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Association study of A2a adenosine receptor genetic polymorphism in panic disorder
Neuroscience Letters 378 (2005) 98–101
Association study of A2a adenosine receptor genetic polymorphism in panic disorder Pok Lama , Chen-Jee Hongb,c , Shih-Jen Tsaib,c,∗ a
c
Department of Psychiatry, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan ROC b Department of Psychiatry, Taipei Veterans General Hospital, Taipei, Taiwan ROC Division of Psychiatry, School of Medicine, National Yang-Ming University, Taipei, Taiwan ROC
Received 10 November 2004; received in revised form 4 December 2004; accepted 7 December 2004
Abstract The adenosine A2a receptor (A2aAR) is thought to be implicated in the pathogenesis of panic disorder because caffeine, a potent antagonist for A2aAR, can precipitate panic attacks, and because disruption of the A2aAR gene increased anxiety-behaviors in mice. Recent studies demonstrated that the A2aAR 1976T>C genetic variant confers susceptibility to panic disorder though not by all studies. The present study tested the hypothesis that the A2aAR 1976T>C genetic variant confers susceptibility to panic disorder using a Chinese population of 104 panic disorder patients and 192 normal controls. We also tested whether the A2aAR 1976T>C polymorphism relates to the age of onset or subtype of panic disorders. Neither the distribution of the A2aAR 1976T>C genotypes (P = 0.296) or alleles (P = 0.864), nor the age of onset (P = 0.719) were significantly different among genotype groups. Furthermore, no association was demonstrated between this A2aAR polymorphism and either mitral-valve prolapse or agoraphobia in panic-disorder patients. These findings suggested that it is unlikely that the A2aAR 1976T>C polymorphism plays a major role in panic disorder pathogenesis in the Chinese population. The positive association between this polymorphism and panic disorder found in western population but not in Asian population suggests that this association could be ethnicity-dependent. The 1976C>T polymorphism may be in linkage disequilibrium with a functional variant that affects panic disorder, and the extent of this linkage disequilibrium is not similar for all ethnic populations. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Association study; Agoraphobia; Polymorphism; Adenosine A2a receptor; Panic disorder; Mitral-valve prolapse
Panic disorder, the most common anxiety disorder seen in clinical practice, is characterized by recurrent, unexpected, unprovoked attacks of anxiety or fear. The etiology of panic disorder is currently unknown. Research over the past two decades using challenge studies with a variety of panicogenic agents (e.g. sodium lactate, caffeine, yohimbine, serotoninergic agents and cholecystokinin) has suggested the pathogenesis of panic disorders may involve dysregulation of multiple neuronal systems [3,10]. In addition, evidences from the results of molecular epidemiological studies suggest a family component to this disorder and indicate the possible involvement of genetic factors in the etiology of panic disorder [6].
∗ Corresponding author. Tel.: +886 2 2875 7027x267; fax: +886 2 2872 5643.
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.12.012
Adenosine, a purine nucleotide, has been long known to act as a neuromodulator and regulate many physiological functions through several subtypes of G-protein-coupled receptors. Currently, four subtypes of adenosine receptors have been identified and classified as the A1, A2a, A2b and A3 types [17]. Of these, the adenosine A2a receptor (A2aAR) is potentially an attractive candidate gene for panic disorder from evidence of animal as well as clinical studies. The possible involvement of A2aAR in panic disorder was revealed by the finding that the knockout mice specific for this receptor showed increased anxiety-related behaviors [13]. In humans, the study of the consumption of caffeine, a potent antagonist for A2aAR, indicated that patients with panic disorder had an increased sensitivity to the effects of caffeine [2]. Furthermore, caffeine produced significantly greater increases in subject-related anxiety, nervousness, fear, nausea,
P. Lam et al. / Neuroscience Letters 378 (2005) 98–101
palpitations, restlessness, and tremors in the panic-disorder patients compared with healthy subjects [4]. The A2aAR locus is located on human chromosome 22q11.2 [12]. In 1998, Deckert et al. [5] first showed that a silent coding polymorphism (1976T>C, formerly 1083T>C) in exon 2 of A2aAR was associated with panic disorder in a German population. This finding was partially replicated by a recent study, which Hamilton et al. [7] provided evidence for linkage between the A2aAR locus and panic disorder in a Caucasian sample of Western European background. However, this association was not replicated by a Japanese group [21]. Given the possible implication of the A2aAR 1976T>C polymorphism in panic disorder, the relationship between this polymorphism and panic disorder and its onset was examined in a Chinese sample population. Since panic-disorder patients often are co-morbid with mitral-valve prolapse or agoraphobia, we also tested if this A2aAR polymorphism is associated with mitral-valve prolapse or agoraphobia in the patient group. This is an extended study of our previous report [11]. The study group included 104 psychiatric outpatients who met the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria for panic disorder. Each patient was assessed by a board-certified psychiatrist using the MiniInternational Neuropsychiatric Interview, a short, structured diagnostic interview [18]. Patients with psychotic symptoms were not included in this study. Thyroid function was evaluated for each patient to rule out hyperthyroidism. Of the 104 patients, 97 underwent M-mode and two-dimensional echocardiogram evaluation. The criteria for diagnosis of mitral-valve prolapse were: a posterior excursion of 2 mm or more, in one or both leaflets during systole on M-mode examination [16]; and/or, a backward displacement of one or both mitral-valve leaflets beyond an imaginary line drawn across the atrioventricular ring during systole on two-dimensional examination [14]. A total of 192 normal volunteers without psychotic or mood disorders as evaluated by a psychiatrist were recruited from the medical staff and general community to serve as normal controls. The study population consisted entirely of ethnic Chinese who had given written, informed consents.
99
For the A2aAR 1976T>C genotyping, peripheral venous blood was withdrawn from the study subjects after informed consents had been obtained. DNA fragments of interest were amplified by polymerase chain reaction, with annealing temperature set at 63 ◦ C. The primer sequences were 5 -CAG AAG GAG GTC CAT GCT GCC-3 and 5 -GAG AAT ATG TGA GGG TCC ACT CAC CAG CCC CAG TGC-3 . The 1976T>C polymorphism was differentiated by identifying the presence or absence of the HpyCH4V site. Contaminated amplification was monitored by inclusion of a blank reaction in each batch of the laboratory work. The categorical data were analyzed using the χ2 -test, or the Fisher’s exact test where necessary. For continuous variables, the differences were evaluated using the Student’s t-test or one-way analysis of variance. Data are presented as mean (S.D.). Comparing the panic-disorder and control populations, the mean age (39.1 ± 10.8 years and 40.6 ± 11.3 years, respectively) and sex distributions (male/female 43/61 and 88/104, respectively) were similar (P = 0.266 and 0.465, respectively). The genotype and allele distributions for the A2aAR 1976C>T polymorphism for the two groups are presented in Table 1. The A2aAR genotype distributions for these two groups were in Hardy-Weinberg equilibrium. Neither genotype (P = 0.296) nor allele frequencies (P = 0.864) were statistically different between the two groups. In the panic-disorder patient group, no significant differences were demonstrated comparing the three A2aAR genotypic subgroups for onset age (34.2 ± 11.4 years, 36.3 ± 10.5 years and 35.7 ± 10.9 years for 1976C/C, 1976C/T and 1976T/T, respectively; P = 0.719). Further, statistically significant association was not demonstrated comparing genotype distribution for panic disorder with or without agoraphobia, and with or without mitral-valve prolapse (Table 1). Central A2aAR may play an important role in the anxietyrelated behaviors and the pathogenesis of panic disorder. Two studies in western population had suggested genetic linkage between the A2aAR 1976C>T polymorphism and panic disorder [5,7]. However, our study found no association between panic disorder and the A2aAR 1976C>T genetic polymorphism in the population studied. Further, we found no asso-
Table 1 Genotype distribution and allele frequencies for the A2aAR (1976C>T) genetic polymorphism for panic-disorder patients and control subjects Patient Group
n
Genotype (%) C/C
Panic disorders With agoraphobia Without agoraphobia With mitral-valve prolapse Without mitral-valve prolapse
104 36 68 32 65
29 (27.9) 9 (25.0) 20 (29.4) 9 (28.1) 19 (29.2)
Controls
192
43 (22.4)
a
Allele frequency (%) C/T 46 (44.2) 14 (38.9) 32 (47.1) 15 (46.9) 28 (43.1)
T/T 29 (27.9) 13 (36.1) 16 (23.5) 8 (25.0) 18 (27.7)
103 (53.6) 46 (24.0)
P
C
T
P
0.296a
104 (50.0)
104 (50.0)
0.864a
189 (49.2)
195 (50.8)
0.395b 0.934c
Compared with the control group. Compared with panic disorder patients without agoraphobia. c Compared with panic-disorder patients without mitral-valve prolapse. Of the 104 patients, 97 underwent M-mode and two-dimensional echocardiogram evaluation. b
100
P. Lam et al. / Neuroscience Letters 378 (2005) 98–101
ciation between age onset of panic disorder and the studied A2aAR genetic polymorphism. Our negative findings suggest no involvement for this A2aAR polymorphism in the pathogenesis of panic disorder in Chinese population; however, this is consistent with the Japanese report [16]. The two negative studies in the Asian population and two positive finding studies in western population suggest that the association between this A2aAR polymorphism and panic disorder might be ethnicity-dependent. The 1976C>T polymorphism may be in linkage disequilibrium with a functional variant that affects panic disorder, and the extent of this linkage disequilibrium is not similar for all ethnic populations. This is less likely, however, since in the study by Decker et al. [5] positive association was noted with the T allele of this polymorphism, while in the study by Hamilton et al. [7], they found C allele was more common in panic-disorder patients, indicating that further replication studies are needed to confirm their positive finding. In this study, our sample size was large enough to detect a moderate allelic association; the size of effect that could be detected with 80% power at the 5% significance level is odds ratio of 1.5 for the panic disorder. Given the assumption that panic disorder is probably multi-determined, if the A2aAR is an uncommon disease locus or one of small effect, our power to detect the gene must consequently be reduced and may result in false negative finding, especially in the sub-group analysis. It is clear, however, that a negative finding for a single polymorphism does not exclude the involvement of a whole locus. It is still possible that molecular variants within the regulatory region or the A2aAR-gene coding area may be associated with panic disorder. However, Decker et al. [5] had screened the A2aAR coding region for polymorphism in panic-disorder patients and found no other A2aAR polymorphism was associated with panic disorder. In a study by Hamilton et al. [7], they used five polymorphisms in or near the A2aAR-gene and also showed no association with panic disorder. Another interpretation of our negative results was that while the A2aAR-gene 1976C>T polymorphism may not be involved in the primary etiological mechanism of panic disorder, it may affect disease subtype. For example, in a study of 394 panic-disorder patients, it was found that 55% of the panic-disorder patients co-morbided with agoraphobia [9]. Therefore, we further analyzed the relationship between the 1976C>T polymorphism and panic-disorder patients stratified according to agoraphobia (Table 1). Statistically significant association was not demonstrated, however. Furthermore, cardiac disorders, such as mitral-valve prolapse, share significant co-morbidity with panic disorder. In a meta-analysis study, it was concluded that a significant association exists between panic disorder and mitral-valve prolapse with overall weighted relative risk 2.3 (confidence interval, 1.6–3.5) [8]. In this study, 97 patients underwent Mmode and two-dimensional echocardiogram evaluation and, among them, 32 had mitral-valve prolapse. We stratified the
panic-disorder patients according to the presence or absence of mitral-valve prolapse, however, the A2aAR-gene 1976C>T genotype distribution was similar between the two panic disorder groups (Table 1). There are several limitations in this study. First, the control samples were not screened for panic disorder. Epidemiological studies have shown that panic disorder occurs in adults with a lifetime prevalence of about 2% [20]. Although epidemiological study in Taiwan showed a relatively low prevalence of panic disorder (0.4%) in community population [19], and, according to Owen et al. [15], a uncommon disorders with a life-time risk of 1% or below, the chance inclusion of a few affected cases in the control group will have minimal effect on statistical power, a false negative result due to inclusion of panic disorder in the control group was still likely. Second, panic disorder patients co-morbided with depression were not excluded in this study, which may confound our result. This effect may be minimal, however, because we found the A2aAR-gene 1976C>T polymorphism is not associated with mood disorders (our unpublished report). In summary, our study in the Chinese population did not replicate a previous association found between A2aARgene 1976C>T polymorphism and panic disorders. We further demonstrated that this polymorphism was not related to age of onset, agoraphobia or mitral-valve prolapse in panicdisorder patients. In a recent association study, it was reported that the A2aAR-gene 1976C>T polymorphism was related to self-reported anxiety after an acute dose of caffeine [1]. It would be of interest to test the association between the A2aAR-genotype and the subtype of panic disorder stratifying according to the caffeine challenge test.
Acknowledgments This work was supported by grant on. NSC 91-2314-B075-048 from the National Science Council, Taiwan, ROC, and Grant VGH-90-163 from the Taipei Veterans General Hospital.
References [1] K. Alsene, J. Deckert, P. Sand, H. de Wit, Association between A2a receptor gene polymorphisms and caffeine-induced anxiety, Neuropsychopharmacology 28 (2003) 1694–1702. [2] J.P. Boulenger, T.W. Uhde, E.A. Wolff 3rd., R.M. Post, Increased sensitivity to caffeine in patients with panic disorders. Preliminary evidence, Arch. Gen. Psychiatry 41 (1984) 1067–1071. [3] M. Bourin, G.B. Baker, J. Bradwejn, Neurobiology of panic disorder, J. Psychosom. Res. 44 (1988) 163–180. [4] D.S. Charney, G.R. Heninger, P.I. Jatlow, Increased anxiogenic effects of caffeine in panic disorders, Arch. Gen. Psychiatry 42 (1985) 233–243. [5] J. Deckert, M.M. Nothen, P. Franke, C. Delmo, J. Fritze, M. Knapp, W. Maier, H. Beckmann, P. Propping, Systematic mutation screening and association study of the A1 and A2a adenosine receptor genes
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in panic disorder suggest a contribution of the A2a gene to the development of disease, Mol. Psychiatry 3 (1998) 81–85. C.T. Finn, J.W. Smoller, The genetics of panic disorder, Curr. Psychiatry Rep. 3 (2001) 131–137. S.P. Hamilton, S.L. Slager, A.B. De Leon, G.A. Heiman, D.F. Klein, S.E. Hodge, M.M. Weissman, A.J. Fyer, J.A. Knowles, Evidence for genetic linkage between a polymorphism in the adenosine 2A receptor and panic disorder, Neuropsychopharmacology 29 (2004) 558–565. D.A. Katerndahl, Panic and prolapse. Meta-analysis, J. Nerv. Ment. Dis. 181 (1993) 539–544. M.B. Keller, K.A. Yonkers, M.G. Warshaw, L.A. Pratt, J.K. Gollan, A.O. Massion, K. White, A.R. Swartz, J. Reich, P.W. Lavori, Remission and relapse in subjects with panic disorder and panic with agoraphobia: a prospective short-interval naturalistic follow-up, J. Nerv. Ment. Dis. 182 (1994) 290–296. J.H. Krystal, D.N. Deutsch, D.S. Charney, The biological basis of panic disorder, J. Clin. Psychiatry 57 (Suppl. 10) (1996) 23–31. P. Lam, C.Y. Cheng, C.J. Hong, S.J. Tsai, Association study of a brain-derived neurotrophic factor (Val66Met) genetic polymorphism and panic disorder, Neuropsychobiology 49 (2004) 178–181. F. Le, A. Townsend-Nicholson, E. Baker, G.R. Sutherland, P.R. Schofield, Characterization and chromosomal localization of the human A2a adenosine receptor gene: ADORA2A, Biochem. Biophys. Res. Commun. 223 (1996) 461–467. C. Ledent, J.M. Vaugeois, S.N. Schiffmann, T. Pedrazzini, M. El Yacoubi, J.J. Vanderhaeghen, J. Costentin, J.K. Heath, G. Vassart, M. Parmentier, Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor, Nature 388 (1997) 674–678.
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[14] J. Morganroth, T.J. Mardelli, M. Naito, C.C. Chen, Apical crosssectional echocardiography. Standard for the diagnosis of idiopathic mitral valve prolapse syndrome, Chest 79 (1981) 23–28. [15] M.J. Owen, P. Holmans, P. McGuffin, Association studies in psychiatric genetics, Mol. Psychiatry 2 (1997) 270–273. [16] R.L. Popp, O.R. Brown, J.F. Silverman, D.C. Harrison, Echocardiographic abnormalities in the mitral valve prolapse syndrome, Circulation 49 (1974) 428–433. [17] J.A. Ribeiro, A.M. Sebastiao, A. de Mendonca, Adenosine receptors in the nervous system: pathophysiological implications, Prog. Neurobiol. 68 (2002) 377–392. [18] D.V. Sheehan, Y. Lecrubier, K.H. Sheehan, P. Amorim, J. Janavs, E. Weiller, T. Hergueta, R. Baker, G.C. Dunbar, The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSMIV and ICD-10, J. Clin. Psychiatry 59 (Suppl. 20) (1998) 22– 33. [19] M.M. Weissman, R.C. Bland, G.J. Canino, C. Faravelli, S. Greenwald, H.G. Hwu, P.R. Joyce, E.G. Karam, C.K. Lee, J. Lellouch, J.P. Lepine, S.C. Newman, M.A. Oakley-Browne, M. Rubio-Stipec, J.E. Wells, P.J. Wickramaratne, H.U. Wittchen, E.K. Yeh, The crossnational epidemiology of panic disorder, Arch. Gen. Psychiatry 54 (1997) 305–309. [20] H.U. Wittchen, C.A. Essau, Epidemiology of panic disorder: progress and unresolved issues, J. Psychiatr. Res. 27 (Suppl. 1) (1993) 47– 68. [21] K. Yamada, E. Hattori, M. Shimizu, A. Sugaya, H. Shibuya, T. Yoshikawa, Association studies of the cholecystokinin B receptor and A2a adenosine receptor genes in panic disorder, J. Neural. Transm. 108 (2001) 837–848.
Association study of A2a adenosine receptor genetic polymorphism in panic disorder Pok Lama , Chen-Jee Hongb,c , Shih-Jen Tsaib,c,∗ a
c
Department of Psychiatry, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan ROC b Department of Psychiatry, Taipei Veterans General Hospital, Taipei, Taiwan ROC Division of Psychiatry, School of Medicine, National Yang-Ming University, Taipei, Taiwan ROC
Received 10 November 2004; received in revised form 4 December 2004; accepted 7 December 2004
Abstract The adenosine A2a receptor (A2aAR) is thought to be implicated in the pathogenesis of panic disorder because caffeine, a potent antagonist for A2aAR, can precipitate panic attacks, and because disruption of the A2aAR gene increased anxiety-behaviors in mice. Recent studies demonstrated that the A2aAR 1976T>C genetic variant confers susceptibility to panic disorder though not by all studies. The present study tested the hypothesis that the A2aAR 1976T>C genetic variant confers susceptibility to panic disorder using a Chinese population of 104 panic disorder patients and 192 normal controls. We also tested whether the A2aAR 1976T>C polymorphism relates to the age of onset or subtype of panic disorders. Neither the distribution of the A2aAR 1976T>C genotypes (P = 0.296) or alleles (P = 0.864), nor the age of onset (P = 0.719) were significantly different among genotype groups. Furthermore, no association was demonstrated between this A2aAR polymorphism and either mitral-valve prolapse or agoraphobia in panic-disorder patients. These findings suggested that it is unlikely that the A2aAR 1976T>C polymorphism plays a major role in panic disorder pathogenesis in the Chinese population. The positive association between this polymorphism and panic disorder found in western population but not in Asian population suggests that this association could be ethnicity-dependent. The 1976C>T polymorphism may be in linkage disequilibrium with a functional variant that affects panic disorder, and the extent of this linkage disequilibrium is not similar for all ethnic populations. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Association study; Agoraphobia; Polymorphism; Adenosine A2a receptor; Panic disorder; Mitral-valve prolapse
Panic disorder, the most common anxiety disorder seen in clinical practice, is characterized by recurrent, unexpected, unprovoked attacks of anxiety or fear. The etiology of panic disorder is currently unknown. Research over the past two decades using challenge studies with a variety of panicogenic agents (e.g. sodium lactate, caffeine, yohimbine, serotoninergic agents and cholecystokinin) has suggested the pathogenesis of panic disorders may involve dysregulation of multiple neuronal systems [3,10]. In addition, evidences from the results of molecular epidemiological studies suggest a family component to this disorder and indicate the possible involvement of genetic factors in the etiology of panic disorder [6].
∗ Corresponding author. Tel.: +886 2 2875 7027x267; fax: +886 2 2872 5643.
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.12.012
Adenosine, a purine nucleotide, has been long known to act as a neuromodulator and regulate many physiological functions through several subtypes of G-protein-coupled receptors. Currently, four subtypes of adenosine receptors have been identified and classified as the A1, A2a, A2b and A3 types [17]. Of these, the adenosine A2a receptor (A2aAR) is potentially an attractive candidate gene for panic disorder from evidence of animal as well as clinical studies. The possible involvement of A2aAR in panic disorder was revealed by the finding that the knockout mice specific for this receptor showed increased anxiety-related behaviors [13]. In humans, the study of the consumption of caffeine, a potent antagonist for A2aAR, indicated that patients with panic disorder had an increased sensitivity to the effects of caffeine [2]. Furthermore, caffeine produced significantly greater increases in subject-related anxiety, nervousness, fear, nausea,
P. Lam et al. / Neuroscience Letters 378 (2005) 98–101
palpitations, restlessness, and tremors in the panic-disorder patients compared with healthy subjects [4]. The A2aAR locus is located on human chromosome 22q11.2 [12]. In 1998, Deckert et al. [5] first showed that a silent coding polymorphism (1976T>C, formerly 1083T>C) in exon 2 of A2aAR was associated with panic disorder in a German population. This finding was partially replicated by a recent study, which Hamilton et al. [7] provided evidence for linkage between the A2aAR locus and panic disorder in a Caucasian sample of Western European background. However, this association was not replicated by a Japanese group [21]. Given the possible implication of the A2aAR 1976T>C polymorphism in panic disorder, the relationship between this polymorphism and panic disorder and its onset was examined in a Chinese sample population. Since panic-disorder patients often are co-morbid with mitral-valve prolapse or agoraphobia, we also tested if this A2aAR polymorphism is associated with mitral-valve prolapse or agoraphobia in the patient group. This is an extended study of our previous report [11]. The study group included 104 psychiatric outpatients who met the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria for panic disorder. Each patient was assessed by a board-certified psychiatrist using the MiniInternational Neuropsychiatric Interview, a short, structured diagnostic interview [18]. Patients with psychotic symptoms were not included in this study. Thyroid function was evaluated for each patient to rule out hyperthyroidism. Of the 104 patients, 97 underwent M-mode and two-dimensional echocardiogram evaluation. The criteria for diagnosis of mitral-valve prolapse were: a posterior excursion of 2 mm or more, in one or both leaflets during systole on M-mode examination [16]; and/or, a backward displacement of one or both mitral-valve leaflets beyond an imaginary line drawn across the atrioventricular ring during systole on two-dimensional examination [14]. A total of 192 normal volunteers without psychotic or mood disorders as evaluated by a psychiatrist were recruited from the medical staff and general community to serve as normal controls. The study population consisted entirely of ethnic Chinese who had given written, informed consents.
99
For the A2aAR 1976T>C genotyping, peripheral venous blood was withdrawn from the study subjects after informed consents had been obtained. DNA fragments of interest were amplified by polymerase chain reaction, with annealing temperature set at 63 ◦ C. The primer sequences were 5 -CAG AAG GAG GTC CAT GCT GCC-3 and 5 -GAG AAT ATG TGA GGG TCC ACT CAC CAG CCC CAG TGC-3 . The 1976T>C polymorphism was differentiated by identifying the presence or absence of the HpyCH4V site. Contaminated amplification was monitored by inclusion of a blank reaction in each batch of the laboratory work. The categorical data were analyzed using the χ2 -test, or the Fisher’s exact test where necessary. For continuous variables, the differences were evaluated using the Student’s t-test or one-way analysis of variance. Data are presented as mean (S.D.). Comparing the panic-disorder and control populations, the mean age (39.1 ± 10.8 years and 40.6 ± 11.3 years, respectively) and sex distributions (male/female 43/61 and 88/104, respectively) were similar (P = 0.266 and 0.465, respectively). The genotype and allele distributions for the A2aAR 1976C>T polymorphism for the two groups are presented in Table 1. The A2aAR genotype distributions for these two groups were in Hardy-Weinberg equilibrium. Neither genotype (P = 0.296) nor allele frequencies (P = 0.864) were statistically different between the two groups. In the panic-disorder patient group, no significant differences were demonstrated comparing the three A2aAR genotypic subgroups for onset age (34.2 ± 11.4 years, 36.3 ± 10.5 years and 35.7 ± 10.9 years for 1976C/C, 1976C/T and 1976T/T, respectively; P = 0.719). Further, statistically significant association was not demonstrated comparing genotype distribution for panic disorder with or without agoraphobia, and with or without mitral-valve prolapse (Table 1). Central A2aAR may play an important role in the anxietyrelated behaviors and the pathogenesis of panic disorder. Two studies in western population had suggested genetic linkage between the A2aAR 1976C>T polymorphism and panic disorder [5,7]. However, our study found no association between panic disorder and the A2aAR 1976C>T genetic polymorphism in the population studied. Further, we found no asso-
Table 1 Genotype distribution and allele frequencies for the A2aAR (1976C>T) genetic polymorphism for panic-disorder patients and control subjects Patient Group
n
Genotype (%) C/C
Panic disorders With agoraphobia Without agoraphobia With mitral-valve prolapse Without mitral-valve prolapse
104 36 68 32 65
29 (27.9) 9 (25.0) 20 (29.4) 9 (28.1) 19 (29.2)
Controls
192
43 (22.4)
a
Allele frequency (%) C/T 46 (44.2) 14 (38.9) 32 (47.1) 15 (46.9) 28 (43.1)
T/T 29 (27.9) 13 (36.1) 16 (23.5) 8 (25.0) 18 (27.7)
103 (53.6) 46 (24.0)
P
C
T
P
0.296a
104 (50.0)
104 (50.0)
0.864a
189 (49.2)
195 (50.8)
0.395b 0.934c
Compared with the control group. Compared with panic disorder patients without agoraphobia. c Compared with panic-disorder patients without mitral-valve prolapse. Of the 104 patients, 97 underwent M-mode and two-dimensional echocardiogram evaluation. b
100
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ciation between age onset of panic disorder and the studied A2aAR genetic polymorphism. Our negative findings suggest no involvement for this A2aAR polymorphism in the pathogenesis of panic disorder in Chinese population; however, this is consistent with the Japanese report [16]. The two negative studies in the Asian population and two positive finding studies in western population suggest that the association between this A2aAR polymorphism and panic disorder might be ethnicity-dependent. The 1976C>T polymorphism may be in linkage disequilibrium with a functional variant that affects panic disorder, and the extent of this linkage disequilibrium is not similar for all ethnic populations. This is less likely, however, since in the study by Decker et al. [5] positive association was noted with the T allele of this polymorphism, while in the study by Hamilton et al. [7], they found C allele was more common in panic-disorder patients, indicating that further replication studies are needed to confirm their positive finding. In this study, our sample size was large enough to detect a moderate allelic association; the size of effect that could be detected with 80% power at the 5% significance level is odds ratio of 1.5 for the panic disorder. Given the assumption that panic disorder is probably multi-determined, if the A2aAR is an uncommon disease locus or one of small effect, our power to detect the gene must consequently be reduced and may result in false negative finding, especially in the sub-group analysis. It is clear, however, that a negative finding for a single polymorphism does not exclude the involvement of a whole locus. It is still possible that molecular variants within the regulatory region or the A2aAR-gene coding area may be associated with panic disorder. However, Decker et al. [5] had screened the A2aAR coding region for polymorphism in panic-disorder patients and found no other A2aAR polymorphism was associated with panic disorder. In a study by Hamilton et al. [7], they used five polymorphisms in or near the A2aAR-gene and also showed no association with panic disorder. Another interpretation of our negative results was that while the A2aAR-gene 1976C>T polymorphism may not be involved in the primary etiological mechanism of panic disorder, it may affect disease subtype. For example, in a study of 394 panic-disorder patients, it was found that 55% of the panic-disorder patients co-morbided with agoraphobia [9]. Therefore, we further analyzed the relationship between the 1976C>T polymorphism and panic-disorder patients stratified according to agoraphobia (Table 1). Statistically significant association was not demonstrated, however. Furthermore, cardiac disorders, such as mitral-valve prolapse, share significant co-morbidity with panic disorder. In a meta-analysis study, it was concluded that a significant association exists between panic disorder and mitral-valve prolapse with overall weighted relative risk 2.3 (confidence interval, 1.6–3.5) [8]. In this study, 97 patients underwent Mmode and two-dimensional echocardiogram evaluation and, among them, 32 had mitral-valve prolapse. We stratified the
panic-disorder patients according to the presence or absence of mitral-valve prolapse, however, the A2aAR-gene 1976C>T genotype distribution was similar between the two panic disorder groups (Table 1). There are several limitations in this study. First, the control samples were not screened for panic disorder. Epidemiological studies have shown that panic disorder occurs in adults with a lifetime prevalence of about 2% [20]. Although epidemiological study in Taiwan showed a relatively low prevalence of panic disorder (0.4%) in community population [19], and, according to Owen et al. [15], a uncommon disorders with a life-time risk of 1% or below, the chance inclusion of a few affected cases in the control group will have minimal effect on statistical power, a false negative result due to inclusion of panic disorder in the control group was still likely. Second, panic disorder patients co-morbided with depression were not excluded in this study, which may confound our result. This effect may be minimal, however, because we found the A2aAR-gene 1976C>T polymorphism is not associated with mood disorders (our unpublished report). In summary, our study in the Chinese population did not replicate a previous association found between A2aARgene 1976C>T polymorphism and panic disorders. We further demonstrated that this polymorphism was not related to age of onset, agoraphobia or mitral-valve prolapse in panicdisorder patients. In a recent association study, it was reported that the A2aAR-gene 1976C>T polymorphism was related to self-reported anxiety after an acute dose of caffeine [1]. It would be of interest to test the association between the A2aAR-genotype and the subtype of panic disorder stratifying according to the caffeine challenge test.
Acknowledgments This work was supported by grant on. NSC 91-2314-B075-048 from the National Science Council, Taiwan, ROC, and Grant VGH-90-163 from the Taipei Veterans General Hospital.
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