Association study of serotonin-2A receptor gene polymorphism and panic disorder in patients from Canada and Germany

Neuroscience Letters 363 (2004) 276–279 www.elsevier.com/locate/neulet

Association study of serotonin-2A receptor gene polymorphism and panic disorder in patients from Canada and Germany C. Rothea,b, D. Koszyckic, J. Bradwejnc, N. Kinga, V. De Lucaa, S. Shaikha, P. Franked, H. Garritsene, J. Fritzef, J. Deckertb, J.L. Kennedya,* a

Clarke Division, Center for Addiction and Mental Health, University of Toronto, Toronto, Canada b Department of Psychiatry, University of Mu¨nster, Mu¨nster, Germany c Department of Psychiatry, University of Ottawa and the University of Ottawa Institute of Mental Health Research, Royal Ottawa Hospital, Ottawa, Canada d Department of Psychiatry, University of Bonn, Bonn, Germany e Institute of Transfusion Medicine, University of Mu¨nster, Mu¨nster, Germany f Department of Psychiatry, University of Frankfurt, Frankfurt, Germany Received 21 February 2004; received in revised form 31 March 2004; accepted 2 April 2004

Abstract The T102C serotonin-2A (5-HT2A) receptor gene polymorphism has been studied extensively in a number of complex psychiatric conditions with mixed results. Recently a genetic association has been described between this polymorphism and panic disorder in a Japanese sample. To evaluate the impact of the T102C polymorphism in panic disorder we genotyped triad families (panic disorder patient and parents), and cases with controls in Canadian and German samples. No significant transmission disequilibrium was observed between the alleles of the T102C 5-HT2A receptor gene polymorphism and panic disorder, nor was a significant excess of either allele found in the case control analysis. Our data suggest thus that this polymorphism is unlikely to play a major role in the pathogenesis of panic disorder. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Panic disorder; Serotonin-2A receptor gene; Association study; Triad families; Transmission disequilibrium test; Genetics

Panic disorder (PD) is an anxiety disorder defined by unexpected recurrent panic attacks and anticipatory anxiety. This severe psychiatric condition is often associated with agoraphobia. It has a life-time prevalence of 1– 3% [18]. Family and twin studies suggest a strong genetic contribution to the pathogenesis of PD with an estimated heritability of 48% [9]. No simple Mendelian pattern of inheritance was identified in segregation studies [17]. This is consistent with the notion that PD can be considered genetically complex with a polygenic mode of inheritance. Many genes of small effect may contribute to the disease susceptibility. Dysregulation of the serotonergic system has been hypothesized to play a role in the pathogenesis of PD as evidenced by biological and pharmacological research. * Corresponding author. Neuroscience Research CAMH, Department of Psychiatry and Institute of Medical Science, University of Toronto, 250 College Street, Toronto, Ontario, M5T 1R8, Canada. Tel.: þ 1-416-9794987; fax: þ 1-416-979-4666. E-mail address: [email protected] (J.L. Kennedy).

Several studies demonstrated serotonergic abnormalities in patients with PD (e.g. Ref. [2]). First line treatment in pharmacotherapy of PD is selective serotonin reuptake inhibitors (SSRIs). Drugs with both serotonin-2A (5-HT2A) and SSRI properties modulate anxiety [5]. Although there are inconclusive results, allelic association has been reported between genetic variants in the 5-HT2A receptor (5-HT2AR) gene and depressive disorder [1,6]. Depression and PD with agoraphobia show clinically a high comorbidity, which is substantially larger than between depression and other phobias [3]. Therefore, the 5-HT2A receptor can be considered a good candidate gene for PD. The 5-HT2AR gene consists of three exons separated by two introns and spans over 20 kb [4]. It is located on chromosome 13q14-q21 in humans [13]. Different variants of the 5-HT2AR gene have been found by systematic screening [7]. The silent single nucleotide polymorphism (SNP) T102C is in complete linkage disequilibrium with the 2 1438 promotor polymorphism [15]. In addition significant linkage disequilibrium has been observed between SNP

0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.04.010

C. Rothe et al. / Neuroscience Letters 363 (2004) 276–279

T102C and C74A, thus indicating a haplotype block in the 50 region from 2 1438 to position 102 [12]. The C allele of the T102C 5-HTR2A gene polymorphism has lower expression than the T allele indicating a functional role for this polymorphism [11]. We therefore tested the T102C SNP because of its putative functional role in gene expression, and the fact that it tags a rather large haplotype block across the 50 region of the gene. The His452Tyr variant of the 5HT2AR gene shows low allele frequency for the Tyr allele [7] thus generally providing limited statistical power. Recently, a linkage region for PD syndrome was described on chromosome 13q in an American sample [8] and a significant association between PD patients and SNP T102C of the 5-HT2AR was found in a Japanese population (n ¼ 63) [10]. Based on these positive results and the neurobiological rationale we investigated samples of patients with PD, independently collected from Canada and Germany, for a possible association with the 5-HT2AR gene polymorphism. The Canadian sample consisted of triads each containing a PD patient with both parents (n ¼ 73), diads containing a patient with one parent (n ¼ 29) and 13 small families having a patient with parents and one or more siblings. A second independent Canadian sample of 94 cases (female, n ¼ 61; male, n ¼ 33) and matched controls was also examined. The sample with patients of German origin included 86 cases (female, n ¼ 55; male, n ¼ 31) and matched controls. The Canadian samples were recruited via referrals or advertising from two university-based anxiety research units. Diagnosis of PD was made by a psychiatrist using DSM-IV criteria (American Psychiatric Association, 1994) and independently confirmed by a psychologist using a structured clinical interview (SCID, Structured Clinical Interview for DSM-IV). The control group consisted of healthy individuals responding to local advertisements and screened for the absence of major psychiatric disorders. The PD patients (61 females and 33 males) were carefully matched with controls for age, gender, and ethnic ancestry. The German patients were recruited from three university hospitals in Germany. PD diagnosis was made by experienced psychiatrists using DSM-IIIR criteria (American Psychiatric Association, 1987) on the basis of structured interviews (SADS-LA, Schedule for Affective Disorders and Schizophrenia – Lifetime Anxiety; CIDI, Composite International Diagnostic Interview) and medical records. The controls were unrelated blood donors of German descent, who were matched according to age and gender. In both samples only patients with predominant PD were included. Comorbidity with other anxiety disorders or depression was allowed as long as the diagnosis of PD was primary and predominant. All participating subjects had given their informed written consent. The study design complied with the revised Declaration of Helsinki and was approved by the local ethics committees. Genomic DNA was extracted using standard high salt methods. The SNP of the 5-HT2A receptor at position 102

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was genotyped using an Assay by Designw (ABI Applied Biosystems, Foster City, USA). The resulting PCR product was 72 bp. Allelic discrimination and analysis was carried out on an ABI Prism 7000 sequence detection system (SDS) (ABI Corporation, USA) using allele specific fluorescent labeled probes. Analysis of Hardy– Weinberg equilibrium and the x2-test were performed with a significance level set at P ¼ 0:05 using an online resource (http://kursus.kvl.dk/ shares/vetgen/_Popgen/genetik/applets/kitest.htm). The transmission disequilibrium test (TDT) was used to analyze the triads [14]. The statistical analyses were basically explorative and, therefore, the results were not adjusted for multiple testing. The results of the case control study are summarized in Table 1. The distribution of the genotypes of the SNP T102C did not differ significantly from those predicted by the Hardy –Weinberg equilibrium in controls (x2 ¼ 1:23, d:f: ¼ 1, P ¼ 0:27) as well as patients (x2 ¼ 0:94, d:f: ¼ 1, P ¼ 0:33). No significant differences were observed in genotype and allele frequency of the T102C polymorphism between PD and control subjects in either the Canadian sample (genotype frequency, P ¼ 0:14; allele frequency, P ¼ 1) or the German sample (genotype frequency, P ¼ 0:24; allele frequency, P ¼ 0:23). Further, no significant differences in genotype frequency were observed between the subgroup of PD patients with agoraphobia and controls (Canadian sample: genotype frequency, P ¼ 0:84; German sample: genotype frequency, P ¼ 0:46). Results remained negative when both samples were combined (genotype frequency, P ¼ 0:22; PD with agoraphobia: genotype frequency, P ¼ 0:39). Results for the triad families are summarized in Table 2. We failed to detect significant transmission disequilibrium between the T102C polymorphism and PD (TDT test: x2 ¼ 2:45, P ¼ 0:12). Recently, a significant association between the SNP T102C of the 5-HT2AR and PD was detected in a sample of 63 Japanese patients [10]. This association was particularly evident in PD patients with coexisting agoraphobia. In the present study, we attempted to replicate these findings in two large and independent samples from Canada and Germany. In contrast to the Japanese study, we failed to detect an association between the SNP T102C of the 5HT2AR and PD in either of our samples. Moreover, no significant association was found in the subgroup of PD patients with agoraphobia (Canadian sample: x2 ¼ 0:04, P ¼ 0:84; German sample: x2 ¼ 0:54, P ¼ 0:46) in contrast to the significant finding in the Japanese sample (P ¼ 0:016). Several factors may account for discrepant findings between our study and that of Inada et al. [10]. First, the sample size of the Japanese study was small and this may have contributed to a chance positive finding. On the other hand, the size of both of our samples and in particular the combined sample had very good power to detect an effect of the size suggested by the Japanese study. Despite the suggestive neurobiological rationale, the 5-

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Table 1 Genotype and allele counts of the T102C 5-HT2A polymorphism in PD patients and controls Sample (n)

x2

Genotypes

PD Canadian sample Patients (94) Controls (94) German sample Patients (86) Controls (86) PD with agoraphobia Canadian sample Patients (74) Controls (74) German sample Patients (59) Controls (59)

T/T

T/C

C/C

22 17

42 52

30 25

2.1594

17 13

40 37

29 36

17 17

38 37

12 9

26 27

HTR2A Transmitted Untransmitted

T

C

33 47

47 33

x2

P

C

0.1417

86 86

102 102

0.0000

1

1.4040

0.2361

74 63

98 109

1.4677

0.2257

19 20

0.0389

0.8436

72 71

76 77

0.0135

0.9075

21 23

0.5383

0.4631

50 45

68 73

0.4404

0.5069

Table 2 TDT for transmission disequilibrium between T102C 5-HT2A receptor gene polymorphism and PD Allele

Alleles T

HT2A receptor gene polymorphism thus may not be involved in the etiopathogenesis of PD. This is consistent with a previous study finding no evidence for an association between this polymorphism and another anxiety disorder, generalized social phobia [16]. Second, different ethnic ancestry may well be another reason for the diverse results. This may either cause differential effect sizes of genetic variants or population specific linkage disequilibrium with other, clinically more relevant variants. The effects of ethnicity on genetic variation are well established and it is possible that the SNP T102C of the 5-HT2AR is a risk factor for PD in Asian populations. Finally, the negative association found in the present study may also be attributed to phenotypic heterogeneity of PD, which may be too extensive to permit verification of a minor influence of this polymorphism. As the rationale for an important role of the serotonin system in PD remains strong, further investigation of candidate genes associated with serotonin neurotransmission will likely advance our understanding of the genetic basis of PD. More informative phenotypes for future studies may be the response to SSRIs as well as the comorbidity with other disorders. In summary, the current study found no evidence that the SNP T102C of the 5-HT2AR gene increases liability for PD in Canadian and German patients. We conclude that this

Sample

P

x2

P

2.45 2.45

0.1175 0.1175

polymorphism has no major influence on the pathogenesis of PD.

Acknowledgements This study was supported by a grant from the Canadian Institute of Health Research (CIHR 44085) and by a grant from IMF (DE 520207). We would like to thank Rose-Marie Mueller RN, Ivan Fruminsky, BscN, and Maria Pizzi, BscN for research assistance and Mary Smirniw for her assistance in preparing the manuscript.

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