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Association of a Met88Val diazepam binding inhibitor (DBI) gene polymorphism and anxiety disorders with panic attacks
JOURNAL OF PSYCHIATRIC RESEARCH
Journal of Psychiatric Research 41 (2007) 579–584
www.elsevier.com/locate/jpsychires
Association of a Met88Val diazepam binding inhibitor (DBI) gene polymorphism and anxiety disorders with panic attacks Christoph K. Thoeringer a, Elisabeth B. Binder a,b, Daria Salyakina a, Angelika Erhardt a, Marcus Ising a, Paul G. Unschuld a, Nikola Kern a, Susanne Lucae a, Tanja M. Brueckl a, Marianne B. Mueller a, Brigitte Fuchs a, Benno Puetz a, Roselind Lieb a, Manfred Uhr a, Florian Holsboer a, Bertram Mueller-Myhsok a, Martin E. Keck a,c,* a
Max Planck Institute of Psychiatry, Munich 80804, Germany Emory University School of Medicine, Atlanta, GA 30322, USA University of Zurich, Division of Psychiatry Research, Minervastr. 145, Zurich 8032, Switzerland b
c
Received 20 January 2006; received in revised form 31 May 2006; accepted 8 June 2006
Abstract Several lines of evidence suggest that anxiety disorders have a strong genetic component, but so far only few susceptibility genes have been identified. There is preclinical and clinical evidence for a dysregulation of the central c-aminobutyric acid (GABA)-ergic tone in the pathophysiology of anxiety disorders. Diazepam binding inhibitor (DBI) has been suggested to play a pivotal role in anxiety disorders through direct and indirect, i.e. via synthesis of neuroactive steroids, modulation of GABAA receptor function. These findings suggest that the DBI gene can be postulated as a candidate for a genetic association study in this disorder. Thus, single nucleotide polymorphisms (SNPs) of the DBI gene were investigated for putative disease associations in a German sample of anxiety disorder patients suffering from panic attacks and matched controls. We were able to detect a significant association between a non-synonymous coding variant of DBI with anxiety disorders with panic attacks. The rare allele of this polymorphism was more frequent in controls than in patients (OR = 0.43; 95% CI: 0.19–0.95). In conclusion, these results suggest a central role of DBI genetic variants in the susceptibility for the development of anxiety disorders that are characterized by the occurrence of panic attacks. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Diazepam binding inhibitor (DBI); Case-control genetic association study; Single nucleotide polymorphisms; Panic disorder
1. Introduction The c-aminobutyric acid (GABA) system has repeatedly been implicated in the pathophysiology of anxiety disorders and related psychiatric diseases. It has been hypothesized for instance, that panic disorder might be due to abnormal activity of endogenous ligands of the benzodiazepine receptor (Nutt et al., 1990). This hypothesis is emphasized by the * Corresponding author. Address: University of Zurich, Division of Psychiatry Research, Minervastr. 145, Zurich 8032, Switzerland. Tel.: +41 44 389 1477; fax: +41 44 389 1414. E-mail address: [email protected] (M.E. Keck).
0022-3956/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2006.06.001
clinical efficacy of positive allosteric modulators of GABAA receptors, such as benzodiazepines, promoting rapid and profound anxiolysis in patients suffering from affective disorders (Brambilla et al., 2003). Apart from exogenous modulators of the GABAA/benzodiazepine receptor, an endogenous inverse peptide agonist, the diazepam binding inhibitor (DBI), has been described (Costa and Guidotti, 1991). This 9-kD endozepine is located in peripheral tissues and in neuronal and glial cells of the central nervous system with an abundant expression in the hypothalamus, amygdala, cortex and cerebellum. One of DBI’s major biological actions comprises the displacement of diazepam from GABAA receptors resulting in a negative modulation of
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GABA channel gating. In addition, DBI acts at the mitochondrial benzodiazepine receptor of glial (and peripheral steroidogenic) cells facilitating the transport of fatty acids to the inner mitochondrial membrane and, thus, the formation of cholesterol and other, e.g. neuroactive, steroids (Zisterer and Williams, 1997). These neurosteroids, in turn, have been shown to facilitate activation of GABAA receptor function (Rupprecht and Holsboer, 1999). Along these lines, studies in patients with panic disorder suggest that 3a-reduced neuroactive steroids may play a pivotal role in human anxiety in that they may serve as a counter-regulatory mechanism against the occurrence of spontaneous panic attacks (Stroehle et al., 2003). With respect to either direct or indirect behavioral effects of DBI, intraventricular, but not intraseptal, administrations of the endozepine have been reported to induce anxiety-related behavior in laboratory animals (Guidotti, 1991; Herzog et al., 1996). Notably, genetic differences in brain DBI concentrations have been correlated with high and low anxiety levels in rats (Sudakov et al., 2001). In addition, recent clinical studies suggest that the inverse agonist action of DBI on the GABAA/benzodiazepine receptor may severely impact the development and severity of anxiety disorders and depression, as DBI has been found to be increased in the cerebrospinal fluid (CSF) of patients diagnosed with depression with (Costa and Guidotti, 1991) and without concomitant anxiety (Roy, 1991). Many studies have shown that anxiety disorders have a significant familial aggregation which has been largely explained by genetic components. At this point, it is not entirely clear whether this susceptibility predisposes to the development of a specific anxiety disorder or to any one of the anxiety disorders such as panic disorder with or without agoraphobia, social phobia and specific phobia (Villafuerte and Burmeister, 2003). Panic attacks are circumscribed episodes of severe state anxiety lasting minutes to hours with escalating symptoms. The features are associated with an array of physical symptoms of primarily sympathetic arousal. Panic attacks are the hallmark of panic disorder but also occur regularly in association with other anxiety disorders such as, e.g., social phobia and specific phobia. With respect to the genetic aetiopathogenesis of panic, a recent meta-analysis of data from family and twin studies by Hettema et al. (2001) reported an estimated heritability of 43% for panic disorder. Linkage studies focusing on panic disorder have identified some loci of potential interest but, so far, yielded only inconsistent results (Finn et al., 2003). For example, suggestive loci on chromosome 9q31 have been detected in a genome-wide linkage study of anxiety with panic disorder (Thorgeirsson et al., 2003), and chromosome 13q and 22 have been implicated in a linkage study of a panic disorder syndrome (Hamilton et al., 2003). However, no significant linkage has been reported for chromosome 2p where the DBI gene is located. As the genetic basis of anxiety does not follow a simple pattern of Mendelian inheritance, it is considered that
many genes, all of a small effect, contribute to the disease susceptibility. Numerous association studies have been performed focusing on candidate genes of neurochemical systems and components. For instance, polymorphisms in the gene encoding for the catechol-O-methyltransferase have been reported to be associated with panic disorder (Rothe et al., 2006; Woo et al., 2002); or, a short tandem repeat in the promoter region of the cholecystokinine gene was significantly associated with panic disorder (Hattori et al., 2001). The gene encoding for DBI is located on human chromosome 2p14.2 and comprises several polymorphic regions. With respect to psychiatric genetics, Niu et al. (2004) performed the only case-control association analysis so far using three DBI polymorphisms in 317 schizophrenic patients, but failed to detect a disease association. There is, however, strong evidence suggesting a pathophysiological role of endozepines in anxiety disorders. We, therefore, performed a case-control analysis of DBI genetic variants in a sample of German anxiety disorder patients characterized by the occurrence of panic attacks. 2. Materials and methods 2.1. Patients The present study consists of a dual case-control approach. We, first, investigated a primary study sample including a total number of 126 panic disorder patients with and without agoraphobia (see Table 1) and screened for case-control associations of the diagnosis panic disorder with five single nucleotide polymorphisms (SNPs) of Table 1 Phenotypic characteristics of the case – control samples Panic disorder patients Primary study sample Total number With agoraphobia Without agoraphobia Male:female ratio Mean age at admission Mean age at onset of PD Mean panic scale score
Control subjects 126 88.5% 11.5% 1:2.28 39.6 years 27.2 years 7.39
Anxiety disorder patients with panic attacks Extended study sample Total number Agoraphobia PD with agoraphobia PD without agoraphobia Social phobia Simple phobia Male:female ratio Mean age at admission Mean age at onset Mean panic scale score
176 3.6% 76.2% 13.7% 5.0% 1.7% 1:1.98 38.6 years 28.0 years 7.47
Total number Male:female ratio Mean age
229 1:2.66 40.0 years
Controls
Total number Male:female ratio Mean age
301 1:1.68 40.25 years
PD, panic disorder. Panic scale score, composed of frequency, severity and duration of panic attacks (the Panic and Agoraphobia Scale).
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the DBI gene. As reported in our study, one SNP displayed significant association with panic disorder (see below). In order to assess the impact of this genetic polymorphism on an extended anxiety phenotype, we enlarged our primary study sample with patients suffering from several forms of pathological anxiety and concomitant panic attacks. As delineated in Table 1, the second study sample consists of 176 anxiety disorder patients with primary diagnoses of agoraphobia, panic disorder with and without agoraphobia, and social or simple phobia with panic attacks. In addition, these patients had more than 2 moderate to severe panic attacks per week during their worst episode of the disease as determined by the Panic and Agoraphobia Scale (Bandelow, 1995). Patients were consecutively admitted to our Anxiety Disorders Outpatient Clinic for diagnosis and treatment of an anxiety disorder. The diagnosis was ascertained by trained psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria by use of the Structured Clinical Interviews for DSM-IV (SCID I and II) (Wittchen et al., 1997). Anxiety disorders due to a medical or neurological condition and a comorbid Axis II disorder were exclusion criteria. In addition, ethnicity was recorded using a self-reporting sheet for perceived nationality, mother language and ethnicity of the subject himself and of all four grandparents. All included individuals reported themselves to be ‘‘Caucasian’’ and 84%, and 81% for the extended sample respectively, were of German origin. 2.2. Controls An overall number of 301 subjects was recruited as controls and matched for ethnicity, gender and age (see Table 1). All individuals were randomly selected from a Munichbased community sample and screened for the presence of anxiety and affective disorders using the Composite International Diagnostic-Screener (CID-S) (Wittchen et al., 1999). Only individuals who were not screening positive for the above-named disorders were included in the sample. The study was approved by the local ethics committee, and written informed consent was obtained from all subjects. 2.3. DNA preparation After enrolling patients and controls in the study, we drew 40 ml of EDTA blood from each individual and extracted DNA from fresh blood using the Puregen whole blood DNA-extraction kit (Gentra Systems; Minneapolis). 2.4. SNP selection and genotyping SNP selection and genotyping was largely performed as previously described (Binder et al., 2004). Briefly, a total number of 5 single nucleotide polymorphisms (SNPs) in the DBI gene (NM_020548; 5.4 kb in length and 5 exons)
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with an average distance of 1119 bp on human chromosome 2p14.2 was selected from public databases (dbSNP; http://www.ncbi.nih.gov and CHIP Bioinformatics Tools; http://snpper.chip.org). The SNP search tool developed at the Institute for Human Genetics, Technical University and GSF-National Research Centre for Environment and Health was chosen to download SNP sequences (http:// ihg.gsf.de) using the hg17 built of the Genome Browser of the University of Santa Cruz (http://genome.ucsc.edu). Genotyping was performed on a MALDI-TOF mass spectrometer (MassArray system) using the Spectrodesigner software (Sequenom; San Diego) for primer selection and multiplexing and the homogenous mass-extension (hMe) process for producing primer extension products. Genotyping was completed at the Genetic Research Centre, Munich, Germany. The primer sequences are shown in the online supplementary materials. 2.5. Statistical analysis Hardy Weinberg equilibrium was calculated in patients and controls (see Supplementary materials). To test for population substructure in our sample, we performed analysis with STRUCTURE with 100 SNPs (http:// pritch.bsd.uchicago.edu/software.html). For the analysis of LD pattern, we used HAPLOVIEW (http://www.broad.mit.edu/mpg/haploview/using.php) applying the D’ method previously described by Gabriel et al. (2002). LD calculation was performed in our control sample. The Pearson v2 test was used to assess allele frequency difference. In order to adjust the minimum p-value for multiple testing, Fisher product method for combining tests was used as previously described (Binder et al., 2004). Briefly, if a SNP in a gene is associated with the phenotype, so might be other SNPs in the same gene due to linkage disequilibrium (LD). Thus, the five SNPs are combined into a single test which is further evaluated using permutation (we used 100,000 replicates). The Armitage trend test was additionally performed. Haplotype analyses were performed using COCAPHASE of the UNPHASED package (http://www.mrcbsu.cam.ac.uk/personal/frank/software/unphased/). These analyses were done in sliding windows of sizes 2, 3, 4 and 5. 3. Results In a first approach a total number of 5 single nucleotide polymorphisms were genotyped within the DBI gene in our primary study sample including panic disorder patients and controls. All variants turned out to be polymorphic and, thus, informative in our sample. Analyzing Hardy Weinberg equilibrium revealed no significant deviation in all polymorphisms studied (see Supplementary material). In addition, analysis with STRUCTURE showed no evidence for population substructure.
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With respect to haplotype block delineation, all five SNPs were in one LD block (see Supplementary materials). As shown in Table 2, the G allele of the DBI polymorphism rs8192506 was significantly more frequent in healthy subjects than in patients with panic disorder, indicating a significant association of this variant and panic disorder. This SNP is located in exon 5 of the gene and results in a Valine to Methionine exchange at amino acid position 88. The p-value for association between rs8192506 and panic disorder was 0.021 and became 0.0298 after adjustment for multiple testing (see Section 2 for details). The G allele was overrepresented in controls with an allelic odds ratio (OR) of 0.33 (95% CI: 0.12–0.88). The Armitage trend test showed similar values (common OR = 0.34, v2 = 5.2, p = 0.022). Three other SNPs showed a trend for an association (p 6 0.1). Even though D’ was high among all five SNPs, the r2 between rs8192506 and the other four SNPs was less than 0.2, explaining the range in the p-values for the association in SNPs located in the same LD block. Haplotype analyses showed several haplotypes to have nominally significant associations, but none exceeded the significance obtained when considering rs8192506 alone (see Supplementary materials). In a second approach, we analyzed case-control relations of the significantly associated SNP rs8192506 in an extended sample of anxiety disorder patients with panic attacks versus healthy controls. Similar to the results of the association with panic disorder, rs8192506 displayed a significant association with the extended anxiety phenotype (see Table 3). The over-representation of the G allele was still present with an allelic OR of 0.43 (95% CI: 0.19–0.95). The Armitage trend test of the extended association displayed similar values (common OR = 0.44, v2 = 4.6, p = 0.033). Table 2 Allele frequencies of DBI single nucleotide polymorphisms in patients with panic disorder and controls p-valuea
SNP
N (subjects)
Allele
rs3091405 Patients Controls
124 225
A (%) 26.6 32.4
C (%) 73.4 67.6
0.109
rs3769664 Patients Controls
126 227
C (%) 81.3 75.8
T (%) 18.7 24.2
0.087
rs3769662 Patients Controls
125 227
C (%) 93.2 95.8
G (%) 6.8 4.2
0.132
rs956309 Patients Controls
125 228
C (%) 80.8 74.7
T (%) 19.2 25.3
0.079
rs8192506 Patients Controls
126 229
A (%) 98.0 94.3
G (%) 2.0 5.7
0.021b
a 2 v -test; control subjects versus panic disorder patients; p-values uncorrected. b The frequency of the G allele was significantly higher in control subjects than in patients (v2 = 5.31; p = 0.021; p = 0.0298 after correction).
Table 3 Allele frequencies of the rs8192506 DBI single nucleotide polymorphism in an extended sample of anxiety disorder patients and controls rs8192506
N (subjects)
A (%)
G (%) Allele
p valuea
Anxiety Controls
176 301
97.7 94.8
2.3 5.2
0.032
a
v2-test; control subjects versus anxiety disorder patients (v2 = 4.61).
Analyzing the impact of the rs8192506 genetic polymorphism on the diagnosis of panic disorder with and without agoraphobia alone in the enlarged patient sample (N = 158), the p-value was 0.064 according to the Armitage trend test. 4. Discussion The present candidate gene association study in a sample carefully diagnosed as having an anxiety disorder associated with panic attacks reveals that polymorphisms within the DBI gene are associated with the diagnosis of panic disorder and, in general, anxiety disorders with panic attacks. The G allele of the exonic SNP rs8192506 is overrepresented in controls versus patients with a frequency almost three times higher in healthy subjects than in anxiety patients. The protective effect of that allele has an OR of 0.33 (95% CI: 0.12–0.88) and an OR of 0.43 (95% CI: 0.19–0.95), respectively. Intriguingly, this genetic polymorphism leads to an amino acid change (Valine to Methionine) at amino acid position 88 and, thus, potentially to functional differences in DBI activity. At this point of the analysis, however, we cannot rule out that this SNP is only a marker of LD with the causal SNP. The analysis of the haplotype block delineation revealed a strong intragenic LD. Importantly, one has to note that the inspection of LD pattern in Caucasian populations as presented in Hapmap project data (see www.hapmap.org) indicates that there is no LD between SNPs of the DBI gene and the 3 0 located genes which encode the transmembrane protein 37 and the secretin receptor precursor. This would suggest that the observed association is indeed with DBI and not neighbouring genes. Further fine mapping and functional studies are necessary to resolve that issue. We have to acknowledge that this is a rather small patient sample and this finding has not been replicated in an independent study sample. It is, thus, possible that this preliminary result represents a false positive association. With respect to mouse genetics, the locus of the DBI gene maps to murine chromosome 1E1. Several studies have been carried out describing the chromosomal locations of genetic variants that determine behavioral variation in rodents (Flint et al., 2005). Quantitative trait locus (QTL) analyses yielded consistent results implicating mouse chromosome 1 to strongly influence anxiety-related behavior (Flint et al., 1995; Henderson et al., 2004; Singer et al., 2005; Turri et al., 2001). Intriguingly, high-resolution mapping revealed highest LOD scores for anxiety-associated parameters in the distal end of chromosome 1, i.e. a
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chromosomal region nearby the location of the DBI gene (Talbot et al., 1999; Turri et al., 2001). Based on the approximate homology between murine and human anxiety phenotypes and the genomic homology of mice and men, Smoller et al. (2001) performed a targeted genome screen in a pedigree of panic disorder using mouse QTL mapping results. So far, linkage analysis provided evidence for a locus on human chromosomes 10q and 12q, but not for 2q, where the DBI gene is located. In addition, preclinical data have linked DBI to anxietyrelated behavior making it an attractive pathophysiological candidate for susceptibility to panic disorder. Several studies reported DBI to act as an endogenous anxiogenic peptide (Guidotti, 1991; Sudakov et al., 2001). The available clinical evidence, however, has been controversial so far. DBI levels in lymphocytes of anxiety disorder patients have been reported to be significantly decreased (Ferrarese et al., 1993). Studies investigating DBI concentrations in the CSF of depressed patients with a severe anxiety component found DBI levels to be increased in these patients (Costa and Guidotti, 1991). However, no alterations in endozepine concentrations in the CSF were observed in panic disorder patients (Payeur et al., 1992). In conclusion, the genetic polymorphisms studied within DBI appear to decrease the susceptibility for the development of anxiety disorders that are characterized by the occurrence of panic attacks. Given the sparse and incomplete knowledge about the role of DBI in the neurobiology of anxiety, however, our (preliminary) results need to be replicated in an independent study sample and further analyzed regarding its functional relevance. Acknowledgements This study was supported in part by the Austrian Academy of Sciences (DOC [Doktorandenprogramm]; CKT), the German Federal Ministry of Education and Research (BMBF) in the National Genome Research Network (NGFN; Fo¨rderkennzeichen 01GS0481), and the Bavarian Ministries of Commerce and Research (Bayerischer Habilitationsfo¨rderpreis; MEK). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jpsychires. 2006.06.001. References Bandelow B. Assessing the efficacy of treatments for panic disorder and agoraphobia, II: the panic and Agoraphobia Scale. International Clinical Psychopharmacology 1995;10:73–81. Binder EB, Salyakina D, Lichtner P, Wochnik GM, Ising M, Pu¨tz B, et al. Polymorphisms in FKBP5 are associated with increased recurrence of depressive episodes and rapid response to antidepressant treatment. Nature Genetics 2004;36:1319–25.
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Brambilla P, Perez J, Barale F, Schettini G, Soares JC. GABAergic dysfunction in mood disorders. Molecular Psychiatry 2003;8:721–37. Costa E, Guidotti A. Diazepam binding inhibitor (DBI): A peptide with multiple biological actions. Life Science 1991;49:325–44. Ferrarese C, Appollonio I, Bianchi G, Frigo M, Marzorati C, Pecora N, et al. Benzodiazepine receptors and diazepam binding inhibitor: A possible link between stress, anxiety and the immune system. Psychoneuroendocrinology 1993;18:3–22. Finn DA, Rutledge-Gorman MT, Crabbe JC. Genetic animals of anxiety. Neurogenetics 2003;4:109–35. Flint J, Corley R, Defries JC, Fulker DW, Gray JA, Miller S, et al. A simple genetic-basis for a complex psychological trait in laboratory mice. Science 1995;269:1432–5. Flint J, Valdar W, Shifman S, Mott R. Strategies for mapping and cloning quantitative trait genes in rodents. Nature Reviews Genetics 2005;6:271–86. Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, et al. The structure of haplotype blocks in the human genome. Science 2002;296:2225–9. Guidotti A. Role of DBI in brain and its posttranslational processing products in normal and abnormal behavior. Neuropharmacology 1991;12:1425–33. Hamilton SP, Fyer AJ, Durner M, Heiman GA, de Leon AB, Hodge SE, et al. Further genetic evidence for a panic disorder syndrome mapping to chromosome 13q. Proceedings of the National Academy of Science 2003;100:2550–5. Hattori E, Ebihara M, Yamada K, Ohba H, Shibuya H, Yoshikawa T. Identification of a compound short tandem repeat stretch in the 5 0 upstream region of the cholecystokinin gene, and its association with panic disorder but not with schizophrenia. Molecular Psychiatry 2001;6:465–70. Henderson ND, Turri MG, DeFries J, Flint J. QTL analysis of multiple behavioral measures of anxiety in mice. Behavior Genetics 2004;34:267–93. Herzog CD, Stackman RW, Walsh TJ. Intraseptal Flumazenil enhances, while diazepam binding inhibitor impairs, performance in a working memory task. Neurobiology of Learning and Memory 1996;66:341–52. Hettema JM, Prescott CA, Kendler KS. A population-based twin study of generalized anxiety disorder in men and women. Journal of Nervous and Mental Disease 2001;189:413–20. Niu N, Rice SR, Heston LL, Sobell JL. Multiple missense mutations in the diazepam binding inhibitor (DBI) gene identified in schizophrenia but lack of disease association. American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 2004;125:10–9. Nutt DJ, Glue P, Lawson C, Wilson SG. Flumazenil provocation of panic attacks. Evidence for altered benzodiazepine receptor sensitivity in panic disorder. Archives of the General Psychiatry 1990;47:917–25. Payeur R, Lydiard RB, Ballenger JC, Laraia MT, Fossey MD, Zealberg J. CSF diazepam-binding inhibitor concentrations in panic disorder. Biological Psychiatry 1992;32:712–6. Rothe C, Koszycki D, Bradwejn J, King N, Deluca V, Tharmalingam S, et al. Association of the Val158Met catechol-o-methyltransferase genetic polymorphism with Panic Disorder. Neuropsychopharmacology, 2006 [Epub ahead of print]. Roy A. Cerebrospinal fluid diazepam binding inhibitor in depressed patients and normal controls. Neuropharmacology 1991;30:1441–4. Rupprecht R, Holsboer F. Neuropsychopharmacological properties of neuroactive steroids. Steroids 1999;64:83–91. Singer JB, Hill AE, Nadeau JH, Lander ES. Mapping quantitative trait loci for anxiety in chromosome substitution strains of mice. Genetics 2005;169:855–62. Smoller JW, Acierno JS, Rosenbaum JF, Biederman J, Pollack MH, Meminger S, et al. Targeted genome screen of panic disorder and anxiety disorder proneness using homology to murine QTL regions. American Journal of Medical Genetics 2001;105:195–206. Stroehle A, Romeo E, di Michele F, Pasini A, Hermann B, Gajewsky G, et al. Induced panic attacks shift gamma-aminobutyric acid type A receptor modulatory neuroactive steroid composition in patients with
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Association of a Met88Val diazepam binding inhibitor (DBI) gene polymorphism and anxiety disorders with panic attacks Christoph K. Thoeringer a, Elisabeth B. Binder a,b, Daria Salyakina a, Angelika Erhardt a, Marcus Ising a, Paul G. Unschuld a, Nikola Kern a, Susanne Lucae a, Tanja M. Brueckl a, Marianne B. Mueller a, Brigitte Fuchs a, Benno Puetz a, Roselind Lieb a, Manfred Uhr a, Florian Holsboer a, Bertram Mueller-Myhsok a, Martin E. Keck a,c,* a
Max Planck Institute of Psychiatry, Munich 80804, Germany Emory University School of Medicine, Atlanta, GA 30322, USA University of Zurich, Division of Psychiatry Research, Minervastr. 145, Zurich 8032, Switzerland b
c
Received 20 January 2006; received in revised form 31 May 2006; accepted 8 June 2006
Abstract Several lines of evidence suggest that anxiety disorders have a strong genetic component, but so far only few susceptibility genes have been identified. There is preclinical and clinical evidence for a dysregulation of the central c-aminobutyric acid (GABA)-ergic tone in the pathophysiology of anxiety disorders. Diazepam binding inhibitor (DBI) has been suggested to play a pivotal role in anxiety disorders through direct and indirect, i.e. via synthesis of neuroactive steroids, modulation of GABAA receptor function. These findings suggest that the DBI gene can be postulated as a candidate for a genetic association study in this disorder. Thus, single nucleotide polymorphisms (SNPs) of the DBI gene were investigated for putative disease associations in a German sample of anxiety disorder patients suffering from panic attacks and matched controls. We were able to detect a significant association between a non-synonymous coding variant of DBI with anxiety disorders with panic attacks. The rare allele of this polymorphism was more frequent in controls than in patients (OR = 0.43; 95% CI: 0.19–0.95). In conclusion, these results suggest a central role of DBI genetic variants in the susceptibility for the development of anxiety disorders that are characterized by the occurrence of panic attacks. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Diazepam binding inhibitor (DBI); Case-control genetic association study; Single nucleotide polymorphisms; Panic disorder
1. Introduction The c-aminobutyric acid (GABA) system has repeatedly been implicated in the pathophysiology of anxiety disorders and related psychiatric diseases. It has been hypothesized for instance, that panic disorder might be due to abnormal activity of endogenous ligands of the benzodiazepine receptor (Nutt et al., 1990). This hypothesis is emphasized by the * Corresponding author. Address: University of Zurich, Division of Psychiatry Research, Minervastr. 145, Zurich 8032, Switzerland. Tel.: +41 44 389 1477; fax: +41 44 389 1414. E-mail address: [email protected] (M.E. Keck).
0022-3956/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2006.06.001
clinical efficacy of positive allosteric modulators of GABAA receptors, such as benzodiazepines, promoting rapid and profound anxiolysis in patients suffering from affective disorders (Brambilla et al., 2003). Apart from exogenous modulators of the GABAA/benzodiazepine receptor, an endogenous inverse peptide agonist, the diazepam binding inhibitor (DBI), has been described (Costa and Guidotti, 1991). This 9-kD endozepine is located in peripheral tissues and in neuronal and glial cells of the central nervous system with an abundant expression in the hypothalamus, amygdala, cortex and cerebellum. One of DBI’s major biological actions comprises the displacement of diazepam from GABAA receptors resulting in a negative modulation of
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GABA channel gating. In addition, DBI acts at the mitochondrial benzodiazepine receptor of glial (and peripheral steroidogenic) cells facilitating the transport of fatty acids to the inner mitochondrial membrane and, thus, the formation of cholesterol and other, e.g. neuroactive, steroids (Zisterer and Williams, 1997). These neurosteroids, in turn, have been shown to facilitate activation of GABAA receptor function (Rupprecht and Holsboer, 1999). Along these lines, studies in patients with panic disorder suggest that 3a-reduced neuroactive steroids may play a pivotal role in human anxiety in that they may serve as a counter-regulatory mechanism against the occurrence of spontaneous panic attacks (Stroehle et al., 2003). With respect to either direct or indirect behavioral effects of DBI, intraventricular, but not intraseptal, administrations of the endozepine have been reported to induce anxiety-related behavior in laboratory animals (Guidotti, 1991; Herzog et al., 1996). Notably, genetic differences in brain DBI concentrations have been correlated with high and low anxiety levels in rats (Sudakov et al., 2001). In addition, recent clinical studies suggest that the inverse agonist action of DBI on the GABAA/benzodiazepine receptor may severely impact the development and severity of anxiety disorders and depression, as DBI has been found to be increased in the cerebrospinal fluid (CSF) of patients diagnosed with depression with (Costa and Guidotti, 1991) and without concomitant anxiety (Roy, 1991). Many studies have shown that anxiety disorders have a significant familial aggregation which has been largely explained by genetic components. At this point, it is not entirely clear whether this susceptibility predisposes to the development of a specific anxiety disorder or to any one of the anxiety disorders such as panic disorder with or without agoraphobia, social phobia and specific phobia (Villafuerte and Burmeister, 2003). Panic attacks are circumscribed episodes of severe state anxiety lasting minutes to hours with escalating symptoms. The features are associated with an array of physical symptoms of primarily sympathetic arousal. Panic attacks are the hallmark of panic disorder but also occur regularly in association with other anxiety disorders such as, e.g., social phobia and specific phobia. With respect to the genetic aetiopathogenesis of panic, a recent meta-analysis of data from family and twin studies by Hettema et al. (2001) reported an estimated heritability of 43% for panic disorder. Linkage studies focusing on panic disorder have identified some loci of potential interest but, so far, yielded only inconsistent results (Finn et al., 2003). For example, suggestive loci on chromosome 9q31 have been detected in a genome-wide linkage study of anxiety with panic disorder (Thorgeirsson et al., 2003), and chromosome 13q and 22 have been implicated in a linkage study of a panic disorder syndrome (Hamilton et al., 2003). However, no significant linkage has been reported for chromosome 2p where the DBI gene is located. As the genetic basis of anxiety does not follow a simple pattern of Mendelian inheritance, it is considered that
many genes, all of a small effect, contribute to the disease susceptibility. Numerous association studies have been performed focusing on candidate genes of neurochemical systems and components. For instance, polymorphisms in the gene encoding for the catechol-O-methyltransferase have been reported to be associated with panic disorder (Rothe et al., 2006; Woo et al., 2002); or, a short tandem repeat in the promoter region of the cholecystokinine gene was significantly associated with panic disorder (Hattori et al., 2001). The gene encoding for DBI is located on human chromosome 2p14.2 and comprises several polymorphic regions. With respect to psychiatric genetics, Niu et al. (2004) performed the only case-control association analysis so far using three DBI polymorphisms in 317 schizophrenic patients, but failed to detect a disease association. There is, however, strong evidence suggesting a pathophysiological role of endozepines in anxiety disorders. We, therefore, performed a case-control analysis of DBI genetic variants in a sample of German anxiety disorder patients characterized by the occurrence of panic attacks. 2. Materials and methods 2.1. Patients The present study consists of a dual case-control approach. We, first, investigated a primary study sample including a total number of 126 panic disorder patients with and without agoraphobia (see Table 1) and screened for case-control associations of the diagnosis panic disorder with five single nucleotide polymorphisms (SNPs) of Table 1 Phenotypic characteristics of the case – control samples Panic disorder patients Primary study sample Total number With agoraphobia Without agoraphobia Male:female ratio Mean age at admission Mean age at onset of PD Mean panic scale score
Control subjects 126 88.5% 11.5% 1:2.28 39.6 years 27.2 years 7.39
Anxiety disorder patients with panic attacks Extended study sample Total number Agoraphobia PD with agoraphobia PD without agoraphobia Social phobia Simple phobia Male:female ratio Mean age at admission Mean age at onset Mean panic scale score
176 3.6% 76.2% 13.7% 5.0% 1.7% 1:1.98 38.6 years 28.0 years 7.47
Total number Male:female ratio Mean age
229 1:2.66 40.0 years
Controls
Total number Male:female ratio Mean age
301 1:1.68 40.25 years
PD, panic disorder. Panic scale score, composed of frequency, severity and duration of panic attacks (the Panic and Agoraphobia Scale).
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the DBI gene. As reported in our study, one SNP displayed significant association with panic disorder (see below). In order to assess the impact of this genetic polymorphism on an extended anxiety phenotype, we enlarged our primary study sample with patients suffering from several forms of pathological anxiety and concomitant panic attacks. As delineated in Table 1, the second study sample consists of 176 anxiety disorder patients with primary diagnoses of agoraphobia, panic disorder with and without agoraphobia, and social or simple phobia with panic attacks. In addition, these patients had more than 2 moderate to severe panic attacks per week during their worst episode of the disease as determined by the Panic and Agoraphobia Scale (Bandelow, 1995). Patients were consecutively admitted to our Anxiety Disorders Outpatient Clinic for diagnosis and treatment of an anxiety disorder. The diagnosis was ascertained by trained psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria by use of the Structured Clinical Interviews for DSM-IV (SCID I and II) (Wittchen et al., 1997). Anxiety disorders due to a medical or neurological condition and a comorbid Axis II disorder were exclusion criteria. In addition, ethnicity was recorded using a self-reporting sheet for perceived nationality, mother language and ethnicity of the subject himself and of all four grandparents. All included individuals reported themselves to be ‘‘Caucasian’’ and 84%, and 81% for the extended sample respectively, were of German origin. 2.2. Controls An overall number of 301 subjects was recruited as controls and matched for ethnicity, gender and age (see Table 1). All individuals were randomly selected from a Munichbased community sample and screened for the presence of anxiety and affective disorders using the Composite International Diagnostic-Screener (CID-S) (Wittchen et al., 1999). Only individuals who were not screening positive for the above-named disorders were included in the sample. The study was approved by the local ethics committee, and written informed consent was obtained from all subjects. 2.3. DNA preparation After enrolling patients and controls in the study, we drew 40 ml of EDTA blood from each individual and extracted DNA from fresh blood using the Puregen whole blood DNA-extraction kit (Gentra Systems; Minneapolis). 2.4. SNP selection and genotyping SNP selection and genotyping was largely performed as previously described (Binder et al., 2004). Briefly, a total number of 5 single nucleotide polymorphisms (SNPs) in the DBI gene (NM_020548; 5.4 kb in length and 5 exons)
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with an average distance of 1119 bp on human chromosome 2p14.2 was selected from public databases (dbSNP; http://www.ncbi.nih.gov and CHIP Bioinformatics Tools; http://snpper.chip.org). The SNP search tool developed at the Institute for Human Genetics, Technical University and GSF-National Research Centre for Environment and Health was chosen to download SNP sequences (http:// ihg.gsf.de) using the hg17 built of the Genome Browser of the University of Santa Cruz (http://genome.ucsc.edu). Genotyping was performed on a MALDI-TOF mass spectrometer (MassArray system) using the Spectrodesigner software (Sequenom; San Diego) for primer selection and multiplexing and the homogenous mass-extension (hMe) process for producing primer extension products. Genotyping was completed at the Genetic Research Centre, Munich, Germany. The primer sequences are shown in the online supplementary materials. 2.5. Statistical analysis Hardy Weinberg equilibrium was calculated in patients and controls (see Supplementary materials). To test for population substructure in our sample, we performed analysis with STRUCTURE with 100 SNPs (http:// pritch.bsd.uchicago.edu/software.html). For the analysis of LD pattern, we used HAPLOVIEW (http://www.broad.mit.edu/mpg/haploview/using.php) applying the D’ method previously described by Gabriel et al. (2002). LD calculation was performed in our control sample. The Pearson v2 test was used to assess allele frequency difference. In order to adjust the minimum p-value for multiple testing, Fisher product method for combining tests was used as previously described (Binder et al., 2004). Briefly, if a SNP in a gene is associated with the phenotype, so might be other SNPs in the same gene due to linkage disequilibrium (LD). Thus, the five SNPs are combined into a single test which is further evaluated using permutation (we used 100,000 replicates). The Armitage trend test was additionally performed. Haplotype analyses were performed using COCAPHASE of the UNPHASED package (http://www.mrcbsu.cam.ac.uk/personal/frank/software/unphased/). These analyses were done in sliding windows of sizes 2, 3, 4 and 5. 3. Results In a first approach a total number of 5 single nucleotide polymorphisms were genotyped within the DBI gene in our primary study sample including panic disorder patients and controls. All variants turned out to be polymorphic and, thus, informative in our sample. Analyzing Hardy Weinberg equilibrium revealed no significant deviation in all polymorphisms studied (see Supplementary material). In addition, analysis with STRUCTURE showed no evidence for population substructure.
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With respect to haplotype block delineation, all five SNPs were in one LD block (see Supplementary materials). As shown in Table 2, the G allele of the DBI polymorphism rs8192506 was significantly more frequent in healthy subjects than in patients with panic disorder, indicating a significant association of this variant and panic disorder. This SNP is located in exon 5 of the gene and results in a Valine to Methionine exchange at amino acid position 88. The p-value for association between rs8192506 and panic disorder was 0.021 and became 0.0298 after adjustment for multiple testing (see Section 2 for details). The G allele was overrepresented in controls with an allelic odds ratio (OR) of 0.33 (95% CI: 0.12–0.88). The Armitage trend test showed similar values (common OR = 0.34, v2 = 5.2, p = 0.022). Three other SNPs showed a trend for an association (p 6 0.1). Even though D’ was high among all five SNPs, the r2 between rs8192506 and the other four SNPs was less than 0.2, explaining the range in the p-values for the association in SNPs located in the same LD block. Haplotype analyses showed several haplotypes to have nominally significant associations, but none exceeded the significance obtained when considering rs8192506 alone (see Supplementary materials). In a second approach, we analyzed case-control relations of the significantly associated SNP rs8192506 in an extended sample of anxiety disorder patients with panic attacks versus healthy controls. Similar to the results of the association with panic disorder, rs8192506 displayed a significant association with the extended anxiety phenotype (see Table 3). The over-representation of the G allele was still present with an allelic OR of 0.43 (95% CI: 0.19–0.95). The Armitage trend test of the extended association displayed similar values (common OR = 0.44, v2 = 4.6, p = 0.033). Table 2 Allele frequencies of DBI single nucleotide polymorphisms in patients with panic disorder and controls p-valuea
SNP
N (subjects)
Allele
rs3091405 Patients Controls
124 225
A (%) 26.6 32.4
C (%) 73.4 67.6
0.109
rs3769664 Patients Controls
126 227
C (%) 81.3 75.8
T (%) 18.7 24.2
0.087
rs3769662 Patients Controls
125 227
C (%) 93.2 95.8
G (%) 6.8 4.2
0.132
rs956309 Patients Controls
125 228
C (%) 80.8 74.7
T (%) 19.2 25.3
0.079
rs8192506 Patients Controls
126 229
A (%) 98.0 94.3
G (%) 2.0 5.7
0.021b
a 2 v -test; control subjects versus panic disorder patients; p-values uncorrected. b The frequency of the G allele was significantly higher in control subjects than in patients (v2 = 5.31; p = 0.021; p = 0.0298 after correction).
Table 3 Allele frequencies of the rs8192506 DBI single nucleotide polymorphism in an extended sample of anxiety disorder patients and controls rs8192506
N (subjects)
A (%)
G (%) Allele
p valuea
Anxiety Controls
176 301
97.7 94.8
2.3 5.2
0.032
a
v2-test; control subjects versus anxiety disorder patients (v2 = 4.61).
Analyzing the impact of the rs8192506 genetic polymorphism on the diagnosis of panic disorder with and without agoraphobia alone in the enlarged patient sample (N = 158), the p-value was 0.064 according to the Armitage trend test. 4. Discussion The present candidate gene association study in a sample carefully diagnosed as having an anxiety disorder associated with panic attacks reveals that polymorphisms within the DBI gene are associated with the diagnosis of panic disorder and, in general, anxiety disorders with panic attacks. The G allele of the exonic SNP rs8192506 is overrepresented in controls versus patients with a frequency almost three times higher in healthy subjects than in anxiety patients. The protective effect of that allele has an OR of 0.33 (95% CI: 0.12–0.88) and an OR of 0.43 (95% CI: 0.19–0.95), respectively. Intriguingly, this genetic polymorphism leads to an amino acid change (Valine to Methionine) at amino acid position 88 and, thus, potentially to functional differences in DBI activity. At this point of the analysis, however, we cannot rule out that this SNP is only a marker of LD with the causal SNP. The analysis of the haplotype block delineation revealed a strong intragenic LD. Importantly, one has to note that the inspection of LD pattern in Caucasian populations as presented in Hapmap project data (see www.hapmap.org) indicates that there is no LD between SNPs of the DBI gene and the 3 0 located genes which encode the transmembrane protein 37 and the secretin receptor precursor. This would suggest that the observed association is indeed with DBI and not neighbouring genes. Further fine mapping and functional studies are necessary to resolve that issue. We have to acknowledge that this is a rather small patient sample and this finding has not been replicated in an independent study sample. It is, thus, possible that this preliminary result represents a false positive association. With respect to mouse genetics, the locus of the DBI gene maps to murine chromosome 1E1. Several studies have been carried out describing the chromosomal locations of genetic variants that determine behavioral variation in rodents (Flint et al., 2005). Quantitative trait locus (QTL) analyses yielded consistent results implicating mouse chromosome 1 to strongly influence anxiety-related behavior (Flint et al., 1995; Henderson et al., 2004; Singer et al., 2005; Turri et al., 2001). Intriguingly, high-resolution mapping revealed highest LOD scores for anxiety-associated parameters in the distal end of chromosome 1, i.e. a
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chromosomal region nearby the location of the DBI gene (Talbot et al., 1999; Turri et al., 2001). Based on the approximate homology between murine and human anxiety phenotypes and the genomic homology of mice and men, Smoller et al. (2001) performed a targeted genome screen in a pedigree of panic disorder using mouse QTL mapping results. So far, linkage analysis provided evidence for a locus on human chromosomes 10q and 12q, but not for 2q, where the DBI gene is located. In addition, preclinical data have linked DBI to anxietyrelated behavior making it an attractive pathophysiological candidate for susceptibility to panic disorder. Several studies reported DBI to act as an endogenous anxiogenic peptide (Guidotti, 1991; Sudakov et al., 2001). The available clinical evidence, however, has been controversial so far. DBI levels in lymphocytes of anxiety disorder patients have been reported to be significantly decreased (Ferrarese et al., 1993). Studies investigating DBI concentrations in the CSF of depressed patients with a severe anxiety component found DBI levels to be increased in these patients (Costa and Guidotti, 1991). However, no alterations in endozepine concentrations in the CSF were observed in panic disorder patients (Payeur et al., 1992). In conclusion, the genetic polymorphisms studied within DBI appear to decrease the susceptibility for the development of anxiety disorders that are characterized by the occurrence of panic attacks. Given the sparse and incomplete knowledge about the role of DBI in the neurobiology of anxiety, however, our (preliminary) results need to be replicated in an independent study sample and further analyzed regarding its functional relevance. Acknowledgements This study was supported in part by the Austrian Academy of Sciences (DOC [Doktorandenprogramm]; CKT), the German Federal Ministry of Education and Research (BMBF) in the National Genome Research Network (NGFN; Fo¨rderkennzeichen 01GS0481), and the Bavarian Ministries of Commerce and Research (Bayerischer Habilitationsfo¨rderpreis; MEK). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jpsychires. 2006.06.001. References Bandelow B. Assessing the efficacy of treatments for panic disorder and agoraphobia, II: the panic and Agoraphobia Scale. International Clinical Psychopharmacology 1995;10:73–81. Binder EB, Salyakina D, Lichtner P, Wochnik GM, Ising M, Pu¨tz B, et al. Polymorphisms in FKBP5 are associated with increased recurrence of depressive episodes and rapid response to antidepressant treatment. Nature Genetics 2004;36:1319–25.
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