RESEARCH ARTICLE
Neuropsychiatric Genetics
Association of CRHR1 and CRHR2 with
Major Depressive Disorder and Panic Disorder
in a Japanese Population
Yoshinobu Ishitobi, Shinya Nakayama, Kana Yamaguchi, Masayuki Kanehisa, Haruka Higuma,
Yoshihiro Maruyama, Taiga Ninomiya, Shizuko Okamoto, Yoshihiro Tanaka, Jusen Tsuru,
Hiroaki Hanada, Koichi Isogawa, and Jotaro Akiyoshi*
Department of Neuropsychiatry, Oita University Faculty of Medicine, Hasama-Machi, Yufu-Shi, Oita, Japan
Manuscript Received: 29 June 2011; Manuscript Accepted: 1 March 2012
Major depressive disorder (MDD) and panic disorder (PD) are
common and disabling medical disorders with stress and genetic
components. Dysregulation of the stress response of the
hypothalamic–pituitary–adrenal axis, including the corticotrophin-
releasing hormone (CRH) signaling via primary receptors
(CRHR1 and CRHR2), is considered to play a major role for
onset and recurrence in MDD and PD. To confirm the association
of CRHR1 and CRHR2 with MDD and PD, we investigated 12
single nucleotide polymorphisms (SNPs) (rs4076452, rs7209436,
rs110402, rs242924, rs242940, and rs173365 for CRHR1 and
rs4722999, rs3779250, rs2267710, rs1076292, rs2284217, and
rs226771 for CRHR2) in MDD patients (n ¼ 173), PD patients
(n ¼ 180), and healthy controls (n ¼ 285). The SNP rs110402 and
rs242924 in the CRHR1 gene and the rs3779250 in the CRHR2
gene were associated with MDD. The SNP rs242924 in the
CRHR1 gene was also associated with PD. The T-A-T-G-G
haplotype consisting of rs7209436 and rs173365 in CRHR1
was positively associated with MDD. The T-A haplotype con-
sisting of rs7209436 and rs110402 in CRHR1 was positively
associated with MDD. The C-C haplotype consisting of
rs4722999 and rs37790 in CRHR1 was associated with PD. These
results provide support for an association of CRHR1 and CRHR2
with MDD and PD.
2012 Wiley Periodicals, Inc.
Key words: major depressive disorder; panic disorder; SNP;
CRHR1; CRHR2
INTRODUCTION
Dysfunction of the stress-responsive hypothalamic–pituitary–
adrenal (HPA) axis is a common feature of anxiety and mood
disorders [Bale and Vale, 2004; Nemeroff and Vale, 2005; Swaab
et al., 2005; Hauger et al., 2006]. Activation of the HPA axis is
controlled and regulated by hypothalamic corticotrophin-releasing
hormone (CRH), which activates CRH receptor 1 (CRHR1) in the
anterior pituitary to mediate the production of adrenocorticotro-
phic hormone (ACTH) [Smith et al., 1998; Timpl et al., 1998].
ACTH in turn promotes the synthesis and release of cortisol from
the adrenal gland. Blood cortisol levels are normally regulated by a

2012 Wiley Periodicals, Inc.
How to Cite this Article:
Ishitobi Y, Nakayama S, Yamaguchi K,
Kanehisa M, Higuma H, Maruyama Y,
Ninomiya T, Okamoto S, Tanaka Y, Tsuru J,
Hanada H, Isogawa K, Akiyoshi J. 2012.
Association of CRHR1 and CRHR2 with
Major Depressive Disorder and Panic
Disorder in a Japanese Population.
Am J Med Genet Part B.
multi-point feedback loop at HPA, limbic, brainstem, and pre-
frontal brain areas. Extrahypothalamic effects are primarily medi-
ated directly through CRHR1s in the central amygdala, which
causes downstream effects on the serotonin neurotransmitter
system, among others.
HPA system abnormality is often noticed in major depressive
disorder (MDD) patients presenting anxiety as a symptom. The
responsiveness of ACTH in MDD patients is lower than that
observed in healthy subjects as measured by the CRF test [Pintor
et al., 2007]. When a dexamethasone suppression test is given to
panic disorder (PD) patients, the rate of the nonsuppression is the
same or higher compared to healthy control subjects. In the CRF
test in PD patients, the cortisol response to CRF is blunted [Charney
and Drevets, 2002]. Such phenomena are hypothesized to depend
on chronic over-secretion of CRF. Excessive levels of CRF desensi-
tize CRF receptor function and induce a state of diminished
pituitary sensitivity to CRF. A combined dexamethasone/CRF
Grant sponsor: Japan Society for the Promotion of Science (JSPS); Grant
number: 20591371.
*Correspondence to:
Dr. Jotaro Akiyoshi, MD, PhD, Department of Neuropsychiatry, Oita
University Faculty of Medicine, Hasama-Machi, Oita 879-5593, Japan.
E-mail: akiyoshi@oita-u.ac.jp
Article first published online in Wiley Online Library
(wileyonlinelibrary.com):
DOI 10.1002/ajmg.b.32046
1
2 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
test is widely used investigate MDD patients [K€ unzel et al., 2003].
ACTH and cortisol responsiveness to CRF after dexamethasone
are markedly increased in MDD patients compared to healthy
control subjects [Isogawa et al., 2005]. This phenomenon is
generally explained as a decreasing negative feedback of gluco-
corticoid and vasopressin signaling on the HPA axis [Watson et al.,
2006].
Regarding morphological HPA changes, both pituitary gland
hypertrophy and adrenal gland hypertrophy are reported in MDD
patients [Nemeroff et al., 1992]. ACTH and cortisol blood con-
centrations are also increased in MDD patients [Carroll et al., 2007].
These reports demonstrate an HPA-axis hyper-activation in MDD.
Interestingly, these abnormalities are known to normalize during
remission.
The association between PD and HPA axis function have not
solved yet. Increasing evidence suggests that CRF may play a critical
role in mediating some types of anxiety responses, and that the CRF
system may be a possible therapeutic target for the treatment of
anxiety disorders [Bailey et al., 2011]; Central injections of CRF
activate ‘panic/defense’ responses [Ku et al., 1998] and injections of
the panic-inducing agent doxapram activate CRF neurons [Choi
et al., 2005]. Consistent with these preclinical findings, clinical
evidence suggests that polymorphisms in the CRF1 receptor gene
may be associated with panic [Keck et al., 2008] and that the CRF
system may also be an important therapeutic target for PD
[Risbrough and Stein, 2006]. The fear response without HPA-
axis activation has been activated during naturally occurring,
spontaneous panic attacks [Kellner and Wiedemann, 1998; Abelson
et al., 2007; Preter and Klein, 2008].
The biological effects of CRH and the urocortins (UCN]s are
mediated by two distinct receptors, CRH receptor type 1 (CRHR1)
and 2 (CRHR2), which both belong to the G protein-coupled
receptor superfamily of proteins [Perrin et al., 2006]. Two separate
genes encode the CRH receptors. CRHR1, a 415-amino acid
protein, exhibits high affinity towards CRH and UCN, but low
affinity towards UCN2 and no affinity towards UCN3. CRHR1 is
primarily expressed in the CNS and the anterior pituitary. The
CRHR2 receptor shares 70% sequence identity with CRHR1 and is
expressed primarily in extra-CNS sites. The CRHR2 receptors
exhibit high affinity towards UCNs and no affinity towards CRH
[Bale, 2005].
To comprehensively map the polymorphic genetic variation in
CRHR1 and CRHR2 in relation to MDD and PD, we analyzed
12 single nucleotide polymorphisms (SNPs) in MDD patients, PD
patients, and healthy controls.
MATERIALS AND METHODS
Subjects
All cases and control subjects were ethnically Japanese and were
recruited in the vicinity of Oita prefecture, Japan. Subjects com-
prised 173 unrelated Japanese with MDD (91 males and 82 females;
age ¼ 41.6
16.2 years) and 180 unrelated Japanese with PD
diagnosed according to DSM-IV criteria (79 males and 102 females;
age ¼ 40.9
10.5 years (mean
SD)), while 285 unrelated healthy
volunteers (173 males and 112 females; age ¼ 32.8
8.7 years)
served as controls. Patients in the MDD group had either pure
MDD or MDD comorbid with anxiety disorders (31.8%). Among
the patients with PD, 33.9% had comorbid MDD and 5.6% had
other anxiety disorders. Cases for MDD and PD were recruited
from Oita University Hospital in Oita. All cases were outpatients or
stable in-patients. Each diagnosis was confirmed using the Mini-
International Neuropsychiatric Interview (MINI) [Sheehan et al.,
1998] and clinical records were also reviewed. The controls received
a short interview (MINI) conducted by one of the authors (J.A.) to
exclude a history of major psychiatric illness. Exclusion criteria were
(1) central nervous system disorders and medical disorders clearly
affecting cerebral function, (2) alcoholism or drug dependence, (3)
personality disorder (SCID-II), (4) presence of brain injury or
disease, (5) pregnancy, (6) mental retardation, (7) age <18 years,
(8) immediate danger of suicide, (9) treatment with lithium and/or
carbamazepine, and (10) endocrine disorder of the pituitary or the
adrenal gland. The objective of the present study was clearly
explained and written informed consent was obtained from all
subjects. The study was approved by the Ethical Committee of the
Faculty of Medicine, the University of Oita.
Genotyping
Genomic DNA was extracted from leukocytes using the standard
phenol–chloroform method. The CRHR SNP selection was basi-
cally performed according to the MAF standard (minor allele
frequency, HapMap Project, 2003) with estimates higher than
0.1. We selected six SNPs (rs4076452, rs7209436vrs110402,
rs242924, rs242940, and rs173365) in CRHR1 and six SNPs
(rs4722999, rs3779250, rs2267710, rs1076292, rs2284217, and
rs226771) in CRHR2. Individual genomic DNAs were genotyped
for every SNP on the Taqman PCR SNP genotyping assay, using a
Roche LightCycler480 (Basel, Switzerland). The standard 10 ml PCR
reaction containing 2 ng genomic DNA was carried out using the
Taqman PCR including Universal PCR Master Mix under the
protocol guidelines.
Statistical Analysis
All of the parameter calculations, allele and genotype frequencies,
Hardy–Weinberg equilibrium calculations, pair-wise linkage dis-
equilibrium analyses, haplotype analyses, and P-value permuta-
tions were conducted online at http://analysis.bio-x.cn [Shi and He,
2005; Li et al., 2009], a robust and user-friendly platform with
integrated analysis tools particularly suited to association studies.
P-values were corrected using 10,000-permutation corrections. The
permutations considering both of the two test types (allelic tests and
genotype tests) were performed for each of the two disease analyses
(e.g., MDD vs. control, PD vs. control). The significance level was
set at a ¼ 0.05.
RESULTS
In the 638 Japanese samples, genotype distributions were in
Hardy–Weinberg equilibrium for all of the SNPs. The allele and
genotype frequencies of 11 SNPs in the 2 patient sample groups and
the healthy controls are listed in Tables I and III. The haplotype
ISHITOBI ET AL. 3

TABLE I. Genotype and Allele Frequency of the Six SNPs in the CRHR1 Gene

Genotype distribution
Chromosome
Gene db SNP ID [M/m]a position (bp) Phenotype N MAFb P-value M/M M/m m/m P-value
CRHR1 rs4076452 [C/G] 41211660 Control 285 0.142 4 73 208
Depression 173 0.136 0.791 7 33 133 0.07
Panic 180 0.186 0.074 5 57 118 0.184
rs7209436 [C/T] 41225913 Control 285 0.144 4 74 207
Depression 173 0.13 0.558 4 37 132 0.442
Panic 180 0.181 0.135 6 53 121 0.24
rs110402 [A/G] 41235818 Control 285 0.221 175 94 16
Depression 173 0.237 0.001 130 39 4 0.008
Panic 180 0.2 0.445 115 58 7 0.673
rs242924 [G/T] 41241147 Control 285 0.114 3 59 223
Depression 173 0.116 0.013 8 24 141 0.013
Panic 180 0.167 0.022 8 44 128 0.033
rs242940 [A/G] 41248380 Control 285 0.119 4 60 221
Depression 173 0.095 0.262 4 25 144 0.176
Panic 180 0.114 0.265 5 42 133 0.465
rs173365 [A/G] 41256855 Control 285 0.116 3 60 222
Depression 173 0.095 0.335 4 25 144 0.133
Panic 180 0.144 0.201 4 44 132 0.392
Statistical analysis was performed using c2 test. P-value < 0.05 were indicated in bold.
a
M, major allele; m, minor allele.
b
MAF, minor allele frequency.

analysis of the two patient sample groups and the healthy controls
are shown in Tables II and IV. The linkage disequilibrium among
the eleven SNPs is shown in Figures 1 and 2. The selection criteria
for haplotypes used in the haplotype analyses were adjacent SNPs
with pairwise D0 > 0.85. In our analyses, haplotypes with frequen-
cies above 0.03 were tested.
CRHR1 and Major Depressive Disorder
According to the selection criteria, five SNPs (rs7209436, rs110402,
rs242924, rs242940 and rs173365) of CRHR1 with strong D0 > 0.85
were in one block for MDD (Fig. 1). Two SNPs (rs7209436 and
rs110402) with strong pairwise D0 > 0.85 were in one block and

TABLE II. Haplotype Analysis of the Two Patients Sample Groups and the Control Group in the CRHR1 Gene

Depression Panic
Haplotype Ca-Freq Co-Freq P-value* Global P* Case-Freq Co-Freq P- value* Global P*
rs7209436–rs173365
T-A-T-G-G 0.846 0.768 0.0003 1.14e5 0.771 0.768 0.1354 0.0017
C-G-G-A-A 0.090 0.109 0.9264 0.121 0.109 0.4182
T-G-T-G-G 0.006 0.077 5.82e6 0.020 0.078 0.0004
C-G-T-G-G 0.026 0.026 1.0000 0.035 0.026 0.2419
rs7209436–rs110402
T-A 0.861 0.775 0.0013 3.75e5 0.792 0.775 0.4612 0.0046
C-G 0.127 0.140 0.5760 0.170 0.140 0.2043
T-G 0.009 0.081 9.47e6 0.029 0.081 0.0016
rs242940–rs173365
G-G 0.905 0.881 0.3260 0.3260 0.847 0.881 0.4546 0.4546
A-A 0.095 0.116 0.3260 0.130 0.116 0.4546
*Corrected P-value after 10,000 permutations and the global P was the P value for analysis of all haplotypes in one block. P-value < 0.05 were indicated in bold.
4 AMERICAN JOURNAL OF MEDICAL GENETICS PART B

TABLE III. Genotype and Allele Frequency of the Six SNPs in the CRHR2 Gene

Genotype distribution
Chromosome
Gene db SNP ID [M/m]a position (bp) Phenotype N MAFb P-value M/M M/m m/m P-value
CRHR2 rs4722999 [C/T] 30693775 Control 285 0.444 87 143 55
Depression 173 0.384 0.077 70 73 30 0.090
Panic 180 0.433 0.745 60 84 36 0.748
rs3779250 [T/C] 30694260 Control 285 0.426 92 143 50
Depression 173 0.208 1.75e11 101 72 0 2.08e11
Panic 180 0.394 0.320 66 86 28 0.603
rs2267710 [C/T] 30696190 Control 285 0.398 99 145 41
Depression 173 0.398 0.056 81 68 24 0.029
Panic 180 0.406 0.890 65 84 31 0.598
rs1076292 [C/G] 30712701 Control 285 0.370 113 133 39
Depression 173 0.315 0.090 86 65 22 0.098
Panic 180 0.381 0.799 73 77 30 0.591
rs 2284217 [A/G] 30713608 Control 285 0.574 46 151 88
Depression 173 0.569 0.892 36 77 60 0.188
Panic 180 0.600 0.474 32 80 68 0.186
rs226771 [C/T] 30716643 Control 285 0.798 10 95 180
Depression 173 0.740 0.040 10 70 93 0.112
Panic 180 0.797 0.981 5 63 112 0.866
Statistical analysis was performed using c2 test. P-value < 0.05 were indicated in bold.
a
M, major allele; m, minor allele.
b
MAF, minor allele frequency.

another two SNPs (rs242940 and rs173365) of CRHR1 in another
block for PD (Fig. 2). Regarding CRHR2, two SNPs (rs4722999 and
rs3779250) with strong pairwise D0 > 0.85 were in one block and
another two SNPs (rs1076292 and rs2284217) were in another block
for MDD (Fig. 3) while three SNPs (rs1076292, rs2284217, and
rs2267716) with strong pairwise D0 > 0.85 were in one block for PD
(Fig. 4).
We found that rs110402 and rs242924 in CRHR1 were positively
associated with MDD in terms of both allele and genotype dis-
tributions (rs110402, allele: P ¼ 0.001, genotype: P ¼ 0.008, odds
ratio (OR) ¼ 1.81 [95% CI ¼ 1.25–2.60]; rs242924, allele:
P ¼ 0.013, genotype: P ¼ 0.013, OR ¼ 1.02 [95% CI ¼ 0.07–1.54]-
) (Table I). After a 10,000-permutations correction, rs110402 was
still significant in the genotype distribution (P ¼ 0.008). Haplotype

TABLE IV. Haplotype Analysis of the Two Patients Sample Groups and the Control Group in the CRHR2 Gene

Depression Panic
Haplotype Ca-Freq Co-Freq P-value* Global P* Ca-Freq Co-Freq P-value* Global P*
rs4722999–rs3779250
C-C 0.613 0.554 0.0817 5.55e16 0.469 0.554 0.0115 2.00e15
T-T 0.205 0.425 1.12e11 0.297 0.425 9.40e5
T-C 0.179 0.020 0.00e0 0.136 0.020 1.84e12
rs1076292–rs2284217
C-A 0.431 0.424 0.8620 0.1412 0.383 0.424 0.2882 0.4028
G-G 0.315 0.368 0.1012 0.363 0.368 0.9691
C-G 0.254 0.206 0.0960 0.237 0.206 0.2239
rs1076292–rs2267716
C-A-T 0.424 0.421 0.7896 0.1078 0.383 0.421 0.4762 0.7617
G-G-T 0.304 0.366 0.0749 0.363 0.366 0.7452
C-G-C 0.243 0.197 0.0826 0.203 0.197 0.6310

*Corrected P-value after 10,000 permutations and the global P was the P value for analysis of all haplotypes in one block. P-value < 0.05 were indicated in bold.
ISHITOBI ET AL.
FIG. 1. CRHR1 SNPs and major depressive disorder and interaction
effects with control. SNPs indicate single-nucleotide
polymorphisms. The position of the CRHR1 gene and its introns
(filled rectangles) on chromosome 17 as well as a linkage
disequilibrium (LD) plot of all tested SNPs using r2 as the
measure of LD is also shown. r2 ¼ 1 indicates complete LD and is
depicted by the darkest shade of blue. r2 < 1.0 are printed in the
respective square for compared SNPs, with darker shades of red
representing higher levels of LD.
analysis revealed that the T-A-T-G-G haplotype consisting of
rs7209436 and rs173365 in CRHR1 was positively associated
with MDD (P ¼ 0.0003). The corrected global P value was
1.14e5. Haplotype analysis revealed that the T-A haplotype con-
sisting of rs7209436 and rs110402 in CRHR1 was positively associ-
ated with MDD (P ¼ 0.0013). The corrected global P value was
3.75e5 (Table II).
CRHR2 and Major Depressive Disorder
Furthermore, we found that rs3779250 in CRHR2 was positively
associated with MDD in both allele and genotype distributions
(allele: P ¼ 1.75e11, genotype: P ¼ 2.08e11, odds ratio (OR) ¼ 2.83
[95% CI ¼ 2.08–3.85]) (Table III). After 10,000-permutation cor-
rection, rs3779250 was still significant in the genotype distribution
(P ¼ 0.000). Haplotype analysis revealed that the C-C haplotype
consisting of rs4722999 and rs3779250 in CRHR2 was associated
with MDD. The corrected global P value was 5.55e16 (Table IV).
CRHR1 and Panic disorder
We found that rs242924 in CRHR1 was positively associated with
PD in both allele and genotype distributions (rs242924, allele:
5
FIG. 2. CRHR1 SNPs and panic disorder and interaction effects with
control. SNPs indicate single-nucleotide polymorphisms. The
position of the CRHR1 gene and its introns (filled rectangles) on
chromosome 17 as well as a linkage disequilibrium (LD) plot of
all tested SNPs using r2 as the measure of LD is also shown.
r2 ¼ 1 indicates complete LD and is depicted by the darkest
shade of blue. r2 < 1.0 are printed in the respective square for
compared SNPs, with darker shades of red representing higher
levels of LD.
P ¼ 0.022, genotype: P ¼ 0.033, odds ratio (OR) ¼ 1.55 [95%
CI ¼ 1.06–2.27]) (Table I). Haplotype analysis revealed that the
T-A-T-G-G haplotype consisting of rs7209436 and rs173365 in
CRHR1 was not associated with PD (P ¼ 0.1354). The corrected
global p value was 0.0017. Haplotype analysis revealed that the T-A
haplotype consisting of rs7209436 and rs110402 in CRHR1 was not
associated with PD (P ¼ 0.4612).
CRHR2 and Panic Disorder
Finally, we found that rs3779250 in CRHR2 was not associated with
PD in both allele and genotype distributions (Table III). Haplotype
analysis revealed that the C-C haplotype consisting of rs4722999
and rs37790 in CRHR1 was associated with PD (P ¼ 0.0115). The
corrected global P value was 2.00e15 (Table IV).
DISCUSSION
The main finding of the present study was a significant association
of the CRHR1 and CRHR2 genes with MDD and PD in a Japanese
population. The data suggest that CRHR1 and CRHR2 might be
involved in MDD and PD susceptibilities, and support previous
reports that MDD and PD may have a substantial overlap in terms
6 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
FIG. 3. CRHR2 SNPs and major depressive disorder and interaction
effects with control. SNPs indicate single-nucleotide
polymorphisms. The position of the CRHR2 gene and its introns
(filled rectangles) on chromosome 7 as well as a linkage
disequilibrium (LD) plot of all tested SNPs using r2 as the
measure of LD is also shown. r2 ¼ 1 indicates complete LD and is
depicted by the darkest shade of blue. r2 < 1.0 are printed in the
respective square for compared SNPs, with darker shades of red
representing higher levels of LD.
of pathogenesis [Simon and Fischmann, 2005]. MDD and PD are
severe mental disorders underlying genetic components as well as
contributory experiential and environmental factors [Anisman
et al., 2008]. In our study, following 10,000-permutation correc-
tion, we found that the CRHR1and CRHR2 genes were associated
with MDD and PD.
Liu et al. [2006] first reported that the CRHR1 gene might be a
new susceptibility gene for depression. In their study, they found
significant allele and genotype associations with rs242939, and the
haplotype defined by alleles G-G-T for rs1876828, rs242939, and
rs242941 was significantly over-represented in major depression
patients compared to controls. However, in our study, we used
rs4076452, rs7209436, rs110402, rs242924, rs242940, and rs173365
from Liu’s study. In the present study, we found an association
between two SNPs (rs110402, rs242924) of the CRHR1 gene with
MDD. rs110402 was shown to be associated with an early onset of a
first episode of depression [Papiol et al., 2007]. CRH is a crucial
activator of the HPA axis, binding to receptors that initiate the stress
response, concluding with the release of cortisol from the adrenal
cortex. Recently, CRHR1 polymorphisms was shown to interact
with childhood maltreatment to predict HPA axis reactivity, which
has been linked to both MDD and early life stress [Tyrka et al.,
2009]. Difference in the CRHR1 function have been linked to risk
FIG. 4. CRHR2 SNPs and panic disorder and interaction effects with
control. SNPs indicate single-nucleotide polymorphisms. The
position of the CRHR2 gene and its introns (filled rectangles) on
chromosome 7 as well as a linkage disequilibrium (LD) plot of all
tested SNPs using r2 as the measure of LD is also shown. r2 ¼ 1
indicates complete LD and is depicted by the darkest shade
of blue. r2 < 1.0 are printed in the respective square for
compared SNPs, with darker shades of red representing higher
levels of LD.
for depression, in the presence of childhood maltreatment [Bradley
et al., 2008; Polanczyk et al., 2009]. The TAT haplotype formed by
rs7209436, rs110402, and rs242924 was associated with a significant
protective effect [Bradley et al., 2008; Polanczyk et al., 2009]. We
could find five SNPs (rs7209436, rs110402, rs242924, rs242940, and
rs173365) of CRHR1 with strong D0 > 0.85 were in one block for
MDD (Fig. 1). The T-A-T-G-G haplotype consisting of rs7209436
and rs173365 in CRHR1 was positively associated with MDD. The
T-A haplotype consisting of rs7209436 and rs110402 in CRHR1 was
positively associated with MDD (Table II). We did not find that
the TAT haplotype was associated with MDD. We did not find
that there was strong positive association between the
rs7209436–rs173365 haplotype and MDD (Fig. 1). Another group
examined SNPs in CRHR1 in a large population sample [Grabe
et al., 2010]. They detected a significant moderator effect of the TAT
haplotype on the risk of adult depression symptoms. However, the
haplotype was associated with greater depressive symptoms only
among individuals who experienced moderate-to-severe physical
neglect. We could not examine the relationship between childhood
maltreatment and MDD. Both rs110402 and rs242924 showed a
significant interaction with maltreatment in the prediction of
cortisol response to the DEX/CRH test [Tyrka et al., 2009]. That
the GG genotype of both SNPs was linked to higher cortisol
ISHITOBI ET AL.
responsiveness to the test extends recent findings of an increase in
depressive symptoms among subjects with the GG genotype of these
SNPs who reported a history of childhood maltreatment [Bradley
et al., 2008]. Maltreatment at this critical age might affect our
results, and further studies are needed to determine the relationship
between childhood maltreatment and MDD in the Japanese.
In our study, we found a significant association between
rs3779250 in CRHR2 and MDD. The CRHR2 gene was reported
unlikely to be involved in the genetic liability underlying HPA axis
dysfunction and mood disorders [Villafuerte et al., 2002]. CRHR2
may function endogenously to dampen or modulate the stress
response associated with CRHR1 activation, and therefore CRHR2
may play a counteracting role in the CRHR2 in the HPA axis stress
response [Bale, 2005]. However, ethnicity and sample size may
explain how the Villafuerte et al. results differ from the present
study, as their 89 patients were of Belgian origin. Allele G of
rs2270007 of CRHR2 gene was associated with a worse overall
response to citalopram after treatment follow-up [Papiol et al.,
2007]. Genetic variants like rs2270007, located on genes involved in
HPA axis regulation, could make this axis more adaptable in
presence of antidepressant treatment. Further exploration of the
CRHR1 and CRHR2 findings in large case–control PD samples may
provide more definitive evidence implicating these loci in the
genetic etiology of PD.
We found a significant association between rs242924 in CRHR1
and PD. Some studies have reported an absence of associations of
polymorphisms in CRHR1 in relation to PD [Keck et al., 2008;
Hodges et al., 2009]. The SNP pair in the combined with rs878886 in
CRHR1 and rs28632197 in arginine vasopressin (AVP) 1B was
significant association in PD [Keck et al., 2008]. In these cases,
ethnicity might again explain the differences between their data and
the present study. We did not find the association between CRHR2
and PD. Some studies reported that the CRHR2 polymorphisms
examined did not confer susceptibility to PD [Tharmalingam et al.,
2006; Keck et al., 2008]. Their SNP is different from the SNPs of our
present study. Further studies investigating additional polymor-
phisms in this gene and other components of the CRH signaling
system may prove useful. In conclusion, these results provide
support for an association of CRHR1 and CRHR2 with MDD
and PD.
ACKNOWLEDGMENTS
This study is supported by a Grant-in-Aid for Scientific Research
(C) (No. 20591371) from the Japan Society for the Promotion of
Science (JSPS). We are grateful to Ms. Kazumi Oda for excellent
technical assistance.
REFERENCES
Abelson JL, Khan S, Liberzon I, Young EA. 2007. HPA axis activity in
patients with panic disorder: Review and synthesis of four studies.
Depress Anxiety 241:66–76.
Anisman H, Merali Z, Stead JD. 2008. Experiential and genetic contribu-
tions to depressive- and anxiety-like disorders: Clinical and experimental
studies. Neurosci Biobehav Rev 32:1185–1206.
7
Bailey JE, Papadopoulos A, Diaper A, Phillips S, Schmidt M, van der Ark P,
Dourish CT, Dawson GR, Nutt DJ. 2011. Preliminary evidence of
anxiolytic effects of the CRF1 receptor antagonist R317573 in the
7.5% CO2 proof-of-concept experimental model of human anxiety. J
Psychopharmacol 25:1199–1206.
Bale TL. 2005. Sensitivity to stress: Dysregulation of CRF pathways and
disease development. Horm Behav 48:1–10.
Bale TL, Vale WW. 2004. CRF and CRF receptors: Role in stress respon-
sivity and other behaviors. Annu Rev Pharmacol Toxicol 44:525–557.
Bradley R, Binder E, Epstein M, Tang Y, Nair H, Liu W, Gillespie C, Berg T,
Evces M, Newport J, Stowe Z, Heim C, Nemeroff C, Schwartz A, Cubells J,
Ressler K. 2008. Influence of child abuse on adult major depressive
disorder: Moderation by the corticotropin-releasing hormone receptor
gene. Arch Gen Psychiatry 65:190–200.
Carroll BJ, Cassidy F, Naftolowitz D, Tatham NE, Wilson WH, Iranmanesh
A, Liu PY, Veldhuis JD. 2007. Pathophysiology of hypercortisolism in
depression. Acta Psychiatr Scand Suppl 433:90–103.
Charney DS, Drevets WD. 2002. Neurobiological basis of anxiety disor-
ders. In: Davis KL, Charney D, Coyle JT, et al., editors. Neuropsycho-
pharmacology: The fifth generation of progress. Baltimore, MD:
Lippincott Williams & Wilkins. pp 901–930.
Choi SH, Kim SJ, Park SH, Moon BH, Do E, Chun BG, Lee MS, Shin KH.
2005. Doxapram increases corticotropin-releasing factor immunoreac-
tivity and mRNA expression in the rat central nucleus of the amygdala.
Peptides 26:2246–2251.
Grabe HJ, Schwahn C, Appel K, Mahler J, Schulz A, Spitzer C, Fenske K,
Barnow S, Lucht M, Freyberger HJ, John U, Teumer A, Wallaschofski H,
Nauck M, V€ olzke H. 2010. Childhood maltreatment, the corticotropin-
releasing hormone receptor gene and adult major depressive
disorder in the general population. Am J Med Genet Part B 153B:
1483–1493.
Hauger RL, Risbrough V, Brauns O, Dautzenberg FM. 2006. Corticotropin
releasing factor (CRF) receptor signaling in the central nervous system:
New molecular targets. CNS Neurol Disord Drug Targets 5:453–479.
Hodges LM, Weissman MM, Haghighi F, Costa R, Bravo O, Evgrafov O,
Knowles JA, Fyer AJ, Hamilton SP. 2009. Association and linkage analysis
of candidate genes GRP, GRPR, CRHR1, and TACR1 in panic disorder.
Am J Med Genet Part B 150B:65–73.
Isogawa K, Nagayma H, Tsutsumi T, Kiyota A, Akiyoshi J, Hieda K. 2005.
Simultaneous use of thyrotropin-releasing hormone test and combined
dexamethasone/corticotropine-releasing hormone test for severity eval-
uation and outcome prediction in patients with major depressive disor-
der. J Psychiatr Res 39:467–473.
Keck ME, Kern N, Erhardt A, Unschuld PG, Ising M, Salyakina D, M€ uller
MB, Knorr CC, Lieb R, Hohoff C, Krakowitzky P, Maier W, Bandelow B,
Fritze J, Deckert J, Holsboer F, M€ uller-Myhsok B, Binder EB. 2008.
Combined effects of exonic polymorphisms in CRHR1 and AVPR1B
genes in a case/control study for panic disorder. Am J Med Genet Part B
147B:1196–1204.
Kellner M, Wiedemann K. 1998. Nonresponse of adrenocorticotropic
hormone in firstever lactate-induced panic attacks in healthy volunteers.
Arch Gen Psychiatry 551:85–86.
Ku YH, Tan L, Li LS, Ding X. 1998. Role of corticotropin releasing factor
and substance P in press or responses of nuclei controlling emotion and
stress. Peptides 19:677–682.
K€
unzel HE, Binder EB, Nickel T, Ising M, Fuchs B, Majer M, Pfennig A,
Ernst G, Kern N, Schmid DA, Uhr M, Holsboer F, Modell S. 2003.
Pharmacological and nonpharmacological factors influencing hypothal-
amic–pituitary–adrenocortical axis reactivity in acutely depressed
psychiatric in-patients, measured by the Dex-CRH test. Neuropsycho-
pharmacology 228:2169–2178.
8 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
Li Z, Zhang Z, He Z, Tang W, Li T, Zeng Z, He L, Shi Y. 2009. A partition-
ligation-combination-subdivision EM algorithm for haplotype inference
with multiallelic markers: Update of the SHEsis (http://analysis.bio-x.
cn). Cell Res 19:519–523.
Liu Z, Zhu F, Wang G, Xiao Z, Wang H, Tang J, Wang X, Qiu D, Liu W, Cao
Z, Li W. 2006. Association of corticotropin-releasing hormone receptor1
gene SNP and haplotype with major depression. Neurosci Lett 404:
358–362.
Nemeroff CB, Vale WW. 2005. The neurobiology of major depressive
disorder: Inroads to treatment and new drug discovery. J Clin Psychiatry
66:5–13.
Nemeroff CB, Krishnan KR, Reed D, Leder R, Beam C, Dunnick NR. 1992.
Adrenal gland enlargement in major depression. A computed tomo-
graphic study. Arch Gen Psychiatry 49:384–387.
Papiol S, Arias B, Gast

o C, Guti
errez B, Catal
an R, Fa~
nan
as L. 2007. Genetic
variability at HPA axis in major depressive disorder and clinical response
to antidepressant treatment. J Affect Disord 104:83–90.
Perrin MH, Grace CR, Riek R, Vale WW. 2006. The three-dimensional
structure of the N-terminal domain of corticotropin-releasing factor
receptors: Sushi domains and the B1 family of G protein-coupled
receptors. Ann N Y Acad Sci 1070:105–119.
Pintor L, Torres X, Navarro V, Martinez de Osaba MA, Matrai S, Gast
o
C. 2007. Corticotropin-releasing factor test in melancholic patients in
depressed state versus recovery: A comparative study. Prog Neuropsy-
chopharmacol Biol Psychiatry 31:1027–1033.
Polanczyk G, Caspi A, Williams B, Price TS, Danese A, Sugden K, Uher
R, Poulton R, Moffitt TE. 2009. Protective effect of CRHR1 gene
variants on the development of adult depression following childhood
maltreatment: Replication and extension. Arch Gen Psychiatry 66:
978–985.
Preter M, Klein DF. 2008. Panic, suffocation false alarms, separation
anxiety and endogenous opioids. Prog Neuropsychopharmacol Biol
Psychiatry 323:603–612.
Risbrough VB, Stein MB. 2006. Role of corticotrophin releasing factor in
anxiety disorders: A translational research perspective. Horm Behav
50:550–561.
Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E,
Hergueta T, Baker R, Dunbar GC. 1998. The Mini-International Neu-
ropsychiatric Interview (M.I.N.I.): The development and validation of a
structured diagnostic psychiatric interview for DSM-IV and ICD-10. J
Clin Psychiatry 59(Suppl):22–33.
Shi YY, He L. 2005. SHEsis, a powerful software platform for analyses of
linkage disequilibrium, haplotype construction, and genetic association
at polymorphism loci. Cell Res 15:97–98.
Simon NM, Fischmann D. 2005. The implications of medical and psychi-
atric comorbidity with panic disorder. J Clin Psychiatry 66:8–15.
Smith GW, Aubry JM, Dellu F, Contarino A, Bilezikjian LM, Gold LH,
Chen R, Marchuk Y, Hauser C, Bentley CA, Sawchenko PE, Koob GF,
Vale W, Lee KF. 1998. Corticotropin releasing factor receptor 1-deficient
mice display decreased anxiety, impaired stress response, and aberrant
neuroendocrine development. Neuron 20:1093–1102.
Swaab DF, Bao AM, Lucasson PJ. 2005. The stress system in the human
brain in major depressive disorder and neurogeneration. Ageing Res Rev
4:141–194.
Tharmalingam S, King N, De Luca V, Rothe C, Koszycki D, Bradwejn J,
Macciardi F, Kennedy JL. 2006. Lack of association between the cortico-
trophin-releasing hormone receptor 2 gene and panic disorder. Psychiatr
Genet 16:93–97.
Timpl P, Spanagel R, Sillaber I, Kresse A, Reul JM, Stalla GK, Blanquet V,
Steckler T, Holsboer F, Wurst W. 1998. Impaired stress response and
reduced anxiety in mice lacking a functional corticotropin-releasing
hormone receptor 1. Nat Genet 19:162–166.
Tyrka AR, Price LH, Gelernter J, Schepker C, Anderson GM, Carpenter
LL. 2009. Interaction of childhood maltreatment with the corticotropin-
releasing hormone receptor gene: Effects on hypothalamic–pituitary–
adrenal axis reactivity. Biol Psychiatry 66:681–685.
Villafuerte SM, Del-Favero J, Adolfsson R, Souery D, Massat I, Mendlewicz
J, Van Broeckhoven C, Claes S. 2002. Gene-based SNP genetic association
study of the corticotropin-releasing hormone receptor-2 (CRHR2) in
major depressive disorder. Am J Med Genet 114:222–226.
Watson S, Gallagher P, Smith MS, Ferrier IN, Young AH. 2006. The dex/CRH
test—Is it better than the DST? Psychoneuroendocrinology 31:889–894.