compared with controls and the expression in patients decreased after antidepressant treatment. However, how these isoforms are regulated, their distinct biological function, and their relationship to disease state are still unclear. In our study, the haplotype showing significant association with PD located at the 5′-flanking region of the splice site of PDE4B2. The haplotype C-T-T-A is significantly more prevalent in PD compared with controls (20.7% in cases vs. 12.5% in controls). Investigation of changes in the expression of PDE4B after antidepressant treatment using PD patients would be important to clarify the involvement of PDE4B in PD. Several lines of studies suggest that PDE4 should be considered a prime target for therapeutic treatment in psychiatric disorders including depression and impaired cognition (Zhang, 2009; Rose et al., 2005). The selective inhibitor of PDE4 rolipram has been shown to produce antidepressant- and anxiolytic-like and memory-enhancing effects in animals (Li et al., 2009; Blockland et al., 2005). Chronic treatment with antidepressants such as SSRIs decreased the expression of PDE4B in the mouse hippocampus (Dlaboga et al., 2006). Inhibition of PDE4B with rolipram increased levels of cAMP and phosphorylation of CREB, resulting in neurogenesis in mice (Li et al., 2009). Dysfunction of fear-relevant memory has been proposed as a risk factor for the development of PD (Berksun, 1999). Therefore, it would be speculated that inhibition of PDE4B might improve fearrelevant memory and decrease anxiety (Fig. 2). Although there are no selective inhibitors of individual PDE4 subtypes, PDE4 inhibitors may be effective in treating psychiatric disorders including anxiety disorders. A number of limitations merit consideration. First, sample size might not be adequate. In the present sample size, the statistical power was calculated as 0.85 at the level of α = 0.05 (minor allele frequency = 0.25, odds ratio = 1.7). We might miss loci with statistically smaller effects than OR = 1.7. Second, the effect of
Fig. 2. Scheme of the role of PDE4B in the action of anxiolytic drugs. cAMP is synthesized from ATP by adenylyl cyclase (AC) which is activated by G protein-coupled receptors (GPCRs). The enzyme PDE4 catalyzes cAMP into AMP. Administration of PDE4 inhibitors (such as rolipram) and antidepressants (SSRIs) reduces the function of PDE4B, which increases intracellular concentration of cAMP and phospholylation of CREB in the hippocampus and prefrontal cortex. The activation of cAMP/CREB signaling increases neurogenesis in the hippocampus and produces antidepressant and anxiolytic effects. ATP, adenosine triphosphate.
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population stratification must be taken into account. However, all subjects were recruited in the central area of Japan. The effect of population stratification may be small because population from the main islands in Japan has been reported to be homogenous (Yamaguchi-Kabata et al., 2008). Third, gender and age are not matched between cases and controls. Though no significant difference of gender ratio was observed between the two groups, significant differences of age were observed. The differences of the condition between samples may affect results. Fourth, since family history of PD was explored only in a limited number of patients, TDT of trio samples was not performed in our study. Replication of our findings in investigations of triads or families using TDT would be important. Finally, although controls were checked to exclude history of major psychiatric illness, family history of PD was not screened among controls. Considering high relative risk among first-degree relatives of PD patients, this may have decreased sensitivity. However, given the relatively low prevalence of PD (b2–3%), it was less likely that inclusion of controls with family history of PD has major effect on statistical power. 5. Conclusions Our results suggest that PDE4B may play a role in the pathophysiology of PD in the Japanese population. Replication studies using larger samples will be needed for more reliable conclusions. Our findings could help explain in part the complex pathogenesis of PD and suggest new candidate for therapeutic target for PD. Supplementary materials related to this article can be found online at doi:10.1016/j.pnpbp.2010.12.013. Acknowledgements This work was supported by research grant from the Japan Ministry of Education, Culture, Sports, Science and Technology (No. 17019029). TO was supported by research fellowship from the Japan Society for the Promotion of Science (No. 21-8373). References Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263–5. Berksun OE. Panic disorder and memory: does panic disorder result from memory dysfunction? Eur Psychiatry 1999;14:54–6. Blockland A, Schreiber R, Prickaerts J. Improving memory: a role for phosphodiesterases. Curr Pharm Des 2005;11:3329–34. Bolger G, Michaeli T, Martins T, St John T, Steiner B, Rodgers L, et al. A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs. Mol Cell Biol 1993;13:6558–71. Charney DS, Deutch A. A functional neuroanatomy of anxiety and fear: implications for the pathophysiology and treatment of anxiety disorders. Crit Rev Neurobiol 1996;10:419–46. Cherry JA, Davis RL. Cyclic AMP phosphodiesterases are localized in regions of the mouse brain associated with reinforcement, movement, and affect. J Comp Neurol 1999;407:287–301. Cherry JA, Thompson BE, Pho V. Diazepam and rolipram differentially inhibit cyclic AMP-specific phosphodiesterases PDE4A1 and PDE4B3 in the mouse. Biochim Biophys Acta 2001;1518:27–35. Crowe RR, Noyes R, Pauls DL, Slymen D. A family study of panic disorder. Arch Gen Psychiatry 1983;40:1065–9. Davis RL, Cherry J, Dauwalder B, Han PL, Skoulakis E. The cyclic AMP system and Drosophila learning. Mol Cell Biochem 1995;149–150:271–8. Dlaboga D, Hajjhussein H, O'Donnell JM. Regulation of phosphodiesterase-4 (PDE4) expression in mouse brain by repeated antidepressant treatment: comparison with rolipram. Brain Res 2006;1096:104–12.
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