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Reduced DNA methylation and psychopathology following endogenous hypercortisolism - A genome-wide study

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Patients with Cushing’s Syndrome (CS) in remission were used as a model to test the hypothesis that long-standing excessive cortisol exposure induces changes in DNA methylation that are associated with persisting neuropsychological consequences. Genome-wide DNA methylation was assessed in 48 women with CS in long-term remission (cases) and 16 controls matched for age, gender and education. The Fatigue impact scale and the comprehensive psychopathological rating scale were used to evaluate fatigue, depression and anxiety. Cases had lower average global DNA methylation than controls (81.2% vs 82.7%; p = 0.002). Four hundred and sixty-one differentially methylated regions, containing 3,246 probes mapping to 337 genes were identified. After adjustment for age and smoking, 731 probes in 236 genes were associated with psychopathology (fatigue, depression and/or anxiety). Twenty-four gene ontology terms were associated with psychopathology; terms related to retinoic acid receptor signalling were the most common (adjusted p = 0.0007). One gene in particular, COL11A2, was associated with fatigue following a false discovery rate correction. Our findings indicate that hypomethylation of FKBP5 and retinoic acid receptor related genes serve a potential mechanistic explanation for long-lasting GC-induced psychopathology.
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Scientific RepoRts | 7:44445 | DOI: 10.1038/srep44445
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Reduced DNA methylation and
psychopathology following
endogenous hypercortisolism –
a genome-wide study
Camilla A. M. Glad1, Johanna C. Andersson-Assarsson2, Peter Berglund3,
Ragnhildur Bergthorsdottir1, Oskar Ragnarsson1,* & Gudmundur Johannsson1,*
Patients with Cushing’s Syndrome (CS) in remission were used as a model to test the hypothesis that
long-standing excessive cortisol exposure induces changes in DNA methylation that are associated
with persisting neuropsychological consequences. Genome-wide DNA methylation was assessed
in 48 women with CS in long-term remission (cases) and 16 controls matched for age, gender and
education. The Fatigue impact scale and the comprehensive psychopathological rating scale were
used to evaluate fatigue, depression and anxiety. Cases had lower average global DNA methylation
than controls (81.2% vs 82.7%; p = 0.002). Four hundred and sixty-one dierentially methylated
regions, containing 3,246 probes mapping to 337 genes were identied. After adjustment for age and
smoking, 731 probes in 236 genes were associated with psychopathology (fatigue, depression and/or
anxiety). Twenty-four gene ontology terms were associated with psychopathology; terms related to
retinoic acid receptor signalling were the most common (adjusted p = 0.0007). One gene in particular,
COL11A2, was associated with fatigue following a false discovery rate correction. Our ndings indicate
that hypomethylation of FKBP5 and retinoic acid receptor related genes serve a potential mechanistic
explanation for long-lasting GC-induced psychopathology.
Hyperactivity of the hypothalamus-pituitary-adrenal (HPA)-axis, with subsequent increase in cortisol exposure
at the tissue level1,2, is implicated in neuropsychiatric disorders such as depression, post-traumatic stress disor-
der and anxiety2–9. Cortisol, the predominant glucocorticoid (GC) in humans, aects the central nervous sys-
tem through binding to its two receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor,
encoded by the NR3C1 and NR3C2 genes, respectively. ese receptors are ubiquitously expressed in the brain,
particularly in the hippocampus, prefrontal cortex and the parvocellular nucleus of the hypothalamus10.
Early-life adverse events have been associated with long-lasting dysregulation of the HPA-axis11, which may
play a pathophysiological role in development of stress-related diseases12,13. is early-life molecular program-
ming of the HPA-axis is thought to be conveyed by epigenetic mechanisms14–20. Several studies have shown that
NR3C1 DNA methylation is inuenced by both quality of maternal care (rodents)20 and experience of child-
hood trauma (humans)14,16–19. Furthermore, increased DNA methylation of the NR3C1 gene promoter has been
observed in the hippocampus and prefrontal cortex in suicide victims with a history of childhood abuse15. e
mechanism behind this change in DNA methylation is not known, however it is plausible that the increased cor-
tisol exposure induced by psychological stress may be involved.
Marked chronic excess and attenuation of the endogenous diurnal variation in cortisol secretion causes
Cushing’s syndrome (CS)21, most commonly caused by an ACTH-producing pituitary adenoma (Cushing’s dis-
ease; CD) or a cortisol-producing adrenal adenoma. Subjects with CS display a characteristic clinical pheno-
type including central obesity, muscle and skin atrophy and osteoporosis, as well as marked neuropsychological
1Department of Internal Medicine and Clinical Nutrition, Institute of Medicine at Sahlgrenska Academy, University of
Gothenburg and Department of Endocrinology, Sahlgrenska University Hospital, Gothenburg, Sweden. 2Department
of Molecular and Clinical Medicine, Institute of Medicine at Sahlgrenska Academy, University of Gothenburg,
Gothenburg, Sweden. 3Institute of Neuroscience and Physiology at Sahlgrenska Academy, University of Gothenburg,
Gothenburg, Sweden. *These authors contributed equally to this work. Correspondence and requests for materials
should be addressed to C.A.M.G. (email: camilla.glad@medic.gu.se)
Received: 19 October 2016
Accepted: 08 February 2017
Published: 16 March 2017
OPEN
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complaints such as mental fatigue, anxiety, depression and cognitive impairment22. Following treatment, most
features of the syndrome improve; however, despite long-term remission, we and others have shown that fatigue
and cognitive dysfunction commonly persists23–25. e mechanism for this persistent cognitive impairment is
not known, but the previous excess cortisol exposure is likely to play a mechanistic role26. In fact, in our previous
study there were no associations between aetiology, treatment (surgery and/or pituitary radiation therapy) or
hormone deciency and cognitive dysfunction23.
Due to previous observations of associations between epigenetics and psychopathology14–20, we hypothesized
that long-standing excessive cortisol exposure induces changes in DNA methylation that are associated with
long-lasting fatigue, depression and anxiety. Here, we used patients with CS as a unique human model of endog-
enous hypercortisolism to assess the impact of cortisol on genome-wide DNA methylation and its relation to
psychopathology.
Patients and Methods
Ethical considerations. Informed written consent was obtained from all patients and controls. e local
ethical committee of the University of Gothenburg, Sweden, approved the study. The study was conducted
according to the Declaration of Helsinki.
Design. is was a cross-sectional, case-controlled, single centre study including 55 patients with CS in remis-
sion and 55 controls matched for age, gender and educational level, as previously described23. In this part of the
study the association between DNA methylation and fatigue, depression and anxiety in 48 women with CS in
remission and 16 controls was analysed. e subjects were studied on three occasions, where medical history was
reviewed, physical examination and corticotropin releasing hormone (CRH) stimulation test were performed,
blood samples were drawn and psychopathology was evaluated. A 24-h urinary free cortisol (UFC) sampling
was performed between the second and last visits, and an overnight dexamethasone suppression test was done
following the last visit23.
Patients. e mean age of patients was 53 ± 14 years, and the mean age at diagnosis of CS was 37 ± 14 years
(Table1). irty-seven (77%) patients had CD and 11 (23%) had a cortisol producing adrenal adenoma. To verify
that the initial diagnosis of CD and cortisol producing adrenal adenoma were correct the clinical, biochemical,
radiological and histopathological data from the time of diagnosis were reviewed. In patients with CD in remis-
sion the primary treatment was transsphenoidal pituitary surgery in 25 (68%), radiotherapy in ve (14%) and
bilateral adrenalectomy in seven (19%). Fieen patients needed additional treatment. In total, 29 (78%) patients
with CD were treated with transsphenoidal pituitary surgery, 11 (30%) with radiotherapy and nine (24%) with
bilateral adrenalectomy. All patients with cortisol producing adrenal adenoma had been treated with unilat-
eral adrenalectomy. Eighteen (38%) patients had adrenal insuciency and received replacement therapy with
a mean daily hydrocortisone dose of 24 ± 8 mg/day. e mean urinary free cortisol (UFC) excretion was higher
in patients compared to controls (Table1). Seventeen (35%) patients had central (N = 15) or primary (N = 2)
hypothyroidism and were receiving a mean L-yroxine dose of 104 ± 31 μ g/day. Out of 37 patients with CD, 19
(51%) had growth hormone deciency of whom 15 were on growth hormone replacement therapy. Four out of
20 premenopausal woman (< 52 years) had hypogonadotropic hypogonadism and were receiving estrogen and
progesterone, 2 of 28 postmenopausal women were receiving treatment with oral estrogen. Two women received
replacement with dehydroepiandrosterone.
Controls. Controls to patients, matched for age and gender, were recruited from a random population sam-
ple obtained from the Swedish Tax Agency. Controls were approached through an invitation letter, responding
subjects were interviewed per telephone and those who matched the patient’s educational levels and had no
previously known psychiatric or chronic diseases known to aect cognitive function, were included. In this part
of the study data from one control (N = 16) per three patients (N = 48) were analysed. e mean age was 54 ± 16
years in controls (Table1).
Evaluation of hormone status. All patients were in remission, dened by an adequate suppression of
serum cortisol concentration ( 50 nmol/l) following a 1 mg overnight dexamethasone suppression test. e
median (interquartile range) duration of remission was 13 (5–19) years. A CRH test was performed in order
to evaluate the function of the HPA-axis. Serum cortisol was measured using competitive electrochemilumi-
nescence immunoassay (Cortisol Elecsys, Roche Diagnostics Scandinavia AB). Urinary free cortisol (UFC) was
measured using radioimmunoassay (SpectRia Cortisol 125I, Orion Diagnostica Oy, Finland). yroid function
was evaluated clinically and by measurements of free thyroxin and thyroid stimulating hormone (TSH) in serum.
Gonadal function was evaluated by asking for menstruation pattern and/or age at menopause as well as measure-
ments of estrogen and gonadotropins in serum. Growth hormone status was evaluated by review of previously
performed stimulation tests and measurement of insulin-like growth factor I.
Evaluation of fatigue, depression and anxiety. Fatigue was evaluated using the fatigue impact
scale, a 40 item questionnaire where dierent aspects of fatigue (physical, cognitive and social) are evaluated27.
Depression and anxiety were evaluated using the comprehensive psychopathological rating scale28.
DNA isolation and methylation assessment. DNA was isolated from whole blood using the QIAamp
DNA Blood Maxi kit (QIAGEN, Hilden, GE). DNA methylation was assessed on the Illumina Infinium
HumanMethylation450K BeadChip (Illumina, San Diego, CA, USA), which simultaneously interrogates
> 465,000 CpG sites and covers 99% of RefSeq genes and 96% of CpG islands. Probes are distributed in CpG
islands, shelves, shores, promoter regions, 5 UTRs, rst exon, gene body and 3 UTRs. Methylation assessment
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was performed at the Mutation Analysis Facility (MAF) at Karolinska University Hospital. e procedure is
briey described below:
Bisulte treatment. 500ng of genomic DNA (OD260/280 > 1.8) was bisulte treated using the EZ-96 DNA
Methylation Kit (D5004; Zymo Research, Inc., Irvine, CA, USA). e CT conversion reagent was mixed with
DNA and incubated in the dark at 50 °C for 16 hours. Aer desulfonation and washing steps, the samples were
puried using spin plates, eluted in 12 μ l elution buer and stored at 20 °C prior to processing.
Innium Methylation assay. e Innium Methylation Assay was performed according to the manufactur-
er’s instructions. Briey, 4 μ l of denatured bisulte-treated DNA was isothermally amplied over night at 37 °C,
followed by an enzymatic fragmentation step. e fragmented DNA was precipitated, resuspended and loaded
(using a Tecan EVO robot) on the 12-sample BeadChip, which was then incubated overnight at 48 °C, allowing
the fragmented DNA to hybridize to locus-specic 50-mers. Non-specically hybridized DNA was washed away,
followed by a single-base extension reaction using DNP- and Biotin-labeled ddNTPs (with use of a Tecan EVO
robot). Subsequently, hybridized DNA was removed from the labeled oligonucleotide and chips were dried under
vacuum and imaged using an Illumina iScan scanner.
Statistical analyses. Clinical parameters. Statistical analyses were performed with IBM SPSS statistics,
version 22, or in R version 3.0.3. Data are presented as mean ± standard deviation or median (25–75 percentiles).
For comparison between groups we used unpaired t-test for normally distributed data and Mann-Whitney U-test
for non-normally distributed data. For proportions, Pearson Chi-square or Fishers exact test were used. Pearson’s
correlation was used to determine correlation between methylation and clinical parameters. Linear regression
(with adjustment for age and smoking habits) was used to analyse the eect of methylation on clinical parameters.
Patients Controls p
Age at diagnosis (yr) 37 ± 14 — —
Age at follow-up (yr) 53 ± 14 54 ± 16 0.9
Duration of remission (yr) 13 (5–19) — —
Educational level (%) 1.0
Elementary school 25 25
Upper secondary education 46 44
University education 29 31
Smoking habits (%) 0.8
Non-smoker 53 44
Ex-smoker 36 44
Smoker 11 13
Employment (%) 0.1
Full-time 34 63
Part-time 30 13
Sick leave/ Disability pension 11 —
Retirement 26 25
Psychopathology
Fatigue (total score) 63 (40–88) 25 (6–37) < 0.01
Depression (score) 4 (3–7) 2 (1–3) < 0.01
Anxiety (score) 5 (4–7) 3 (3–6) 0.08
Hormone measurements
S-cortisol – BL (nmol/l)*327 ± 129 305 ± 119 0.6
S-cortisol – Peak (nmol/l)*557 ± 147 584 ± 78 0.5
UFC (nmol/24 h) 202 ± 158 131 ± 59 0.02
FreeT4 (pmol/l) 16.7 ± 3.2 14.8 ± 1.5 < 0.01
IGF-I (μ g/l) 149 ± 78 151 ± 84 0.9
Table 1. Background characteristics, sociodemographic status, psychopathology and hormone
measurements in 48 patients with Cushing’s syndrome in remission and 16 controls, matched for age,
gender and educational level. Data is presented as mean ± standard deviation or median (interquartile range).
*S-cortisol levels were analyzed only in ACTH sucient patients (N = 30). S-cortisol was measured in the
morning, before (baseline; BL) and aer administration of CRH; S-cortisol – peak represents the highest level
measured aer CRH administration. Psychopathology was evaluated through Fatigue impact scale (FIS) and
comprehensive psychopathological rating scale (depression and anxiety).
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DNA methylation analyses. Data was extracted using GenomeStudio (Illumina, Methylation Module v1.9),
which was also used to subtract the background and to normalize staining intensities using internal controls
present on the chip. A beta-value was calculated to estimate the methylation level of each CpG locus using the
ratio of intensities between methylated and unmethylated alleles (0 = unmethylated, 1 = fully methylated). e
performances of the dierent controls used were evaluated and potential outliers identied. Data quality control
and analysis was performed using the ChAMP methylation analysis package (v. 1.4.0)29 in R. Briey, intensity data
from IDAT les were loaded, normalized using default settings (i.e. BMIQ) and corrected for batch eects using
ComBat. Dierentially methylated regions (DMR) were then identied using the Probe Lasso DMR Hunter func-
tion with Benjamini-Hochberg p-value adjustment. Correction for multiple testing was done using the “fdrtool
package (v. 1.2.13) in R.
Gene ontology analyses. Gene ontology analyses were performed in DAVID Bioinformatics Resources 6.7
(NIAID/NIH) using the Functional Annotation Cluster and Functional Annotation Chart functions30,31. DAVID
provides unadjusted p-values as well as p-values adjusted for multiple testing using both the Bonferroni and the
Benjamini methods. Here we present Benjamini-adjusted p-values.
Results
Identication of dierentially methylated regions and overall DNA methylation. Initial quality
control (QC) analyses of the methylation raw data identied one case sample as a technical outlier due to low lev-
els of detected CpG:s (only 38,007 CpG:s were detected with a detection p-value < 0.01), this sample was removed
from further analysis. e nal data set consisted of 47 cases and 16 controls. On average, 485,001 CpG:s were
detected (484,979 in cases and 485,066 in controls, detection p-value < 0.01).
We rst performed DNA methylation analysis in ChAMP, to assess dierences between patients with CS in
long-term remission and matched controls (Table2). Overall, patients had lower average percentage of DNA
methylation than controls (81.2% vs 82.7%, p = 0.002; Fig.1a). ere were 3,903 probes that lay in dieren-
tially methylated regions (DMR:s; n = 461), the majority (n = 3,692; 94.6%) being hypomethylated. Of the 3,903
probes, 3,246 (83.2%) mapped to a gene (n = 337). Of the 337 genes, 278 were exclusively hypomethylated, 7
exclusively hypermethylated and 52 genes contained both hypo- and hypermethylated probes (Supplemental
Table1). Of the 3,903 probes, 55.9% (n = 2,183) had an annotated location; with the most common being gene
body (33.3%), 3 -UTR (3.9%) and TSS15 (within 1,500 base pairs upstream or downstream of the transcriptional
start site, 2.8%; Fig.1b).
Identication of probes associated with fatigue, depression and anxiety. To investigate whether
the epigenetic status of the CS subjects is associated with persistent fatigue, anxiety or depression a regression
analysis adjusted for age and smoking habits was performed. We identied 731 probes in 236 genes that were
associated with at least one of the three clinical traits. Of these 731 probes; 434 were associated with fatigue, 374
with depression, and 452 with anxiety. One hundred and sixty ve probes in 108 genes were associated with all
three traits.
Aer multiple testing correction using false discovery rate (FDR; 10%), four probes remained signicantly
associated with fatigue; cg22890571 (qval: 0.052), cg16479323 (qval: 0.052), cg09502339 (qval: 0.073), and
cg07889869 (qval: 0.087). ese probes are annotated to the following genes: TFDP1, ITPK1, COL11A2, and
DAGLB. Notably all four probes were also associated with depression and anxiety, however the p-values did not
remain signicant following FDR testing (qval: 0.13–0.23).
Functional validation of identied probes through gene ontology analyses. To explore the func-
tional relevance of the identied DMR:s and clinically associated probes, we next performed gene ontology (GO)
analyses using DAVID30,31. We initially performed GO analysis of all 337 genes with probes that lay in DMR:s and
found 202 GO terms, of which 18 were signicant aer Benjamini correction (Fig.2a). Terms related to retinoic acid,
thyroid hormone receptor and hormone/nuclear hormone receptor binding was the most common (Figs2b and 3a).
Genes, probes and clinical traits No
Total no of probes in DMRs 3903
Signicantly associated with Anxiety 527 (75 does not match a gene)
Signicantly associated with Depression 436 (62 does not match a gene)
Signicantly associated with Fatigue 508 (76 does not match a gene)
Total no of probes in genes 3246
Total no of genes with probes 337
No of probes in genes and signicantly
associated with clinical traits 731
No of genes associated with Anxiety 183
No of genes associated with Depression 172
No of genes associated with Fatigue 194
No of genes associated with Anxiety,
Depression and/or Fatigue 236
Table 2. Summaries from DNA methylation analyses.
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When focusing the analysis on the 236 genes that were associated with at least one of the clinical traits fatigue,
depression, and anxiety, 184 terms were identied of which 24 terms were signicant aer Benjamini correction
(Table3). As before, terms related to retinoic acid were among the most common (Fig.3a).
DNA methylation of the GC receptor gene (NR3C1). To explore the potential eect of hypercorti-
solism on DNA methylation of the NR3C1 gene, we analysed specically DNA methylation of this gene. Fieen
out of 49 probes annotated to the NR3C1 gene were signicantly dierentially methylated in CS cases compared
to controls. Aer multiple testing correction using false discovery rate (FDR, 10%), all 15 probes remained signif-
icant (qval 0.00019–0.065). e most signicant dierence was observed for probe cg15645634 (p = 8.31 × 106,
qval: 0.00019), located in intron 8 of the NR3C1 gene. Notably, out of these 15 dierentially methylated probes, 8
probes were specically hypermethylated and 7 probes were hypomethylated (Table4).
Correlation with markers of HPA-axis activity. To validate the functional value of DNA methylation of
the NR3C1 gene and the genes involved in retinoic acid signalling (in total, n = 672 probes), we performed cor-
relation analyses with urinary free cortisol (UFC; n = 47) and the change in serum cortisol concentration (delta
cortisol; n = 24) during a CRH-stimulation test as measures of cortisol exposure and HPA-axis activity, respec-
tively. e CRH-stimulation test was performed in a subgroup of CS patients who had not previously received
Figure 1. (a) Boxplot of average DNA methylation in cases vs controls. (b) Bar graph showing DNA
methylation in dierent regions in cases and controls.
Figure 2. (a) Pie chart showing frequency of DAVID terms. Input data: 337 genes, 18 signicant DAVID terms
aer Benjamini correction. ese 18 terms are grouped into 11 categories as shown above. Genes included in
the GO-term hormone/nuclear hormone receptor binding: ZBTB22, ZBTB9, PHB2, NCOA6, RING1, COL11A2,
DAXX, RGL2. Retinoic acid: RXRB, ZBTB22, ZBTB9, NCOA6, RING1, COL11A2, RGL2. yroid hormone
receptor: ZBTB22, ZBTB9, NCOA6, RING1, COL11A2, RGL2. (b) Venn diagram showing overlap of genes
included in the three most common GO term families: Retinoic acid (in light grey), Nuclear Receptors (in light
blue) and yroid (in dark grey). Numbers reect number of genes in each category.
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pituitary irradiation and who did not receive GC replacement therapy. irty-one probes were signicantly corre-
lated with UFC (SupplementalTable2), with the strongest correlation observed for probe cg02319187 annotated
to the RXRA gene (p = 0.005; Pearsons r = 0.412). Twenty-ve probes were signicantly correlated with change
in serum cortisol in response to CRH (SupplementalTable3). e strongest correlation was observed for probe
cg00629244, located in the NR3C1 gene (p = 0.002; Pearson’s r = 0.598). ree probes (cg01367322, cg03058556
and cg03825390) annotated to the ZBTB22, ZBTB9 and RGL2 genes (respectively) were signicantly associated
with both UFC and the change in serum cortisol. None of the correlations remained signicant aer correction
for multiple testing (FDR, 10%).
Inuence of current GC replacement therapy on DNA methylation. To evaluate the eect of cur-
rent GC replacement therapy on DNA methylation, we performed a subgroup analysis using the entire data-
set, n = 468,149 probes, where patients were stratied by occurrence of current GC replacement therapy. 12,128
probes in 6,186 genes were dierentially methylated between the two groups. e most signicant dierentially
methylated probe (cg03546163, p = 2.99 × 106; Fig.3b) was located in the FKBP5 gene.
In total, there are 34 probes annotated to the FKBP5 gene on the Illumina 450 K methylation chip and four of
these probes showed dierential methylation (cg03546163, cg00058684, cg08586216 and cg25114611) in patients
with, as compared to without, current GC replacement therapy (Table5). Most probes annotated to FKBP5 were
hypomethylated in cases receiving GC replacement therapy; (Fig.3b). No probes remained signicantly dieren-
tially methylated aer multiple testing correction (FDR, 10%). However it is well worth noting that this correction
for multiple testing takes into account a very large number of tests, and that in particular the unadjusted p-value
for FKBP5 probe cg03546163 reached borderline genome-wide signicance (p = 2.99 × 106). Together, this sug-
gests a true relevance of this nding, despite qval > 0.1.
Discussion
Hyperactivity of the HPA-axis may increase susceptibility to neuropsychiatric disorders such as depres-
sion, post-traumatic stress disorder and anxiety2–9. Here we provide evidence for a distinguishable pattern of
genome-wide DNA methylation in patients previously treated for CS and propose a mechanism for the long-term
Figure 3. (a) Bar graph showing DNA methylation in retinoic acid-related genes in cases and controls. Only
probes that were dierentially methylated between cases and controls are included. (b) Bar graphs showing
DNA methylation in the FKBP5 gene in cases receiving glucocorticoid replacement therapy (black bars), in
cases not receiving such therapy (strong grey bars) and in controls (so grey bars). Only probes that were
dierentially methylated between cases receiving replacement therapy and cases not receiving such therapy are
included.
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adverse neuropsychological consequences of endogenous hypercortisolism, which is also commonly observed in
a number of dierent psychiatric disorders.
Aberrations in DNA methylation has been associated with neurological and neuropsychiatric disorders such
as autism32, schizophrenia33 and Alzheimer’s disease34, as well as early life adverse events such as child maltreat-
ment and parental stress17. Childhood adverse events may inuence the life-time set point of the HPA-axis35; one
plausible mechanism for the induction of a programmed HPA-axis is through GC-induced epigenetic changes.
GO-terms No of genes Unadjusted P-value Benjamini-adjusted P-value
Alternative splicing 135 4.91E-07 0.0002
Genomewide Association Study of an AIDS-Nonprogression
Cohort Emphasizes the Role Played by HLA Genes (ANRS
Genomewide Association Study 02)
5 3.36E-06 0.0003
GO:0008134~transcription factor binding 23 2.70E-06 0.0006
GO:0004886~retinoid-X receptor activity 5 4.36E-06 0.0007
GO:0042974~retinoic acid receptor binding 6 2.69E-06 0.0012
GO:0030375~thyroid hormone receptor coactivator activity 5 1.12E-05 0.0013
GO:0010861~thyroid hormone receptor activator activity 5 1.12E-05 0.0013
GO:0003708~retinoic acid receptor activity 5 1.66E-05 0.0015
GO:0051059~NF-kappaB binding 6 4.51E-05 0.0034
GO:0030546~receptor activator activity 5 7.55E-05 0.0042
GO:0046966~thyroid hormone receptor binding 6 7.39E-05 0.0047
GO:0006986~response to unfolded protein 9 4.56E-06 0.0062
Splice variant 131 6.79E-06 0.0063
GO:0030374~ligand-dependent nuclear receptor transcription
coactivator activity 6 1.32E-04 0.0066
GO:0042809~vitamin D receptor binding 5 2.63E-04 0.0117
GO:0030545~receptor regulator activity 5 7.54E-04 0.0278
GO:0035257~nuclear hormone receptor binding 7 7.06E-04 0.0284
GO:0046978~TAP1 binding 3 1.14E-03 0.0386
GO:0046979~TAP2 binding 3 1.14E-03 0.0386
GO:0046977~TAP binding 3 1.14E-03 0.0386
GO:0042288~MHC class I protein binding 4 1.31E-03 0.0410
GO:0046983~protein dimerization activity 18 1.51E-03 0.0416
GO:0051427~hormone receptor binding 7 1.42E-03 0.0417
GO:0042824~MHC class I peptide loading complex 4 1.65E-04 0.0419
Table 3. DAVID gene ontology analysis with 236 genes with probes signicantly associated with at least
one clinical trait.
Probe PositionaStrand Loc ation
Intron
number CasesbControlsc
Delta-
betadpqvale
cg15645634 142783639 F Intron 8 0.0356 0.0261 0.0095 8.31E-06 0.000189
cg14558428 142784982 R Intron 8 0.0396 0.0314 0.0082 0.000396 0.004514
cg17860381 142783569 R Intron 8 0.0412 0.0336 0.0076 0.00306 0.015499
cg26464411 142784222 R Intron 8 0.0556 0.0465 0.0091 0.00406 0.017017
cg18019515 142783385 R Intron 8 0.0235 0.0192 0.0043 0.00437 0.017388
cg18146873 142782827 F Intron 8 0.0279 0.0211 0.0068 0.00550 0.018472
cg07733851 142781498 R Intron 8 0.375 0.407 0.032 0.00573 0.018648
cg25535999 142757312 R Intron 7 0.922 0.935 0.013 0.0110 0.030167
cg18594054 142623446 R Upstream 0.926 0.939 0.013 0.0144 0.035689
cg04097219 142629749 F Upstre am 0.972 0.978 0.006 0.0177 0.040303
cg06770322 142851098 F Downstream 0.960 0.966 0.006 0.0279 0.051311
cg23273257 142658828 R 5 UTR 0.977 0.982 0.005 0.0279 0.051315
cg21702128 142784721 F Intron 8 0.0516 0.0473 0.0043 0.0306 0.053550
cg25781210 142610141 F Upstre am 0.959 0.967 0.008 0.0429 0.064421
cg06521673 142782072 R Intron 8 0.0227 0.0201 0.0026 0.0435 0.064851
Table 4. Methylation in een signicantly dierentially methylated probes in NR3C1. Methylation in
een signicantly dierentially methylated probes (reported as beta-values) in NR3C1 on chromosome 5.
aHuman genome build 37. bCS patients. cControls. dDierence in methylation between cases and controls.
eq-values from multiple correction analysis using a 10% FDR.
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We performed a global DNA methylation analysis to assess dierences between patients with CS in long-term
remission and matched controls. Generally, patients with CS had lower levels of DNA methylation than controls.
Four hundred and sixty-one dierentially methylated regions were identied, with the majority being hypo-
methylated in patients. Also, we identied 731 probes in 236 genes that were associated with at least one of three
psychopathological traits; fatigue, anxiety and/or depression. Gene ontology analyses revealed an enrichment of
genes functioning as retinoic acid receptors, thyroid hormone receptors or hormone/nuclear hormone receptors.
ese receptors all belong to the nuclear receptor superfamily36 and serve as ligand-activated regulators of gene
transcription and stimulators of intracellular pathways. ese genes were hypomethylated in cases as compared
to controls, and associated with psychopathology in the patients.
e DNA methylation of the genes belonging to the retinoic acid receptor family was also correlated with
UFC and change in cortisol concentrations during a CRH-stimulation test, suggesting a functional link between
retinoic acid receptor and HPA-axis activity. e retinoic acid family includes Vitamin A and its derivatives, 13-cis
retinoic acid and all-trans retinoic acid. Retinoic acid is a transcriptionally active compound that regulates gene
expression via binding to specic nuclear receptors termed RARs37, or retinoic X receptors (RXRs)38. Retinoic
acid is crucial during development of the central nervous system39–41 and for neuronal plasticity in adult brain42–44.
Previous data suggests an involvement of the retinoic acid family in the regulation of the HPA-axis. Both chronic
(rats)45 and intermittent (humans)46 retinoic acid treatment has been shown able to induce HPA-axis hyperactiv-
ity and anxiety-like, as well as depressive, behaviour. A plausible mechanism may be that the retinoic acid inter-
rupts the GC receptor induced negative feedback47 by down-regulating 11β-HSD1 expression and by inhibiting
GC receptor transactivation48.
Probe PositionaGCRT - yesbGCRT - nocDelta_betadpqvale
cg03546163 35654363 0.484 0.588 0.104 2.99E-06 1
cg00052684 35694245 0.514 0.550 0.036 0.00159 1
cg08586216 35612351 0.981 0.976 0.0050 0.0122 1
cg25114611 35696870 0.314 0.339 0.025 0.0218 1
cg08915438 35697759 0.559 0.588 0.029 0.0599 1
cg16052510 35603143 0.809 0.783 0.026 0.0995 1
cg20813374 35657180 0.442 0.462 0.020 0.109 1
cg00130530 35657202 0.690 0.710 0.020 0.115 1
cg19226017 35697185 0.752 0.770 0.018 0.137 1
cg10300814 35565116 0.948 0.953 0.0050 0.175 1
cg06087101 35551932 0.418 0.440 0.022 0.197 1
cg14642437 35652521 0.876 0.888 0.012 0.204 1
cg19014730 35635985 0.681 0.695 0.014 0.226 1
cg07843056 35656848 0.0257 0.0229 0.0028 0.305 1
cg07485685 35696061 0.0394 0.0375 0.0019 0.409 1
cg17085721 35645341 0.945 0.949 0.004 0.442 1
cg07061368 35631736 0.894 0.901 0.007 0.485 1
cg17030679 35696300 0.0215 0.0227 0.0012 0.520 1
cg00610228 35695934 0.0368 0.0359 0.0009 0.582 1
cg16012111 35656758 0.0484 0.0500 0.0016 0.585 1
cg23416081 35693573 0.208 0.199 0.009 0.613 1
cg11845071 35695859 0.0209 0.0202 0.0007 0.616 1
cg00140191 35656242 0.0634 0.0615 0.0019 0.661 1
cg08636224 35657921 0.961 0.962 0.001 0.686 1
cg01294490 35656906 0.0934 0.0913 0.0021 0.687 1
cg18726036 35543610 0.946 0.947 0.001 0.707 1
cg03591753 35659141 0.539 0.534 0.005 0.720 1
cg14284211 35570224 0.139 0.134 0.005 0.738 1
cg06937024 35695489 0.0259 0.0264 0.0005 0.791 1
cg00862770 35655764 0.0256 0.0251 0.0005 0.795 1
cg02665568 35544468 0.921 0.923 0.002 0.799 1
cg07633853 35569471 0.155 0.157 0.002 0.829 1
cg15929276 35687457 0.187 0.186 0.001 0.911 1
cg10913456 35656590 0.0175 0.0175 0.000 0.989 1
Table 5. Methylation in FKBP5, grouped based on GC replacement therapy. Summary of methylation
(reported as beta-values) in FKBP5 on chromosome 6. GCRT = glucocorticoid replacement therapy. aHuman
genome build 37. bPatients receiving GCRT. cPatients not receiving GCRT. dDierence in methylation between
patients receiving GCRT and those not receiving such therapy. eq-values from multiple correction analysis using
a 10% FDR.
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Early adverse events and poor maternal care have been linked to changes in the GC receptor (NR3C1) DNA
methylation. To explore the potential eect of hypercortisolism on NR3C1 DNA methylation specically, we
analysed methylation in this gene and found that 15 out of 49 probes annotated to the NR3C1 gene were dier-
entially methylated in cases as compared to controls. Previously, increased levels of NR3C1 methylation has been
observed in post-mortem hippocampal brain tissue from suicide victims who had endured childhood abuse15,
and in peripheral blood from subjects with a history of perinatal stress16–18 and neglect or abuse during child-
hood49,50. Recently, common stressful life events were found to be associated with higher blood DNA methyla-
tion of the NR3C1 gene in adolescents51, suggesting that the NR3C1 DNA methylation is subject to change not
only during childhood. In accordance, herein we report that adults who have endured long-term endogenous
hypercortisolism have a dierential pattern of NR3C1 DNA methylation than matched controls, lending further
support for the importance of excess cortisol exposure as a possible cause in the programming of the HPA-axis
and its psychological consequences.
To evaluate the possibly confounding eect of current GC replacement therapy on our results, we performed
a subgroup analysis dividing the cases into groups of patients receiving GC replacement or not. ese analyses
revealed that the DNA methylation of 12,128 probes in 6,186 genes was inuenced by current GC replacement
therapy. One of the genes that were specically hypomethylated in cases compared to controls, with an additional
reduction in patients currently receiving GC replacement therapy, was the FK506 binding protein 5 (FKBP5).
FKBP5 binds to and negatively regulates GR function, which subsequently reduces anity of the GR to corti-
sol52. Common genetic variants in the FKBP5 gene have been associated with a relative GR resistance, and found
to interact with childhood abuse to predict post-traumatic stress disorder53. Previously, studies in mice have
reported that long-lasting exposure to GC decreases FKBP5 DNA methylation in the hippocampus, hypothala-
mus and blood, and that this demethylation is associated with anxiety-like behavior54,55 and reect previous GC
load55. Consistent with these ndings, herein we show that FKBP5 is indeed hypomethylated in CS patients as
compared to controls, and that the methylation is further reduced in a sub-group of CS patients receiving GC
replacement. ese ndings validate the suitability of CS as study model for GC exposure and further enlighten
the strong eect of GC on DNA methylation.
Despite the rigorous study protocol and adequate study model this project is not without limitations. Firstly,
the DNA methylation was assessed in whole blood and not in brain or any other isolated GC target tissue. A
recent study, however, provided evidence that DNA methylation variation observed in the brain is in fact reected
in the blood56. Secondly, the ndings herein remain to be validated and further explored as for whether the
observed changes in DNA methylation are indeed associated with subsequent changes in mRNA and protein
expression. Lastly, these ndings remain to be validated in studies of patients with specic psychiatric disorders
with HPA-axis hyperactivity.
In conclusion, our ndings suggest that long-standing hypercortisolism reduces global DNA methylation,
specically in genes that are known to attenuate the sensitivity of the GC receptor and therefore may induce
hyperactivity of the HPA-axis. Consequently, this may be of importance for the action of cortisol on the central
nervous system, and by such contribute to the frequent psychopathology observed in our patients. e mecha-
nism proposed might also apply to other disorders with transient or chronic hyperactivation of the HPA-axis that
aects a considerable part of the general population; such as depression, generalised anxiety and post-traumatic
stress.
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Acknowledgements
We would like to extend our gratitude to all of those who aided in performing this study, especially Ann-Charlotte
Olofsson and Jenny Tiberg at the Centre for Endocrinology and Metabolism at Sahlgrenska University Hospital,
for their skilful technical support and to Jessica Lindvall, Kristina Duvefelt and Gunnar Falk at the Mutation
Analysis Facility at Karolinska University Hospital for excellence in planning, running and correspondence
related to the DNA methylation analyses. Last but not least, we are indebted to the patients and controls that
participated in this study. is project has received nancial support from the Swedish federal government
under the LUA/ALF agreement (Drs Ragnarsson and Johannsson), e Health & Medical Care Committee of
the Regional Executive Board, Region Västra Götaland (Dr Ragnarsson), e Swedish Society of Medicine (Dr
Ragnarsson) and e Swedish Society of Endocrinology (Dr Ragnarsson).
Author Contributions
Dr Johannsson had full access to all the data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis. Study concept and design: Glad, Berglund, Bergthorsdottir, Ragnarsson,
Johannsson, Acquisition, analysis, or interpretation of data: All authors. Drafting of the manuscript: Glad,
Ragnarsson. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis:
Glad, Andersson-Assarsson. Obtained funding: Ragnarsson, Johannsson. Administrative, technical, or material
support: Glad, Andersson-Assarsson, Berglund. Study supervision: Ragnarsson, Johannsson.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing Interests: CG, JAA, RB and OR have no disclosures. PB has received lecture fees from Boehringer
Ingelheim and Lundbeck. GJ has received lecture fees from NovoNordisk, Pzer, Otsuka and Shire, and has
been a consultant for Viropharma/Shire and Astra Zeneca.
How to cite this article: Glad, C. A. M. et al. Reduced DNA methylation and psychopathology following
endogenous hypercortisolism – a genome-wide study. Sci. Rep. 7, 44445; doi: 10.1038/srep44445 (2017).
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... To this end, it has been shown that acute high-dose DEX treatment in a dental procedure significantly decreased CpG island methylation on the Crabp1 gene promoter [15], suggesting that GR or acute stress could upregulate Crabp1 expression. Of noteworthy mention is that patients with Cushing's syndrome (hypercortisolism) generally showed reduced levels of DNA methylation in genes related to RAR binding, RAR activity, and RXR activity [24]. Glucocorticoid replacement therapy also resulted in hypomethylation in the Fkbp5 gene [24]. ...
... Of noteworthy mention is that patients with Cushing's syndrome (hypercortisolism) generally showed reduced levels of DNA methylation in genes related to RAR binding, RAR activity, and RXR activity [24]. Glucocorticoid replacement therapy also resulted in hypomethylation in the Fkbp5 gene [24]. Moreover, studies in mice have showed that long-tern exposure to Glucocorticoid reduced DNA methylation of Fkbp5 in the hippocampus and hypothalamus, and the demethylation was associated with anxiety-like behavior [25,26]. ...
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Retinoic acid (RA), the principal active metabolite of vitamin A, is known to be involved in stress-related disorders. However, its mechanism of action in this regard remains unclear. This study reports that, in mice, endogenous cellular RA binding protein 1 (Crabp1) is highly expressed in the hypothalamus and pituitary glands. Crabp1 knockout (CKO) mice exhibit reduced anxiety-like behaviors accompanied by a lowered stress induced-corticosterone level. Furthermore, CRH/DEX tests show an increased sensitivity (hypersensitivity) of their feedback inhibition in the hypothalamic–pituitary–adrenal (HPA) axis. Gene expression studies show reduced FKBP5 expression in CKO mice; this would decrease the suppression of glucocorticoid receptor (GR) signaling thereby enhancing their feedback inhibition, consistent with their dampened corticosterone level and anxiety-like behaviors upon stress induction. In AtT20, a pituitary gland adenoma cell line elevating or reducing Crabp1 level correspondingly increases or decreases FKBP5 expression, and its endogenous Crabp1 level is elevated by GR agonist dexamethasone or RA treatment. This study shows, for the first time, that Crabp1 regulates feedback inhibition of the the HPA axis by modulating FKBP5 expression. Furthermore, RA and stress can increase Crabp1 level, which would up-regulate FKBP5 thereby de-sensitizing feedback inhibition of HPA axis (by decreasing GR signaling) and increasing the risk of stress-related disorders.
... Downstream effects of benign functioning adrenal masses include subclinical hypercortisolism [10], Cushing's syndrome [11] hyperaldosteronism [11], hyperandrogenism [12], local mass effects [13], and potential for malignancy [14]. Further removed downstream effects of hypercortisolism, potentially resulting from an adrenal incidentaloma, include obesity [15], weight loss [16], lipolysis [17], skin changes [18], muscle weakness [19], fatigue [20], hypertension [21], high blood glucose and sequelae [22], osteoporosis [23], menstrual irregularities [24], and insulin resistance [25]. Importantly, insulin resistance itself has a host of detrimental effects on the conditions of the body's metabolic system, including reflex hyperinsulinemia [26], type 2 diabetes [27], high blood glucose and sequelae [28], atherosclerosis [29], metabolic syndrome [30], cardiovascular disease [31], and lipolysis [32]. ...
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Abstract: Adrenal incidentalomas are incidentally discovered adrenal masses greater than one centimeter in diameter. An association between insulin resistance and adrenal incidentalomas has been established. However, the pathophysiological link between these two conditions remains incompletely characterized. This review examines the literature on the interrelationship between insulin resistance and adrenal masses, their subtypes, and related pathophysiology. Some studies show that functional and non-functional adrenal masses elicit systemic insulin resistance, whereas others conclude the inverse. Insulin resistance, hyperinsulinemia, and the anabolic effects on adrenal gland tissue, which have insulin and insulin-like growth factor-1 receptors, offer possible pathophysiological links. Conversely, autonomous adrenal cortisol secretion generates visceral fat accumulation and insulin resistance. Further investigation into the mechanisms and timing of these two pathologies as they relate to one another is needed and could be valuable in the prevention, detection, and treatment of both conditions.
... We observed that blood DNA hypomethylation progressively recovers in the years following remission. Similarly, a subtle DNA hypomethylation was observed by Glad et al. several years after Cushing's syndrome correction (43). The authors could correlate some blood methylation levels at specific genomic regions with long-term Cushing-associated neuropsychological sequels. ...
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Objective: Cushing’s syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing’s syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing’s syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers. Design: We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing’s syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. Methods: Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850000 CpG sites). Both unsupervised (Principal Component Analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. Results: Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing’s syndrome correction. In Cushing’s syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. Conclusions: Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assess glucocorticoid action beyond hormone assays.
... Another intriguing possibility is that prolonged increases of cortisol levels are affecting DNA methylation levels, and vice-versa. In a study with 48 women with Cushing's syndrome (chronically increased cortisol), genome-wide global DNA methylation levels were significantly reduced, as well as specific genes related to fatigue and psychopathology (Glad et al., 2017). DNA methylation of genes associated with psychiatric disorders is known to be affected by childhood trauma and can affect cortisol response in adulthood (Nätt et al., 2015;Houtepen et al., 2016). ...
Article
The incidence of hypoxia in water bodies is increasing more rapidly than aquatic life can adapt. This study aimed to determine the effects of hypoxia on fish physiology, as well as protein expression through proteomics. To do this, 40 rainbow trout were divided into normoxic control (11.5 mg/L dissolved oxygen) and hypoxic treatment (5 mg/L dissolved oxygen) tanks for a period of 7 days. Fish were then anesthetized and blood was sampled. Fish were then euthanized and heart and liver samples were taken. Blood glucose, cortisol and lipid, body and liver mass, fork length, hematocrit and, blood cell counts and global heart methylation were measured. Red blood cell counts were significantly lower, while hematocrit and mean corpuscular volume were significantly higher in the hypoxic treatment. Global DNA methylation was significantly decreased in hypoxic heart tissue. Plasma cortisol and 18:1 monoacylglyerol increased, while 15:0–18:1 phosphatidylethanolamine, and 18:1 lysophosphatidylethanolamine decreased in plasma of rainbow trout under hypoxic conditions. Plasma proteomics revealed 70 significantly altered proteins (p < 0.05) in the hypoxia treatment (Data are available via ProteomeXchange with identifier PXD026589). Many of these molecular changes appear to be related to the observed increase in red blood cell volume and epigenetic modifications, as well as to angiogenesis, lipid, and glucose metabolism. This study highlights a range of cellular and molecular responses in the blood and plasma of freshwater fish that may be phenotypic adaptions to hypoxia, and that could aid in diagnosing the health status of wild fish populations using several, potential, discovered biomarkers.
... The probe cg03546163, located at the promoter of gene FKBP5, showed the strongest association in the MOMENT meta-analysis of neurodegenerative disorders (p META = 3.42 × 10 −12 , decreased blood DNA methylation in cases compared to controls). Demethylation at this site has previously been reported in patients with Cushing's syndrome (marked by chronic excess and attenuation of the endogenous diurnal variation in cortisol secretion) [52] and Behçet's disease compared to controls, in the same direction of effect as for neurodegenerative disorders. Especially relevant to neurodegenerative disorders is the finding that FKBP5 expression has been shown to progressively increase with normal aging, concomitant with reduced FKBP5 DNA methylation [53], which correlated with Braak staging in human brains and increased tau pathology both in vitro and in mouse models of AD [53,54]. ...
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Background People with neurodegenerative disorders show diverse clinical syndromes, genetic heterogeneity, and distinct brain pathological changes, but studies report overlap between these features. DNA methylation (DNAm) provides a way to explore this overlap and heterogeneity as it is determined by the combined effects of genetic variation and the environment. In this study, we aim to identify shared blood DNAm differences between controls and people with Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. Results We use a mixed-linear model method (MOMENT) that accounts for the effect of (un)known confounders, to test for the association of each DNAm site with each disorder. While only three probes are found to be genome-wide significant in each MOMENT association analysis of amyotrophic lateral sclerosis and Parkinson’s disease (and none with Alzheimer’s disease), a fixed-effects meta-analysis of the three disorders results in 12 genome-wide significant differentially methylated positions. Predicted immune cell-type proportions are disrupted across all neurodegenerative disorders. Protein inflammatory markers are correlated with profile sum-scores derived from disease-associated immune cell-type proportions in a healthy aging cohort. In contrast, they are not correlated with MOMENT DNAm-derived profile sum-scores, calculated using effect sizes of the 12 differentially methylated positions as weights. Conclusions We identify shared differentially methylated positions in whole blood between neurodegenerative disorders that point to shared pathogenic mechanisms. These shared differentially methylated positions may reflect causes or consequences of disease, but they are unlikely to reflect cell-type proportion differences.
... Generally, our high-confidence genes have been previously associated with psychological and developmental problems, inflammation, and stress responses. Molecular changes were shown at TAPBP for major depressive disorder and suicide (Murphy et al., 2017), TAPBP and DOCK4 for schizophrenia (Alkelai et al., 2012;Lee, Kim, & Song, 2013;Zhang et al., 2020), ZBTB22 for intellectual disability (Agapite et al., 2020) and psychopathologies following hypercortisolism (Glad et al., 2017), and DOCK4 for autism and dyslexia Maestrini et al., 2010). Enrichment for pathways including Dock4 has been repeatedly associated with stress-related responses in mice (Lee et al., 2005;Lisowski et al., 2011;Papale, Madrid, Li, & Alisch, 2017), while ZBTB12 DNAm is related to markers of inflammation (e.g. ...
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Background. Experimental work in animals has shown that DNA methylation (DNAm), an epigenetic mechanism regulating gene expression, is influenced by typical variation in maternal care. While emerging research in humans supports a similar association, studies to date have been limited to candidate gene and cross-sectional approaches, with a focus on extreme deviations in the caregiving environment. Methods. Here, we explored the prospective association between typical variation in maternal sensitivity and offspring epigenome-wide DNAm, in a population-based cohort of children (N = 235). Maternal sensitivity was observed when children were 3- and 4-years-old. DNAm, quantified with the Infinium 450 K array, was extracted at age 6 (whole blood). The influence of methylation quantitative trait loci (mQTLs), DNAm at birth (cord blood), and confounders (socioeconomic status, maternal psychopathology) was considered in follow-up analyses. Results. Genome-wide significant associations between maternal sensitivity and offspring DNAm were observed at 13 regions ( p < 1.06 × 10−07), but not at single sites. Follow-up analyses indicated that associations at these regions were in part related to genetic factors, confounders, and baseline DNAm levels at birth, as evidenced by the presence of mQTLs at five regions and estimate attenuations. Robust associations with maternal sensitivity were found at four regions, annotated to ZBTB22, TAPBP, ZBTB12, and DOCK4. Conclusions. These findings provide novel leads into the relationship between typical variation in maternal caregiving and offspring DNAm in humans, highlighting robust regions of associations, previously implicated in psychological and developmental problems, immune functioning, and stress responses.
... Additional evidence has suggested the interference of GCs with DNA methylation of target genes encoding for key proteins of steroidassociated pathways [91,92]. ...
Article
Autoimmune rheumatic diseases (ARDs) are chronic conditions with a striking female predominance, frequently affecting women of childbearing age. Sex hormones and gender dimorphism of immune response are major determinants in the multifactorial pathogenesis of ARDs, with significant implications throughout reproductive life. Particularly, pregnancy represents a challenging condition in the context of autoimmunity, baring profound hormonal and immunologic changes, which are responsible for the bi-directional interaction between ARDs outcome and pregnancy course. In the latest years epigenetics has proven to be an important player in ARDs pathogenesis, finely modulating major immune functions and variably tuning the significant gender effects in autoimmunity. Additionally, epigenetics is a recognised influencer of the physiological dynamic modifications occurring during pregnancy. Still, there is currently little evidence on the pregnancy-related epigenetic modulation of immune response in ARDs patients. This review aims to overview the current knowledge of the role of epigenetics in the context of autoimmunity, as well as during physiologic and pathologic pregnancy, discussing under-regarded aspects in the interplay between ARDs and pregnancy pathology. The outline of a new ongoing European project will be presented.
Article
Context & Objective Chronic glucocorticoid (GC) overexposure, resulting from endogenous Cushing’s syndrome (CS) or exogenous GC therapy, causes several adverse outcomes, including persistent central fat accumulation associated with a low-grade inflammation. However, no previous multi-omics studies in visceral adipose tissue (VAT) from patients exposed to high levels of unsuppressed GC during active CS or after remission are available yet. Methods We employed a translational approach combining high-throughput data on endogenous CS patients and a reversible CS mouse model. We performed RNA-seq and ChIP-seq on histone modifications (H3K4me3, H3K27ac and H3K27me3) to identify persistent transcriptional and epigenetic signatures in VAT produced during active CS and maintained after remission. Results VAT dysfunction was associated with low-grade pro-inflammatory status, macrophage infiltration and extracellular matrix remodeling. Most notably, chronic hypercortisolism caused a persistent circadian rhythm disruption in VAT through core clock genes modulation. Importantly, changes in the levels of two histone modifications associated to gene transcriptional activation (H3K4me3 and H3K27ac) correlated with the observed differences in gene expression during active CS and after CS remission. Conclusion We identified for the first time, the persistent transcriptional and epigenetic signatures induced by hypercortisolism in VAT, providing a novel integrated view of molecular components driving the long-term VAT impairment associated with CS.
Chapter
Glucocorticoids (GCs) play an important role in the function and homeostasis of the central nervous system. Acute cortisol increase is an adaptative body mechanism helpful to deal with stressful situations (including illness); however, chronic exposure to hypercortisolism can have deleterious consequences. These may include anatomical brain changes, an increased prevalence of psychiatric diseases, cognitive impairment, mood alterations, and sleep disturbances. If high GC exposure occurs in early life, it can also adversely program the hypothalamic pituitary adrenal (HPA) axis and increase susceptibility to develop metabolic, neuropsychiatric, and neurodegenerative disorders. Genetic risk is also important contributing to affective disorders in hypercortisolim. In addition to altering gene expression, epigenetic mechanisms represent a means through which stress and GCs can leave long-lasting “memories” of past experiences that can contribute to modulate physical and mental health. These changes have also been associated with the development of stress-related psychiatric disorders. After chronic exposure, correction of hypercortisolism results in improvement of brain volume, cognitive function, and mood disorders, but not complete normalization. New studies are necessary to develop strategies to improve these persistent long-term symptoms of chronic hypercortisolism.
Article
Measurement of Health-Related Quality of Life (HRQoL) is emerging as an important clinical endpoint which complements diagnostic workup and contributes to place patients at the centre of the decision-making process through the recognition of their needs, concerns, goals and expectations. Chronic excessive cortisol exposure in Cushing’s syndrome (CS) causes severe physical and psychological morbidity which invariably affects HRQoL during the active phase of the disease and even after successful treatment. This sustained deterioration of patient’s wellbeing is partly related to the persistence of several features associated with prior cortisol excess, including affective disorders, cognitive dysfunctions and negative illness perception. The aim of this review is to summarize the most recent evidence on HRQoL in CS, including the main determinants of its impairment and the results of some educational programs specifically addressed to improve patient’s coping abilities. The preliminary results of an unpublished survey on patient’s unmet needs will also be presented.
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DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
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Children exposed to extreme stress are at heightened risk for developing mental and physical disorders. However, little is known about mechanisms underlying these associations in humans. An emerging insight is that children's social environments change gene expression, which contributes to biological vulnerabilities for behavioral problems. Epigenetic changes in the glucocorticoid receptor gene, a critical component of stress regulation, were examined in whole blood from 56 children aged 11–14 years. Children exposed to physical maltreatment had greater methylation within exon 1F in the NR3C1 promoter region of the gene compared to nonmaltreated children, including the putative NGFI-A (nerve growth factor) binding site. These results highlight molecular mechanisms linking childhood stress with biological changes that may lead to mental and physical disorders.
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Stress early in life is a known risk factor for the development of affective disorders later in life. Epigenetic mechanisms, such as DNA methylation, may have an important role in mediating that risk. Recent epigenetic research reported on the long-term relationship between traumatic stress in childhood and DNA methylation in adulthood. In this study, we examined the impact of various types of stress (perinatal stress, stressful life events (SLEs) and traumatic youth experiences) on methylation of the glucocorticoid receptor gene (NR3C1) in the blood of a population sample of 468 adolescents (50.4% female, mean age 16.1 years). Second, we determined whether stress at different ages was associated with higher NR3C1 methylation. NR3C1 methylation rates were higher after exposure to SLEs and after exposure to traumatic youth experiences. NR3C1 methylation in adolescence was not higher after exposure to perinatal stress. Experience of SLEs in adolescence was associated with a higher NR3C1 methylation, independently of childhood SLEs. We demonstrate that not only traumatic youth experiences but also (more common) SLEs are associated with higher NR3C1 methylation. In addition, our findings underline the relevance of adolescent stress for epigenetic changes in the NR3C1 gene.
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Clinical reports have highlighted a role for retinoids in the etiology of mood disorders. Although we had shown that recruitment of the nuclear receptor retinoic acid receptor-α (RAR-α) to corticotropin-releasing hormone (CRH) promoter is implicated in activation of the hypothalamus-pituitary-adrenal (HPA) axis, further insight into how retinoids modulate HPA axis activity is lacking. Here we show that all-trans retinoic acid (RA)-induced HPA activation involves impairments in glucocorticoid receptor (GR) negative feedback. RA was applied to rats chronically through intracerebroventricular injection. A 19-day RA exposure induced potent HPA axis activation and typical depression-like behavior. Dexamethasone failed to suppress basal corticosterone (CORT) secretion, which is indicative of a disturbed GR negative feedback. In the hypothalamic paraventricular nucleus, increased CRH(+) and c-fos(+) cells were found while a negative R-2(+)/ER(+) correlation was present between the number of RAR-α(+) and GR(+) cells. This was paralleled by increased RAR-α and decreased GR protein expression in the hypothalamus. Additional in vitro studies confirmed that RA abolished GR-mediated glucocorticoid-induced suppression of CRH expression, indicating a negative cross-talk between RAR-α and GR signaling pathways. Finally, the above changes could be rapidly normalized by treatment with GR antagonist mifepristone. We conclude that in addition to the 'classic' RAR-α-mediated transcriptional control of CRH expression, disturbances in GR negative feedback constitute a novel pathway that underlies RA-induced HPA axis hyperactivity. The rapid normalization by mifepristone may be of potential clinical interest in this respect.
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Unlabelled: The Illumina Infinium HumanMethylation450 BeadChip is a new platform for high-throughput DNA methylation analysis. Several methods for normalization and processing of these data have been published recently. Here we present an integrated analysis pipeline offering a choice of the most popular normalization methods while also introducing new methods for calling differentially methylated regions and detecting copy number aberrations. Availability and implementation: ChAMP is implemented as a Bioconductor package in R. The package and the vignette can be downloaded at bioconductor.org
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Prenatal exposure to maternal stress can have lifelong implications for psychological function, such as behavioral problems and even the development of mental illness. Previous research suggests that this is due to transgenerational epigenetic programming of genes operating in the hypothalamic-pituitary-adrenal axis, such as the glucocorticoid receptor (GR). However, it is not known whether intrauterine exposure to maternal stress affects the epigenetic state of these genes beyond infancy. Here, we analyze the methylation status of the GR gene in mothers and their children, at 10-19 years after birth. We combine these data with a retrospective evaluation of maternal exposure to intimate partner violence (IPV). Methylation of the mother's GR gene was not affected by IPV. For the first time, we show that methylation status of the GR gene of adolescent children is influenced by their mother's experience of IPV during pregnancy. As these sustained epigenetic modifications are established in utero, we consider this to be a plausible mechanism by which prenatal stress may program adult psychosocial function.
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Childhood maltreatment, through epigenetic modification of the glucocorticoid receptor gene (NR3C1), influences the hypothalamic–pituitary–adrenal axis (HPA axis). We investigated whether childhood maltreatment and its severity were associated with increased methylation of the exon 1F NR3C1 promoter, in 101 borderline personality disorder (BPD) and 99 major depressive disorder (MDD) subjects with, respectively, a high and low rate of childhood maltreatment, and 15 MDD subjects with comorbid post-traumatic stress disorder (PTSD). Childhood sexual abuse, its severity and the number of type of maltreatments positively correlated with NR3C1 methylation (P=6.16 × 10−8, 5.18 × 10−7 and 1.25 × 10−9, respectively). In BPD, repetition of abuses and sexual abuse with penetration correlated with a higher methylation percentage. Peripheral blood might therefore serve as a proxy for environmental effects on epigenetic processes. These findings suggest that early life events may permanently impact on the HPA axis though epigenetic modifications of the NR3C1. This is a mechanism by which childhood maltreatment may lead to adulthood psychopathology.