Progress in Neuro-Psychopharmacology & Biological Psychiatry 51 (2014) 119–125
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Gene × gene × gender interaction of BDNF and COMT genotypes associated with panic disorder Yoshiaki Konishi a, Hisashi Tanii a,⁎, Takeshi Otowa b, Tsukasa Sasaki c, Mamoru Tochigi d, Tadashi Umekage e, Eishi Motomura a, Takashi Shiroyama a, Hisanobu Kaiya f, Yuji Okazaki g, Motohiro Okada a a
Department of Psychiatry, Division of Neuroscience, Graduate School of Medicine, Brain Science and Animal Model Research Center (BSAM), Mie University, Mie, Japan Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Laboratory of Health Education, Graduate School of Education, The University of Tokyo, Tokyo, Japan d Department of Psychiatry, Teikyo University School of Medicine, Tokyo, Japan e Division for Environment, Health and Safety, The University of Tokyo, Tokyo, Japan f Research Center for Panic Disorder, Nagoya Mental Clinic, Aichi, Japan g Department of Psychiatry, Tokyo Metropolitan Matsuzawa Hospital, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 8 November 2013 Received in revised form 29 January 2014 Accepted 29 January 2014 Available online 4 February 2014 Keywords: BDNF COMT Gender Gene × gene × gender interaction Panic disorder
a b s t r a c t Genetic and gender differences are among the factors that have a role in the etiology of panic disorder (PD). It is thought that PD is related to neurotransmitter pathways, such as brain-derived neurotrophic factor (BDNF) and catechol-O-methyltransferase (COMT), both of which are involved in the regulation of the monoamine mechanism. We examined the interactions of BDNF, COMT and gender differences in terms of personality characteristics in PD. The subjects were 470 patients (178 men, 292 women) with a DSM-IV diagnosis of PD, and 458 healthy controls (195 men, 263 women). The subjects were further clinically characterized using the Revised NEO Personality Inventory (NEO-PI-R) and State-Trait Anxiety Inventory (STAI). COMT Val158Met polymorphisms (rs4680) and BDNF Val66Met (rs6265) polymorphisms were genotyped using allelic discrimination by a real-time PCR assay. A multivariate analysis of covariance (MANCOVA) was performed with STAI and NEOPI-R scores as the dependent factor, gender and genotyping groups (BDNF and COMT) as fixed factors, and the covariate of age in the PD and healthy control groups. Post hoc MANCOVA tests were conducted to evaluate COMT × BDNF interactions. An interaction of BDNF × COMT × gender was confirmed in the PD group by MANCOVA on STAI scores and NEO-PI-R Neuroticism and Extraversion scores, whereas no association of such interactions was observed in the healthy controls. The anxiety sensitivity of the COMT Met + BDNF Val/Val carriers was higher than that of the COMT Val/Val + BDNF Val/Val carriers by post hoc MANCOVA. A significant BDNF × COMT × gender interaction was observed in the PD patients but not in the controls. Our findings partly demonstrated the involvement of a gene × gene × gender interaction in the pathogenesis of PD. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Panic disorder (PD) is an anxiety disorder characterized by frequent, unexpected panic attacks and anticipatory anxiety. Epidemiological studies have detected gender differences in the symptomatology of PD. Women are affected with PD approximately twice as often as men, Abbreviations: BDNF, brain-derived neurotrophic factor; COMT, catechol-Omethyltransferase; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, 4th edition; HA, harm avoidance; MANCOVA, multivariate analysis of covariance; Met, methionine; MINI 24, Mini International Neuropsychiatric Interview 5.0.0; NEO-PI-R, NEO Inventory; PCR, polymerase chain reaction; PD, panic disorder; PTSD, posttraumatic stress disorder; SPSS, Statistical Package for Social Science; STAI, The State-Trait Anxiety Inventory; Val, valine. ⁎ Corresponding author at: Department of Psychiatry, Division of Neuroscience, Graduate School of Medicine, Brain Science and Animal Model Research Center (BSAM), Mie University, 2-174, Edobashi, Tsu, Mie, Japan. Tel.: +81 59 231 5018; fax: +81 59 231 5208. E-mail address: [email protected] (H. Tanii). 0278-5846/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2014.01.020
and experience more panic symptoms than men do (Crowe et al., 1983). Female patients with PD are more likely to have respiratory symptoms (shortness of breath, feeling smothered, choking, difficulty swallowing) and faintness during panic attacks (Weissman, 1993). It has been suggested that genetic factors may influence vulnerability to PD, as many current patients have a relative with the disorder (Crowe et al., 1983; Hettema et al., 2001; Weissman, 1993). Several genes related to neurotransmission and neurotrophic systems may contribute to the genetic variation of PD-related traits, and they may modify the phenotypic expression of pathologic anxiety. One plausible genetic risk factor involves brain-derived neurotrophic factor (BDNF), a protein hypothesized to limit or repair the damage caused by stress, and catechol-O-methyltransferase (COMT) (Domschke et al., 2007), both of which has been investigated as the typical gene polymorphisms related to anxiety disorders such as panic disorder. BDNF Met carriers may play a crucial role in increased sensitivity to anxiety, and persons with the Met/Met carriers polymorphism may be
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more sensitive to the stress-induced down-regulation of BDNF (Elzinga et al., 2011). The symptoms of Met/Met carriers tended to be more serious compared to those of Val/Val or Val/Met carriers (Monteleone et al., 2006). COMT Val158Met polymorphisms have been associated with “worrier” (Met allele) tendencies based on findings that the Met allele has been broadly associated with anxiety-related phenotypes (Domschke et al., 2004; McGrath et al., 2004). Studies of personality traits in anxiety disorders have clarified a relationship between neuroticism (Ormel et al., 2004) and extraversion (Jylha et al., 2009). Another recent study reported that resilience was influenced by COMT and BDNF polymorphisms in male — but not female — healthy college students (Kang et al., 2013). Resilience was reported to be negatively associated with neuroticism, and positively related to extraversion in young healthy adults (Campbell-Sills et al., 2006). Regarding gene–gene interaction with anxiety disorder, associations between BDNF and dopamine receptor 3 gene polymorphism have been shown in bipolar disorder comorbid with anxiety disorder (Chang et al., 2013). Hemmings et al. (2013) reported that the interaction of BDNF Val66Met and DRD2 Taq1A polymorphisms influences posttraumatic stress disorder (PTSD) symptom severity. An interaction of promoter variants of the cannabinoid receptor 1 gene (CNR1) and 5-HTTLPR affects the anxious phenotype (Lazary et al., 2009). From the viewpoint of pharmacogenomics and gene–gene interactions, an association between polymorphisms in serotonin transporter (SLC6A4) and serotonin receptor 2A (HTR2A) genes was reported to predict treatment response to venlafaxine XR in generalized anxiety disorder (Lohoff et al., 2013). In addition Lee et al. (2013) reported a significant interaction effect for the Val/Val carriers of the BDNF Val66Met polymorphism and the Met carriers of the COMT Val158Met polymorphism in a comparative study between patients with bipolar II disorder without anxiety disorder and controls. With regard to the gender difference observed in PD, several studies have investigated BDNF and COMT as representative polymorphisms. Shalev et al. (2009) reported that in male subjects, BDNF Val/Val homozygotes showed a greater increase in salivary cortisol than Val/Met heterozygotes. In female subjects, the opposite trend was observed; that is, the Val/Val homozygotes had the lowest increase (Shalev et al., 2009). The female subjects also displayed significantly lower platelet BDNF levels compared to the males. Lommatzsch et al. (2005) found that platelet BDNF levels changed during the menstrual cycle. The 196G/A (Val66Met) polymorphism of the BDNF gene was revealed to be significantly associated with the severity of binge eating behavior in women with bulimia nervosa or other binge eating disorders. It has been suggested that the role of gender in the relationship between the COMT gene and personality may be due to an interaction between estrogen and COMT activity (Harrison and Tunbridge, 2008). An earlier study showed that women with high estrogen levels had lower COMT activity (Briggs and Briggs, 1973). Estrogen was reported to inhibit COMT mRNA expression and reduce its activity (Jiang et al., 2003; Xie et al., 1999). COMT plays an important role in metabolizing catechol estrogens, thereby lowering their levels (Creveling, 2003). Thus, gender differences may interact with the COMT gene to affect personality. As noted above, gender differences in BDNF and COMT have been investigated by many researchers, but the potential interaction of BDNF, COMT and gender is not clear, and no definitive results were obtained in the studies of the relationship between PD and BDNF and COMT We suspected that it would be worthwhile to investigate the gender differences between PD and healthy controls from the viewpoint of gender × genotype interaction. Based on previous studies, we hypothesized that the gene–gene interaction of BDNF Met66 allele and COMT Met158 allele would predispose PD individuals to higher anxiety sensitivity, and would show a gender difference. The aim of our study was to explore the possible association between the gender-specific characteristics of PD-related anxiety traits and two functional polymorphisms of the BDNF and COMT genes. This
study focused particularly on a gene × gene interaction of BDNF and COMT. 2. Methods 2.1. Subjects The study subjects were 470 PD patients (178 men and 292 women) and 458 healthy controls (195 men and 263 women). The study was explained to all subjects prior to their participation, and they each provided written informed consent to participate. In the healthy control group, the inclusion criteria were as follows: drug-free, no previous diagnosis of a psychiatric disorder, and no family history of psychiatric disorder. We excluded individuals with a history of major physical illness, neurological disorder, alcohol abuse, substance abuse, or loss of consciousness due to head injury. The healthy control subjects were screened for the presence or absence of a Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) axis I disorder using the Japanese version of the Mini International Neuropsychiatric Interview 5.0.0 (MINI, 24) (Otsubo et al., 2005). All of the patients with a DSM-IV diagnosis of PD were outpatients at the Nagoya Mental Clinic in Japan, and were diagnosed by at least two doctors. The MINI is a structured interview used to assess psychiatric illness. Screenings were completed for psychiatric illnesses including PD. Agoraphobia was found in 65.8% (n = 192) of the female patients and 51.7% (n = 92) of the male patients. Depression was found in 33.5% (n = 98) of the female patients and 27.5% (n = 49) of the male patients. Bipolar disorder was found in 16.1% (n = 47) of the female patients and 11.2% (n = 20) of the male patients. Most of the 470 patients were on medication and took antidepressants (n = 401), anxiolytics (n = 357), or a mood stabilizer including antipsychotic drugs (n = 160). Most of the patients were being treated with an antidepressant drug such as a serotonin reuptake inhibitor, and the average imipramine equivalent was 74.4 ± 3.2 mg (standard error: SE) (Bollini et al., 1999). This study was approved by the institutional ethics committees of the Mie University Graduate School of Medicine and the Warakukai Nagoya Mental Clinic. 2.2. Psychological tests Psychological tests were administered to the participants by questionnaire and by interview. The questionnaire was used to collect clinical information including basic data on family members with PD and genetic factors. The collected data included gender, age, height, weight, blood type, birthplace, growth history, body weight at birth, marital status, drinking habits, smoking habits, menstruation, medical health history, family medical history, and disease under treatment. The questionnaire concerned the individual's experience with PD, including symptoms of first panic attack, frequency of attacks, and avoidance during the previous month of situations in which a panic attack might occur. The State-Trait Anxiety Inventory (STAI) was administered to all of the subjects The STAI has 20 questions to assess state anxiety, and 20 for trait anxiety (Nakazato and Mizuguchi, 1982; Spielberger et al., 1983). “Trait anxiety” represents stable individual differences in the propensity for anxiety, and refers to a tendency to respond to life situations with “general” anxiety. The Revised NEO Personality Inventory (NEO-PI-R) was also administered to all of the subjects The NEO-PI-R is a standard instrument for measuring the personality traits of individuals over a wide range of ages, from the elderly to the young (Costa and McCrae, 1992; Shimonaka, 1997). The inventory is based on a fivefactor model of character: Neuroticism (N), Extroversion (E), Openness to Experience (O), Agreeableness (A), and Conscientiousness (C). The subjects responded to the 240 items on a 5-point scale.
Y. Konishi et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 51 (2014) 119–125
170 (40%) 207 (39%) 377 (40%) 149 (42%) 259 (44%) 408 (43%) 0.897 0.129 0.418
250 (60%) 319 (61%) 569 (60%) 207 (58%) 325 (56%) 532 (57%) 38 (20%) 40 (15%) 78 (17%) 32 (18%) 64 (22%) 96 (20%) 94 (48%) 127 (48%) 221 (48%) 85 (48%) 131 (45%) 216 (46%) 0.163 0.123 0.787
63 (32%) 96 (37%) 159 (35%) 61 (34%) 97 (33%) 158 (34%) 0.114 0.137 0.941 0.160 0.118 470 PD patients
Abbreviations: COMT = catechol-O-methyltransferase; BDNF = brain-derived neurotrophic factor; Val = valine; Met = methionine. PD = panic disorder; SD = standard deviation. ⁎ Relative to the control group.
113 (29%) 172 (33%) 285 (31%) 121 (34%) 165 (28%) 286 (30%) 277 (71%) 354 (67%) 631 (69%) 235 (66%) 419 (72%) 654 (70%) 19 (10%) 24 (9%) 43 (9%) 17 (9%) 25 (9%) 42 (9%) 75 (38%) 124 (47%) 199 (43%) 87 (49%) 115 (39%) 202 (43%) 101 (52%) 115 (44%) 216 (47%) 74 (42%) 152 (52%) 226 (48%) Men (195) Women (263) Total (458) Men (178) Women (292) Total (470) 36.63 ± 13.96 458 Controls
37.93 ± 11.10
Val Val/Met Val/Val Met Val
COMT allele: n (%)
P⁎ Met/Met Val/Met
COMT genotypes: n (%)
Val/Val P⁎ Sex (n) P⁎ Age Mean ± SD
The average age of the patients in the PD group was 37.93 ± 11.10 years, and that of the healthy control group was 36.63 ± 13.96 years (t = 1.566, p = 0.118). The gender ratios of the PD group (178 men, 292 women) and controls (195 men and 263 women) showed no significant difference (χ2 value = 1.942, p = 0.163) (Table 1). The COMT Val158Met polymorphism followed Hardy–Weinberg equilibrium in each subgroup (p = 0.742 for PD, p = 0.770 for healthy control; p = 0.289 for male, PD, p = 0.360 for male healthy control; p = 0.356 for female PD, p = 0.248 for female healthy control). The Hardy–Weinberg equilibrium also applied to the BDNF Val66Met polymorphism in each subgroup (p = 0.161 for PD, p = 0.935 for healthy control; p = 0.800 for male PD, p = 0.782 for male healthy control; p = 0.119 for female, PD, p = 0.850 for female healthy control). No significant difference was observed in the genotypic or allele frequencies of COMT and BDNF polymorphisms between the PD and healthy control groups (Table 1). Equivalent doses to that of the standard imipramine did not differ significantly among the COMT Val158Met or BDNF Val66Met polymorphism groups (all, male, or female PD patients; Supplementary Table 1). There was a significant health status effect on STAI and NEO-PI-R scores, and there were significant interactions between healthy status, gender, COMT and BDNF polymorphisms on the STAI, Neuroticism and Extraversion scores (Table 2).
n
3. Results
Group
The Hardy–Weinberg equilibrium for genotyping frequencies was calculated using χ2 tests. Statistical calculations were carried out using SPSS for Windows (Release 17.0)®. A multivariate analysis of covariance (MANCOVA) was performed with STAI and NEO-PI-R scores as the dependent factor, and health status (PD patients and control subjects), gender and genotyping groups (BDNF and COMT) as fixed factors, and the covariate of age. As a sub-analysis, we performed a MANCOVA with STAI and NEO-PI-R scores as the dependent factor, gender and genotyping groups (BDNF and COMT) as fixed factors, and the covariate of age in the PD patients and healthy controls. Post hoc MANCOVA tests were conducted to evaluate COMT × BDNF interactions. The statistical power of effect size was used for comparative effects in the power analysis. Comparisons of gender-linked genotype distributions and allele frequencies between PD and healthy controls were performed by the χ2 test. Student's t-test was used for differences in age. All tests were performed with a two-tailed type-I error rate of p b 0.05.
Table 1 Comparison of the mean age, gender, COMT Val158Met, BDNF Val66Met genotype distribution, and allele frequency between the PD patient and control groups.
2.4. Statistical analyses
P⁎
BDNF genotypes: n (%)
Met/Met
P⁎
BDNF allele: n (%)
For each subject, a 7-mL blood sample was centrifuged at 2000 rpm × 10 min in a collection tube and separated into the plasma layer, buffy-coat layer (containing white blood corpuscles), and red blood corpuscle layer. A 150-μL sample of the buffy coat was used for automatic blood DNA extraction. DNA was extracted using a BioRobot EZ1 (Qiagen, Hilden, Germany). BDNF Val66Met (rs6265 (assay ID C__11592758_10)) and COMT Val158Met (rs4680 (Assay ID C__25746809_50)) polymorphisms were genotyped with allelic discrimination using the TaqMan real-time polymerase chain reaction (PCR) assay (Life Technologies Japan, Tokyo). The assay includes a sequence-specific fluorogenic minor groove binder probe for each allele. Each probe is 5′-labeled with a different reporter fluorescent dye (4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein and 6-carboxyfluorescein) to differentiate the amplification of each allele. Primer–probe sets for the detection of the polymorphisms were purchased from Applied Biosystems (Foster City, CA, USA). PCR was carried out on an ABI StepOne +™ Real-Time PCR System according to the manufacturer's protocol (Applied Biosystems).
Met
P⁎
0.714 0.100 0.123
2.3. SNP genotyping
121
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Table 2 Effects of health status, gender, COMT polymorphism and BDNF polymorphism on STAI and NEO-PI-R scores. Category of groups
Response variables
Health status (HS) Gender (G) COMT polymorphism (CP) BDNF polymorphism (BP) HS × G HS × CP HS × BP BP × CP HS × G × CP HS × G × BP HS × CP × BP G × CP × BP HS × G × CP × BP
STAI state
STAI trait
Neuroticism
Extraversion
Openness
Agreeableness
Consciousness
b0.001⁎⁎⁎ 0.989 0.546 0.536 0.798 0.001⁎⁎ 0.679 0.774 0.051 0.465 0.021⁎ 0.058 0.042⁎
b0.001⁎⁎⁎ 0.180 0.362 0.275 0.789 0.034⁎ 0.131 0.896 0.098 0.640 0.051 0.300 0.022⁎
b0.001⁎⁎⁎ 0.001⁎⁎ 0.940 0.814 0.942 0.007⁎⁎ 0.056 0.953 0.216 0.938 0.045⁎ 0.212 0.030⁎
b0.001⁎⁎⁎ 0.063 0.960 0.107 0.328 0.008⁎⁎ 0.551 0.39 0.006⁎⁎ 0.742 b0.001⁎⁎⁎ 0.008⁎⁎ 0.001⁎⁎
b0.001⁎⁎⁎ 0.675 0.811 0.893 0.259 0.004⁎⁎ 0.629 0.365 0.027⁎ 0.716 0.045⁎ 0.777 0.938
0.024⁎ b0.001⁎⁎⁎ 0.738 0.392 0.929 0.999 0.053 0.671 0.585 0.976 0.666 0.890 0.723
0.001⁎⁎ 0.617 0.370 0.567 0.663 0.109 0.035⁎ 0.552 0.413 0.396 0.141 0.603 0.122
Abbreviations: COMT = catechol-O-methyltransferase; BDNF = brain-derived neurotrophic factor; STAI = State-Trait Anxiety Inventory; NEO-PI-R = Revised NEO Personality Inventory; MANCOVA was performed with STAI and NEO-PI-R scores as dependent factor, and health status, gender and genotyping groups as fixed factors and covariate of age. ⁎ P b 0.05. ⁎⁎ P b 0.01. ⁎⁎⁎ P b 0.001.
Following the sub-analysis, Tables 3 and 4 summarize the interaction of gene × gender revealed by the MANCOVA on STAI and NEOPI-R scores. In the PD patients, BDNF × COMT × gender showed a significant effect on STAI state (F = 5.70, p = 0.018, power = 0.662), STAI trait (F = 4.47, p = 0.035, power = 0.559), and the Neuroticism (F = 5.22, p = 0.023, power = 0.625), and Extraversion scores (F = 12.3, p = 0.001, power = 0.938). No interactive effects of the BDNF × COMT × gender interaction on STAI or NEO-PI-R scores were detected in the healthy controls, however. Particularly in the male PD patients, our results indicated a significant gene × gene interaction by a post hoc MANCOVA on STAI state and the Neuroticism and Extraversion scores (Fig. 1, Suppl. Table 2). The post hoc MANCOVA revealed that among the male PD patients, COMT Met + BDNF Val/Val carriers had significantly higher STAI state and Neuroticism scores than the COMT Val/Val + BDNF Val/Val carriers (STAI state; p = 0.009, Neuroticism; p = 0.014). Among the male PD patients, the COMT Val/Val + BDNF Val/Val carriers and the COMT Met + BDNF Met carriers also had significantly higher Extraversion scores than the COMT Met + BDNF Val/Val carriers (p b 0.001, p = 0.007). Finally, among the male PD patients, the COMT Val/Val + BDNF Val/Val carriers had significantly higher Extraversion scores than the COMT Val/Val + BDNF Met carriers (p = 0.048).
4. Discussion Our results showed that BDNF × COMT × gender had a significant effect on STAI state, STAI trait, and the NEO-PI-R Neuroticism and Extraversion scores in PD patients. No interactive effects of the BDNF × COMT × gender interaction on STAI or NEO-PI-R scores were detected in the healthy controls. The gene effect was observed particularly in the men with PD. As shown in previous studies of personality traits in anxiety disorders, we found that higher anxiety scores are associated with higher Neuroticism (Ormel et al., 2004) and lower Extraversion scores (Jylha et al., 2009). In the present study, the male PD patients who were COMT Met carriers had higher STAI state and Neuroticism scores and lower Extraversion scores than the COMT Val/Val carriers, especially in the BDNF Val/Val carrier group (Fig. 1), corresponding to opposite gender effects on BDNF Val/Val homozygotes (Shalev et al., 2009), and these effects were enhanced in COMT Met carriers. The results of our study suggest the possibility of BDNF–COMT– gender involvement in anxiety traits, personality, and panic disorder, without significant differences of genotype distribution related to gene–gene interaction. Gender differences may interact with the BDNF and COMT genes' effects on anxiety sensitivity and related personality
Table 3 Effects of gender, BDNF polymorphism, and COMT polymorphism on STAI scores in the PD and healthy control groups. Panic disorder
STAI state
STAI trait
Gender (G) BDNF polymorphism (BP) COMT polymorphism (CP) G × BP G × CP BP × CP G × BP × CP Gender (G) BDNF polymorphism (BP) COMT polymorphism (CP) G × BP G × CP BP × CP G × BP × CP
Healthy control
F
P
Power
F
P
Power
0.05 0.56 4.72 0.27 2.53 1.15 5.70 0.69 2.71 0.56 0.00 3.55 1.32 4.47
0.822 0.456 0.031⁎ 0.604 0.113 0.283 0.018⁎ 0.408 0.101 0.456 0.972 0.061 0.251 0.035⁎
0.056 0.115 0.581 0.081 0.354 0.188 0.662 0.131 0.375 0.116 0.050 0.467 0.209 0.559
0.03 0.27 6.81 0.47 1.86 6.24 0.01 0.80 0.01 7.96 0.43 0.09 2.65 1.02
0.869 0.601 0.009⁎⁎ 0.496 0.173 0.013⁎ 0.942 0.372 0.921 0.005⁎⁎ 0.513 0.763 0.104 0.312
0.053 0.082 0.740 0.104 0.275 0.702 0.051 0.145 0.051 0.804 0.100 0.060 0.368 0.172
Abbreviations: BDNF = brain-derived neurotrophic factor; COMT = catechol-O-methyltransferase; STAI = State-Trait Anxiety Inventory. MANCOVA was performed with STAI scores as dependent factor, gender and genotyping groups as fixed factors and covariate of age. ⁎ P b 0.05. ⁎⁎ P b 0.01.
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Table 4 Effects of gender, BDNF polymorphism, and COMT polymorphism on NEO-PI-R (Neuroticism and Extraversion) scores in the PD and healthy control groups. Panic disorder
Neuroticism
Gender (G) BDNF polymorphism (BP) COMT polymorphism (CP) G × BP G × CP BP × CP G × BP × CP Gender (G) BDNF polymorphism (BP) COMT polymorphism (CP) G × BP G × CP BP × CP G × BP × CP
Extraversion
Healthy control
F
P
Power
F
P
Power
4.05 2.12 2.79 0.08 4.17 1.59 5.22 2.66 0.42 2.47 0.15 7.20 7.13 12.3
0.045⁎ 0.146 0.096 0.776 0.042⁎ 0.208 0.023⁎ 0.104 0.515 0.117 0.695 0.008⁎⁎ 0.008⁎⁎ 0.001⁎⁎
0.518 0.306 0.384 0.059 0.530 0.242 0.625 0.369 0.100 0.347 0.068 0.762 0.758 0.938
7.86 2.18 5.09 0.17 0.23 2.36 0.43 0.68 3.60 5.23 0.00 0.52 5.96 0.59
0.005⁎⁎ 0.141 0.025⁎ 0.682 0.630 0.125 0.514 0.412 0.059 0.023⁎ 0.976 0.469 0.015⁎ 0.445
0.799 0.313 0.614 0.069 0.077 0.335 0.100 0.130 0.473 0.626 0.050 0.112 0.683 0.119
Abbreviations: BDNF = brain-derived neurotrophic factor; COMT = catechol-O-methyltransferase; NEO-PI-R = Revised NEO Personality Inventory; MANCOVA was performed with STAI scores as dependent factor, gender and genotyping groups as fixed factors and covariate of age. ⁎ P b 0.05. ⁎⁎ P b 0.01.
characteristics in the pathogenesis of this disorder through the monoamine system comprised of serotonergic, norepinephrine and dopaminergic systems. Previous studies of the potential involvement of gene–gene interactions with PD examined COMT and serotonin receptor polymorphisms. Freitag et al. (2006) reported a nominally significant interaction between the serotonin receptor 1A and COMT polymorphisms. Interaction of serotonin-2A receptor and COMT polymorphism (Karacetin et al., 2012) was reported with no significant interaction. Gene–gene interactions in PD have not been extensively studied.
**
140 BDNF Val/Val BDNF Met Carrier
STAI-state
50 40 30 20 10
BDNF Val/Val BDNF Met Carrier
100 80 60 40 20
0 COMT Val/Val
COMT Met Carrier
m
*** *
COMT Val/Val
0
COMT Met Carrier
COMT Val/Val COMT Met COMT Val/Val COMT Met Carrier Carrier
f
m
f
** BDNF Val/Val BDNF Met Carrier
100
Extraversion
*
120
Neuroticism
60
120
Most brain imaging studies of patients with PD have consistently shown abnormalities in the limbic system and ventromedial prefrontal cortex associated with emotional arousal (Gorman et al., 2000). In a study of patients with PD, we observed hypoactivation in the lateral prefrontal cortex (PFC), which is responsible for executive function through the dopaminergic system (Nishimura et al., 2007). A hypothetical inverted U-shaped curve was proposed for the relationship between working memory performance and prefrontal dopaminergic activity (Goldman-Rakic et al., 2000). In the present study, significant genetic effects by COMT genotype were observed in healthy control subjects
80 60 40 20 0 COMT Val/Val
m
COMT Met Carrier
COMT Val/Val
COMT Met Carrier
f
Fig. 1. Comparison between genotype groups with post hoc test in male and female PD patients. A gene effect was observed, particularly in the male PD patients, on the STAI state (COMT Val/Val + BDNF Val/Val carriers = 40.2 ± 1.8, COMT Met + BDNF Val/Val carriers = 50.8 ± 2.6), the NEO-PI-R factor Neuroticism scores (COMT Val/Val + BDNF Val/Val carriers = 98.5 ± 4.5, COMT Met + BDNF Val/Val carriers = 117.2 ± 6.8), and the NEO-PI-R factor Extraversion scores (COMT Val/Val + BDNF Val/Val carriers = 93.5 ± 2.7, COMT Val/Val + BDNF Met carriers = 88.1 ± 4.1, COMT Met + BDNF Val/Val carriers = 73.6 ± 5.1, COMT Met + BDNF Met carriers = 93.5 ± 2.7). Error bars: standard error of the mean. ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001. PD, panic disorder; COMT, catechol-O-methyltransferase; BDNF, brain-derived neurotrophic factor; Val, valine; Met, methionine; STAI, State-Trait Anxiety Inventory; m, males; f, females.
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(Tables 3, 4; Suppl. Table 3), but the genotype effects of COMT were the opposite between the PD patients and the healthy controls. The modulation of the COMT genotype on anxiety-related parameters seems to be different in PD patients. Our present data on the distinct effect of the COMT genotype could be explained by the application of an inverted U-shaped hypothesis regarding the prefrontal dopamine level. In accord with this inverted U-shaped hypothesis, the PFC dopamine levels of Val158 homozygotes and Met carriers fell into an inverted U-shaped curve in accord with the increase/decrease of COMT activity and showed more complex results than the simple ‘warrior/worrier’ hypothesis of COMT genotype (Mier et al., 2010). As prefrontal dopamine could not be measured directly, this speculation is only a working hypothesis, but it is thought that anxiety sensitivity might be indirectly related to PD through the dopamine neurotransmission of the PFC. There are few previous studies of gene–gene interactions related to gender differences. Niewold et al. (2010) reported a gene–gene–sex interaction in cytokine gene polymorphisms in juvenile dermatomyositis; to the best of our knowledge, such an interaction has not yet been reported in psychiatric disease. We studied the gene × gene × gender interaction of BDNF and COMT genotypes associated with panic disorder, and we suspect that the gender specificity of COMT Val158Met influences personality traits in male PD patients. Whether the interaction of the BDNF and COMT genes leads to an elevation of anxiety sensitivity and how such an interaction affects the etiology of PD requires further clarification. The present study has several limitations. First, only one representative functional SNP within the BDNF and COMT was examined. It would have been desirable to study all tagging SNPs within BDNF and COMT, as well as other variants associated with dopaminergic neurotransmission. Second, a self-reported questionnaire assessment measure was used to evaluate personality-related PD symptoms. Additional assessments by clinicians may be more accurate and/or reliable. The personality inventory scores may reflect effects from the symptoms, and the STAI state may correspond to the severity of the symptoms. Further data for the severity of symptoms of patients with PD are necessary. Third, we included PD patients whose comorbidities included agoraphobia, depression or bipolar disorder as a composite group of PD. For replication studies, it is desirable to collect more detailed information and a homogeneous group with larger sample sizes that could lead to more specific evidence about anxiety disorders such as PD. 5. Conclusion Gene × gene × gender interactions were observed in the association of the BDNF and COMT polymorphisms with STAI scores and with the NEO-PI-R Neuroticism and Extraversion scores in the male PD group. Our results show the possibility of a gender effect in anxiety disorders such as PD, which has a higher prevalence rate in women. The BDNF and COMT interaction appears to exhibit gender differences in personality scores, with potential implications for gender differences in the pathogenesis of PD. Contributors Y.K. executed the experimental protocols and statistical analyses, and wrote the first draft of the manuscript. Y.K., Y.O., and H.T. were responsible for the study concept and design. M.T., T.U., E.M., T.S., H.K. and Y.O. contributed to the acquisition of clinical data. Y.K., E.M. and M.O. conducted the data analysis. T.O., T.S. and M.O. interpreted the findings and provided critical revisions of the manuscript. Acknowledgments This work was supported by KAKENHI (a Grant-in-Aid for Scientific Research) in the Priority Area of Applied Genomics from the Ministry
of Education, Culture, Sports, Science and Technology of Japan, (No. 17019029; No. 21591483) awarded to H. Tanii and Y. Okazaki. We thank the staff of the Department of Psychiatry at the Mie University Graduate School of Medicine and the Nagoya Mental Clinic for their enthusiastic cooperation. We are grateful to all of the PD patients and others who answered the questionnaire and allowed their blood to be sampled. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.pnpbp.2014.01.020. References Bollini P, Pampallona S, Tibaldi G, Kupelnick B, Munizza C. Effectiveness of antidepressants. Meta-analysis of dose–effect relationships in randomised clinical trials. Br J Psychiatry 1999;174:297–303. Briggs MH, Briggs M. Hormonal influences on erythrocyte catechol-O-methyl transferase activity in humans. Experientia 1973;29:278–80. Campbell-Sills L, Cohan SL, Stein MB. 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Kang JI, Kim SJ, Song YY, Namkoong K, An SK. Genetic influence of COMT and BDNF gene polymorphisms on resilience in healthy college students. Neuropsychobiology 2013;68:174–80. Karacetin G, Bayoglu B, Cengiz M, Demir T, Kocabasoglu N, Uysal O, et al. Serotonin-2A receptor and catechol-O-methyltransferase polymorphisms in panic disorder. Prog Neuropsychopharmacol Biol Psychiatry 2012;36:5–10. Lazary J, Lazary A, Gonda X, Benko A, Molnar E, Hunyady L, et al. Promoter variants of the cannabinoid receptor 1 gene (CNR1) in interaction with 5-HTTLPR affect the anxious phenotype. Am J Med Genet B Neuropsychiatr Genet 2009;150B:1118–27. Lee SY, Chen SL, Wang YS, Chang YH, Huang SY, Tzeng NS, et al. COMT and BDNF interacted in bipolar II disorder not comorbid with anxiety disorder. Behav Brain Res 2013;237:243–8. Lohoff FW, Narasimhan S, Rickels K. Interaction between polymorphisms in serotonin transporter (SLC6A4) and serotonin receptor 2A (HTR2A) genes predict treatment response to venlafaxine XR in generalized anxiety disorder. Pharmacogenomics J 2013;13:464–9. Lommatzsch M, Zingler D, Schuhbaeck K, Schloetcke K, Zingler C, Schuff-Werner P, et al. The impact of age, weight and gender on BDNF levels in human platelets and plasma. Neurobiol Aging 2005;26:115–23.
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Gene × gene × gender interaction of BDNF and COMT genotypes associated with panic disorder Yoshiaki Konishi a, Hisashi Tanii a,⁎, Takeshi Otowa b, Tsukasa Sasaki c, Mamoru Tochigi d, Tadashi Umekage e, Eishi Motomura a, Takashi Shiroyama a, Hisanobu Kaiya f, Yuji Okazaki g, Motohiro Okada a a
Department of Psychiatry, Division of Neuroscience, Graduate School of Medicine, Brain Science and Animal Model Research Center (BSAM), Mie University, Mie, Japan Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Laboratory of Health Education, Graduate School of Education, The University of Tokyo, Tokyo, Japan d Department of Psychiatry, Teikyo University School of Medicine, Tokyo, Japan e Division for Environment, Health and Safety, The University of Tokyo, Tokyo, Japan f Research Center for Panic Disorder, Nagoya Mental Clinic, Aichi, Japan g Department of Psychiatry, Tokyo Metropolitan Matsuzawa Hospital, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 8 November 2013 Received in revised form 29 January 2014 Accepted 29 January 2014 Available online 4 February 2014 Keywords: BDNF COMT Gender Gene × gene × gender interaction Panic disorder
a b s t r a c t Genetic and gender differences are among the factors that have a role in the etiology of panic disorder (PD). It is thought that PD is related to neurotransmitter pathways, such as brain-derived neurotrophic factor (BDNF) and catechol-O-methyltransferase (COMT), both of which are involved in the regulation of the monoamine mechanism. We examined the interactions of BDNF, COMT and gender differences in terms of personality characteristics in PD. The subjects were 470 patients (178 men, 292 women) with a DSM-IV diagnosis of PD, and 458 healthy controls (195 men, 263 women). The subjects were further clinically characterized using the Revised NEO Personality Inventory (NEO-PI-R) and State-Trait Anxiety Inventory (STAI). COMT Val158Met polymorphisms (rs4680) and BDNF Val66Met (rs6265) polymorphisms were genotyped using allelic discrimination by a real-time PCR assay. A multivariate analysis of covariance (MANCOVA) was performed with STAI and NEOPI-R scores as the dependent factor, gender and genotyping groups (BDNF and COMT) as fixed factors, and the covariate of age in the PD and healthy control groups. Post hoc MANCOVA tests were conducted to evaluate COMT × BDNF interactions. An interaction of BDNF × COMT × gender was confirmed in the PD group by MANCOVA on STAI scores and NEO-PI-R Neuroticism and Extraversion scores, whereas no association of such interactions was observed in the healthy controls. The anxiety sensitivity of the COMT Met + BDNF Val/Val carriers was higher than that of the COMT Val/Val + BDNF Val/Val carriers by post hoc MANCOVA. A significant BDNF × COMT × gender interaction was observed in the PD patients but not in the controls. Our findings partly demonstrated the involvement of a gene × gene × gender interaction in the pathogenesis of PD. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Panic disorder (PD) is an anxiety disorder characterized by frequent, unexpected panic attacks and anticipatory anxiety. Epidemiological studies have detected gender differences in the symptomatology of PD. Women are affected with PD approximately twice as often as men, Abbreviations: BDNF, brain-derived neurotrophic factor; COMT, catechol-Omethyltransferase; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, 4th edition; HA, harm avoidance; MANCOVA, multivariate analysis of covariance; Met, methionine; MINI 24, Mini International Neuropsychiatric Interview 5.0.0; NEO-PI-R, NEO Inventory; PCR, polymerase chain reaction; PD, panic disorder; PTSD, posttraumatic stress disorder; SPSS, Statistical Package for Social Science; STAI, The State-Trait Anxiety Inventory; Val, valine. ⁎ Corresponding author at: Department of Psychiatry, Division of Neuroscience, Graduate School of Medicine, Brain Science and Animal Model Research Center (BSAM), Mie University, 2-174, Edobashi, Tsu, Mie, Japan. Tel.: +81 59 231 5018; fax: +81 59 231 5208. E-mail address: [email protected] (H. Tanii). 0278-5846/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2014.01.020
and experience more panic symptoms than men do (Crowe et al., 1983). Female patients with PD are more likely to have respiratory symptoms (shortness of breath, feeling smothered, choking, difficulty swallowing) and faintness during panic attacks (Weissman, 1993). It has been suggested that genetic factors may influence vulnerability to PD, as many current patients have a relative with the disorder (Crowe et al., 1983; Hettema et al., 2001; Weissman, 1993). Several genes related to neurotransmission and neurotrophic systems may contribute to the genetic variation of PD-related traits, and they may modify the phenotypic expression of pathologic anxiety. One plausible genetic risk factor involves brain-derived neurotrophic factor (BDNF), a protein hypothesized to limit or repair the damage caused by stress, and catechol-O-methyltransferase (COMT) (Domschke et al., 2007), both of which has been investigated as the typical gene polymorphisms related to anxiety disorders such as panic disorder. BDNF Met carriers may play a crucial role in increased sensitivity to anxiety, and persons with the Met/Met carriers polymorphism may be
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more sensitive to the stress-induced down-regulation of BDNF (Elzinga et al., 2011). The symptoms of Met/Met carriers tended to be more serious compared to those of Val/Val or Val/Met carriers (Monteleone et al., 2006). COMT Val158Met polymorphisms have been associated with “worrier” (Met allele) tendencies based on findings that the Met allele has been broadly associated with anxiety-related phenotypes (Domschke et al., 2004; McGrath et al., 2004). Studies of personality traits in anxiety disorders have clarified a relationship between neuroticism (Ormel et al., 2004) and extraversion (Jylha et al., 2009). Another recent study reported that resilience was influenced by COMT and BDNF polymorphisms in male — but not female — healthy college students (Kang et al., 2013). Resilience was reported to be negatively associated with neuroticism, and positively related to extraversion in young healthy adults (Campbell-Sills et al., 2006). Regarding gene–gene interaction with anxiety disorder, associations between BDNF and dopamine receptor 3 gene polymorphism have been shown in bipolar disorder comorbid with anxiety disorder (Chang et al., 2013). Hemmings et al. (2013) reported that the interaction of BDNF Val66Met and DRD2 Taq1A polymorphisms influences posttraumatic stress disorder (PTSD) symptom severity. An interaction of promoter variants of the cannabinoid receptor 1 gene (CNR1) and 5-HTTLPR affects the anxious phenotype (Lazary et al., 2009). From the viewpoint of pharmacogenomics and gene–gene interactions, an association between polymorphisms in serotonin transporter (SLC6A4) and serotonin receptor 2A (HTR2A) genes was reported to predict treatment response to venlafaxine XR in generalized anxiety disorder (Lohoff et al., 2013). In addition Lee et al. (2013) reported a significant interaction effect for the Val/Val carriers of the BDNF Val66Met polymorphism and the Met carriers of the COMT Val158Met polymorphism in a comparative study between patients with bipolar II disorder without anxiety disorder and controls. With regard to the gender difference observed in PD, several studies have investigated BDNF and COMT as representative polymorphisms. Shalev et al. (2009) reported that in male subjects, BDNF Val/Val homozygotes showed a greater increase in salivary cortisol than Val/Met heterozygotes. In female subjects, the opposite trend was observed; that is, the Val/Val homozygotes had the lowest increase (Shalev et al., 2009). The female subjects also displayed significantly lower platelet BDNF levels compared to the males. Lommatzsch et al. (2005) found that platelet BDNF levels changed during the menstrual cycle. The 196G/A (Val66Met) polymorphism of the BDNF gene was revealed to be significantly associated with the severity of binge eating behavior in women with bulimia nervosa or other binge eating disorders. It has been suggested that the role of gender in the relationship between the COMT gene and personality may be due to an interaction between estrogen and COMT activity (Harrison and Tunbridge, 2008). An earlier study showed that women with high estrogen levels had lower COMT activity (Briggs and Briggs, 1973). Estrogen was reported to inhibit COMT mRNA expression and reduce its activity (Jiang et al., 2003; Xie et al., 1999). COMT plays an important role in metabolizing catechol estrogens, thereby lowering their levels (Creveling, 2003). Thus, gender differences may interact with the COMT gene to affect personality. As noted above, gender differences in BDNF and COMT have been investigated by many researchers, but the potential interaction of BDNF, COMT and gender is not clear, and no definitive results were obtained in the studies of the relationship between PD and BDNF and COMT We suspected that it would be worthwhile to investigate the gender differences between PD and healthy controls from the viewpoint of gender × genotype interaction. Based on previous studies, we hypothesized that the gene–gene interaction of BDNF Met66 allele and COMT Met158 allele would predispose PD individuals to higher anxiety sensitivity, and would show a gender difference. The aim of our study was to explore the possible association between the gender-specific characteristics of PD-related anxiety traits and two functional polymorphisms of the BDNF and COMT genes. This
study focused particularly on a gene × gene interaction of BDNF and COMT. 2. Methods 2.1. Subjects The study subjects were 470 PD patients (178 men and 292 women) and 458 healthy controls (195 men and 263 women). The study was explained to all subjects prior to their participation, and they each provided written informed consent to participate. In the healthy control group, the inclusion criteria were as follows: drug-free, no previous diagnosis of a psychiatric disorder, and no family history of psychiatric disorder. We excluded individuals with a history of major physical illness, neurological disorder, alcohol abuse, substance abuse, or loss of consciousness due to head injury. The healthy control subjects were screened for the presence or absence of a Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) axis I disorder using the Japanese version of the Mini International Neuropsychiatric Interview 5.0.0 (MINI, 24) (Otsubo et al., 2005). All of the patients with a DSM-IV diagnosis of PD were outpatients at the Nagoya Mental Clinic in Japan, and were diagnosed by at least two doctors. The MINI is a structured interview used to assess psychiatric illness. Screenings were completed for psychiatric illnesses including PD. Agoraphobia was found in 65.8% (n = 192) of the female patients and 51.7% (n = 92) of the male patients. Depression was found in 33.5% (n = 98) of the female patients and 27.5% (n = 49) of the male patients. Bipolar disorder was found in 16.1% (n = 47) of the female patients and 11.2% (n = 20) of the male patients. Most of the 470 patients were on medication and took antidepressants (n = 401), anxiolytics (n = 357), or a mood stabilizer including antipsychotic drugs (n = 160). Most of the patients were being treated with an antidepressant drug such as a serotonin reuptake inhibitor, and the average imipramine equivalent was 74.4 ± 3.2 mg (standard error: SE) (Bollini et al., 1999). This study was approved by the institutional ethics committees of the Mie University Graduate School of Medicine and the Warakukai Nagoya Mental Clinic. 2.2. Psychological tests Psychological tests were administered to the participants by questionnaire and by interview. The questionnaire was used to collect clinical information including basic data on family members with PD and genetic factors. The collected data included gender, age, height, weight, blood type, birthplace, growth history, body weight at birth, marital status, drinking habits, smoking habits, menstruation, medical health history, family medical history, and disease under treatment. The questionnaire concerned the individual's experience with PD, including symptoms of first panic attack, frequency of attacks, and avoidance during the previous month of situations in which a panic attack might occur. The State-Trait Anxiety Inventory (STAI) was administered to all of the subjects The STAI has 20 questions to assess state anxiety, and 20 for trait anxiety (Nakazato and Mizuguchi, 1982; Spielberger et al., 1983). “Trait anxiety” represents stable individual differences in the propensity for anxiety, and refers to a tendency to respond to life situations with “general” anxiety. The Revised NEO Personality Inventory (NEO-PI-R) was also administered to all of the subjects The NEO-PI-R is a standard instrument for measuring the personality traits of individuals over a wide range of ages, from the elderly to the young (Costa and McCrae, 1992; Shimonaka, 1997). The inventory is based on a fivefactor model of character: Neuroticism (N), Extroversion (E), Openness to Experience (O), Agreeableness (A), and Conscientiousness (C). The subjects responded to the 240 items on a 5-point scale.
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170 (40%) 207 (39%) 377 (40%) 149 (42%) 259 (44%) 408 (43%) 0.897 0.129 0.418
250 (60%) 319 (61%) 569 (60%) 207 (58%) 325 (56%) 532 (57%) 38 (20%) 40 (15%) 78 (17%) 32 (18%) 64 (22%) 96 (20%) 94 (48%) 127 (48%) 221 (48%) 85 (48%) 131 (45%) 216 (46%) 0.163 0.123 0.787
63 (32%) 96 (37%) 159 (35%) 61 (34%) 97 (33%) 158 (34%) 0.114 0.137 0.941 0.160 0.118 470 PD patients
Abbreviations: COMT = catechol-O-methyltransferase; BDNF = brain-derived neurotrophic factor; Val = valine; Met = methionine. PD = panic disorder; SD = standard deviation. ⁎ Relative to the control group.
113 (29%) 172 (33%) 285 (31%) 121 (34%) 165 (28%) 286 (30%) 277 (71%) 354 (67%) 631 (69%) 235 (66%) 419 (72%) 654 (70%) 19 (10%) 24 (9%) 43 (9%) 17 (9%) 25 (9%) 42 (9%) 75 (38%) 124 (47%) 199 (43%) 87 (49%) 115 (39%) 202 (43%) 101 (52%) 115 (44%) 216 (47%) 74 (42%) 152 (52%) 226 (48%) Men (195) Women (263) Total (458) Men (178) Women (292) Total (470) 36.63 ± 13.96 458 Controls
37.93 ± 11.10
Val Val/Met Val/Val Met Val
COMT allele: n (%)
P⁎ Met/Met Val/Met
COMT genotypes: n (%)
Val/Val P⁎ Sex (n) P⁎ Age Mean ± SD
The average age of the patients in the PD group was 37.93 ± 11.10 years, and that of the healthy control group was 36.63 ± 13.96 years (t = 1.566, p = 0.118). The gender ratios of the PD group (178 men, 292 women) and controls (195 men and 263 women) showed no significant difference (χ2 value = 1.942, p = 0.163) (Table 1). The COMT Val158Met polymorphism followed Hardy–Weinberg equilibrium in each subgroup (p = 0.742 for PD, p = 0.770 for healthy control; p = 0.289 for male, PD, p = 0.360 for male healthy control; p = 0.356 for female PD, p = 0.248 for female healthy control). The Hardy–Weinberg equilibrium also applied to the BDNF Val66Met polymorphism in each subgroup (p = 0.161 for PD, p = 0.935 for healthy control; p = 0.800 for male PD, p = 0.782 for male healthy control; p = 0.119 for female, PD, p = 0.850 for female healthy control). No significant difference was observed in the genotypic or allele frequencies of COMT and BDNF polymorphisms between the PD and healthy control groups (Table 1). Equivalent doses to that of the standard imipramine did not differ significantly among the COMT Val158Met or BDNF Val66Met polymorphism groups (all, male, or female PD patients; Supplementary Table 1). There was a significant health status effect on STAI and NEO-PI-R scores, and there were significant interactions between healthy status, gender, COMT and BDNF polymorphisms on the STAI, Neuroticism and Extraversion scores (Table 2).
n
3. Results
Group
The Hardy–Weinberg equilibrium for genotyping frequencies was calculated using χ2 tests. Statistical calculations were carried out using SPSS for Windows (Release 17.0)®. A multivariate analysis of covariance (MANCOVA) was performed with STAI and NEO-PI-R scores as the dependent factor, and health status (PD patients and control subjects), gender and genotyping groups (BDNF and COMT) as fixed factors, and the covariate of age. As a sub-analysis, we performed a MANCOVA with STAI and NEO-PI-R scores as the dependent factor, gender and genotyping groups (BDNF and COMT) as fixed factors, and the covariate of age in the PD patients and healthy controls. Post hoc MANCOVA tests were conducted to evaluate COMT × BDNF interactions. The statistical power of effect size was used for comparative effects in the power analysis. Comparisons of gender-linked genotype distributions and allele frequencies between PD and healthy controls were performed by the χ2 test. Student's t-test was used for differences in age. All tests were performed with a two-tailed type-I error rate of p b 0.05.
Table 1 Comparison of the mean age, gender, COMT Val158Met, BDNF Val66Met genotype distribution, and allele frequency between the PD patient and control groups.
2.4. Statistical analyses
P⁎
BDNF genotypes: n (%)
Met/Met
P⁎
BDNF allele: n (%)
For each subject, a 7-mL blood sample was centrifuged at 2000 rpm × 10 min in a collection tube and separated into the plasma layer, buffy-coat layer (containing white blood corpuscles), and red blood corpuscle layer. A 150-μL sample of the buffy coat was used for automatic blood DNA extraction. DNA was extracted using a BioRobot EZ1 (Qiagen, Hilden, Germany). BDNF Val66Met (rs6265 (assay ID C__11592758_10)) and COMT Val158Met (rs4680 (Assay ID C__25746809_50)) polymorphisms were genotyped with allelic discrimination using the TaqMan real-time polymerase chain reaction (PCR) assay (Life Technologies Japan, Tokyo). The assay includes a sequence-specific fluorogenic minor groove binder probe for each allele. Each probe is 5′-labeled with a different reporter fluorescent dye (4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein and 6-carboxyfluorescein) to differentiate the amplification of each allele. Primer–probe sets for the detection of the polymorphisms were purchased from Applied Biosystems (Foster City, CA, USA). PCR was carried out on an ABI StepOne +™ Real-Time PCR System according to the manufacturer's protocol (Applied Biosystems).
Met
P⁎
0.714 0.100 0.123
2.3. SNP genotyping
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Table 2 Effects of health status, gender, COMT polymorphism and BDNF polymorphism on STAI and NEO-PI-R scores. Category of groups
Response variables
Health status (HS) Gender (G) COMT polymorphism (CP) BDNF polymorphism (BP) HS × G HS × CP HS × BP BP × CP HS × G × CP HS × G × BP HS × CP × BP G × CP × BP HS × G × CP × BP
STAI state
STAI trait
Neuroticism
Extraversion
Openness
Agreeableness
Consciousness
b0.001⁎⁎⁎ 0.989 0.546 0.536 0.798 0.001⁎⁎ 0.679 0.774 0.051 0.465 0.021⁎ 0.058 0.042⁎
b0.001⁎⁎⁎ 0.180 0.362 0.275 0.789 0.034⁎ 0.131 0.896 0.098 0.640 0.051 0.300 0.022⁎
b0.001⁎⁎⁎ 0.001⁎⁎ 0.940 0.814 0.942 0.007⁎⁎ 0.056 0.953 0.216 0.938 0.045⁎ 0.212 0.030⁎
b0.001⁎⁎⁎ 0.063 0.960 0.107 0.328 0.008⁎⁎ 0.551 0.39 0.006⁎⁎ 0.742 b0.001⁎⁎⁎ 0.008⁎⁎ 0.001⁎⁎
b0.001⁎⁎⁎ 0.675 0.811 0.893 0.259 0.004⁎⁎ 0.629 0.365 0.027⁎ 0.716 0.045⁎ 0.777 0.938
0.024⁎ b0.001⁎⁎⁎ 0.738 0.392 0.929 0.999 0.053 0.671 0.585 0.976 0.666 0.890 0.723
0.001⁎⁎ 0.617 0.370 0.567 0.663 0.109 0.035⁎ 0.552 0.413 0.396 0.141 0.603 0.122
Abbreviations: COMT = catechol-O-methyltransferase; BDNF = brain-derived neurotrophic factor; STAI = State-Trait Anxiety Inventory; NEO-PI-R = Revised NEO Personality Inventory; MANCOVA was performed with STAI and NEO-PI-R scores as dependent factor, and health status, gender and genotyping groups as fixed factors and covariate of age. ⁎ P b 0.05. ⁎⁎ P b 0.01. ⁎⁎⁎ P b 0.001.
Following the sub-analysis, Tables 3 and 4 summarize the interaction of gene × gender revealed by the MANCOVA on STAI and NEOPI-R scores. In the PD patients, BDNF × COMT × gender showed a significant effect on STAI state (F = 5.70, p = 0.018, power = 0.662), STAI trait (F = 4.47, p = 0.035, power = 0.559), and the Neuroticism (F = 5.22, p = 0.023, power = 0.625), and Extraversion scores (F = 12.3, p = 0.001, power = 0.938). No interactive effects of the BDNF × COMT × gender interaction on STAI or NEO-PI-R scores were detected in the healthy controls, however. Particularly in the male PD patients, our results indicated a significant gene × gene interaction by a post hoc MANCOVA on STAI state and the Neuroticism and Extraversion scores (Fig. 1, Suppl. Table 2). The post hoc MANCOVA revealed that among the male PD patients, COMT Met + BDNF Val/Val carriers had significantly higher STAI state and Neuroticism scores than the COMT Val/Val + BDNF Val/Val carriers (STAI state; p = 0.009, Neuroticism; p = 0.014). Among the male PD patients, the COMT Val/Val + BDNF Val/Val carriers and the COMT Met + BDNF Met carriers also had significantly higher Extraversion scores than the COMT Met + BDNF Val/Val carriers (p b 0.001, p = 0.007). Finally, among the male PD patients, the COMT Val/Val + BDNF Val/Val carriers had significantly higher Extraversion scores than the COMT Val/Val + BDNF Met carriers (p = 0.048).
4. Discussion Our results showed that BDNF × COMT × gender had a significant effect on STAI state, STAI trait, and the NEO-PI-R Neuroticism and Extraversion scores in PD patients. No interactive effects of the BDNF × COMT × gender interaction on STAI or NEO-PI-R scores were detected in the healthy controls. The gene effect was observed particularly in the men with PD. As shown in previous studies of personality traits in anxiety disorders, we found that higher anxiety scores are associated with higher Neuroticism (Ormel et al., 2004) and lower Extraversion scores (Jylha et al., 2009). In the present study, the male PD patients who were COMT Met carriers had higher STAI state and Neuroticism scores and lower Extraversion scores than the COMT Val/Val carriers, especially in the BDNF Val/Val carrier group (Fig. 1), corresponding to opposite gender effects on BDNF Val/Val homozygotes (Shalev et al., 2009), and these effects were enhanced in COMT Met carriers. The results of our study suggest the possibility of BDNF–COMT– gender involvement in anxiety traits, personality, and panic disorder, without significant differences of genotype distribution related to gene–gene interaction. Gender differences may interact with the BDNF and COMT genes' effects on anxiety sensitivity and related personality
Table 3 Effects of gender, BDNF polymorphism, and COMT polymorphism on STAI scores in the PD and healthy control groups. Panic disorder
STAI state
STAI trait
Gender (G) BDNF polymorphism (BP) COMT polymorphism (CP) G × BP G × CP BP × CP G × BP × CP Gender (G) BDNF polymorphism (BP) COMT polymorphism (CP) G × BP G × CP BP × CP G × BP × CP
Healthy control
F
P
Power
F
P
Power
0.05 0.56 4.72 0.27 2.53 1.15 5.70 0.69 2.71 0.56 0.00 3.55 1.32 4.47
0.822 0.456 0.031⁎ 0.604 0.113 0.283 0.018⁎ 0.408 0.101 0.456 0.972 0.061 0.251 0.035⁎
0.056 0.115 0.581 0.081 0.354 0.188 0.662 0.131 0.375 0.116 0.050 0.467 0.209 0.559
0.03 0.27 6.81 0.47 1.86 6.24 0.01 0.80 0.01 7.96 0.43 0.09 2.65 1.02
0.869 0.601 0.009⁎⁎ 0.496 0.173 0.013⁎ 0.942 0.372 0.921 0.005⁎⁎ 0.513 0.763 0.104 0.312
0.053 0.082 0.740 0.104 0.275 0.702 0.051 0.145 0.051 0.804 0.100 0.060 0.368 0.172
Abbreviations: BDNF = brain-derived neurotrophic factor; COMT = catechol-O-methyltransferase; STAI = State-Trait Anxiety Inventory. MANCOVA was performed with STAI scores as dependent factor, gender and genotyping groups as fixed factors and covariate of age. ⁎ P b 0.05. ⁎⁎ P b 0.01.
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Table 4 Effects of gender, BDNF polymorphism, and COMT polymorphism on NEO-PI-R (Neuroticism and Extraversion) scores in the PD and healthy control groups. Panic disorder
Neuroticism
Gender (G) BDNF polymorphism (BP) COMT polymorphism (CP) G × BP G × CP BP × CP G × BP × CP Gender (G) BDNF polymorphism (BP) COMT polymorphism (CP) G × BP G × CP BP × CP G × BP × CP
Extraversion
Healthy control
F
P
Power
F
P
Power
4.05 2.12 2.79 0.08 4.17 1.59 5.22 2.66 0.42 2.47 0.15 7.20 7.13 12.3
0.045⁎ 0.146 0.096 0.776 0.042⁎ 0.208 0.023⁎ 0.104 0.515 0.117 0.695 0.008⁎⁎ 0.008⁎⁎ 0.001⁎⁎
0.518 0.306 0.384 0.059 0.530 0.242 0.625 0.369 0.100 0.347 0.068 0.762 0.758 0.938
7.86 2.18 5.09 0.17 0.23 2.36 0.43 0.68 3.60 5.23 0.00 0.52 5.96 0.59
0.005⁎⁎ 0.141 0.025⁎ 0.682 0.630 0.125 0.514 0.412 0.059 0.023⁎ 0.976 0.469 0.015⁎ 0.445
0.799 0.313 0.614 0.069 0.077 0.335 0.100 0.130 0.473 0.626 0.050 0.112 0.683 0.119
Abbreviations: BDNF = brain-derived neurotrophic factor; COMT = catechol-O-methyltransferase; NEO-PI-R = Revised NEO Personality Inventory; MANCOVA was performed with STAI scores as dependent factor, gender and genotyping groups as fixed factors and covariate of age. ⁎ P b 0.05. ⁎⁎ P b 0.01.
characteristics in the pathogenesis of this disorder through the monoamine system comprised of serotonergic, norepinephrine and dopaminergic systems. Previous studies of the potential involvement of gene–gene interactions with PD examined COMT and serotonin receptor polymorphisms. Freitag et al. (2006) reported a nominally significant interaction between the serotonin receptor 1A and COMT polymorphisms. Interaction of serotonin-2A receptor and COMT polymorphism (Karacetin et al., 2012) was reported with no significant interaction. Gene–gene interactions in PD have not been extensively studied.
**
140 BDNF Val/Val BDNF Met Carrier
STAI-state
50 40 30 20 10
BDNF Val/Val BDNF Met Carrier
100 80 60 40 20
0 COMT Val/Val
COMT Met Carrier
m
*** *
COMT Val/Val
0
COMT Met Carrier
COMT Val/Val COMT Met COMT Val/Val COMT Met Carrier Carrier
f
m
f
** BDNF Val/Val BDNF Met Carrier
100
Extraversion
*
120
Neuroticism
60
120
Most brain imaging studies of patients with PD have consistently shown abnormalities in the limbic system and ventromedial prefrontal cortex associated with emotional arousal (Gorman et al., 2000). In a study of patients with PD, we observed hypoactivation in the lateral prefrontal cortex (PFC), which is responsible for executive function through the dopaminergic system (Nishimura et al., 2007). A hypothetical inverted U-shaped curve was proposed for the relationship between working memory performance and prefrontal dopaminergic activity (Goldman-Rakic et al., 2000). In the present study, significant genetic effects by COMT genotype were observed in healthy control subjects
80 60 40 20 0 COMT Val/Val
m
COMT Met Carrier
COMT Val/Val
COMT Met Carrier
f
Fig. 1. Comparison between genotype groups with post hoc test in male and female PD patients. A gene effect was observed, particularly in the male PD patients, on the STAI state (COMT Val/Val + BDNF Val/Val carriers = 40.2 ± 1.8, COMT Met + BDNF Val/Val carriers = 50.8 ± 2.6), the NEO-PI-R factor Neuroticism scores (COMT Val/Val + BDNF Val/Val carriers = 98.5 ± 4.5, COMT Met + BDNF Val/Val carriers = 117.2 ± 6.8), and the NEO-PI-R factor Extraversion scores (COMT Val/Val + BDNF Val/Val carriers = 93.5 ± 2.7, COMT Val/Val + BDNF Met carriers = 88.1 ± 4.1, COMT Met + BDNF Val/Val carriers = 73.6 ± 5.1, COMT Met + BDNF Met carriers = 93.5 ± 2.7). Error bars: standard error of the mean. ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001. PD, panic disorder; COMT, catechol-O-methyltransferase; BDNF, brain-derived neurotrophic factor; Val, valine; Met, methionine; STAI, State-Trait Anxiety Inventory; m, males; f, females.
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(Tables 3, 4; Suppl. Table 3), but the genotype effects of COMT were the opposite between the PD patients and the healthy controls. The modulation of the COMT genotype on anxiety-related parameters seems to be different in PD patients. Our present data on the distinct effect of the COMT genotype could be explained by the application of an inverted U-shaped hypothesis regarding the prefrontal dopamine level. In accord with this inverted U-shaped hypothesis, the PFC dopamine levels of Val158 homozygotes and Met carriers fell into an inverted U-shaped curve in accord with the increase/decrease of COMT activity and showed more complex results than the simple ‘warrior/worrier’ hypothesis of COMT genotype (Mier et al., 2010). As prefrontal dopamine could not be measured directly, this speculation is only a working hypothesis, but it is thought that anxiety sensitivity might be indirectly related to PD through the dopamine neurotransmission of the PFC. There are few previous studies of gene–gene interactions related to gender differences. Niewold et al. (2010) reported a gene–gene–sex interaction in cytokine gene polymorphisms in juvenile dermatomyositis; to the best of our knowledge, such an interaction has not yet been reported in psychiatric disease. We studied the gene × gene × gender interaction of BDNF and COMT genotypes associated with panic disorder, and we suspect that the gender specificity of COMT Val158Met influences personality traits in male PD patients. Whether the interaction of the BDNF and COMT genes leads to an elevation of anxiety sensitivity and how such an interaction affects the etiology of PD requires further clarification. The present study has several limitations. First, only one representative functional SNP within the BDNF and COMT was examined. It would have been desirable to study all tagging SNPs within BDNF and COMT, as well as other variants associated with dopaminergic neurotransmission. Second, a self-reported questionnaire assessment measure was used to evaluate personality-related PD symptoms. Additional assessments by clinicians may be more accurate and/or reliable. The personality inventory scores may reflect effects from the symptoms, and the STAI state may correspond to the severity of the symptoms. Further data for the severity of symptoms of patients with PD are necessary. Third, we included PD patients whose comorbidities included agoraphobia, depression or bipolar disorder as a composite group of PD. For replication studies, it is desirable to collect more detailed information and a homogeneous group with larger sample sizes that could lead to more specific evidence about anxiety disorders such as PD. 5. Conclusion Gene × gene × gender interactions were observed in the association of the BDNF and COMT polymorphisms with STAI scores and with the NEO-PI-R Neuroticism and Extraversion scores in the male PD group. Our results show the possibility of a gender effect in anxiety disorders such as PD, which has a higher prevalence rate in women. The BDNF and COMT interaction appears to exhibit gender differences in personality scores, with potential implications for gender differences in the pathogenesis of PD. Contributors Y.K. executed the experimental protocols and statistical analyses, and wrote the first draft of the manuscript. Y.K., Y.O., and H.T. were responsible for the study concept and design. M.T., T.U., E.M., T.S., H.K. and Y.O. contributed to the acquisition of clinical data. Y.K., E.M. and M.O. conducted the data analysis. T.O., T.S. and M.O. interpreted the findings and provided critical revisions of the manuscript. Acknowledgments This work was supported by KAKENHI (a Grant-in-Aid for Scientific Research) in the Priority Area of Applied Genomics from the Ministry
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