Correspondence : Kang-Seob Oh, MD, PhD, Department of Psychiatry, Kangbuk Samsung Hospital, Sungkyunkwan University College of Medicine, 108 Pyeong-dong, Jongno-gu, Seoul 110-746, Korea
Tel : +82-2-2001-2478, Fax : +82-2-2001-2211, E-mail : ks2485@empal.com

Introduction

   Panic disorder (PD) is a common anxiety disorder which can be a significant burden to patients. However, the etiology of PD is not yet fully established. Family and twin studies have indicated that certain genes might influence the susceptibility to PD.¹ The overall lifetime morbidity risk in first-degree relatives of PD patients is almost eight times higher than that in the relatives of healthy individuals.² However, the results of genetic association studies provide only tentative evidence for the specific involvement of some candidate genes, such as catechol-O-methyltransferase (COMT).^3,4
Recently, BDNF, the most abundant neurotrophin in the brain, has gained attention for its association with some psychiatric illnesses such as depression^5,6 and obsessive-compulsive disorder.⁷ The amount of evidence that BDNF is involved in hippocampal plasticity and hippocampal-dependent memory in cell models and in animals is quite substantial.^8,9 Bremner et al.¹⁰ reported that patients with PD showed a decrease in measures of benzodiazepine receptor binding in the hippocampus. There have also been some neuroimaging studies which found abnormalities in glucose metabolism in the hippocampus of PD patients.^11,12 On the basis of these previous studies, we hypothesized that the BDNF gene might be a candidate for susceptibility to PD. In this study, we investigated the possible association of the val66met polymorphism (rs 6265) in exon XIIIA of chromosome 11q in patients with PD by using a case-control design, because this SNP has been reported to be functional and associated with a activity-dependent secretion of BDNF.¹³ To our knowledge, there have only been two previous association studies^14,15 concerning the BDNF val66met polymorphism and panic disorder. However, these studies were simple association studies which only compared the frequencies of the genotypes and alleles in a case-control design. In this study, we also investigated the possible association of the BDNF polymorphism and the severity of anxiety in the PD patients by measuring the clinical rating scales.

Methods

Subjects 
   This study was approved by the ethics committee of Kangbuk Samsung Hospital. One hundred and six unrelated patients with panic disorder (63 males and 43 females) were recruited from the department of psychiatry at Kangbuk Samsung Hospital. All of the subjects were Korean adults aged at least 18 years [age: 41.87±10.23 (mean±S.D.)] and met the criteria for panic disorder according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV).¹⁶ We excluded patients with comorbid current major depression, bipolar disorder, post-traumatic stress disorder, or any kind of psychotic disorder, including schizophrenia, by administrating the Mini-International Neuropsychiatric Interview. Information about the symptomatic characteristics of these patients was gathered by measuring the Hamilton anxiety rating scale (HAMA), Beck anxiety inventory (BAI), Spielberg statetrait anxiety inventory (STAI), and anxiety sensitivity index (ASI). 
The normal control group consisted of 160 healthy volunteers [male 52, female 108, age: 31.61±9.04 (mean±S.D.)] who visited the hospital for their regular health screening.

　

Genotyping
Venous blood samples were obtained after the completion of informed consent forms. The genomic DNA was extracted from the white blood cells by standard procedures. The BDNF val66met SNP was assayed by PCR using constructed primers (forward: 5'-GGTGAGAAGAGTGATGACCA-3' and reverse: 5'-GCCAGCCAATTCTCTTTTTG-3'). The PCR products were digested at 37℃ with the restriction enzyme PmaCI (Takara Shuzo Ltd., Kyoto, Japan), followed by 2% agarose gel-electrophoresis, as described previously.¹⁷

Data analysis
Differences in the frequency distribution of the genotype, allele, and allele carrier were examined by cross-tabulation and Pearson chi-square tests using SPSS version ¹³.0 for Microsoft Windows. Hardy-Weinberg equilibrium for the genotype frequencies in the PD patients and normal control samples were tested with chi-square tests. The possible effects of the variation in the BDNF polymorphism on the clinical characteristics of the PD patients were examined with ANOVA or t-tests by comparing the mean scores which were measured by clinical scales. The significance level for the results was set at p<0.05. 

Results

In the comparison of the control group and the patients with panic disorder group, the mean ages [control: 31.61±9.04 vs. patients 41.87±10.23 years, p<0.001] and sex distributions [male/female: 51/108 (control) and 63/43 (patients), p<0.001] were significantly different. The distribution of the genotypes, allele, and allele carrier frequencies for the BDNF Val66Met polymorphism in the patients and controls are shown in Table 1. Since it has been reported that the Met66 allele is associated with lower activity-dependent secretion of BDNF and abnormal hippocampal function,13 we divided the BDNF Val66Met polymorphism into two groups, namely carriers of the Met66 allele (Met/Met and Met/Val genotypes) and carriers of the Val/Val genotype. The observed genotype distributions of the patient and control groups did not differ significantly from the expected rate of Hardy-Weinberg equilibrium (Patients: χ²=1.81, df=1, p=0.18, Controls: χ²=0.19, df=1, p=0.65) There were no significant differences in the frequencies of the genotypes (χ²=4.16, df=2, p=0.13), alleles (χ²=0.79, df=1, p=0.37) or allele carriers (χ²=0.28, df=1, p=0.59) between the patients and controls. We also compared those panic patients with comorbid agoraphobia with the controls, because the comorbidity of agoraphobia in patients with panic disorder causes more severe disturbance and it has been reported that agoraphobia can exist without a history of panic attack.¹⁸ However, the result was also negative. When the above comparisons were conducted separately by gender, we did not find any differences between the two groups (Statistics not shown).
In addition, a one-way ANOVA between the genotype groups and independent t-tests between the met66 allele carriers and Val/Val carriers failed to show any significant difference with respect to the clinical variables or measured scores of the clinical rating scales (Table 2). 

Discussion

We investigated the possible association between panic disorder and the BDNF 196G>A (val66met) polymorphism. Although we failed to find any significant association, our negative finding suggests that the BDNF genetic polymorphism does not play a major role in the susceptibility to or severity of panic disorder in our Korean sample. However, it is possible that other variants of the BDNF gene may play a role in the development of panic disorder or in determining the severity of this illness. The results of the present study correspond with those of earlier studies which reported that there was no association between PD and the BDNF polymorphism.^14,15
However, our findings are somewhat different from those of another previous study¹⁹ which reported that the Val/Val genotype is associated with a higher trait anxiety score in the STAI. This inconsistency may originate from differences in the samples used in the two studies. In the present study, the subjects were composed of panic patients who demonstrated more severe anxiety (mean trait score of STAI; 51.04±9.82) in comparison with the subjects from the normal population (mean trait score of STAI; 35.01±7.4) which were used in the study of Lang et al.¹⁹
There are several limitations to this study. First, the sex and age distributions were significantly different between the patient and control groups. Therefore, it is possible that some of the subjects in the control group might develop PD in later life. However, given the relatively low prevalence of PD in an epidemiological study of a community population in Korea,²⁰ the chance inclusion of a few affected cases in the control group will have little effect on the statistical power.²¹ Considering that negative results were also obtained when we performed all of the statistical analyses in this study separately by gender, it is not likely that the difference in the sex distribution in our sample would have caused any false-negative results. Second, the relatively small sample size limits the generalization of our results. These data need to be confirmed in a much larger sample. 
In conclusion, the results of the present study suggest that the BDNF (val66met) polymorphism does not play a major role in the susceptibility to or severity of panic disorder in our Korean samples. 

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