Association of glucocorticoid and type 1 corticotropin-releasing

hormone receptors gene variants and risk for depression during

pregnancy and post-partum
Neelam Engineer
a
, Lucy Darwin
b
, Deole Nishigandh
a
, Kandala Ngianga-Bakwin
c
,
Steve C. Smith
b
, Dimitris K. Grammatopoulos
b, c,
*
a
Dept of Obstetrics and Gynaecology, UHCW NHS Trust, Coventry, UK
b
Dept of Clinical Biochemistry, UHCW NHS Trust, Coventry, UK
c
Division of Metabolic and Vascular Heallth, Warwick Medical School, University of Warwick, Coventry, UK
a r t i c l e i n f o
Article history:
Received 29 December 2012
Received in revised form
27 March 2013
Accepted 6 May 2013
Keywords:
HPA axis
Postnatal depression
CRH receptor
GR
Variant
a b s t r a c t
Women with postnatal depression (PND) appear to have abnormal hypothalamic pituitary adrenal (HPA)
axis responses to stress, which might involve a genetic variability component. We investigated associ-
ation of genetic variants in the glucocorticoid receptor (GR, NR3C1) and corticotropin releasing hormone
receptor 1 (CRHR1) genes with increased risk for PND. Two hundred pregnant women were recruited
prospectively and PND risk was assessed by the Edinburgh Postnatal Depression Scale (EPDS) during
pregnancy and again 2e8 weeks post-natally (CW-GAPND study). The BclI and ER22/23EK single
nucleotide polymorphisms (SNPs) of the GR and the haplotype-tagged rs1876828, rs242939 and
rs242941 SNPs of the CRHR1 associated with genetic risk to depressive disorders were genotyped. A cut-
off score of 10 was used to detect increased risk of PND. Association analysis was carried out in 140
patients that completed the study protocol. The BclI and rs242939 SNPs were over-represented in
women with postnatal EPDS score 10 with signicant allele association (p 0.011 and <0.001,
respectively) and risk ratios of 2.9 (95% CI: 1.2e6.9) for BclI, 4.9 (2e12) for rs242939 and 5.48 (2.13
e14.10) for both. The rs242939 SNP was also associated with increased EPDS values during pregnancy.
Moreover, the G-G-T haplotype of the CRHR1 was signicantly over-represented in patients with high
EPDS scores, with risk ratio of 3.22 (95% CI: 1.91e5.42). This is the rst evidence that specic SNPs of
genes involved in stress responses might contribute in the genetics of high-risk for depression during
pregnancy and postpartum.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
In Western countries postnatal depression (PND) affects
approximately 1 in 7 women, whereas in non-Western populations
prevalence ranges between 5 and 60% (OHara and Swain, 1996;
Cooper et al., 1999; Klainin and Arthur, 2009). If left untreated, PND
has profound consequences on the quality of family life, social
functioning as well as in the long-term emotional and cognitive
development of the baby (Grace et al., 2003; Payne, 2007). Most
importantly, severe forms of PND, such as puerperal psychosis,
which affects 0.1e0.2% of new mothers, are linked to suicide and
infanticide (Spinelli, 2009).
Despite detailed prevalence statistics, high probabilities of re-
experience with future pregnancies, and well documented conse-
quences of the disorder, PND remains difcult to identify. Routine
assessment for either prenatal depression or PND is not universal.
Screening guidelines for PND assessment across healthcare in-
stitutions are inconsistent. As a result fewer than half of PND cases
are detected by primary healthcare professionals in routine clinical
practice (Hearn et al., 1998) and in the UK screening policies rely on
opportunistic case nding. The Edinburgh Postnatal Depression
Scale (EPDS) which detects high levels of PNDsymptoms, is a widely
used screening tool, however, it cannot be used for prospective
identication of women at risk and is not considered to be cost-
effective (Paulden et al., 2009). Clearly, PND management and out-
comes would benet substantially from early identication of
women at risk and clinically effective treatments available. There is
alsoinconclusive dataabout thelong-termoutcomeof PNDpatients;
most cases last around3 months and resolve spontaneously without
* Corresponding author. Warwick Medical School, The University of Warwick,
Gibbet Hill Road, Coventry, CV4 7AL, UK. Tel.: 44 2476 968661; fax: 44 2476
574637.
E-mail address: d.grammatopoulos@warwick.ac.uk (D.K. Grammatopoulos).
Contents lists available at SciVerse ScienceDirect
Journal of Psychiatric Research
j ournal homepage: www. el sevi er. com/ l ocat e/ psychi res
0022-3956/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jpsychires.2013.05.003
Journal of Psychiatric Research 47 (2013) 1166e1173
treatment. However, several studies demonstrated the presence
of depression, with over 50% lasting over 6 months and some still
being present up to 24 months postpartum (Goodman, 2004).
Several psychosocial risk factors for PND have been identied:
the strongest predictors are past history of depression and psy-
chological disturbance during pregnancy, as well as poor marital
relationship and low social support, and stressful life events
(OHara and Swain, 1996). A family history of depression is another
risk factor for PND (Murphy-Eberenz et al., 2006), in agreement
with the view that depressive illnesses are associated with
high heritability, although individual episodes are frequently trig-
gered by stressful life events. The later implicates stress-driven
hormonal responses in disease pathogenesis (Spijker & van
Rossum, 2009). The maternal hypothalamic-pituitary-adrenal
(HPA) axis undergoes gradual changes during pregnancy because
of an increasing production of placental corticotrophin-releasing
hormone (CRH) (Chrousos et al., 1998). The abrupt withdrawal of
placental CRH at birth results in a re-equilibration of the maternal
HPA axis in the days post-delivery. Indeed, women with PND
appear to have abnormal hypothalamic pituitary adrenal (HPA) axis
responses to stress, possibly due to enhanced sensitivity to gonadal
steroids during pregnancy (Magiakou et al., 1996; Bloch et al.,
2005). It has been hypothesized that in women with symptoms of
postpartum blues or depression, the HPA axis function fails to
return to normal post-delivery and exhibits blunted adrenocorti-
cotropic hormone (ACTH) responses to CRH challenge at 6 and 12
weeks postpartum suggesting prolonged HPA suppression
(Magiakou et al., 1996; Rich-Edwards et al., 2008). Recent studies
(OKeane et al., 2011) investigating hormonal changes in the HPA
axis in the days after delivery in relation to daily mood changes
suggest that postpartum blues positively correlated with ACTH;
and negatively correlated with oestriol levels during the post-
partum days, and with the reduction in CRH concentrations from
pregnancy. These ndings support the hypothesis that the reac-
tivation of hypothalamic ACTH secretagogue peptides may be
involved in the aetiology of the disease.
Impaired signalling of key molecules of the HPA axis such as the
glucocorticoid receptor (GR) and corticotrophin releasing hormone
receptor type 1 (CRH-R1) has been implicated in the pathogenesis
of various depressive disorders including PND (Wisner and Stowe,
1997; Kammerer et al., 2006; Brummelte and Galea, 2010). HPA
dysfunction has been identied in a wide spectrum of postpartum
psychiatric disorders; for example it has been shown that increased
circulating ACTH levels but not CRH or cortisol, are associated with
the presence of postpartum thoughts of harming the infant (Labad
et al., 2011). Measurement of maternal circulating CRH levels at
mid-pregnancy has been proposed as a specic biomarker of
maternal depression (Rich-Edwards et al., 2008) and this was
supported by other studies suggested that the activity of the HPA
axis during pregnancy is associated with maternal HPA respon-
siveness to stress postpartum(Meinlschmidt et al., 2010). However,
recent studies yielded contradictory results with positive or no
association with either prenatal or postpartum depression (Yim
et al., 2009; Meltzer-Brody et al., 2011).
The functional anomalies of the HPA axis in depressive disorders
appear to include an inherited component due to genetic variability
(Spijker & van Rossum, 2009). The genetic contribution to PND and
postpartum psychosis is strongly supported by family and twin
studies and genome-wide linkage analysis in conditions such as
bipolar disorder and history of mood or anxiety disorder (Jones and
Craddock, 2001; Forty et al., 2006; Jones et al., 2007; Kumar et al.,
2007; Payne, 2007; Friedman, 2009; Mahon et al., 2009). While
there has been much interest in the identication of genes
responsible for psychiatric illness, there remains a paucity of in-
formation relating to molecular markers and detection of PND risk.
The objective of our study was to investigate association be-
tween genetic variations (single nucleotide polymorphisms-SNPs)
in the GR and CRHR1 genes, previously shown signicant correla-
tion with depression (Liu et al., 2006; Claes, 2009; Spijker & van
Rossum, 2009), and increased risk for PND as determined by a
raised EPDS score. To address this, a prospective cohort study was
designed involving a secondary care University Hospital setting. To
our knowledge, this represents the rst prospective investigation of
genetic variation of genes involved in HPA activity and PND risk.
2. Materials and methods
2.1. Patients-study design
For the Coventry and Warwickshire Genetic Risk for PostNatal
Depression (CW-GAPND) study, 200 Caucasian women were
recruited during antenatal clinic visits. The study focused on Cau-
casians to ensure ethnic background homogeneity of the cohort
since ethnicity-related differences in PND prevalence have been
reported (OHara and Swain, 1996; Patel et al., 2002). Symptoms
were assessed using the Edinburgh Postnatal Depression Scale
(EPDS) questionnaire, which was completed upon the hospital visit
between 20 and 28 weeks gestation (mean 25.4 1.9) and again 2e
8 weeks post-delivery (mean 4.6 2.1). The choice of this time-
period was based on previous studies suggesting that incidence
of PND peaks at around 4e6 weeks (Cox et al., 1993; Evans et al.,
2001; Verkerk et al., 2003). From the 200 women invited to
participate, questionnaires were obtained from 140 subjects for a
return rate of 70%. There was no signicant difference in the socio-
demographic characteristics (educational qualication, marital
status, employment, support from partner, subsequent pregnancy)
of responders and non-responders. Following current clinical
practice, there was no follow-up of women who returned EPDS
scores below the cut-off of 10.
Recruited patients were assessed for risk factors such as family
history (rst degree) of depression, personal history of depression,
and depressive symptomatology at recruitment using the EPDS,
however, there was no stratication to high or low-risk groups in
accordance with current clinical practice. According to standard
clinical practice all women were assessed for presence of anaemia
(by full blood count and haemoglobin measurement) and thyroid
disease (by TSH measurement) and women with abnormal results
were excluded fromthe study. Women with a history of depression
before pregnancy are especially vulnerable to depressive symptoms
during or after pregnancy (Rich-Edwards et al., 2006). Therefore, in
order to ensure pregnancy-specic and exclude non-pregnancy
related depressive illness, women with pre-existing mental ill-
nesses or on antidepressant medications or any other medications
that can inuence risk of developing PND were excluded from the
study. A cut-off EPDS score of 10 was used for the identication of
high-risk patients for PND. This reported performance character-
istics at this cut-off point is: sensitivity of 0.81 and specicity of
0.86 for detection of both major and minor depression and a
sensitivity of 0.917 and specicity of 0.77 for major depression only
(Hewitt et al., 2009). The study was approved by the local ethics
committee and informed consent was obtained from all subjects.
Similar to previous studies (Fasching et al., 2012; Mehta et al.,
2012), the CW-GAPND study protocol included antenatal use of
the EPDS questionnaire to assess depressive symptoms during
pregnancy between 20 and 28 weeks gestation. Several studies
validated the use of EPDS as a pre-screening tool for depression
during pregnancy especially in the research setting (Rubertsson
et al., 2011; Milgrom et al., 2008) although, it is not currently
used in routine antenatal care in the UK. In this study it was used
exclusively as a research instrument to screen for symptoms of PND
N. Engineer et al. / Journal of Psychiatric Research 47 (2013) 1166e1173 1167
at antenatal clinics and thus identify presence of pregnancy-related
underlying risk.
2.2. Genomic DNA extraction
5 mL of peripheral venous blood was collected fromeach subject
in an EDTA tube. Genomic DNA was extracted using the QIAamp
DNA Mini Kit (Qiagen, UK). The quality of the DNA was determined
by spectrophotometric analysis using the A
260
/A
280
ratio. Typically
values of intact, high-purity DNA were approximately 1.7e1.8. In
contrast, extracted DNA with ratios below 1.6 or above 2.0 indi-
cating possible DNA contamination were excluded.
2.3. SNP selection and genotyping
The glucocorticoid receptor SNPs genotyped were the BclI and
ER22/23EK (dbSNP NCBI identiers rs41423247 and rs6190
respectively) whereas the 3 CRHR1 SNPs were the rs1876828,
rs242939 and rs242941. These were selected from previous reports
that demonstrated an association with depressive disorders (Liu
et al., 2006; van Rossum et al., 2006; Spijker & van Rossum,
2009). Genotyping was performed using allele-specic PCR and
melting curve analysis on the LightCycler (Roche Diagnostics Ltd,
Burgess Hill, UK). Primers and probes were designed and syn-
thesised by TIB MolBiol (Berlin, Germany). The amplication
mixture included 5 mL of genomic DNA as template with LightCycler
FastStart DNA Master HybProbe mastermix, primers and probes.
LightCycler parameters for PCR and melting curve analysis con-
sisted of an initial denaturation step of 95

C for 10 min followed by

an amplication stage of 40 cycles of 10 s at 95

C, 10 s at 53

C and
20 s at 72

C. After amplication, the melting curve analysis was
performed in one cycle of 95

C for 20 s followed by 40

C for 20 s
and then ramping to 85

C. A single cooling cycle of 40

C for 30 s
was then employed. A temperature transition rate of 20

C/s was
used at each step. All laboratory procedures were carried out blind
to EPDS data status. At least 10% of all samples were analysed twice
to conrm accuracy of allele-specic PCR results.
Following determination of each SNP genotype (wild type,
heterozygous or homozygous) haplotype frequency of the htSNPs
rs1876828, rs242939 and rs242941 predicted haplotypes (G-A-G,
G-A-T, G-G-G, G-G-T, A-A-G, A-A-T, A-G-G, A-G-T) was determined
by using the SNPStats web tool (http://bioinfo.iconcologia.net/
SNPstats) (Sol et al., 2006). The software implements the
expectation-maximization (EM) algorithm coded into haplo.stats
package to calculate the estimated relative frequencies for each
haplotype. Haplotype association analyses for SNPs were per-
formed with unconditional logistic regression using the default
setting of a log-additive model and expressed in terms of ORs and
95% CIs.
2.4. Sequencing
Genotype status of the SNPs was conrmed by sequencing,
Sequencing samples and run on the 3130xl Genetic Analyser
(Applied Biosystems, PE, USA). Data were collected and analysed
using the Applied Biosystems Sequence Scanner Version 1.0 soft-
ware. At least 20 random samples for each SNP were sequenced to
validate allele-specic PCR results.
Fig. 1. Characteristics of EPDS scores in the cohort of the study. (a) Mean prenatal or postnatal EPDS scores, across all subjects investigated. (b) Prenatal EPDS scores of patients
classied as high-risk for PND. Only 44% of these patients also exhibited increased EPDS scores prenatally. (c) Postnatal EPDS scores of subjects presented with raised EPDS scores
prenatally. 52% of these patients exhibited raised postnatal EPDS scores.
N. Engineer et al. / Journal of Psychiatric Research 47 (2013) 1166e1173 1168
2.5. Statistical data analysis
Possible correlations between the population characteristics
and EPDS scores were assessed by the Spearman rank
correlation test. To test whether there were differences in subject
characteristics between women classied as high PND risk
(EPDS score 10) and low PND risk (EPDS score <10), the
ManneWhitney U-tests and c
2
tests were used.
A formal sample size calculation was undertaken to power this
study, as it is apparent in the signicance test of the SNPs examined.
For instance, a statistically signicant association of raised EPDS
score and G allele of the BclI SNP was identied despite the small
sample size of the study. Allele (presence/absence of minor allele)
and haplotype frequencies for the cohort were calculated. These
matched those reported in the literature for all SNPs (van Rossum
et al., 2006) and were consistent with HardyeWeinberg equilib-
rium (HWE), indicating that no population selection bias occurred.
For HWE assessment of each SNP, the Pearson chi-square (c
2
) sta-
tistic was used. For continuous and categorical variables, respec-
tively, ManneWhitney U-tests and c
2
tests were used to determine
the statistical signicance of any difference in the distribution of
SNPs examined across categories of high or low EPDS scores in the
sample. In the multivariate analysis, a logistic regression analysis
were performed to calculate the relative risk of EPDS score 10
associated with the selected genetic variants (odds ratios and 95%
CI). Since we multiple adjusted for SNPs, a Bonferroni correction
was employed to correct for multiple adjustment of SNPs. All an-
alyses were carried out using STATA, version 11, software (Stata-
Corp, 4905 Lakeway Drive, College Station, Texas 77845 USA).
3. Results
3.1. EPDS score characteristics of the study cohort
For the 140 patients that completed the study protocol, there
was no difference in the mean EPDS score obtained either
Table 1
Patient demographics and SNP distribution in the CW-GAPND study cohort. HET:
Heterozygous; HOM: homozygous; WT: wild type.
Variable Low PND risk
number (%)
High PND risk
number (%)
p-Value
Total number 106 (75.7) 34 (24.3)
Mean age in years 30.1 (5.0) 28.1 (5.5) 0.047*
BMI >30 29 (27) 13 (38) 0.13
Unemployed 23 (22) 6 (18) 0.59
Social housing 6 (5) 2 (6) 1.00
Smoking 11 (10) 2 (6) 0.43
Single nucleotide polymorphisms p-Value
GR-BclI
HET/HOM 51 (48.6) 25 (73.5) 0.011*
WT 54 (51.4) 9 (26.5)
GR-ER22/23EK
HET/HOM 9 (8.6) 2 (5.9) 0.61
WT 96 (91.4) 32 (94.1)
CRHR1
(1)
-rs1876828
HET/HOM 34 (32.4) 14 (41.2) 0.359
WT 71 (67.6) 20 (58.8)
CRHR1
(2)
-rs242939
HET/HOM 13 (12.4) 14 (41.2) <0.001*
WT 92 (87.6) 20 (58.8)
CRHR1
(3)
-rs242941
HET/HOM 62 (59.6) 26 (76.5) 0.076
WT 42 (40.4) 8 (23.5)
* Statistically signicant.
Fig. 2. Relationship of prenatal or postnatal EPDS scores and individual SNPs. (): Subjects positive for the minor allele; (): Subjects negative for the minor allele. Data represent
the mean SEM EPDS score for each SNP and analysed for statistical signicance by analysis of variance.
N. Engineer et al. / Journal of Psychiatric Research 47 (2013) 1166e1173 1169
prenatally or postnatally (4.69 SEM 0.28; 5.19 SEM 0.41,
p > 0.05). The majority of the recruited patients (n 111, 80%) had
an EPDS score below 10 prenatally (Fig. 1a). Postnatal assessment
revealed an EPDS score of 10 or more in thirty-four patients (24%),
indicative of high-risk for postpartum depressive illness (high-risk
for PND patients); 44% of these patients also exhibited increased
EPDS scores prenatally (Fig. 1b). In patients with prenatal EPDS
score 10 (n 29) only 52% progressed to raised EPDS score
postnatally (Fig. 1c). Similarly, investigation of relevant dichoto-
mous variables such as smoking, medical history, educational level,
employment status and housing and continuous variables such as
BMI, duration of gestation failed to detect any signicant difference
between the two groups (EPDS 10 vs <10) (Table 1). Only age
during booking showed a small but statistically signicant differ-
ence between groups.
3.2. Association between SNP and EPDS scores
Data were also analysed for potential association between in-
dividual SNPs genotyped and EPDS scores. Results showed that the
presence of the minor allele of the polymorphisms BclI, ER22/23EK
(R23K) and CRHR1
(1)
(rs1876828), had no effect on the mean pre-
natal (or postnatal) EPDS score of all subjects included in the study
(Fig. 2a,b). However, a signicant association was found between
the CRHR1
(2)
(rs242939) SNP and EPDS score; pregnant women
with the Gallele exhibited an increased mean EPDS score compared
to the A allele, either prenatally (9.29 vs 5.69, p < 0.0001) or
postnatally (9.03 vs 5.40, p < 0.0001) (Fig. 2b) suggesting that the
association is not specic for postpartum-onset depression. Inter-
estingly, a weak but statistically signicant association (p 0.03)
was identied between the T allele of the CRHR1
(3)
(rs242941) SNP
and the postnatal EPDS score only (Fig. 2b), suggesting a positive
effect of this SNP on EPDS score only during the postnatal period.
We next focused on the potential association between single
SNP and increased EPDS score (high-risk PND patients). We
observed signicant allele association with the BclI and CRHR1
(2)
SNPs (p 0.011 and 0.003, respectively), which were over-
represented in high-risk PND patients. No association was found
between PND risk and the allele frequencies of the ER22/23EK,
CRHR1
(1)
and CRHR1
(3)
SNPs (Table 1). Women with the BclI (C/G)
and CRHR1
(2)
(A/G) minor alleles exhibited signicantly increased
likehood of EPDS score 10 (OR: 2.9, 95% CI 1.2 to 6.9 and 4.9, 2 to
12, respectively) (Fig. 3). Furthermore, the risk ratio for high EPDS
score associated with simultaneous presence of BclI and CRHR1
(2)
was increased to 5.48 (2.13e14.10) suggesting independent
contribution of the two SNPs. The risk of high EPDS score remained
statistically signicant and was not altered when either of BclI or
CRHR1
(2)
were present along with ER22/23EK, CRHR1
(1)
and
CRHR1
(3)
SNPs suggesting absence of any synergistic or antago-
nistic interactions.
3.3. Association of haplotype-tag SNPs and EPDS score
Moreover, since the CRHR1 SNPs (rs1876828, rs242939 and
rs242941) are haplotype-tag SNPs (htSNPs) we investigated the
association of the various haplotypes for the SNPs rs1876828,
rs242939 and rs242941 with high EPDS score. Estimation of
haplotype distribution showed that only the G-A-G, G-A-T, A-A-G
and G-G-T haplotypes were present in >10% of the cohort (40, 29,
21 and 11% respectively) (Fig. 4a). The common (major allele)
haplotype G-A-G showed a population frequency of 0.4 (0.43 for
the low-risk and 0.29 for the high-risk) and the variant (minor
allele) haplotype G-G-T had a population frequency of 0.11 (0.066
for the low-risk and 0.21 for the high-risk). Using the chi-square
test, a global test of haplotype distribution of these four
haplotypes showed a signicant difference between patients with
high and low EPDS scores (c
2
29.091, d.f. 3, p < 0.001).
Compared to non-G-A-G and non-G-G-T groups respectively, pa-
tients with the G-A-G haplotype had signicantly lower and with
the G-G-T haplotype increased likehood of EPDS 10 (p 0.01 and
<0.0001, respectively) with odds ratio (OR) & 95% CI of 0.72 (0.53e
0.97) and 3.22 (1.91e5.42) respectively (Fig. 4b). Almost 50%
(48.6%) of the G-G-T haplotypes were found in subjects with EPDS
10 (high-risk PND group), whereas only 18% of the G-A-G
haplotype were in the high-risk group (Fig. 4c).
4. Discussion
The CW-GAPND study protocol was designed to test at the
clinical setting the potential use of SNPs in prospectively identi-
fying women with high (10) EPDS score and thus increased risk
for PND. We focused on GR and CRHR1 gene SNPs that might
impact on responses to psychobiological stressors during preg-
nancy or the postpartum period. A positive association with high
EPDS score was identied for the BclI but not the ER22/23EK
polymorphism. Moreover, investigation of the haplotype tagged
SNPs rs1876828, rs242939 and rs242941 of the CRHR1 gene, sug-
gested that patients carrying the minor allele of the rs242939 SNP
present with signicantly higher EPDS either pre- or postnatally.
Most importantly, the haplotype G-G-T and independently the (G)
minor allele of the rs242939 showed a strong associationwith EPDS
Fig. 3. Forrest plot of calculated risk ratios for high EPDS score 10 associated with
increased risk for PND associated with presence of the minor allele independently or in
combination. ES: effect size.
N. Engineer et al. / Journal of Psychiatric Research 47 (2013) 1166e1173 1170
score 10 and high-risk for PND, whereas the G-A-G haplotype
appeared to exert a protective effect on EPDS score.
There are limited studies investigated the genetic impact on risk
for PND across all pregnant women. Genetic polymorphisms of the
serotonergic transmission and monoamine-related genes have
been previously associated with the development of postnatal
depression (Doornbos et al., 2009; Costas et al., 2010; Fasching
et al., 2012; Mehta et al., 2012). Thus, the focus on molecules
regulating stress responses and prospective cohort design of the
study are the two main strengths. The prospective recruitment of
pregnant women who were not clinically depressed and longitu-
dinal assessment for PND risk in the early postpartum period,
preserved the temporality of the observed associations, and also
minimise the potential confounding effects of unmeasured factors
(such as residual confounding), and also establish specicity of
SNPs with PND. We acknowledge that the cohort size of the study
does not provide adequate power for all SNPs associations and this
is a weakness of the study; hence further work using a larger cohort
would be necessary. The CW-GAPND study was based on a con-
venience sample of xed number of participants, and there was no
attempt for formal assessment of the power calculation based on
the expected effect size of the primary outcome of interest. The
power of the study for the three associations with these patient
numbers was calculated at 44%, 86% and 21% for the BclI, rs242939
and rs242941 polymorphisms respectively. A minimum of 80% is
considered as an adequate study power and this was achieved for
one SNP only. To achieve this power for all SNPs a cohort of mini-
mum 594 patients would be required, although other factors that
inuence association studies such as differences in allele fre-
quencies, age and ethnicity should also be taken into account. Use
of a larger cohort will be able to determine the potential impact of
other important parameters such as of allele dosage effect (i.e.
homozygous vs heterozygous). Another potential issue of the study
is the identication strategy used for determining PND risk, and the
lack of an instrument capable of providing denite diagnosis. The
EPDS questionnaire is an established tool, most frequently used in
clinical practice, and the chosen cut-off score of 10 is widely used
and offers reasonable performance characteristics with both
sensitivity and specicity above 80% for detection of both major and
minor depression (Cox et al., 1993; Schaper et al., 1994; Hewitt et al.,
2009). However, different cut-off points could lead to variations in
measures of accuracy of the test (i.e. sensitivity and specicity) and
thus difculties may arise in results interpretation and impact
assessment. This might explain the higher prevalence of high-PND
risk of 24% observed in the cohort population, which may also
reect the higher rate of adverse socio-economic factors in our
population including poor social support, teenage pregnancies,
immigrant population and unemployment. Certainly, further
renement would be required by investigating performance of the
SNP association analysis with different EPDS scores or different
identication strategies.
We focused our approach on genetic variants of the HPA axis
previously associated with depression. The HPA axis serves as a key
interface between environmental stressors and the development of
depression and functional differences due to genetic variation may
alter individual responses to stressful events and may regulate
vulnerability to stress-related psychiatric disorders. The presence
of depressive symptoms during pregnancy appears to be associated
with altered regulation of GR sensitivity (Katz et al., 2012). GR
sensitivity appears diminished with progression of pregnancy and
increasing maternal depressive symptoms; peripheral expression
of GR co-chaperone genes may serve as a biomarker for risk of
Fig. 4. Relationship between specic CRHR1 haplotype and risk for PND. (a) Distribution of different CRHR1 haplotypes among the CW-GAPND cohort. Rare haplotypes with
frequency <1% are excluded. (b) Forrest plot of risk ratios for increased risk for PND associated with presence of different CRHR1 haplotypes. (c) Distribution of different CRHR1
haplotype among groups classied as high or low ris for development of PND based on an EPDS score 10.
N. Engineer et al. / Journal of Psychiatric Research 47 (2013) 1166e1173 1171
developing depressive symptoms during pregnancy. The GR gene
SNPs investigated such as the BclI and ER22/23EK are linked with
clinical phenotypes, possibly due to altered function of GR and
therefore may increase the risk of depressive disorders by altering
cortisol responses to psychosocial stress (Spijker & van Rossum,
2009). In particular the ER22/23EK SNP has been reported to
relate to decreased cortisol suppression to dexamethasone
administration (van Rossum and Lamberts, 2004), whereas the BclI
polymorphism seems to be associated with attenuated cortisol
responses. Interestingly, our study identied a signicant associa-
tion with high PND risk of only the G minor allele of the BclI SNP,
found despite no effect on the prenatal or postnatal EPDS score, a
nding that is consistent with results from non-pregnant
depressed population (van Rossum et al., 2006). Similar ndings
have also reported in a group of pre-menopausal women and
suggested that homozygous carriers of both BclI and ER22/23EK
SNPs have increased risk of developing a major depressive episode
(van Rossum and Lamberts, 2004; Krishnamurthy et al., 2008). The
lack of association between the ER22/23EK SNP and high EPDS
score in our study suggests that patients at risk for PND might
represent a subgroup with distinct geneeenvironment interactions
and unique genetic characteristics that exhibit increased vulnera-
bility to pregnancy-associated triggers. The high-risk PND group of
the CW-GAPND cohort also exhibited a strong association with
rs242939, a CRHR1 SNP previously been associated with major
depression in a group of Han Chinese patients (Liu et al., 2006). In
fact this SNP was associated with raised EPDS values both before
and after delivery and were thus not specic for postpartum-onset
depression. It is increasingly recognised that multiple genetic var-
iations with discrete functional effects in a number of genes regu-
lating the HPA axis may alter susceptibility to stress-related
psychiatric disorders and a system genetics approach is required to
fully elucidate the impact of gene environment interactions.
There is already evidence for the association of genetic variants in
genes intricately tied to the CRH system with depression and
anxiety, such as the GR gene or the serotonin transporter (Ressler
et al., 2010). In agreement with this, we noted a combined effect
of the simultaneous presence of the BclI and rs242939 SNPs that
appeared to further increase the odds of raised EPDS score and high
PND risk, raising the possibility of gene gene interactions be-
tween CRHR1 and GR loci.
The CW-GAPND study provides the rst preliminary evidence of
genetic association between gene variants of the stress endocrine
axis and risk for depression during pregnancy and post-partum. It
also offers additional benets in understanding the biological basis
of disease. Polymorphisms of genes regulating stress responses
appear to be promising targets for further investigations. Certainly,
a larger prospective study of a replication sample that will include a
structured diagnostic interview is required to fully explore the
magnitude of this genetic effect and potential usefulness in
detection and management of postnatal depression.
Funding body agreements and policies
No specic requirements as specied as conditions of the grant
awards supported this research.
Contributions
N.E contributed to the design of study, recruitment of patients
and collection of clinical details, data analysis and manuscript
preparation; L.D contributed to the molecular methods develop-
ment, performed the experiments and data analysis and managed
the literature searches; D.N contributed to the recruitment of
patients, collection of clinical details and data analysis; K.N-B
undertook data statistical analysis and manuscript preparation;
S.C.S contributed to the design of study, data analysis and manu-
script preparation; D.K.G. contributed to the design of the study,
guided data interpretation and manuscript writing. All authors
have approved the submitted version.
Author disclosure
The authors have no conict of interest to disclose.
Acknowledgements
The principal investigator conrms that he had full access to all
of the data in the study and takes responsibility for the integrity of
the data and the accuracy of the data analysis. Research supported
by the Robert Gaddie Memorial Fund and the Birmingham-War-
wick Science City Research Alliance.
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