Accepted Manuscript

DNA methylation differences at the glucocorticoid receptor gene indepression are related to functional alterations inhypothalamic-pituitary-adrenal axis activity and to early life emotional
abuse
Chloё Farrell , Kelly Doolin , Niamh O’ Leary , Chaitra Jairaj ,Darren Roddy , Leonardo Tozzi , Derek Morris , Andrew Harkin ,Thomas Frodl , Zsófia Nemoda , Moshe Szyf , Linda Booij ,Veronica O’Keane
PII:
DOI:
Reference:

S0165-1781(17)32062-0
10.1016/j.psychres.2018.04.064
PSY 11400

To appear in:

Psychiatry ResearchReceived date:
Revised date:
Accepted date:

8 November 201722 April 201824 April 2018

Please cite this article as: Chloё Farrell , Kelly Doolin , Niamh O’ Leary , Chaitra Jairaj ,Darren Roddy , Leonardo Tozzi , Derek Morris , Andrew Harkin , Thomas Frodl , Zsófia Nemoda ,Moshe Szyf , Linda Booij , Veronica O’Keane , DNA methylation differences at the glucocorticoid receptor gene in depression are related to functional alterations in hypothalamic-pituitaryadrenal axis activity and to early life emotional abuse, Psychiatry Research (2018), doi:
10.1016/j.psychres.2018.04.064

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Highlights
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Differences in DNA methylation are observed at the NR3C1 exon 1F in depression.
The degree of DNA methylation at the NR3C1 exon 1F is correlated with increases inmorning cortisol concentrations and childhood emotional abuse severity.
No differences in DNA methylation are observed at the FKBP5 sites measured indepression.

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DNA methylation differences at the glucocorticoid receptor gene in depression are related to functional
alterations in hypothalamic-pituitary-adrenal axis activity and to early life emotional abuse
Authors: Chloё Farrell1, Kelly Doolin1, Niamh O’ Leary1, Chaitra Jairaj1, Darren Roddy1, Leonardo Tozzi2,
Derek Morris3, Andrew Harkin4, Thomas Frodl2, Zsófia Nemoda5, Moshe Szyf6, Linda Booij7,8 and Veronica
O’Keane1,9.
1Dublin, Dublin 2, Ireland
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Department of Psychiatry, School of Medicine, Trinity College Institute of Neuroscience, Trinity College

Department of Psychiatry and Psychotherapy, Otto von Guericke University Magdeburg, Magdeburg,

Germany
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Discipline of Biochemistry, NUI Galway, Galway, Ireland

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School of Pharmacy and Pharmaceutical Studies, Trinity College Institute of Neuroscience, Trinity College

Dublin, Dublin 2, Ireland
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Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University,

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Budapest, Hungary
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Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

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Department of Psychology, Concordia University, Montreal, Quebec, Canada

8Sainte-Justine Hospital Research Centre, Montreal, Quebec, Canada

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Trinity Centre for Health Sciences, AMNCH (Tallaght Hospital), Tallaght, Dublin 24, Ireland

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Corresponding author: Chloё Farrell, Ph.D
Trinity College Institute of NeuroscienceAC

The Lloyd Institute, Room 336
Trinity College Dublin, Dublin 2, Ireland
Phone: +353 1 896 4234
Email: farrelc6@tcd.ie

Key Words:

Epigenetics; depression; early life adversity; hypothalamic-pituitary-adrenalaxis; glucocorticoids; stress.

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ABSTRACT
Depression is associated with alterations in hypothalamic-pituitary-adrenal (HPA) axis activity. A
proposed mechanism to explain these alterations are changes in DNA methylation levels, secondary toearly life adversity (ELA), at stress-related genes. Two gene regions that have been implicated in theliterature, the glucocorticoid receptor gene (NR3C1) exon 1F and the FKBP5 gene intron 7 were examined

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in 67 individuals (33 depressed patients and 34 controls). We investigated whether cortisol
concentrations, evaluated in 25 depressed patients and 20 controls, and measures of ELA wereassociated with the degree of methylation at these candidate gene regions. Mean NR3C1 exon 1F DNA
methylation levels were significantly increased in the depressed cohort and the degree of methylation

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was found to be positively associated with morning cortisol concentrations. DNA methylation levels at
specific CG sites within the NR3C1 exon 1F were related to childhood emotional abuse severity. DNA
methylation at CG38 was related to both HPA axis and childhood emotional abuse measures in the
depressed group. No FKBP5 differences were revealed. Our findings suggest that hypermethylation at theM

NR3C1 exon 1F may occur in depression. This locus-specific epigenetic change is associated with higher

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basal HPA axis activity, possibly reflecting acquired glucocorticoid receptor resistance.

INTRODUCTION

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Major depressive disorder (MDD) is a debilitating mood disorder with a lifetime prevalence rate of
approximately 17% (Kessler et al., 2005). Despite extensive research, there are no established biologicalmarkers for the diagnosis of depression (Mossner et al., 2007). Longitudinal studies indicate that many
adulthood-onset diseases are associated with adverse developmental or biological events experienced
early in life. The latency between exposure to adversity and development of a disorder can span years,

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and even decades in some instances (Shonkoff et al., 2009). Exposure to early life adversity (ELA) has
been identified as a major risk factor in the development of almost all psychiatric disorders (Kessler et al.,2010), including MDD (Kessler and Magee, 1993). It has been reported that adult patients with MDD havea two-fold higher rate of ELA compared to healthy controls and that ELA has been shown to predictANUSearlier-onset depression, a higher risk of recurrent depression, more severe course of illness, greater
chronicity and suicide (Williams et al., 2016). MDD patients with a history of ELA show different
treatment responses reinforcing the idea of diverse pathways to the loose clinical syndrome of
depression, one of which is ELA (Nemeroff et al., 2003).

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The hypothalamic-pituitary-adrenal (HPA) axis is the main stress response system (Bellavance and Rivest,2014). Through glucocorticoid receptors (GR) cortisol mediates a myriad of tissue-specific effects (YoungED

et al., 2003). Importantly, cortisol signaling via GR is responsible for negative feedback inhibition of the
HPA axis. A frequently replicated finding in depression research is the association of depression withPT

hyperactivity of the HPA axis (Pariante and Lightman, 2008). It has been hypothesized that the increased

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HPA axis activity results from dysfunctional feedback because of reduced efficacy of central GR function(Young et al., 2003). The cortisol awakening response (CAR), the brisk increase of cortisol levels within 20-AC

30 minutes of awakening in the morning (Fries et al., 2009), provides a naturalistic test for a cortisol
stress response. Several studies have demonstrated an increased CAR in currently depressed individuals(Suzuki et al., 2013; Vreeburg et al., 2009), as well as in recovered patients with depression (Bhagwagar
et al., 2003).
The GR co-chaperone protein FK506 binding protein 5 (FKBP5) is an immunophillin protein that
accompanies the inactive GR complex in the cytoplasm. FKBP5 reduces the affinity of the GR complex for
cortisol and thus decreases overall GR signaling (Rao et al., 2016). It is hypothesized that genetic variants

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within the FKBP5 gene may influence individuals’ susceptibility to depression, with certain variants
associated with increased FKBP5 protein levels (Szczepankiewicz et al., 2014), and consequently withrelative decrease in GR complex activity. A recent meta-analysis supports the association between
specific FKBP5 genetic variants and an increased risk of depression (Rao et al., 2016).
Epigenetic mechanisms are essential for the regulation of gene expression as they can modulate events

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at the level of transcription and translation (Portela and Esteller, 2010). Epigenetic mechanisms may
provide an etiological link between ELA and the syndrome of depression (Dalton et al., 2014). The
addition of a methyl group to the cytosine within a CG site in a critical regulatory region can inhibittranscriptional activity (Farrell and O’Keane, 2016).

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In relation to MDD and alterations in HPA axis activity, an interesting epigenetic modification is
methylation of a nerve growth factor-inducible protein A (NGFI-A) binding site located in exon 1F of the
GR gene, the nuclear receptor subfamily 3 group C member 1 (NR3C1) (Swirnoff and Milbrandt, 1995),
that is associated with ELA. Alterations at or near this NGFI-A binding site may explain one of theMbiological links between ELA and the development of depressive illnesses (Smart et al., 2015). NR3C1
exon 1F hypermethylation in relation to ELA has been demonstrated in peripheral tissues, including cordEDblood, white blood cells and whole blood (Oberlander et al., 2008; Perroud et al., 2011; Radtke et al.,2011; Radtke et al., 2015) and would potentially result in dysregulation of HPA axis negative feedbackPT

were it occurring in the brain. It is possible that this hypermethylation is indeed occurring centrally asCEincreased DNA methylation has been reported in the hippocampus of the orthologue 17 site in stressed
rats (Weaver et al., 2004) and in the hippocampus of abuse victims who died by suicide (McGowan et al.,AC2009).

FKBP5 has recently attracted attention as a protein possibly implicated in the area of epigeneticmediated gene-childhood trauma interaction leading to adult mental illness. Klengel et al. performed thefirst human study examining gene-ELA interaction and methylation at FKBP5. They showed that
individuals suffering from post-traumatic stress disorder (PTSD) who were exposed to ELA and who
carried a functional FKBP5 risk T allele at rs1360780 had decreased levels of methylation at intron 7 of
the FKBP5 gene. This intronic demethylation was associated with an increase in FKBP5 induction by GR

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activation, leading to symptoms of GR resistance (Klengel et al., 2013). One study examining FKBP5
methylation in individuals with the risk T allele and remitted depression revealed a (non-significant) trend
towards higher methylation levels compared to healthy controls (Höhne et al., 2015). There are a few
more studies that examined FKBP5 methylation association to ELA but the direction of effect wasinconsistent (Needham et al., 2015; Tyrka et al., 2015).

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No studies, to our knowledge, have examined NR3C1 and FKBP5 methylation in the same individuals, andthe possible associations between their DNA methylation levels and HPA axis function. The aims of thisstudy were to determine if DNA methylation at the NR3C1 exon 1F and/or FKBP5 intron 7 were altered inindividuals with depression compared to controls. We also explore possible relationships between NR3C1

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and FKBP5 DNA methylation and HPA axis function as assessed through the CAR.

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METHODS AND MATERIALS
Patient Recruitment and Assessment
Depressed patients were recruited from the Tallaght Mental Health Services. Any patient presenting withdepression was considered for the study. They were screened for eligibility by a consultant psychiatrist.

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Screening involved assessing whether patients fulfilled criteria for a Major Depressive Episode (MDE)from the Mini International Neuropsychiatric Interview (M.I.N.I) (Sheehan et al., 1997); scored >17 on the
Hamilton Depression Rating 21-Item Scale (HAM-D-21) (Hamilton, 1960), indicative of moderate
depression; were not suffering from a psychotic or substance abuse disorder or from any chronic medicalANUSillnesses and were not taking any steroid medications, excluding the contraceptive pill. Healthy, control
individuals were screened and recruited from the general population. Screening involved establishingthat individuals had never experienced a mental health disorder, scored ≤7 on the HAM-D-21, were notsuffering from any chronic medical illnesses, and were not taking any steroid medications, except the oralM

contraceptive pill. For both groups, 45 years, considered the beginning of middle age (Stevenson, 2010),
was selected as the maximum age for inclusion to reduce the possibility of age-related effects on

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biological data. The groups were balanced for age and sex. If individuals met inclusion and exclusion
criteria, they were invited to participate. If, following a thorough explanation of the study, and theyPTremained willing to participate, they gave written informed consent. As this was not an intervention

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study, all treatment was continued as usual. Detailed demographic information was collected. The
International Standard Classification of Education (ISCED) is a standardized scale used to gatherAC

information on educational achievement (UNESCO, 2011) and was used to grade participants’ level of
education. Participants were then administered a battery of clinical instruments: Centre for
Epidemiological Studies Depression Rating Scale (CES-D) (Orme et al., 1986), suicidality module (ModuleC) of the M.I.N.I (Sheehan et al., 1997) and the Childhood Trauma Questionnaire (CTQ) (Bernstein, et al.,1997). The CTQ is a retrospective, self-report measure of ELA. The CTQ contains five subscales; threeassessing abuse (emotional, physical and sexual) and two assessing neglect (emotional and physical).
Other traumatic events (deaths, bullying etc.) that may have occurred during childhood were not

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assessed. The study was approved by the Tallaght Hospital/St. James’s Hospital Joint Research Ethics
Committee (2013/23/02) following the principles in the 1964 Declaration of Helsinki(World Medical
Association, 2013).

DNA methylation analysis of HPA axis genes

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DNA methylation of the NR3C1 exon 1F and FKBP5 intron 7 was measured in whole blood bypyrosequencing, performed on bisulfite-treated DNA. A schematic representation of both regions
investigated can be found at Figure S1. Blood was collected into PAXgene® Blood DNA tubes and DNA
was extracted using the FlexiGene® DNA Kit (Qiagen, Valencia, California) using the manufacturer’sANUS

recommended protocol. 1 μg DNA was bisulfite-treated using the EZ DNA Methylation Gold™ Kit (Zymo
Research, Irvine, California) using the manufacturer’s recommended protocol. A 319bp fragment
corresponding to our region of interest within the NR3C1 exon 1F was amplified by touchdown PCR using
the following primers: NR3C1F-FW-biotin: 5Biosg/GTTTTTAATTTTTTAGGAAAAAGGGT and NR3C1F-REV:M

ACCCCTTTCCAAATAACACACTTC. A 341bp fragment corresponding to our region of interest within the
FKBP5 intron 7 was amplified by standard PCR using the following primers: FKBP5-FW:
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TGGGATAATAATTTGGAGTTATAGTGTAGGFKBP5-REV-biotin:

5Biosg/AAATTTATCTCTTACCTCCAACACTAC. For both PCR reactions, a master mix solution was made up

PTusing EpiMark® Hot Start Taq DNA Polymerase (NEB® Inc., Ontario, Canada). A no template control wasCE

included to monitor contamination of extraneous nucleic acids. The pyrosequencing reaction was
performed using the PyroMark Q96 platform (Qiagen, Valencia, California). Sequencing primers usedAC

were as follows: NR3C1F_LR_S3: AACTCCCCAATAAATCTAAAAC (NR3C1 exon 1F CG36-CG39),
NR3C1F_LR_S4:

AAAAAACTCCATAACCCTCT(NR3C1

exon

1F

CG41)

and

FKBP5_S4:

GTTGATATATAGGAATAAAATAAGA (FKBP5 intron 7 CG6-CG7). Percentage methylation levels and quality
control were analyzed using PyroMark CpG Software 1.0.1.1 (Qiagen, Valencia, California).

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FKBP5 rs1360780 genotyping
The rs1360780 allele was genotyped from whole blood DNA using a Taqman SNP Genotyping Assay
performed on a 7900HT Sequence Detection System (Applied Biosystems, Foster City, California) as

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previously described (Tozzi et al., 2016).

Salivary cortisol analysis

Saliva samples were collected within one week of recruitment into the study. Participants were
instructed not to eat, drink or brush their teeth 30 minutes prior to saliva collection. Saliva samples wereAN
UScollected in Salivette® tubes (Sarstedt, Nümbrecht, Germany) at five time points throughout the day.
These time points were 0, 30 and 60 minutes after waking (to assess CAR), and 12 and 12.5 hours after
waking (to assess diurnal variation). Cortisol concentrations were measured by liquid chromatography-Mtandem mass spectrometry (LC-MS/MS) as previously described (Jones et al., 2012).

Statistical Analysis

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Data are presented as mean with standard deviation (SD) or standard error of the mean (SEM) whereappropriate. All data were tested for normality using the Shapiro-Wilk test. DNA methylation and cortisol

PTvalues were log transformed to normalize distribution. DNA methylation and cortisol data were analyzed

CEusing analyses of variance with covariates where appropriate. In our preliminary analysis it was observedthat smoking and education were driving differences in DNA methylation between the two groups, andACwere therefore included as covariates in the final analysis. Similarly, in the preliminary cortisol analysis, it
was observed that BMI and education were driving differences between the two groups, and therefore
were included as covariates in the final analysis. Non-parametric data were analyzed using the MannWhitney U test. Associations between categorical variables were tested using the Chi-Square (χ2) test.
Correlational analysis was carried out using Spearman’s rho statistics. Given the exploratory nature of thecorrelational analyses, Bonferroni corrections were not applied due to the heightened risk of Type IIerrors associated with such corrections. Statistical analyses were considered significant when p≤0.05.

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Graphs were generated using GraphPad Prism Software Version 6.00 (La Jolla, California). All data were

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analyzed using SPSS Version 22 (IBM, Armonk, New York).

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RESULTS
Demographic and clinical data of patients with depression and healthy controls
There were no significant differences in age, gender or BMI between the groups. The depressed group
had a higher prevalence of smokers (p=0.02) and lower duration of education (p<0.001), compared tothat of the control group. Consistent with a diagnosis of depression, patients with MDD had significantly

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higher HAM-D-21 and CES-D scores compared to healthy controls. A score of 19-22 in the HAM-D-21 isconsidered to represent severe depression. The mean HAM-D score in the patient group was 21.94(SD=5.49) indicating that overall this group was severely depressed. Furthermore, a score of 9-16 in thesuicidality module of the M.I.N.I indicated that there was a moderate suicide risk. The mean score in theANUSpatient group was 10.06 (SD=12.16), thus indicating a moderate risk for a suicide. Any participant who
met criteria for “moderate” severity on any of the CTQ subscales was considered to have experienced
ELA. The depressed group had a higher prevalence of individuals who had ELA, and had significantly
higher CTQ global, emotional abuse, physical abuse, emotional neglect and physical neglect compared toM

the control group. Within the depressed sample, 20 individuals were receiving pharmacologicaltreatment for depression at the time of recruitment. Of these, 14 were taking selective serotoninED

reuptake inhibitors (SSRIs), two were taking serotonin-noradrenaline reuptake inhibitors (SNRIs) and fourPT

were receiving other antidepressant treatments [Table 1].

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DNA methylation levels of NR3C1 exon 1F in depressed patients and healthy controls
Mean DNA methylation at NR3C1 exon 1F was significantly higher in the depressed patients relative toAChealthy controls (F=3.89, d=0.49, p=0.05). There were no significant differences in DNA methylation at
any of the individual CG sites measured [Table 2].

DNA methylation levels of FKBP5 intron 7 promoter in depressed patients and healthy controls
There were no statistically significant differences in overall, or individual CG site, DNA methylation at
FKBP5 intron 7 in depressed patients relative to healthy controls [Table 2]. At the analysis of rs1360780
the homozygous TT samples were rare in our group, therefore they were grouped with CT samples. In the11

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depressed group, 16 possessed the CC genotype and 17 possessed the CT/TT genotype. In the control
group, 21 possessed the CC genotype and 13 possessed the CT/TT genotype. There was no significant
difference in the prevalence of the risk allele between the groups. There were no significant differencesin methylation when individuals were divided based on their FKBP5 rs1360780 allele status [Table 3].

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Salivary cortisol levels in depressed patients and healthy controls
Out of the 67 participants recruited into the study with DNA methylation data available, 45 (25 depressed
patients and 20 healthy controls) had corresponding cortisol data [Table S1]. Twenty-four participants
provided insufficient sample quantity or quality, and were excluded from the analysis. Analysis revealed aANUSsignificant increase in wakening (T0) cortisol concentrations and overall morning cortisol output indepressed patients compared to healthy controls. There were no differences in evening cortisol values
between the groups [Table 4].

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Associations between cortisol concentrations and NR3C1 DNA methylation

There was a significant relationship between methylation at the NR3C1 exon 1F CG38 site and cortisolEDlevels at T0, and with average morning cortisol output [Table 5].

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Associations between ELA and NR3C1 DNA methylation

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Spearman’s rho correlation analysis revealed a significant positive association between the severity ofemotional abuse experienced during childhood and the degree of DNA methylation at NR3C1 exon 1F

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CG37 (r=0.53, p=0.01) and CG38 (r=0.43, p=0.04) [Figure 2]. This finding was only present in depressedindividuals who reported ELA and not in the overall sample. No associations were revealed between
NR3C1 DNA methylation and physical/sexual abuse or emotional/physical neglect. No associations wererevealed between FKBP5 DNA methylation and any adversity parameter examined.

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DISCUSSION
This study sought to determine whether there were differences in DNA methylation levels at the NR3C1
exon 1F and/or the FKBP5 intron 7 were present in a sample of depressed individuals, and if so, whether
these differences were associated with changes in HPA axis measures, as measured by the CAR/diurnal
cortisol-HPA axis test. The overall percentage of methylation at the NR3C1 exon 1F region was

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significantly increased in the depressed patients relative to healthy controls. The degree of thismethylation was related to increased cortisol concentrations at T0, as well as overall morning cortisol
secretion in the entire study cohort. Within the NR3C1 exon 1F, DNA methylation levels at CG38 wererelated to the same two cortisol measures. CG38 DNA methylation was also related to childhood

ANUSemotional abuse severity in the depressed group. No significant differences between depressed andcontrol groups, in the total group and when stratifying for a risk polymorphism, were seen in FKBP5 DNA
methylation measures.

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NR3C1 DNA methylation and the HPA axis

Although NR3C1 DNA methylation levels were low in general, the overall percentage methylation at theED

NR3C1 exon 1F region was significantly increased in the depressed patients relative to healthy controls.
This finding is in keeping with the literature generally (Palma-Gudiel et al., 2015), with the same exon 1F

PTregion being previously implicated in various stress-related psychopathologies including depression,CEbipolar disorder and PTSD (Argentieri et al., 2017). The mechanism(s) underlying how ELA produces
targeted methylation of specific gene regions is currently unknown. We know that the epigenome isAC

responsive to external influences and it has been hypothesized that this plasticity enables optimal
adaptation to changing environmental conditions (Ryan et al., 2016). It is possible that this plasticity gives
trauma the opportunity to impact the epigenome in a manner that explains the long-term effects oftrauma on the risk of developing psychiatric disorders. Future studies should attempt to elucidate themolecular mechanisms underlying trauma-induced DNA methylation changes at specific gene regions.
The depressed group had increased cortisol concentrations upon wakening, as well as an overall increasein morning cortisol output relative to the healthy controls. Although not all previous studies have been

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consistent (Gunnar and Vazquez, 2001), elevated wakening cortisol levels have often been found in asignificant number of previous studies examining the CAR tests in depression (Bhagwagar et al., 2005;
Suzuki et al., 2013;,Vreeburg et al., 2009). While there were no significant methylation differences
between the depressed and control groups at individual NR3C1 exon 1F CG sites, significant relationships
were revealed between individual CG sites and CAR parameters, particularly at the CG38 site.

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Interestingly, the relationship between methylation at the CG38 site and the CAR measures existed forthe two CAR values that distinguished the depressed from the control group (i.e. T0 and morning
average). It is worth highlighting that CG37 and CCG38 are at the location of the NGFI-A binding site
(Palma-Gudiel et al., 2015) that has been previously associated with depression pathophysiology(Smart etof the NR3C1 exon 1F and HPA axis activity.

NR3C1 methylation and ELA

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USal., 2015). This indicates a possible functional relationship between DNA methylation alterations at CG38M

As previously mentioned, ELA has been identified as a major risk factor in the development of almost all
psychiatric disorders (Kessler et al., 2010), including MDD (Kessler and Magee, 1993). To date, a numberEDof studies have shown that increases in DNA methylation at the glucocorticoid receptor gene have been
associated with exposure to childhood abuse (McGowan et al., 2009; Perroud et al., 2011; Radtke et al.,PT2015). Overall, these studies have revealed positive associations with the degree of methylation at theCEexon 1F region and the severity of abuse exposure during childhood. In our study, we observed asignificant positive association between the severity of emotional abuse experienced during childhoodACand the degree of DNA methylation at NR3C1 exon 1F CG37 and CG38 sites. This finding is particularly
noteworthy as a diagnosis of depression is more likely to be related to reports of childhood emotional
abuse than to physical or sexual abuse (Gibb et al., 2007). This finding was only present in depressed
patients who reported ELA and was not present in the overall cohort, suggesting an association between
ELA severity, rather than depression per se, and methylation status of the CG37 and CG38 sites NR3C1
exon 1F. As previously mentioned, the CG37 and CG38 sites are at the NGFI-A binding site, which has
been implicated in childhood trauma-associated DNA methylation changes(McGowan et al., 2009). The

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findings of our present study solidifies these previous finding. Overall, although causality cannot be
determined, the NR3C1 methylation findings in this group of depressed patients suggests that the CG38
site of exon 1F is methylated in association with emotional abuse in childhood and that this may lead toincreased morning HPA axis activity.

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FKBP5 intron 7 DNA methylation in depression
As FKBP5 intron 7 methylation alterations have previously been observed in individuals with a history of
ELA who suffer with post-traumatic stress disorder (PTSD) (Klengel et al., 2013), we sought to assess
differences in methylation at the same region in our depressed cohort. Analysis revealed no significant

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differences in DNA methylation at FKBP5 intron 7 in depressed patients relative to healthy controls. As
the previous methylation findings have been allele-specific in nature, the functional FKBP5 SNP
rs1360780 was genotyped and groups were divided according to whether or not they possessed the risk
T-allele. This allele is associated with increased FKBP5 induction by glucocorticoids (Fujii et al., 2014). InM

our cohort, no significant allele-specific differences in DNA methylation were observed in depressed
patients relative to healthy controls; nor did the risk-allele status separate out depressed from control

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groups. No associations between FKBP5 methylation and cortisol concentrations or CAR parameters wereobserved. Overall, this study did not reveal significant differences in FKBP5 intron 7 methylation inPT

depressed individuals and healthy controls, even when considering the presence of a risk polymorphism.

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These findings suggest that although FKBP5 DNA methylation is implicated in certain stress-relatedACdisorders, it may not be a candidate marker for state depression.

Study limitations and strengths
A limitation of this study is its cross-sectional design. Findings from cross-sectional studies need to beinterpreted with caution as it cannot be assumed that model parameters are stable over time (Bowen
and Wiersema, 1999). There is extensive evidence that the depressive phenotype changes over thecourse of an illness (O’Keane et al., 2012), therefore, future longitudinal studies are necessary to solidify
these findings. Although this study is at least comparable to similar studies in terms of sample size, the15ACCEPTED MANUSCRIPT
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statistical power is limited because of the relatively small sample size. Three saliva samples werecollected in the morning to assess individual CAR measurements. We found that subjects were less likely
to comply if more than three samples were required. Three samples are deemed to be adequate for theassessment of the CAR (Stalder et al., 2016), but additional morning samples can provide a betteroverview of HPA axis activity. Furthermore, due to lapses in participant compliance, not all participants

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had cortisol concentration values. This needs to be considered when interpreting the results ofcorrelation analyses. The study’s relatively small sample size needs to be highlighted as a limitation in thecontext of statistical power. Underpowered statistical analyses run the risk of Type II errors (i.e. false
negative results) and this should be considered when interpreting the results presented in thisANUSmanuscript. Additionally, given the novelty of DNA methylation analysis in psychiatry research, much of
the work presented is exploratory in nature. As a result of this exploratory work, no corrections weremade to address multiple comparisons, which run the risk of Type I errors (i.e. false positive results). Thisshould be considered when interpreting the results. Future work should attempt to (i) increase sample

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size to increase statistical power, and (ii) use conservative post-hoc corrections (i.e. Bonferroni) to ensure
the reporting of robust, statistically significant, positive findings. We did not include antidepressant useED

as a covariate in our analyses as they did not impact upon the DNA methylation or cortisol results acrossthe groups. This could be due to of the relatively small sample size, and needs to be considered whenPTinterpreting the findings. Finally, this study would have benefited from the inclusion of a higher numberCEof individuals exposed to early life adversity. Future studies should attempt to increase the number of
non-depressed controls who have been exposed to early life adversity, in an attempt to disentangle theACeffects of early life adversity in the pathogenesis of depression.
This study had a number of strengths. To our knowledge, this is the first study to assess both NR3C1 and

FKBP5 DNA methylation in the same sample and to examine this methylation in relation to HPA axis
activity and ELA in clinically depressed and healthy samples. Another strength of the study is the use of
LC-MS/MS for quantification of cortisol concentrations. More commonly used immunoassays are lessspecific and measure the inactive metabolite cortisone, resulting in false-high cortisol levels being
reported in the literature. LC-MS/MS is considered the best available technique for the specific analysis of16

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salivary cortisol (Antonelli et al., 2015). A final strength of the study is the use of whole blood samples.
Whole blood is easily accessible in the clinical environment, and may be potentially useful in the futurefor diagnosis and prognosis of depression. The use of whole blood should also be considered as alimitation as it is known that DNA methylation patterns differ across tissues (Kriebel et al., 2016), and this

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should be considered when interpreting the findings presented in this manuscript.

Conclusion

While there were no differences in DNA methylation levels at the FKBP5 sites of interest, methylation at
the NR3C1 exon 1F region was significantly increased in the depressed group. Our findings of increases inANUSmorning cortisol concentrations, in a group with MDD compared to controls, are compatible with overallfindings of HPA axis hyperactivity in individuals suffering with MDD. This study provides evidence that this
hyperactivity is associated with increased methylation at the NR3C1 exon 1F, within which the CG38 was
identified as being particularly associated with CAR over-activity. The degree of methylation at CG38 wasMalso specifically associated with childhood emotional abuse severity in depressed patients. The CG38 site
resides at a NGFI-A site that has been previously investigated in relation to HPA axis alterations inEDdepression. These results suggest that epigenetic alterations at the NR3C1 gene, but not the FKBP5 gene,may contribute to the finding of both increased morning cortisol levels in depression and ELA. The studyCEPT

provides novel insights into the molecular basis of depression and how this may evolve from ELA.

Acknowledgements and Disclosures

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This work was supported by a grant awarded by the Health Research Board (HRA-POR-2015-1317). We
sincerely thank all of the individuals who participated in this study. We thank Prof. Brian Keevil(University Hospital of South Manchester) for cortisol analysis by LC-MS/MS. The authors report nobiomedical financial interests or potential conflicts of interest.

Author Contributions

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Veronica O’Keane, Andrew Harkin, Linda Booij, and Thomas Frodl conceived and designed theexperiments; Veronica O’Keane screened and recruited all study participants; Chloё Farrell, Kelly Doolin,
Niamh O’Leary, Chaitra Jairaj, Darren Roddy, and Leonardo Tozzi performed sample collection; Zsófia
Nemoda designed primers for DNA methylation analysis; Moshe Szyf provided expert advice for all
epigenetic methodology and analysis; Derek Morris performed FKBP5 genotyping; Chloё Farrell

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performed DNA methylation analysis; Chloё Farrell analysed the data; Chloё Farrell wrote the

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manuscript.

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Tables and Figures

Controls (n=34)

Age (years)

28.27 (8.77)

28.21 (7.47)

Gender (M/F)

9/24

13/21

BMI

26.31 (6.89)

23.15 (3.87)

Smoking (Y/N)

12/21

ISCED Education

3.12 (1.58)

HAM-D-21

21.94 (5.49)

CES-D

38.39 (9.54)

CTQ Global Score

42.30 (15.31)

CTQ EACTQ PA

Statistics (p-value)
Z=-0.08, p=0.94
χ2=0.91, p=0.44

Z=-1.65, p=0.10

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Table 1. Demographic and clinical data for all recruited depressed patients and healthy controls

χ2=5.58, p=0.02*

6.00 (1.97)

Z=-4.85, p<0.001***

2.16 (2.93)

Z=-6.88, p<0.001***

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4/30

Z=-6.53, p<0.001***

29.35 (7.81)

Z=-4.01, p<0.001***

10.25 (4.28)

6.15 (2.22)

Z=-4.39, p<0.001***

6.88 (2.98)

5.50 (1.33)

Z=-2.41, p=0.02*

7.47 (6.26)

5.53 (3.09)

Z=-1.27, p=0.20

11.06 (5.14)

6.74 (2.21)

Z=-3.35, p=0.001***

7.03 (2.72)

5.50 (1.58)

Z=-2.81, p=0.004**

ELA (Y/N)

15/18

3/31

χ2=11.44, p=0.001***

Suicidality

10.06 (12.16)

0.00 (0.00)

Z=-5.86, p<0.001***

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6.82 (8.77)

CTQ ENAC

CTQ PNCE

CTQ SA

Data expressed as mean with SD in parentheses. Statistical analysis was performed using a Mann
Whitney U test (Age, BMI, Education, HAM-D, CES-D, PSQI, CTQ Global Score, Suicidality) and Chi squared(χ2) test (Gender, Smoking, ELA, Antidepressant use). BMI=Body Mass Index; ISCED=International
Standard Classification of Education; HAM-D=Hamilton Rating Scale for Depression; CES-D=Center for

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Epidemiologic Studies Depression Scale; CTQ=Childhood Trauma Questionnaire; EA=Emotional Abuse;
PA=Physical Abuse; SA=Sexual Abuse; EN=Emotional Neglect; PN=Physical Neglect. *p≤0.05, **p≤0.01,***p≤0.001 vs. control.

Table 2. NR3C1 exon 1F and FKBP5 intron 7 methylation data for depressed patients and healthy

Controls (n=34)

Mean NR3C1F*

4.07 (0.57)

3.81 (0.48)

NR3C1F CG36

2.72 (1.24)

2.44 (1.00)

NR3C1F CG37

3.12 (1.02)

3.27 (1.23)

NR3C1F CG38

5.58 (0.94)

NR3C1F CG39

5.38 (0.76)

NR3C1F CG41

3.57 (1.74)

Mean FKBP5

60.11 (4.50)

FKBP5 CG6

60.35 (11.57)

FKBP5 CG7

57.99 (5.43)

Statistics (p-value)

F=3.89, d=0.49, p=0.05
F=1.80, d=0.25, p=0.19
F=0.02, d=0.13, p=0.90

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F=0.11, d=0.10, p=0.75

5.27 (0.77)

F=0.84, d=0.14, p=0.36

2.92 (0.95)

F=3.07, d=0.46, p=0.09

60.03 (6.09)

F=0.28, d=0.02, p=0.60

EDM

5.49 (0.93)

62.32 (5.24)

F=0.10, d=0.22, p=0.92

57.74 (8.64)

F=0.34, d=0.04, p=0.57

Data are expressed as mean whole blood DNA methylation percentage with SD in parentheses. StatisticalPT

analysis was performed using analysis of variance, with smoking and education as covariates, on log

ACCEtransformed methylation data. d=Cohen’s d.

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Patients CT/TT (n=17)

Controls CC (n=21)

Controls CT/TT (n=13)

Statistics (p-value)

Mean FKBP5

60.88 (3.89)

59.40 (5.25)

60.82 (6.99)

58.90 (4.52)

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F=0.79, p=0.51

FKBP5 CG6

58.61 (16.72)

61.57 (4.30)

62.82 (5.62)

61.61 (4.76)

F=0.31, p=0.82

FKBP5 CG7

58.97 (4.13)

57.24 (6.67)

58.83 (10.51)

56.18 (4.86)

F=0.85, p=0.47

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Table 3. FKBP5 intron 7 methylation data for depressed patients and healthy controls, categorized on their rs1360780 allele status

Data are expressed as mean methylation percentage with SD in parentheses. Statistical analysis was performed using analysis of variance, with smoking and

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Table 4. Morning and evening cortisol concentrations in depressed patients and healthy controls
Patients (n=25)

Controls (n=20)

Statistics (p-value)

Cortisol T0*

10.88 (6.42)

7.01 (3.98)

F=4.60, d=0.72, p=0.04

Cortisol T30

13.86 (7.27)

10.07 (4.87)

F=2.75, d=0.61, p=0.11

Cortisol T60

8.59 (5.70)

7.65 (4.38)

Cortisol AUCi

100.94 (266.61)

101.67 (245.08)

Cortisol AM average*

11.41 (5.97)

8.54 (3.47)

Cortisol T750

2.48 (2.49)

Cortisol PM average

2.05 (1.63)

F=0.53, d=0.18, p=0.47
F=0.72, d=0.01, p=0.40

F=3.96, d=0.59, p=0.05

ANUS

2.15 (1.99)

3.00 (4.89)

F=0.05, d=0.23, p=0.83

3.48 (5.36)

F=1.13, d=0.24, p=0.30

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Evening cortisol
Cortisol T720

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CAR

3.13 (4.83)

F=0.18, d=0.30, p=0.68

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Data are expressed as mean cortisol concentrations (nMol/L) with SD in parentheses. Statistical analysis

ACCE

d=Cohen’s d.

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Table 5. Correlational analysis of NR3C1 exon 1F DNA methylation and cortisol concentrations in all participants.
NR3C1F CG37

NR3C1F CG38

NR3C1F CG39

NR3C1F CG41

meanNR3C1F

Cortisol T0

r=0.29 (p=0.07)

r=0.18 (p=0.26)

r=0.32 (p=0.04)*

r=-0.13 (p=0.47)

r=-0.06 (p=0.73)

r=0.25 (p=0.12)

Cortisol AM

r=0.34 (p=0.03)*

r=0.16 (p=0.32)

r=0.37 (p=0.02)*

r=-0.02 (p=0.90)

r=0.03 (p=0.88)

r=0.31 (p=0.04)*

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NR3C1F CG36

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Statistical analysis was performed using the Spearman’s rho correlation, with *p≤0.05 considered significant.

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Figure 1. Morning and evening cortisol concentrations in depressed patients and healthy controls. Cortisol concentrations at three morning and two evening timeCEpoints in depressed patients and healthy controls. Data are expressed as mean cortisol concentrations (nMol/L) with SEM, analysis of variance, with BMI andAC

education as covariates on log transformed data, *p≤0.05 vs. controls.

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Figure 2. Relationship between NR3C1 exon 1F DNA methylation and early life severity. A significant

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relationship exists between (a) NR3C1 exon 1F CG37 DNA methylation and emotional abuse and (b)AC

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NR3C1 exon 1F CG38 DNA methylation and emotional abuse.

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