A Tryptophan Hydroxylase 2 Gene Polymorphism is Associated with Panic Disorder
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- Kim, Y., Lee, H., Yang, J. et al. Behav Genet (2009) 39: 170. doi:10.1007/s10519-008-9254-8
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Panic disorder (PD) is a complex and heterogeneous psychiatric condition. Dysfunction within the serotonergic system has been hypothesized to play an important role in PD. The novel brain-specific serotonin synthesizing enzyme, tryptophan hydroxylase 2 (TPH2), which represents the rate-limiting enzyme of serotonin production in the brain, may therefore be of particular importance in PD. We investigated the TPH2 703G/T SNP for association with PD. Patients with PD (n = 108), and control subjects (n = 247), were genotyped for rs4570625 (TPH2 703G/T). Male and female subjects were analyzed separately. The severity of their symptoms was measured using the Spielberger state-trait anxiety inventory (STAI), panic disorder severity scale (PDSS), anxiety sensitivity index (ASI), acute panic inventory (API), and Hamilton’s rating scale for depression (HAMD). The genotype and allele frequencies of the PD patients and controls were analyzed using χ2 statistics. There was a significant difference in the allele frequency in rs4570625 between the PD patients and normal controls. The T allele was significantly less frequent in the PD patients. We also found a significant association with rs4570625 in the female subgroup. There was no difference in symptom severity among the genotypes of this polymorphism. This result suggests that rs4570625 polymorphism may play a significant role in the pathogenesis of PD. Moreover, rs4570625 may have a gender-dependent effect on susceptibility to PD. Further studies are needed to replicate the association that we observed.
KeywordsPanic disorderPolymorphismTryptophan hydroxylase 2Gender-dependent effect
Panic disorder (PD) is a complex and heterogeneous psychiatric condition. PD is a potentially disabling disorder defined by recurrent unexpected attacks of intense somatic and cognitive symptoms of anxiety, anticipatory anxiety, and phobic avoidance. Various factors may be involved in the pathogenesis of PD, among these, the genetic factor has come into the spotlight recently. Substantial evidence supports the idea that there is a genetic component to PD. PD has a worldwide lifetime prevalence of 1–5% and a female:male ratio among those affected of 2:1 (Eaton et al. 1994; Grant et al. 2006; Weissman et al. 1997). Family and twin studies suggest a genetic component in the pathogenesis of PD with an estimated heritability of up to 46% (Hettema et al. 2001).
Several lines of evidence indicate that disturbances of the serotonin system are involved in the neurobiology of PD. Selective serotonin reuptake inhibitors (SSRIs) and other serotonin-modulating medications can attenuate the anxiety symptoms of PD (De Angelis 2002; Nutt 1998). Furthermore, several challenge studies have shown that 5-HT level is important in the modulation of panic symptoms (Schruers et al. 2000, 2002). Therefore, serotonin-related genes are good candidates for the study of PD.
Tryptophan hydroxylase (TPH) is the rate limiting enzyme in the biosynthesis of serotonin, converting the amino-acid tryptophan to 5-hydroxy tryptophan, which is further decarboxylated into serotonin. Thus, TPH has received much attention in the genetics of anxiety disorders. Until recently, it was thought that only one gene encodes TPH. Unexpectedly, however, Walther et al. (2003) found a new brain-specific TPH isoform, which was termed TPH2. Indeed, this novel TPH2 was identified exclusively in the brain, while the classical TPH isoform (TPH1) was detected in the periphery (Walther and Bader 2003; Walther et al. 2003). Moreover, Zill et al. (2004a) have established that human TPH1 and TPH2 are expressed in nearly equal amounts in several brain regions, such as the frontal cortex, thalamus, hippocampus, hypothalamus and amygdale. However, TPH2 is predominant in the brain stem, the major locus of the 5-HT producing neurons, and TPH1 mRNA is exclusively present in peripheral tissues.
Many studies have investigated an association between the TPH1 gene and PD, but found none (Fehr et al. 2001; Han et al. 1999; Kim et al. 2006; Maron et al. 2005). There were only two studies of the association between TPH2 and PD. Mössner et al. (2006a) investigated an association between two SNPs (rs4570625 and rs4565946) of the TPH2 gene and PD in a German sample, but found no association. Maron et al. (2007) found no association with TPH2 gene SNPs (rs1386494 and rs1386483) in their total sample of PD patients. However, they found an association with rs1386494 in the female subgroup.
We previously investigated the 5-HT2A receptor and TPH1 genes for an association with PD. We found a significant association between panic symptom severity and the serotonin 2A receptor gene. However, we found no association with the TPH1 gene. At the end of our article, we commented that the relationship between the TPH2 gene and PD must be investigated (Yoon et al. 2008).
We hypothesize that the TPH2 gene regulates the serotonergic system and contributes to the susceptibility to PD. rs4570625 is one of the potentially functional variants in the upstream regulatory region of TPH2, and functional studies depict the impact of rs4570625 on amygdala responses to emotional stimuli (Brown et al. 2005; Canli et al. 2005). Therefore, rs4570625 is expected to be a good candidate.
The aim of the present study is to detect a possible association between rs4570625 and PD. Moreover, we aim to examine the association in male and female subgroups. A positive association will help to establish the pathogenesis of PD.
Materials and methods
Subjects and assessments
One hundred and eight patients diagnosed with PD (58 males, 50 females) participated in this study. All of the PD patients met the DSM-IV criteria for PD. Each patient was given a diagnostic assessment based on clinical interviews using the Structured Clinical Interview for DSM-IV (SCID) (First et al. 1998). Patients with comorbid mood disorder, any kind of psychotic disorder (including schizophrenia), or other anxiety disorders were excluded.
The normal control group consisted of randomly selected healthy individuals who visited the Korea University Ansan hospital for regular health screenings. Subjects were excluded if they had any self-reported personal or familial psychiatric history, or a psychotropic medication history. Finally, 247 normal controls (125 males, 122 females) were selected. They were carefully matched with the patient group in terms of age and sex. All patients and control subjects were biologically unrelated native Koreans. Written informed consent was obtained from all subjects. The study protocol was approved by the Ethics Committee of Korea University.
In order to assess the severity of the patients’ symptoms, we administered the Spielberger state-trait anxiety inventory (STAI) (Spielberger 1983), panic disorder severity scale (PDSS) (Lim et al. 2007; Shear et al. 1997, 2001), anxiety sensitivity index (ASI) (Reiss et al. 1986; Taylor et al. 1991; Telch et al. 1989), acute panic inventory (API) (Dillon et al. 1987; Papp et al. 1997), and Hamilton’s Rating Scale for Depression (HAMD) (Hamilton 1960).
DNA analysis and genotyping
rs4570625 is located in the putative transcriptional control region of TPH2. Genotyping was performed as described by Mössner et al. (2006a). DNA was extracted from blood leukocytes by using a commercial DNA extraction kit, Wizard Genomic DNA purification kit (Promega, USA). Polymerase Chain Reaction (PCR) was performed with the forward primer 5′-TTT TAT GAA AGC CAT TAC ACA T-3′ and the reverse primer 5′-TTC CAC TCT TCC AGT TAT TTT A-3′. The amplification mixture contained 1 μl of 100 ng/μl DNA, 5 μl of 10 × Ex Taq buffer, 4 μl of 2.5 mM Ex dNTP mixture, 2 μl primer, 37.75 μl distilled water, and 0.25 μl Taq polymerase (TaKaRa, Japan). Samples were amplified using a Thermocycler (GeneAmp PCR system 2700, Applied Biosystems, Foster city CA94404 USA) for 36 cycles. After an initial 5 min at 95°C, each subsequent cycle consisted of 45 s at 95°C, 45 s at 51.9°C, and 45 s at 72°C. After a final 3 min at 72°C, the reaction was terminated at 4°C. The amplified DNA was digested with the restriction enzyme PsiI (New England Biolabs), which cuts at the −703T site. The product was electrophoresed in 3% agarose gels and stained with ethidium bromide. The undigested PCR product carries the G variant, whereas the digested product with two fragments of 55 and 149 bp contains the T allele. Homozygous genotypes were identified by the presence of a single 204 bp band (G/G), or bands of 55 and 149 bp (T/T). The heterozygous genotype had three bands: 204, 55, and 149 bp (G/T).
Genotyping completion rate was 97% (355/362). All laboratory procedures were carried out blind to case-control status and clinical status.
The presence of Hardy–Weinberg equilibrium was tested using a χ2-test for goodness of fit. The genotype and allele frequencies of the PD patients and the controls were analyzed using χ2 statistics. Male subjects and female subjects were analyzed separately. The possible effect of the genotype on symptom severity in the polymorphism was examined using analysis of variance (ANOVA) by comparing the mean scores of each genotype. All statistical analyses were performed with SPSS version 12.0 for Windows.
The mean age of the PD patients was 41.08 ± 10.08 years, and the mean age of the control subjects was 39.44 ± 9.03 years. Age of onset and duration of illness of PD patients were 33.01 ± 11.56 and 22.08 ± 10.35 years, respectively. Twenty-five PD patients (23.1%) had a family history of PD.
Genotype and allele frequencies of the TPH 2 703G/T in panic disorder (PD) patients and controls
Male PD patients
Female PD patients
Male PD patients
Female PD patients
For testing the hypothesis that rs4570625 has a gender-dependent effect on susceptibility to PD, genotype and allele frequencies of male patients, female patients, and controls were compared. In the male subgroup, there was no difference between PD patients and controls. In the female subgroup, there were significant differences in the genotype and allele frequencies between PD patients and normal controls (Table 1). However, there was no significant difference between male and female PD patients. Instead, genotype and allele frequency is different between male control and female control. T allele was significantly more frequent in female controls (OR = 1.58).
Comparison of symptom severity in patients with panic disorder according to TPH2 703G/T genotype
G/G (N = 29)
G/T (N = 55)
T/T (N = 24)
53.35 ± 8.24
54.25 ± 10.18
55.33 ± 15.19
50.88 ± 6.95
51.03 ± 10.82
49.11 ± 13.71
25.07 ± 8.00
24.06 ± 10.24
26.22 ± 7.63
41.53 ± 12.83
38.03 ± 15.05
40.01 ± 13.73
18.29 ± 3.58
17.59 ± 4.23
17.32 ± 4.77
12.01 ± 8.19
10.25 ± 3.56
8.87 ± 3.65
Panic disorder is thought to be generated by a complex interaction of environmental and biological factors with a number of genes involving distinct neural circuits, such as the brain serotonin system.
We found a significant association of rs4570625 with PD. There was a significant statistical difference in the allele frequency of rs4570625 polymorphism between the PD patients and normal controls. However, we failed to verify the hypothesis that rs4570625 has a gender-dependent effect on susceptibility to PD.
The TPH gene is a promising candidate for anxiety disorder because it is the rate-limiting enzyme of serotonin synthesis. A second TPH isoform, TPH2, has been especially studied in effort to determine its role in increasing vulnerability to psychiatric disorder. The TPH2 polymorphisms have been associated with many psychiatric disorders (De Luca et al. 2005; Higashi et al. 2007; Lin et al. 2007; Lopez de Lara et al. 2007; Mössner et al. 2006b, 2007; Sheehan et al. 2005; Zill et al. 2004b).
In a few studies, functional magnetic resonance imaging (fMRI) was employed to provide evidence that one of the potentially functional variants in the upstream regulatory region of TPH2 (rs4570625) alters the responsiveness of the amygdala, a structure critically involved in the modulation of emotional behaviors (Brown et al. 2005; Canli et al. 2005). These studies emphasized the impact of rs4570625 on amygdala responses to emotional stimuli. The lateral nucleus of the amygdala receives excitatory glutamatergic inputs from thalamic and cortical sensory processing areas and is believed to be involved in the evaluation of the affective significance of sensory events (LeDoux et al. 1990; Savander et al. 1997). Serotonin modulates the processing of sensory information within the lateral nucleus of the amygdala and thus may regulate amygdala-related functions (Chaouloff 1993; Kawahara et al. 1993; Stutzmann et al. 1998).
Two recent studies show that the T allele of rs4570625 is associated with a low anxiety level (Gutknecht et al. 2007; Reuter et al. 2007). These results are in line with our results. Taken together, these findings suggest that rs4570625 affects amygdala response to emotional stimuli by modifying gene expression, and it may be one of the key variants in the transcriptional control region.
Recently, a positive association with the rs1386494 SNP in female PD patients was reported (Maron et al. 2007). Nishizawa et al. (1997) measured rates of serotonin synthesis in the human brain using positron emission tomography, and found that the mean rate of synthesis in normal males was found to be 52% higher than in normal females. Greater sensitivity to cholecystokinin-tetrapeptide (CCK-4) effects in females has been reported in PD patients. We had expected that rs4570625 might also have a gender-dependent effect on susceptibility to PD, and we examined our hypothesis. There were significant results in the female subgroup. However, because genotype and allele frequencies were similar between male and female patients, it was hard to conclude that gender-dependent hypothesis is right. Instead, allele frequency is different between male control and female control, and T allele was significantly more frequent in female controls (OR = 1.58). This result suggested that T allele of rs4570625 might be an autosomal marker that potentially differentiates males from females.
There are some limitations in the present study. First, the relatively small sample size limits the generalization of our results. These data need to be confirmed in a much larger sample. Second, we investigated only one SNP. Single SNP can only provide a partial insight into the genetic basis of complex disorders such as panic disorder and major depressive disorder at best. Multiple different genes or neurotransmitter systems have been implicated in the PD phenotype. Ideally, association studies should aim to look at possible interactions among multiple genes or multiple polymorphisms. Possible interactions between TPH1 and TPH2 should also be considered as modulating mechanisms in the pathogenesis of PD.
Besides the genetic factors, other processes may be involved in the pathogenesis of PD, including neurobiological processes (e.g., neural systems that emphasize the amygdala and related structures as part of a dysfunctional anxiety assessment and response system) and psychopathological processes (e.g., anxiety sensitivity, the belief that anxiety could cause deleterious physical, social, and psychological consequences that extend beyond any immediate physical discomfort during a panic attack). Because these processes may interact with each other, these interactions can act as confounding factors, making it very difficult to interpret the effect of only one factor. An understanding of this heterogeneity will be facilitated by the continued development and application of genetic, neurobiological, neuroimaging, and cognitive-behavioral approaches that can refine PD phenotypes and elucidate the genotypes associated with this disorder.
In conclusion, to our knowledge, this is the first report of a positive association with genetic variants of the TPH2 gene in the entire sample of PD patients studied. Our result suggests that rs4570625 polymorphism may play a significant role in the pathogenesis of PD. Moreover, rs4570625 may have a gender-dependent effect on susceptibility to PD. Further studies are needed to replicate the association that we observed, and to demonstrate that rs4570625 is indeed in the transcriptional control region.
This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A040042).