Tryptophan hydroxylase and serotonin transporter gene polymorphism does not affect the diagnosis, clinical features and treatment outcome of panic disorder in the Korean population

Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) 1413 – 1418 www.elsevier.com/locate/pnpbp

Tryptophan hydroxylase and serotonin transporter gene polymorphism does not affect the diagnosis, clinical features and treatment outcome of panic disorder in the Korean population Won Kim a , Young Hee Choi b , Kyung-Sik Yoon c , Dae-Yeon Cho d , Chi-Un Pae e , Jong-Min Woo a,⁎ a

c

Department of Psychiatry and Stress Research Institute, Seoul Paik Hospital, Inje University, Seoul, Republic of Korea b Mettaa Institute of Cognitive Behavior therapy and Psychiatry, Seoul, Republic of Korea Department of Biochemistry and Molecular Biology, School of Medicine, KyungHee University, Seoul, Republic of Korea d LabGenomics Clinical Research Institute, LabGenomics, Seoul, Republic of Korea e Department of Psychiatry, Kangnam St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea Received 1 February 2006; received in revised form 28 April 2006; accepted 18 May 2006 Available online 5 July 2006

Abstract Panic disorder may be associated with defective serotonin (5-HT) neurotransmission. This study was to investigate the association between the tryptophan hydroxylase (TPH) gene and a serotonin transporter gene promoter polymorphism (5-HTTLPR), with panic disorder in a Korean population. 244 Korean patients with panic disorder and the 227 controls were genotyped by a polymerase chain reaction-based method. The severity of panic disorders was assessed by number of panic attacks during the previous 1 month, as well as scores for anticipatory anxiety, panic distress, and agoraphobic distress, as determined by a visual analogue scale (VAS). All the subjects completed the assessment measures including Spielberger State-Trait Anxiety Inventory-State (STAI-S), Spielberger State-Trait Anxiety Inventory-Trait (STAI-T), Beck Depression Inventory (BDI), Symptom Checklist-90-Revised (SCL-90-R), Revised Anxiety Sensitivity Index (ASI-R), Clinical Global Impression Scale – Severity of Illness (CGI-S), Panic Disorder Severity Scale (PDSS), and the Hamilton Depression Rating Scale (HAMD). Responder analyses were conducted based on changes in CGI-I scores after 10 weeks of treatment. We found no significant differences in the genotype and allele frequencies in TPH A218C and 5-HTTLPR polymorphisms between the panic patients and the control group. Subgroup analyses in terms of comorbidities, response, and other primary clinical variables, indicated no differences in these polymorphisms. Our findings suggest that the TPH A218C polymorphism and 5-HTTLPR play no significant roles in the pathogenesis and clinical symptomatologies, at least in a Korean population. © 2006 Elsevier Inc. All rights reserved. Keywords: Korean; Panic disorder; Polymorphism; Serotonin transporter gene; Tryptophan hydroxylase gene

1. Introduction Panic disorder is prevalent and characterized by recurrent panic attacks and anticipatory anxiety (American psychiatric association, 1994). According to the results of family-based studies, the lifetime prevalence is estimated approximately ⁎ Corresponding author. Department of Neuropsychiatry, Seoul Paik Hospital, Inje University 85, Jeo-Dong-2-Ga, Jung-Gu, Seoul 100-032, Republic of Korea. Tel.: +82 2 2270 0064; fax: +82 2 2270 0344. E-mail address: [email protected] (J.-M. Woo). 0278-5846/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2006.05.017

2.9%, and 7.9–17.3% for the 1-year incidence (Weissman et al., 1997; Noyes et al., 1986; Crowe et al., 1983). Several twin studies demonstrated that monozygotic twins carry about a twofold relative risk for panic disorder as do dizygotic twins, and that the familial aggregation of panic disorder might be attributed principally to genetic factors (Perna et al., 1997; Skre et al., 1993; Torgerson, 1990). Kendler et al. (1993) estimated the heritability of liability to be within a range from 30% to 40%. Genetic and biological factors may play important roles in the etiopathogenesis of panic disorder, although the etiology of

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panic disorder has not yet been clearly revealed. An increasing body of evidence suggests that serotonergic neurotransmission may be linked with the modulation of anxiety via serotonin metabolism, as well as alterations in serotonin reuptake and activity, etc., in the brain, under the influence of a variety of external and internal stimuli. Therefore, genetic variants of the serotonergic system would be reasonable candidate for study of panic disorder (Maron and Shlik, 2006). Tryptophan hydroxylase (TPH) is a rate-limiting enzyme in serotonin synthesis, and two common polymorphisms (A218C, A779C) have been reported in intron 7 of the TPH gene (Heinz et al., 2000; Heils et al., 1996). TPH polymorphisms do not directly alter amino acid sequence of TPH, and the functional significance of these polymorphisms has not yet been determined. The serotonin transporter, which is encoded by a serotonin transporter gene located on chromosome 17q12, is a key enzyme in the process of serotonin recycling and the reaccumulation of serotonin into the presynaptic terminals (Lesch et al., 1994). The serotonin transporter promoter polymorphism, 5HTTLPR, is the functional polymorphism of the serotonin transporter, and can be designated by the long (L) or short (S) allele (Heils et al., 1996). The in vitro basal 5-HTT activity in the 5-HTTLPR long allele carriers has been determined to be more than twice as high as that seen in the short (S) allele variant, suggesting that the transcription of the serotonin transporter gene is probably modulated by such variants (Heils et al., 1996). Several studies have reported an association between the S allele of the 5-HTTLPR and anxiety-related traits (Greenberg et al., 2000; Lesch et al., 1996; Mazzanti et al., 1998). Perna et al. (2005) also reported that the presence of the long allelic variant was associated with a better response to paroxetine, although other recent studies failed to find its association with panic disorder (Olesen et al., 2005; Sand et al., 2000; Hamilton et al., 1999). However, these previous ones have focused primarily on case–control studies, without consideration of the subclinical phenotypes of panic disorder. Furthermore, the TPH A218C polymorphism has not yet been investigated within an Asian population. Therefore, this study was undertaken to investigate not only the association of the TPH A218C and 5-HTTLPR polymorphisms with the diagnosis of panic disorder, but also with clinical phenotypes, including other clinical variables, in a Korean population. 2. Methods 2.1. Subject Two hundred and forty-four outpatients diagnosed with panic disorder participated in this study. All subjects were interviewed with The Anxiety Disorders Interview Schedule– Panic Attack & Agoraphobia (ADIS–P&I) (Brown, 1994) and diagnosed based on the criteria established in the DSM-IV, by the consensus of two psychiatrists (YHC, JMW). Patients with comorbid major depressive disorder were also included in the study. Subjects exhibiting any medicosurgical illnesses, or any

Axis I disorders other than panic disorder or major depressive disorder were excluded from this study. Two hundred and twenty-seven age- and sex-matched controls were enrolled in the study. The controls were interviewed by two psychiatrists (YHC, JMW), via personal interviews, in order to determine whether or not they had a current or past history of psychiatric disorders or neurological diseases. The patients and control groups were biologically unrelated native Koreans, and all subjects provided written informed consent. The institutional review board of Seoul Paik Hospital, at Inje University, reviewed the study protocol and approved the study. 2.2. Clinical variables and measurements We collected the clinical variables of all of the patients, including age of onset, duration of illness, and number of panic attacks within the previous 1 month. In order to assess the severity of the patients' symptoms, patients were asked to score, on a visual analogue scale (VAS), the severity of their anticipatory anxiety, subjective distress regarding panic symptoms, and subjective distress regarding agoraphobic symptoms (from 0: absent to 8: severely concerned). Self-rating scales, including the Spielberger State-Trait Anxiety Inventory (STAI) (Spielberger et al., 1970), the Beck Depression Inventory (BDI) (Beck et al., 1961) and the Revised Anxiety Sensitivity Index (ASI-R) (Taylor and Cox, 1988) were also used to assess the patients. Finally, clinician-administered scales, including the Panic Disorder Severity Scale (PDSS) (Shear et al., 1997) and the Hamilton Depression Rating Scale (HAMD) were also assessed (Hamilton, 1960). All the patients were treated with paroxetine and were evaluated by the Clinical Global Impression Scale-Improvement (CGI-I) after 10 weeks of treatment. Patients were given paroxetine with a flexible dosing schedule (10–40 mg/day), under the discretion of the clinicians and the patients' clinical responses. Clonazepam up to 0.25 mg/day was allowed for control of acute anxiety on an as-needed basis. 2.3. DNA analysis Genomic DNA was extracted from the patients' whole blood samples, using a DNA extraction kit (Accuprep Genomic DNA extraction kit, Bioneer, Korea). In order to analyze the TPH A218C polymorphism, we applied a chip-based matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry platform (Sequenom, Inc., CA) (Blondal et al., 2003). The patients' genomic DNA was amplified via polymerase chain reaction (PCR), using the sense primer, 5′–ACG TTG GAT GAG AAT GGT ACC TGG CAT GA–3′, and the antisense primer, 5′– ACG TTG GAT GCA GTG TTA CAT TCC CTA TGC TC–3′. The reaction mixture consisted of 2.5 mM of MgCl2, 0.2 mM of dNTP, 0.1 U of HotStart Taq polymerase (Quiagen GmbH, Germany), 100 nM of each primer, 4.0 ng of DNA, and 5 μl of 1 × PCR buffer (Takara, Japan). The PCR products were treated with shrimp alkaline phosphatase for 20 min at 37 °C. Homogenous MassEXTEND reactions were conducted in reaction

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mixtures consisting of homogenous MassEXTEND enzyme (Thermosequenase; Amersham Pharmacia Biotech, UK), termination mix, and 5 μM of primer (5′–CCC TAT GCT CAG AAT AGC AGC–3′). The products were spotted on 384-well SpectroCHIPs (Sequenom, Inc., CA) using SpectroJET (Sequenom, Inc., CA). These SpectroCHIPs were analyzed via an automated MALDI-TOF Mass Array System (Sequenom, Inc., CA). In order to conduct the modified PCR genotyping of the 5HTT promoter region polymorphisms, we used the following primers: forward, 5′–GGC GTT GCC GCT CTG AAT TGC–3′ and reverse, 5′–GAG GGA CTG AGC TGG ACA ACC CAC– 3′, to amplify the 528-bp (L) and 484-bp (S) alleles, in accordance with the method reported elsewhere (Heils et al., 1996). 2.4. Statistics Genotype distributions in each polymorphism were compared via Chi-squared tests. Monte Carlo methods were applied in the assessment of the contingency table, for small cell counts. Continuous variables were evaluated via Mann–Whitney and Kruskal–Wallis tests between the comparisons, where appropriate, as the Kolmogorov–Smirnov test indicated a skew of the data from normality. P values of <0.01 (0.05/5) were considered to be significant considering a Bonferroni correction for multiple confrontation. All of the statistical tests were conducted using the SPSS v10.0 program (SPSS Inc., Chicago, IL). The power of our sample to detect differences between variants was also calculated, using a two-tailed α value of 0.05. Using these parameters, considering the allele frequencies in our sample, our power analyses indicated that our sample size had the power (0.80) to detect fairly small effect sizes (w = 0.14), corresponding to a 13% difference between the two alleles (Odds Ratio [OR] = 1.84). 3. Results 3.1. Demographic characteristics of subjects 143 (58.6%) and 101 women (41.4%) comprised the patient group, and 102 men (44.9%) and 125 women (55.1%) comprised the control group. The mean age of the patients group was 36.1 ± 9.0 years, and the mean age of the controls was 33.1 ± 9.1 years.

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CGI-S score was 5.46 ± 0.98 (n = 244) at the baseline, and was 2.13 ± 1.12 (n = 143) at the end of 10-week treatment with paroxetine. CGI-S score was not different according to genotype at the baseline, and also after 10-week pharmacotherapy. 3.2. TPH A218C polymorphism and clinical variables The genotypic distribution of TPH A218C in all of the panic patients (χ2 = 2.218, df = 2, p = 0.345) and controls (χ2 = 0.044, df = 2, p = 0.978) was not significantly different from those expected based on Hardy–Weinberg equilibrium (HWE). The genotypes and allele frequencies also did not significantly differ between the two groups (Table 1). Among the total 244 panic disorder patients, 47 exhibited comorbid major depressive disorder. Genotypic frequencies between the panic patients with or without comorbid major depressive disorder and the controls did not significantly differ (Table 1). With regard to the subgroup analysis of agoraphobia, 55 patients were determined to suffer from agoraphobia. Whether or not a given panic patient exhibited agoraphobia had no effects on the genotypic distribution (Table 1). We compared the clinical features and measurements of panic symptoms among the genotypes. We determined there to be no differences in the various clinical features among the genotype (Table 2). 3.3. 5-HTTLPR polymorphism and clinical variables The genotypic distributions of 5-HTTLPR in all of the panic patients (χ2 = 0.127, df = 2, p = 0.939) and the controls (χ2 = 0.004, df = 2, p = 0.998) did not differ significantly from those expected on the basis of HWE. The genotypes and allele frequencies also did not significantly differ between the two groups (Table 3). Genotypic frequencies also did not differ between the panic patients with or without comorbid major depressive disorder and the controls (Table 3). With regard to the subgroup analysis of agoraphobia, comorbidity of agoraphobia had no effects on genotypic distributions. The genotypic frequencies between the panic patients with or without agoraphobia and the controls did not differ (Table 3). We determined there to be no differences in the various clinical features among the genotype (Table 4).

Table 1 Genotypes and allele frequencies of TPH A218C polymorphism in panic patients and normal controls TPH A218C

Genotype A/A

Allele C/A

C/C

Analysis

All panic patients (n = 244) Panic patients with major depression (n = 47) Panic patients without major depression (n = 197) Panic patients with agoraphobia (n = 187) Panic patients without agoraphobia (n = 55) Controls (n = 227) Data represent number (percentage).

62 (25.4%) 13 (27.6%) 49 (24.9%) 48 (25.7%) 14 (25.5%) 53 (23.3%)

133 (54.5%) 23 (48.9%) 109 (55.3%) 101 (54.0%) 30 (54.5%) 115 (50.7%)

49 (20.1%) 11 (23.5%) 39 (19.8%) 38 (20.3%) 11 (20.0%) 59 (26.0%)

A

χ

p

2.326 0.425 2.288 1.850 0.853

0.313 0.808 0.319 0.396 0.653

2

257 (52.7%) 49 (52.1%) 207 (52.5%) 197 (52.7%) 58 (52.7%) 221 (48.7%)

C

231 (47.3%) 45 (47.9%) 187 (47.5%) 177 (47.3%) 52 (47.3%) 233 (51.3%)

Analysis χ2

p

1.495 0.371 1.261 1.310 0.581

0.221 0.543 0.262 0.252 0.446

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Table 2 Data in total panic patients by TPH A218C polymorphism

Table 4 Data in total panic patients by 5-HTTLPR polymorphism

Genotype A/A (n = 62)

Genotype C/A (n = 133)

C/C (n = 49)

p

Age of onset 32.92 (10.34) 32.80 (9.80) 34.29 (9.91) Duration of illness 35.32 (55.38) 38.14 (54.75) 44.04 (64.46) No. of Panic attacks 2.44 (4.84) 2.05 (3.34) 2.41 (4.61) during the last one month Anticipatory anxiety 5.10 (1.83) 5.04 (1.68) 5.55 (1.52) Panic distress 5.52 (2.24) 5.43 (1.94) 5.31 (1.78) Agoraphobic distress 3.58 (2.49) 3.93 (2.62) 3.90 (2.44) STAI-S 54.10 (11.15) 54.92 (11.03) 56.78 (8.09) STAI-T 54.10 (10.44) 54.61 (10.26) 54.22 (9.39) BDI 16.87 (8.32) 17.66 (9.76) 17.51 (6.95) SCL-90-R 13.69 (9.22) 14.24 (10.20) 17.27 (10.33) ASI-R 103.61 (33.63) 105.83 (29.48) 103.27 (33.92) CGI-S 5.44 (0.92) 5.50 (0.97) 5.39 (0.81) PDSS 13.39 (4.70) 13.53 (5.06) 13.18 (4.55) HAMD 15.97 (7.04) 14.54 (7.93) 15.47 (6.95)

0.659 0.719 0.766

0.183 0.858 0.657 0.400 0.939 0.845 0.128 0.842 0.763 0.909 0.434

S/S (n = 159)

L/S (n = 77)

L/L (n = 8)

Age of onset 33.77 (10.56) 31.71 (8.77) 33.88 (6.11) Duration of illness 39.87 (61.69) 37.42 (47.92) 24.75 (28.56) No. of panic attacks 2.31 (4.17) 2.04 (3.79) 2.13 (3.36) during the last one month Anticipatory anxiety 5.15 (1.77) 5.18 (1.54) 5.00 (1.69) Panic distress 5.44 (2.07) 5.27 (1.77) 6.63 (2.00) Agoraphobic distress 3.99 (2.54) 3.60 (2.58) 3.13 (2.10) STAI-S 54.99 (10.23) 55.05 (11.33) 57.25 (8.36) STAI-T 53.75 (9.81) 55.90 (10.64) 51.00 (9.89) BDI 17.21 (8.35) 17.96 (9.92) 16.75 (9.51) SCL-90-R 14.43 (9.76) 15.27 (10.43) 14.75 (12.59) ASI-R 105.13 (30.28) 104.05 (33.63) 103.88 (34.76) CGI-S 5.52 (.089) 5.38 (1.01) 5.13 (0.84) PDSS 13.60 (4.88) 13.13 (4.88) 12.75 (4.62) HAMD 15.18 (7.44) 14.97 (7.94) 14.38 (5.42)

p 0.326 0.747 0.889

0.958 0.183 0.395 0.840 0.216 0.811 0.835 0.967 0.327 0.723 0.942

Mean (S.D.). Kruskal–Wallis tests. There was no statistical significance. STAI-S (State-Trait Anxiety Inventory-State), STAI-T (State-Trait Anxiety Inventory-Trait), BDI (Beck Depression Inventory), SCL-90-R (Symptom Checklist-90-Revised), ASI-R (Revised Anxiety Sensitivity Index), CGI-S (Clinical Global Impression Scale – Severity of Illness), PDSS (Panic Disorder Severity Scale), HAMD (Hamilton Depression Rating Scale).

Mean (S.D.). Kruskal–Wallis tests. There was no statistical significance. STAI-S (State-Trait Anxiety Inventory-State), STAI-T (State-Trait Anxiety Inventory-Trait), BDI (Beck Depression Inventory), SCL-90-R (Symptom Checklist-90-Revised), ASI-R (Revised Anxiety Sensitivity Index), CGI-S (Clinical Global Impression Scale – Severity of Illness), PDSS (Panic Disorder Severity Scale), HAMD (Hamilton Depression Rating Scale).

3.4. Comparisons of CGI-I scores and responder analysis according to the genotypes of each polymorphism

HTTLPR polymorphism and panic disorder. Moreover, comorbidity with major depressive disorder and the presence of agoraphobia did not contribute to the association between each of the polymorphisms and panic disorder. The clinical variables and panic symptom measurements did not differ based on the genotypes of both genes. These findings indicate that the TPH and 5-HTTLPR genes do not appear to constitute primary candidate genes for panic disorder, at least not in a Korean population. As for the TPH A218C polymorphism, our finding is in line with some previous studies reposting no association with panic disorder (Fehr et al., 2001; Rodonto et al., 2002). However, there has been paucity with regard to association of TPH polymorphisms with panic disorder in the Asian population, and thus this is the first to report in Asian sample. As to the 5-HTTLPR polymorphism and panic disorder, another groups failed to find any association of the 5-HTTLPR polymorphism with panic disorders in Caucasians (Sand et al.,

Out of the 244 patients with panic disorder, 143 patients completed a 10-week pharmacotherapy with paroxetine, and were subsequently included in the responder analysis. CGI-I scores did not significantly differ among the genotypes of the TPH A218C and 5-HTTLPR polymorphisms, as shown in Table 5. When we subdivided the patients into responders (CGII: 1–2) and non-responders (CGI-I: 3-7), we found no differences between the groups with regard to genotypic and allelic frequencies. 4. Discussion In the present study, we found no significant associations between the TPH A218C polymorphism and panic disorder. We also failed to find any significant associations between the 5-

Table 3 Genotypes and allele frequencies of 5-HTTLPR polymorphism in panic patients and normal controls 5-HTTLPR

Genotype S/S

Allele L/S

L/L

Analysis

All panic patients (n = 244) Panic patients with major depression (n = 47) Panic patients without major depression (n = 197) Panic patients with agoraphobia (n = 187) Panic patients without agoraphobia (n = 55) Controls (n = 227) Data represent number (percentage).

159 (65.2%) 28 (59.6%) 131 (66.5%) 124 (66.3%) 33 (60.0%) 141 (62.1%)

77 (31.6%) 19 (40.4%) 58 (29.4%) 56 (29.9%) 21 (38.8%) 76 (33.5%)

8 (3.3%) 0 (0%) 8 (4.1%) 7 (3.7%) 1 (1.8%) 10 (4.4%)

S

χ

p

0.696 2.652 0.890 0.793 1.083

0.706 0.265 0.641 0.673 0.584

2

395 (80.9%) 75 (79.8%) 320 (81.2%) 304 (81.3%) 87 (79.1%) 358 (78.9%)

L

93 (19.1%) 19 (20.2%) 74 (18.8%) 70 (18.7%) 23 (20.9%) 96 (21.1%)

Analysis χ2

p

0.639 0.041 0.735 0.755 0.003

0.424 0.840 0.391 0.385 0.957

W. Kim et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 30 (2006) 1413–1418 Table 5 Response to 10-week paroxetine treatment in panic patients according to TPH A218C and 5-HTTLPR polymorphism Genotype (TPH A218C)

CGI-I

Analysis

A/A (n = 34)

C/A (n = 82)

C/C (n = 27)

p

2.20 (0.84)

2.16 (0.61)

1.96 (0.71)

0.352

Genotype (5-HTTLPR)

CGI-I

Analysis

S/S (n = 92)

L/S (n = 45)

L/L (n = 6)

p

2.16 (0.63)

2.07 (0.84)

2.16 (0.41)

0.745

Mean (S.D.). Kruskal–Wallis tests. CGI-I, Clinical Global Impression Scale – Global Improvement.

2000; Hamilton et al., 1999; Deckert et al., 1997) as well as Japanese (Ishiguro et al., 1997) as our results. Ethnic differences are well-known with regard to the 5-HTTLPR polymorphism. The short allele (S) is less frequent in the Asian population than in Caucasians, indicating that different genetic backgrounds of the 5-HTTLPR polymorphism do exist among populations (Kim et al., 2000; Matsushita et al., 1997; Lesch et al., 1996). Coupled with the previous studies, our results indicate that 5HTTLPR may not be significantly involved in the pathogenesis of panic disorder, regardless of any ethnic differences which may indeed exist. Panic disorder is commonly accompanied by many other psychiatric comorbidities such as major depressive disorder (Breier et al., 1984) and agoraphobia, resulting in more severe symptomatic manifestation and lesser treatment response (Scheibe and Albus, 1994; Reich et al., 1993; Vollrath et al., 1990; Cassano et al., 1989; Charney et al., 1986). We resumed that comorbid major depressive disorder and agoraphobia may constitute a putative phenotype of more severe panic disorder. However, our results did not support our assumption that those two polymorphisms may be associated with more severe form of panic disorder. We also tried to find any associations of the two polymorphisms with major clinical variables without effect, including age of onset, a parameter of genetic disposition in a host of psychiatric disorders. Some studies have reported the gender-related difference regarding serotonin involvement with anxiety. Greenberg et al. (2000) showed an association between 5-HTTLPR polymorphism and personality traits in a female population sample, and Perna et al. (2005) reported an association of long allele of 5-HTTLPR with the better response to paroxetine only in female patients with panic disorder. In this study, we also examined the gender effects additionally, but the comparisons controlling for gender made no impact on our results. Moreover, patients' responses to a 10-week treatment regimen of paroxetine also did not significantly differ according to genotypes. Compared to previous studies that reported different antidepressants responses according to genotype of TPH A218C and 5-HTTLPR in cases of major depressive disorder (Serretti et al., 2001; Zanardi et al., 2000), our results suggest that the primary therapeutic mechanisms exploited by antidepressants in cases of panic disorder may be different from those in cases of major depressive disorder.

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In addition, we determined the relative contribution of demographic, clinical and genetic factors to CGI-I scores after 10-week pharmacotherapy through a linear regression model. As the demographic factors, age and sex were recruited in the first model. As the clinical, number of panic attacks during the last 1 month and HAM-D score were recruited in the second, and the genotypes of two polymorphic site, in the third. The two demographic factors explained 7.9% of the response of 10-week pharmacotherapy (p = 0.003), the two clinical factors did 9.5% (p = 0.001), and the two polymorphisms explain no more that model (p = 0.379). In the final third model, age, number of panic attacks during the last 1 month, and HAM-D score was significant variables which explain the response to pharmacotherapy (p = 0.007; p = 0.005; p = 0.035). Younger age, more attacks during the last 1 month, and higher HAM-D score was the predictors of the good response to pharmacotherapy. We have some limitation in the present study. First, we did not consider the other isoform of TPH, named TPH2. Actually, TPH investigated in the previous association studies and the present study is TPH1. However, Walther et al. (2003) reported that TPH1 mRNA was expressed in the pineal gland and the periphery and barely detected in the brain. On the contrary, TPH2 was demonstrated to be expressed in the brain (Patel et al., 2004). Therefore, the contribution of TPH1 to 5-HT synthesis in the brain is not known, and TPH2 has the possibility to play a role in the 5-HT metabolism, even if a recent article seems to re-evaluate the role of TPH1 (Nakamura et al., 2006). In the future, the study investigating the role of TPH2 in 5-HT metabolism and anxiety disorders will be needed. Second, the subjects with panic disorder were not homogeneous with the medication history. 47 out of 244 patients had no history of previous medication, and others had been prescribed with various agents such as SSRI or benzodiazepines. This may influence our outcome. 5. Conclusion In this study, we found no significant differences in the genotype and allele frequencies in the TPH A218C and 5HTTLPR polymorphisms between the panic patients and the controls as well as subgroup analyses. Our findings suggest that the TPH A218C polymorphism and 5-HTTLPR play no significant roles in the pathogenesis and clinical symptomatologies, at least in a Korean population. Adequately powered subsequent studies will be mandatory to confirm our findings. Acknowledgement This study was supported by a grant from the Korea Science and Engineering Foundation (R01-2002-000-00327-0(2003)). References American psychiatric association. Diagnostic and statistical manual of mental disorder. 4th ed. Washington: American Psychiatric Association Publishing; 1994. Beck A, Mendelson M, Mock J. Inventory for measuring depression. Arch Gen Psychiatry 1961;4:561–71.

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