The novel brain-specific tryptophan hydroxylase

Original Papers
J
Psychopharm
The novel brain-specific tryptophan
hydroxylase-2 gene in panic disorder
Journal of Psychopharmacology
20(4) (2006) 547–552
© 2006 British Association
for Psychopharmacology
ISSN 0269-8811
SAGE Publications Ltd,
London, Thousand Oaks,
CA and New Delhi
10.1177/0269881106059704
Rainald Mössner Department of Psychiatry and Psychotherapy, University of Würzburg, Würzburg, Germany.
Christine M. Freitag Department of Child and Adolescent Psychiatry and Psychotherapy, University of Homburg, Homburg, Germany.
Lise Gutknecht Department of Psychiatry and Psychotherapy, University of Würzburg, Würzburg, Germany.
Andreas Reif Department of Psychiatry and Psychotherapy, University of Würzburg, Würzburg, Germany.
Ralf Tauber Department of Psychiatry, University of Jena, Jena, Germany.
Petra Franke Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany.
Jürgen Fritze Department of Psychiatry and Psychotherapy, University of Frankfurt, Frankfurt, Germany.
Gerd Wagner Department of Psychiatry, University of Jena, Jena, Germany.
Gregor Peikert Department of Psychiatry, University of Jena, Jena, Germany.
Berit Wenda Department of Psychiatry, University of Jena, Jena, Germany.
Philipp Sand Department of Psychiatry and Psychotherapy, University of Würzburg, Würzburg, Germany.
Marcella Rietschel Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany.
Henk Garritsen Institute of Transfusion Medicine and Transplantation Immunology, University of Münster, Münster, Germany.
Christian Jacob Department of Psychiatry and Psychotherapy, University of Würzburg, Würzburg, Germany.
K. Peter Lesch Department of Psychiatry and Psychotherapy, University of Würzburg, Würzburg, Germany.
Jürgen Deckert Department of Psychiatry and Psychotherapy, University of Münster, Münster, Germany.
Abstract
Panic disorder is a common psychiatric disorder characterized by
recurrent anxiety attacks and anticipatory anxiety. Due to the severity of
the symptoms of the panic attacks and the frequent additional
occurrence of agoraphobia, panic disorder is an often debilitating
disease. Elevation of central serotonin levels by drugs such as
clomipramine represents one of the most effective treatment options for
panic disorder. This points to an important role of dysregulation of the
serotonergic system in the genetic etiology of panic disorder. The novel
brain-specific 5-HT synthesizing enzyme, tryptophan hydroxylase-2
(TPH2), which represents the rate-limiting enzyme of 5-HT production in
the brain, may therefore be of particular importance in panic disorder.
We focused on the putative transcriptional control region of TPH2 and
identified two novel common single nucleotide polymorphisms (SNPs) of
TPH2 in and close to this region. Moreover, a recently described loss-offunction mutation of TPH2 which results in an 80% reduction of
serotonin production, was assessed. In an analysis of the putative
transcriptional control region SNPs in a sample of panic disorder patients
and controls no association of the disorder with the TPH2 SNPs or
haplotypes was found. Moreover, the loss-of-function R441H mutation of
TPH2 was not present in the panic disorder patients. The results of this
first study of TPH2 in panic disorder argue against an importance of
allelic variation of TPH2 in the pathogenesis of panic disorder with or
without agoraphobia.
Keywords
panic disorder, agoraphobia, serotonin, tryptophan hydroxylase-2
Corresponding author: R. Mössner, MD, Department of Psychiatry and Psychotherapy, University of Würzburg, Füchsleinstr. 15, 97080 Würzburg, Germany.
Email: [email protected]
548
TPH2 gene in panic disorder
Introduction
Panic disorder is a severe anxiety disorder characterized by recurrent panic attacks and anticipatory anxiety. Panic attacks occur
spontaneously and suddenly and are characterized by autonomic
and cognitive symptoms. During a panic attack, patients
experience a range of autonomic nervous system symptoms, such
as chest pain, palpitations, shortness of breath, sweating, and
vertigo. Typical cognitive symptoms are the fear of death, especially the imagined fear of dying from a heart attack, a stroke, or
from suffocation. Moreover, many patients experience the fear of
losing control. A panic attack typically lasts for 10–30 min. Many
panic disorder patients develop agoraphobia, where anxiety
attacks occur in certain situations, typically in crowds, in large
public places and squares as well as on public transport such as
trains. Without treatment, only approximately 20% of panic disorder patients remit spontaneously. The lifetime prevalence of
panic disorder with or without agoraphobia is approximately
1–3%, with women being affected twice as often as men (Weissman et al., 1997; Bandelow, 2001).
Due to the severity and common occurrence of panic disorder,
the pathogenesis of the disorder is of considerable interest.
Environmental as well as genetic factors are thought to contribute
to the development of panic disorder (Hettema et al., 2001). An
increasing body of evidence points to an involvement of the serotonergic system in the pathophysiology of panic disorder. Dysregulation of the serotonergic system in panic disorder is indicated
especially by the current pharmacological treatment of the disorder. Amongst the best pharmacological treatments available for
panic disorder, clomipramine and selective serotonin reuptake
inhibitors such as fluoxetine modulate synaptic serotonin (5-HT)
levels by way of inhibiting the 5-HT transporter (Bandelow,
2001). Moreover, panic disorder patients display a blunted responsitivity to stimulation of the 5-HT1A receptor subtype (Lesch et al.,
1992; Broocks et al., 2000).
Serotonin influences many brain functions such as emotions,
cognition, motor function, and pain as well as circadian and neuroendocrine functions including food intake, sleep, and reproductive activity (Essmann, 1978; Eide and Hole, 1993; Lesch and
Mössner, 1998). While there are multiple presynaptic and postsynaptic 5-HT receptor subtypes mediating these complex actions of
5-HT, a major factor in the availability of 5-HT is the synthesis
rate of this neurotransmitter. 5-HT is synthesized in a two-step
process, with the enzyme tryptophan hydroxylase (TPH) representing the rate-limiting step in the synthesis of 5-HT (GrahameSmith, 1964; Lovenberg et al., 1967). Until very recently, TPH
was thought to be a single enzyme, responsible for synthesizing 5HT both in the serotonergic neurons of the midbrain raphe as well
as in the periphery, where it influences smooth muscle tone of
blood vessels and of the gastrointestinal tract, platelet activity, and
immune responses (Essmann, 1978; Mössner and Lesch, 1998).
Unexpectedly, however, mice lacking TPH continue to produce
normal concentrations of 5-HT in serotonergic neurons of the
brain (Walther et al., 2003). In contrast, 5-HT levels in peripheral
tissues of these knockout mice was reduced to less than 4% of
normal levels. This pointed to the existence of a second, brain-spe-
cific form of TPH, which was termed TPH2. Indeed, this novel
TPH2 was identified exclusively in the brain, while the classical
TPH isoform, now termed TPH1, was detected in the periphery,
especially in the duodenum and in blood, but not in the brain,
apart from the pineal gland (Walther and Bader, 2003; Walther et
al., 2003).
This finding of a dichotomy of the classical TPH1 synthesizing
5-HT in peripheral tissues, and the novel TPH2 synthesizing 5-HT
in serotonergic neurons of the midbrain raphe, may shed light on
the pathophysiology and pathogenesis of a number of psychiatric
disorders. In this respect, studies of TPH2 in panic disorder are
urgently warranted to elucidate the role of 5-HT synthesis in the
pathophysiology of this disorder. Another aspect pointing to a possible importance of TPH2 variants in panic disorder are the findings from a number of studies showing that influencing the
availability of 5-HT by way of the tryptophan depletion test or
administration of the 5-HT precursor L-5-hydroxytryptophan has
important modulatory effects on the panic challenge (Miller et al.,
2000; Schruers et al., 2000; Bell et al., 2002; Schruers et al.,
2002; Maron et al., 2004).
We have searched for and identified single nucleotide polymorphisms of TPH2 in and close to the putative transcriptional control
region of the TPH2 gene. Given that the transcriptional control
region is a major determinant of gene expression, variants in this
region are expected to be of particular importance for TPH2
expression. Moreover, very recently, a functional variant of TPH2
in exon 11 (G1463A) leading to an amino acid exchange of arginine to histidine at amino acid position 441 of TPH2 was
described (Zhang et al., 2005). This R441H mutation leads to a
reduction of serotonin production by approximately 80% (Zhang
et al., 2005). We therefore analysed these TPH2 variants in a
sample of panic disorder patients with or without agoraphobia.
Methods
Study sample
Our study sample consisted of 134 unrelated patients with panic
disorder (49 males, 85 females). Panic disorder was diagnosed by
experienced psychiatrists according to DSM-IIIR/DSM-IV on the
basis of structured interviews (SADS-LA (Mannuzza et al., 1986),
CIDI (Robins et al., 1988), IDCL (Hiller, 1997)) and medical
records. Only patients with predominant panic disorder were
included. Comorbidity with other anxiety disorders or depression
was allowed as long as the diagnosis of panic disorder was
primary and predominant. The 134 controls were unrelated,
anonymous blood donors. Screening of blood donors did not
include assessment of personal or family history of psychiatric
disorders. All patients and controls were of German descent. They
were matched for gender and age (average age of patients
45.7 11.4 years and of controls 45.2 10.2 years, mean standard error). The study design was approved by the local Ethics
Committees. All participating subjects had given their informed
written consent. Genomic DNA was extracted from EDTA-anticoagulated whole blood using QIAamp DNA Mini kits.
TPH2 gene in panic disorder 549
TPH2 polymorphisms and genotyping
Screening of the human TPH2 gene by single strand conformation
polymorphism (SSCP) analysis including coding, untranslated,
and putative transcriptional control regions in 120 chromosomes
revealed or confirmed several common single nucleotide polymorphisms (SNPs) (Gutknecht et al., manuscript in preparation). Two
SNPs which were found to represent common allelic variants of
TPH2 in the general population were chosen for association analyses. Both SNPs were found to be listed in the SNP database of the
National Center of Biotechnology Information (NCBI) but had not
been verified and tested for allele frequencies. SNP rs4570625 is
located in the putative transcriptional control region of TPH2, at
position 703 with respect to the transcription start site (1).
SNP rs4565946 is located in intron 2 of TPH2. Genotyping of the
two SNPs was performed as described by Gutknecht et al. (manuscript in preparation). Briefly, for detection of SNP rs4570625, a
204-bp PCR product containing the SNP was amplified by polymerase chain reaction (PCR) using the following reaction mix:
20 ng of genomic DNA in 75 mM Tris-HCl (pH 9.0), 20 mM
ammonium sulfate, 0.01% Tween 20, 1.5 mM magnesium chloride, 0.4 M of each of the primers, forward (5-TTT TAT GAA
AGC CAT TAC ACA T) and reverse (5-TTC CAC TCT TCC
AGT TAT TTT A), 0.25 mM dNTP, and 1U Taq polymerase.
After an initial denaturation for 5 min at 95 °C, 36 cycles of denaturating at 95 °C for 45 s, annealing at 51.9 °C for 45 s and extension of 72 °C for 45 s were performed, followed by a final
extension of 72 °C for 3 min. PCR products were digested with
PsiI at 37 °C for 16 h. PCR products were visualized on a 3%
agarose gel containing 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. Determination of the SNP rs4565946 was performed in a similar PCR procedure, with 0.24 M each of the forward primer 5-CAT CCA
AGG CTG TGT CCA TA and reverse primer 5-TGT GTC ACG
TTG GGC TTT TA yielding a 225 bp product. Annealing temperature was 56.3 °C. PCR products were digested with Bpu10I. The
undigested PCR product carries the T variant, whereas the
digested product with two fragments of 93 and 132 bp contains the
C allele. For genotyping of the G1463A mutation, PCR reactions
were carried out as described by Zhang et al. (2005), employing
the primers hOuter/Forward (5-ATG TGT GAA AGC CTT TGA
CCC AAA GAC A), hOuter/Reverse (5-TGC GTT ATA TGA
CAT TGA CTG AAC TGC T), and 5-TAG GGA TTG AAG
TAT ACT GAG AAG GCA T specific for the A allele. PCR was
carried out for 40 cycles: 30 s at 94 °C, 30 s at 63 °C, 30 s at 72 °C.
Patient samples heterozygous or homozygous for the 1463A allele
(a kind gift of M. G. Caron, Duke University) served as controls.
Statistics
Statistical analysis was performed by Armitage trend test for
genotype frequency and 2 test for allele frequency using the SAS
statistical package (SAS/STAT, version 8.1, Cary, NC: SAS Institute Inc., 1999). Assessment of linkage disequilibrium was carried
out by the program FastEHPlus (Zhao and Sham, 2002). Haplo-
type frequency was assessed by GENECOUNTING, version 1.3
(Zhao et al., 2002). Hardy-Weinberg equilibrium was assessed by
the program FINETTI (T. Wienker, personal communication). No
adjustment for multiple testing was made.
Results
We identified and confirmed two common SNPs located in the
putative transcriptional control region (SNP rs4570625) and in
intron 2 (rs4565946) of TPH2. The distribution of both SNPs did
not differ significantly from those predicted by Hardy-Weinberg
equilibrium in controls as well as patients.
No association of SNP rs4570625 with panic disorder in the
total panic disorder sample or in the subgroup of panic disorder
patients with agoraphobia was observed (Table 1). Similarly, no
association of SNP rs4565946 with panic disorder was found in
the total panic disorder sample or in the subgroup of panic disorder patients with agoraphobia (Table 2). SNPs rs4570625 and
rs4565946 were in strong linkage disequilibrium (D 0.97, SD
0.02). Haplotype analysis did not show an association with panic
disorder or agoraphobia (Table 3).
The loss-of-function allele 1463A was not present in any of the
panic disorder patients. Thus, all panic disorder patients with or
without agoraphobia were homozygous for the G1463 allele. DNA
samples from patients with unipolar major depression who were
heterozygous or homozygous for the 1463A allele served as positive controls.
Discussion
Panic disorder is a severe disorder, which due to recurrent and
unforseeable anxiety attacks, frequently leads to great suffering of
the patients, and patients afflicted with panic disorder often lose
their workplace. Heritability of panic disorder is rather high, with
twin studies indicating that approximately 40% of the variance in
the development of panic disorder is due to heritable factors
(Hettema et al., 2001). It is therefore imperative to elucidate the
genetic basis of panic disorder.
This study is the first report of TPH2, the rate-limiting 5-HT
synthesizing enzyme in the brain, in panic disorder patients. A
sample of 134 patients with panic disorder and 134 controls was
assessed. Using SSCP screening, several novel SNPs in TPH2 were
identified, of which two, one located in the putative transcriptional
control region and the other in intron 2, were common and suitable
for haplotype association analysis. Since disease risk is frequently
modulated by functional variants in regions which control expression or stability of gene transcripts (Lesch et al., 1996), common
polymorphic variants in or close to the upstream regulatory region
of TPH2 were selected for analysis in panic disorder.
In the present study no association of either SNP with panic
disorder was observed. The two SNPs were found to be in almost
complete linkage disequilibrium. Therefore, haplotype analysis
did not result in a positive association finding with panic disorder,
with or without agoraphobia. Moreover, the loss-of-function allele
550
TPH2 gene in panic disorder
Table 1 Genotype and allele frequencies of SNP rs4570625 in panic disorder patients and controls
Genotypes
Panic disorder (n 134)
Panic disorder with agoraphobia (n 102)
Controls (n 134)
Armitage Trend Test – two sided
GG
GT
TT
85
(63.4%)
64
(62.8%)
79
(59.0%)
45
(33.6%)
35
(34.3%)
48
(35.8%)
4
(3.0%)
3
(2.9%)
7
(5.2%)
Panic disorder with agoraphobia (n 102)
Controls (n 134)
Z 0.80, p 0.422
Chi2-test
Alleles
Panic disorder (n 134)
Z 0.96, p 0.336
G
T
215
(80.2%)
163
(79.9%)
206
(76.9%)
53
(19.8%)
41
(20.1%)
62
(23.1%)
2 0.90; 1 DF, p 0.344
2 0.63; 1 DF, p 0.429
DF degree of freedom
Table 2 Genotype and allele frequencies of SNP rs4565946 in panic disorder patients and controls
Genotypes
Panic disorder (n 134)
Panic disorder with agoraphobia (n 102)
Controls (n 134)
CC
CT
37
(27.6%)
28
(27.5%)
44
(32.8%)
66
(49.3%)
50
(49.0%)
68
(50.8%)
Armitage Trend Test – two sided
TT
31
(23.1%)
24
(23.5%)
22
(16.4%)
Panic disorder with agoraphobia (n 102)
Controls (n 134)
C
T
140
(52.2%)
106
(52.0%)
156
(58.2%)
128
(47.8%)
98
(48.0%)
112
(41.8%)
1463A, leading to an exchange of arginine with histidine at amino
acid position 441, was not present in any of the panic disorder
patients. This R441H mutation is the only functional variant of
TPH2 known to date. In a previous study, the 1463A allele was
detected in 10.3% of patients with major depression, but in none
of 60 bipolar disorder patients (Zhang et al., 2005).
Z 1.36, p 0.173
Chi2-test
Alleles
Panic disorder (n 134)
Z 1.40, p 0.162
2 1.93; 1 DF, p 0.165
2 1.83; 1 DF, p 0.176
Our findings thus argue against a pathogenetic role of TPH2
genetic polymorphisms in the development of panic disorder.
However, it cannot be excluded that other TPH2 gene variants
which we did not detect in our SSCP analysis may contribute to
the pathogenesis of panic disorder. Factors other than the rate of
synthesis of 5-HT appear to be of greater importance in the patho-
TPH2 gene in panic disorder 551
Table 3 Results of the Haplotype analysis
Haplotype
rs4570625–
rs4565946
Panic disorder:
estimated
frequency
G–C
G–T
T–C
T–T
0.33
0.47
0.19
0.01
Panic disorder
compared to
controls: empirical
p-value
p 0.598
p 0.207
p 0.265
p 0.336
genesis of panic disorder. Thus, a functional polymorphism in the
promoter region of the 5-HT1A receptor gene, which alters 5-HT1A
receptor gene expression, was associated with panic disorder with
agoraphobia (Rothe et al., 2004b). This finding was not replicated in
a subsequent smaller study, in which, moreover, 65% of patients
had comorbid major depression or bipolar depression (Maron et al.,
2005). Another 5-HT1A receptor gene polymorphism (294G/A) has
been investigated in a Japanese panic disorder sample with negative
results (Inada et al., 2003). Moreover, polymorphisms in other serotonergic candidate genes have been assessed. Thus, studies of polymorphisms in the 5-HT2A receptor gene (Fehr et al., 2001; Inada et
al., 2003; Rothe et al., 2004a; Maron et al., 2005) and the 5-HT2C
receptor gene (Deckert et al., 2000; Fehr et al., 2000a; Inada et al.,
2003; Maron et al., 2005) have yielded conflicting results. No
association with panic disorder was observed for the serotonin
transporter promoter polymorphism (5HTTLPR) (Deckert et al.,
1997; Hamilton et al., 1999; Maron et al., 2005), the 5-HT1B and 5HT3A receptor genes (Fehr et al., 2000b; Maron et al., 2005) or for
TPH1 (Fehr et al., 2001; Maron et al., 2005). Finally, high-activity
monoamine oxidase A (MAOA) gene promoter alleles have been
shown to be associated with panic disorder in female patients
(Deckert et al., 1999), although this was not found in a later study
(Hamilton et al., 2000).
In summary, this is the first report to assess TPH2 in panic disorder. In a sample of panic patients with or without agoraphobia,
and matched controls, we did not find TPH2 variations linked to
the development of panic disorder. Given the recent findings on
the interaction of stressful life events and allelic variation of serotonergic gene expression in the pathogenesis of depression and
anxiety (Champoux et al., 2002; Caspi et al., 2003), it will be
interesting to conduct similar studies of environmental-genetic
interactions in panic disorder in the future.
Acknowledgements
We thank all probands for their participation. We would like to thank G.
Ortega, N. Döring, N. Steigerwald, and L. Bogusch for excellent technical
assistance. The Bundesministerium für Bildung und Forschung (BMBF,
01KS9603, 01KW006; 01GS0118), the Deutsche Forschungsgemeinschaft
(SFB 581; KFO 125/1-1), and the Interdisziplinäres Zentrum für Klinische
Forschung (IZKF Würzburg, N-2) supported this work.
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