Corresponding author: A. Menke, M.D., Max Planck Institute of Psychiatry, Kraepelinstr. 10, D-80804 Munich, Germany. E-mail: email@example.com
The FK506 binding protein 51 or FKBP5 has been implicated in the regulation of glucocorticoid receptor (GR) sensitivity, and genetic variants in this gene have been associated with mood and anxiety disorders. GR resistance and associated stress hormone dysregulation are among the most robust biological findings in major depression, the extent of which may be moderated by FKBP5 polymorphisms. FKBP5 mRNA expression in peripheral blood cells (baseline and following in vivo GR stimulation with 1.5 mg dexamethasone p.o.) was analyzed together with plasma cortisol, ACTH, dexamethasone levels and the FKBP5 polymorphism rs1360780 in 68 depressed patients and 87 healthy controls. We observed a significant (P = 0.02) interaction between disease status and FKBP5 risk allele carrier status (minor allele T) on GR-stimulated FKBP5 mRNA expression. Patients carrying the risk T allele, but not the CC genotype, showed a reduced induction of FKBP5 mRNA. This FKBP5 polymorphism by disease status interaction was paralleled by the extent of plasma cortisol and ACTH suppression following dexamethasone administration, with a reduced suppression only observed in depressed patients carrying the T allele. Only depressed patients carrying the FKBP5 rs1360780 risk allele showed significant GR resistance compared with healthy controls, as measured by dexamethasone-induced FKBP5 mRNA induction in peripheral blood cells and suppression of plasma cortisol and ACTH concentrations. This finding suggests that endocrine alterations in depressed patients are determined by genetic variants and may allow identification of specific subgroups.
The FK506 binding protein 51 or FKBP5 was first described in a complex with the steroid hormone receptors 20 years ago (Sanchez 1990; Smith et al. 1990), and has emerged as one as the key players in stress-related phenotypes (Binder 2009). FKBP5 is a heat shock protein 90 (HSP90) co-chaperone which modifies steroid receptor hormone sensitivity, and interacts with the glucocorticoid receptor (GR), the progesterone receptor (PR) and the androgen receptor (AR) (Storer et al. 2011). FKBP5 is a negative regulator of GR function; when bound to the receptor complex, cortisol binds with lower affinity and the nuclear translocation of the receptor is less efficient. GR activation induces FKBP5 mRNA and protein expression and this provides an ultra-short feedback loop for GR sensitivity (Binder 2009, Jaaskelainen et al. 2011; Reynolds et al. 1999). Polymorphisms in FKBP5 have been shown to be associated with an altered induction of FKBP5 mRNA with GR activation (Binder et al. 2004) and by that interfere with the negative feedback of the stress hormone system. Individuals carrying the alleles associated with a higher induction have also been shown to have a more prolonged cortisol response to psychological stressors (Ising et al. 2008; Luijk et al. 2010), indicative of GR resistance. This altered responsiveness to stress seems to predispose to psychiatric disorders, and a number of studies have shown association of these FKBP5 polymorphisms with an increased susceptibility to major depression (Lavebratt et al. 2010; Lekman et al. 2008; Zimmermann et al. 2011; Zobel et al. 2010), bipolar disorder (Willour et al. 2008) and posttraumatic stress disorder (PTSD) (Appel et al. 2011; Binder et al. 2008; Mehta et al. 2011; Sarapas et al. 2011; Xie et al. 2010) as well as an increased suicide risk (Brent et al. 2010; Roy et al. 2012; Supriyanto et al. 2010), especially in interaction with exposure to early trauma.
In fact, a dysregulation of the stress hormone is one of the most robust biological findings in stress-related psychiatric disorders, including major depression and PTSD with changes in GR sensitivity being often reported (Binder 2009; de Kloet et al. 2005; Heim & Nemeroff 2001; Holsboer 2000; Pariante & Miller 2001). A decrease in GR sensitivity has been shown at multiple levels in patients with major depression, whereas an increase in sensitivity has been reported in patients with PTSD (Holsboer 2000; Pariante & Miller 2001; Yehuda et al. 2004) in some but not all studies.
We have recently shown that FKBP5 genotype maybe associated with biologically distinct subtypes of PTSD. In fact, only carriers of the risk alleles showed the previously described GR supersensitivity after administration of a low dose of dexamethasone, a specific agonist of the GR (Binder et al. 2008; Mehta et al. 2011). This finding was paralleled by genotype-specific gene expression changes with PTSD. In this study, we wanted to test if such FKBP5-dependent differences in GR sensitivity would also be observed in patients with major depression and could thus provide a possibility to stratify patients with depression according to biological differences. To this aim, we compared FKBP5 gene expression differences in whole blood and plasma cortisol and ACTH concentrations in healthy controls and depressed patients before and 3 h after ingestion of 1.5 mg dexamethasone in dependence of the FKBP5 single-nucleotide polymorphism (SNP) rs1360780.
Materials and methods
We enrolled 68 patients aged 18–65 years (65.3% males) who were admitted as inpatients to the Max Planck Institute of Psychiatry (MPI), Munich, Germany, for treatment of a depressive episode. All participants were of Caucasian origin. They were enrolled into the Munich Antidepressant Response Signature (MARS) project (www.mars-depression.de) (Hennings et al. 2009; Ising et al. 2009; Kohli et al. 2011). Data from some of these patients have been reported in a study that described dexamethasone-induced genome-wide gene expression (Menke et al. 2012). The patients were treated with antidepressant medications according to the doctor's choice. As carbamazepine is likely associated with an altered metabolism of dexamethasone, patients treated with this drug were excluded (Kunzel et al. 2003). Severity of depressive symptoms was assessed at admission and then weekly until discharge by trained raters using the 21-item Hamilton Depression Rating Scale (HAMD). Patients fulfilling the criteria for at least a moderate depressive episode (HAMD ≥ 14) were eligible. Patients with severe neurological or general medical conditions were excluded (e.g. Parkinson's disease, dementia, stroke, intoxication, severe infection, ischemic heart disease). Blood was taken to examine inflammation markers, liver enzymes, renal function and coagulation; vital signs (blood pressure and pulse) were monitored. The study was approved by the ethics committee of the Ludwig-Maximilans-University in Munich, and written informed consent was obtained from all patients (Nr. 244/01).
Recruitment of controls
The control samples consisted of 87 subjects (64.8% males), matched for age, sex and ethnicity and were also recruited at the MPI. They were screened for the presence of any axis I disorder using the Composite International Diagnostic Interview (CIDI) Screener (Wittchen et al. 1999). Only individuals free of a history of psychiatric disorders as well as major neurological and general medical disorders were included in the sample, which was ensured by examination of the same laboratory measures and monitoring of vital signs as in patients. All control subjects were in good physical condition and free of medication.
Study design and blood collection
Unstimulated peripheral blood samples were collected at 6 pm after 2 h of fasting and abstention from caffeine-containing beverages and physical activity, then 1.5 mg dexamethasone was administered orally. At 7 pm, patients and controls received a standard meal at the hospital. The second blood draw was at 9 pm. At each timepoint whole blood RNA was collected using a PAXgeneTM (Qiagen, Hilden, Germany) whole blood RNA collection tube, as well as serum and whole blood to analyze differential blood count, cortisol and ACTH levels as well as liver enzymes. PAXgeneTM tubes were stored for at least 2.5 h at room temperature, and then transferred to −20°C. In the samples taken at 9 pm plasma dexamethasone concentration was assessed using LC-MSMS on an API4000 (AB Sciex, Framingham, MA, USA). For patients, this test was administered within the first 5 days of in-patient treatments (on average on day 3.4 (SD = 1.1).
RNA extraction and quality assessment
The total RNA was isolated from PAXgeneTM whole blood samples according to the manufacturer's instructions. Samples were processed using the PAXgeneTM Blood RNA Kit with the Qiagen method for column purification of nucleic acids (PreAnalytiX GmbH, Hombrechtikon, Switzerland). Total RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). All samples had an RNA integrity number (RIN) ≥7 (7.0–8.7, mean: 8.1 ± SD: 0.4). Concentration and purity of the total RNA with 260 nm UV absorption and with 260/280 ratios were assessed, respectively (Nanophotometer, Implen, Munich, Germany).
The total RNA was reverse-transcribed using random primers and the Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. Relative FKBP5 mRNA expression levels were measured using TATA-binding protein (TBP) and ribosomal protein L7a (RPL7A) as reference genes and the delta Cq method using the LinRegPCR program (Ruijter et al. 2009) to estimate the crossing thresholds. FKBP5 expression levels were normalized against the mean expression levels of TBP and RPL7A in each sample. qPCR experiments were performed using the Roche 480 LightCycler (Roche Applied Science). qPCR assays were designed using the Roche universal probe library (UPL) (http://qpcr.probefinder.com/organism.jsp) (Table S1). All samples were run in duplicates, and duplicates discordant in crossing points by more than 0.2 cycles were excluded from the analysis. Results are reported in fold change using 2(−delta-delta Cq).
DNA preparation, SNP selection and genotyping
Ethylenediaminetetraacetic acid (EDTA) blood (40 ml) was drawn from each subject. DNA was extracted from fresh blood using the Puregene® whole blood DNA-extraction kit (Gentra Systems Inc., Minneapolis, MN, USA). SNP rs1360780 was genotyped using a hybridization probe-based assay on the light cycler device (Roche Applied Science). This was selected as the tag SNP for the reported association of an FKBP5 haplotype with psychiatric disorders as recent data from our lab, suggesting that these polymorphisms might be the functionally relevant variants (Klengel et al. personal communication). All primer sequences and assay protocols are available upon request. There was no deviation from Hardy–Weinberg equilibrium (HWE) for this SNP in the case or the control group (P > 0.05).
Power calculation was conducted using G*Power 3.1.5 (University of Kiel, Germany) applying a post hoc design for repeated measurements analysis.
Statistical analysis of genetic associations
General linear models (GLMs), with and without repeated measures analysis, were used to test the effects of case status, FKBP5 genotypes (T-allele carrier model) and their interaction term on FKBP5 mRNA expression, cortisol and ACTH at baseline and after dexamethasone stimulation. Statistics are reported for the Greenhouse-Geisser test. All analyses were controlled for co-varying factors age, sex and dexamethasone levels when post-dexamethasone values were included in the analysis. These analyses were performed using SPSS for Windows (Releases 16, SPSS Inc., Chicago, IL, USA).
Baseline FKBP5 mRNA expression
A GLM showed no main effects of disease status, FKBP5 genotype, age or sex, nor a significant interaction effect of disease status and FKBP5 genotype on baseline FKBP5 mRNA expression.
Stimulated FKBP5 mRNA expression
Using a repeated measures GLM and adjusting for sex and age, we identified a main effect of disease status (between subjects effects F1,612 = 6.78; P = 0.01) (see also Fig. 1) as well as a genotype × disease status interaction (within subjects contrasts F1,612 = 5.58; P = 0.02) on the change in mRNA expression from 6 pm and 9 pm. There were also significant contrasts of dexamethasone stimulation and sex (within subjects contrasts F1,612 = 113.52; P < 0.001 and F1,612 = 3.97; P = 0.048). Because male subjects are overrepresented in our study, we performed the analysis only with female subjects and still found a significant interaction effect between disease status and FKBP5 risk allele carrier status on GR-stimulated FKBP5 mRNA expression (F = 4.35; P = 0.043). The explained variance of the disease status alone was 0.015, and increased by adding the interaction with FKBP5 genotype to 0.043. The power to observe these effects with an estimated effect size of 0.222 in this sample was 56%.
Dexamethasone stimulation resulted in a distinct increase in FKBP5 expression in both controls and cases by more than 10-fold. While there were no significant differences between cases and controls in rs1360780 CC genotype carriers (FKBP5 expression increased by 11.9-fold ± 3.9- and 12.7-fold ± 4.3 in controls and cases respectively carrying the CC genotype), depressed cases carrying the T alleles showed a significantly lower FKBP5 mRNA induction than controls with the same genotype (12.7-fold ± 4.3 SD induction in controls and 10.5-fold ± 4.3 SD in cases (post hoc GLM: F1,308 = 10.9; P = 0.002)).
Influence of covariates
We repeated the GLM of the change in gene expression after dexamethasone in dependence of genotype adjusting for additional confounds including baseline cortisol, body mass index, smoking status, type of antidepressant, type of comedication, history of suicide attempt and dexamethasone plasma concentration (in a subset of patients). These covariates did not contribute significantly to the variance in gene expression.
FKBP5 SNP-dependent endocrine alterations
Using a repeated-measures GLM, we found a main effect of disease status (between subjects effects F1,924 = 4.22; P = 0.043) and an interaction effect of genotype × disease status on the cortisol response 3 and 21 h after GR stimulation (within subjects contrasts F1,924 = 4.27; P = 0.041) (Fig. 2). Depressed T-carriers displayed less suppressed cortisol levels in comparison with healthy subjects carrying the T allele. Age, sex or dexamethasone concentrations had no significant interaction or main effects. There was no significant difference between the cortisol levels at 9 pm of cases carrying CC (32.92 ± SE 3.8 ng/ml) and cases carrying the T allele (35.25 ± SE 4.9 ng/ml; P = 0.93). There was also no significant difference between the cortisol levels at baseline of cases carrying CC (82.57 ± SE 7.6 ng/ml) compared with cases carrying the T allele (84.06 ± SE 9.9 ng/ml; P = 0.96).
A repeated-measures GLM also showed a main effect of disease status (between subjects effects F1, 924 = 6.0; P = 0.016) as well as a trend for an interaction of disease status and genotype (within subjects contrasts F1,924 = 3.32; P = 0.071) on ACTH levels following dexamethasone suppression. T-carriers suffering from depression showed elevated ACTH levels compared with control subjects. Sex (between subjects effects P < 0.05), but not age and dexamethasone concentration, had a significant effect on ACTH levels over time (Fig. 2).
Clinical characteristics separated for genotype
To assess potential confounds of this analysis owing to clinical variables known to affect hypothalamic-pituitary-adrenal (HPA)-axis regulation, we compared clinical characteristics of patients according to the FKBP5 rs1360780 T-allele carrier status. Table 1 shows the disease-related and sociodemographic variables of this sample. There was a significant association of FKBP5 genotype with previous suicide attempts. A higher proportion of patients carrying the risk allele had a history (37.5%; P = 0.018) of suicide attempts with an overall higher number of past attempts (mean number = 0.6; P = 0.08) than patients carrying the CC genotype (11.4% and 0.1 respectively). Adding history of suicide attempts as a covariate did not alter the previously reported disease status × FKBP5 SNP interactions. The other sociodemographic and clinical factors listed in Table 1 were not different between the groups. There were also no differences in known confounds of RNA and endocrine measures including age, gender, BMI or smoking status between genotype groups. We also found no correlations between severity of depression as measured with the HAMD with cortisol and ACTH levels at 6 pm and 9 pm and FKBP5 mRNA at 6 pm and 9 pm.
Table 1. Sociodemographic and clinical characteristics of depressed patients and healthy controls separated for FKBP5 genotype
n = 39
n = 29
n = 47
n = 40
Patients vs. controls
CC vs. T carrier
P values of Pearson X2 tests (qualitative data) and univariate analysis of variance (quantitative data) are reported.
BMI, body mass index; HAMD, Hamilton Depression Rating Scale; NARI, noradrenaline reuptake inhibitor; NASSA, noradrenergic and specific serotonergic antidepressant; SNRI, serotonin and noradrenaline reuptake inhibitor; SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressants.
HAMD score at admission
Age onset (years)
Previous episodes (N)
Recurrent depression (%)
Family history of MD (%)
Suicide attempts (%)
Number of suicide attempts
Family history of suicide (%)
Psychotic features (%)
Therapy resistance (%)
Alcohol abuse (%)
Benzodiazepine abuse (%)
Medication at RNA withdrawal
Mood stabilizer (%)
Using an in vivo GR stimulation with dexamethasone, we found evidence that FKBP5 rs1360780 genotype status may define neuroendocrinologically distinct subtypes of major depression. The often reported impaired GR sensitivity in major depression (Holsboer 2000; Pariante & Miller 2001; Vreeburg et al. 2009) was observed only in patients carrying the FKBP5 risk allele, i.e. the minor rs1360780 T allele. This was seen using both FKBP5 mRNA induction in peripheral blood cells as well as plasma cortisol levels as a read out of GR function. These findings might explain why a perturbed HPA axis is not consistently found in patients suffering from major depression (Anacker et al. 2011; Carpenter et al. 2009; Menke et al. 2012) and supports the notion that patients with depression are different regarding underlying causal mechanisms.
While we observed genotype-dependent differences after GR stimulation, we did not observe such differences at baseline. This is in line with animal studies where transgenic mice lacking the FKBP5 gene did not exhibit significant alterations in exploratory behavior or endocrine measurements at baseline, but only after exposure to acute or chronic stressors (Hartmann et al. 2012; Touma et al. 2011).
After stimulation with dexamethasone, we observed differences in both FKBP5 mRNA induction as well as the suppression of plasma cortisol levels, both in the direction that depressed FKBP5 rs1360780 T-allele carriers, but not carriers of the protective CC genotype display GR resistance as compared with healthy controls. FKBP5 mRNA is induced by stress, including restrained stress and food deprivation in a number of tissues, including peripheral blood cells and brain and glucocorticoids mediate this effect (Menke et al. 2012,b; Scharf et al. 2011). In fact, FKBP5 mRNA induction has been proposed as a bioassay for an individual's sensitivity to glucocorticoids (Vermeer et al. 2004) and indeed, in genome-wide gene expression experiments, we could show that reduced FKBP5 mRNA induction by dexamethasone is among the 19 transcripts that most robustly predict depression status in a case/control study (Menke et al. 2012).
The fact that FKBP5 risk alleles are associated with GR resistance has been reported in a number of different studies with healthy individuals. After exposure to a psychosocial stressor, an insufficient recovery of cortisol activation was seen in a sample of healthy individuals carrying the rs1360780 risk genotype (Ising et al. 2008). Healthy volunteers with the same genotype also showed impaired dexamethasone suppression (Touma et al. 2011). On the other hand, the FKBP5 rs1360780 risk allele was associated with a reduced cortisol area under the curve sampled over 1 day in a cohort of elderly individuals (Velders et al. 2011), suggesting that there may be differential effects of the FKBP5 genotype on baseline vs. stimulated endocrine parameters. In contrast to previous studies (Binder et al. 2004, 2008; Touma et al. 2011) we did not observe genotype-dependent differences in either FKBP5 mRNA expression or cortisol levels after dexamethasone stimulation in the control group per se. This may be related to differences in the timing (11 pm vs. 6 pm) and dosage (1.5 vs. 0.5 mg) of dexamethasone administration. More detailed time and dose-response curves, possibly using ex vivo experiments, will be necessary to better characterize the effects of the FKBP5 genotype on dexamethasone-induced FKBP5 mRNA induction and cortisol suppression. In addition, differential effects of dexamethasone and the endogenous corticosteroids induced by stress exposure will need to be explored as they may have different effects on GR receptor activation.
Opposite to the findings presented here, we had previously reported that depressed patients carrying the FKBP5 risk alleles displayed a less resistant GR, as indicated by the more pronounced cortisol suppression after dexamethasone (Binder et al. 2004). In this previous sample, the dexamethasone suppression test was administered within the first 10 days of in-patient treatment, while in this study, the test was administered within the first 5 days of in-patient treatment and on average on day 3 after in-patient admission. As the same FKBP5 genotype has also been shown to be associated with better antidepressant treatment response (Binder et al. 2004; Lekman et al. 2008) and response to antidepressant treatment is associated with a normalization of GR function (Binder et al. 2009; Ising et al. 2007), we speculate that the difference in GR sensitivity in depressed patients carrying the risk allele is likely dependent on the timing after in-patient admission. Even if the difference in treatment duration between the two studies is not very large, the effect of antidepressants on GR mRNA levels has been shown to occur as early as 2.5 h after treatment (Heiske et al. 2003).
Finally, when investigating the effects of FKBP5 genotype on low-dose dexamethasone suppression in patients with PTSD, we observed an enhanced GR sensitivity in PTSD patients with the FKBP5 risk genotypes (Mehta et al. 2011), an effect opposite to the one observed among depressed patients in the sample reported here. This may be related to methodological differences in the suppression test – low dose vs. higher dose of dexamethasone, but could also be related to epigenetic effects of trauma exposure or type of disease on FKBP5 DNA methylation. In fact, exposure to glucocorticoids has been shown to lead to a demethylation of specific CpG dinucleotides flanking enhancer glucocorticoid response elements within the fkbp5 locus in mice, both in peripheral blood cells as well as neuronal and pituitary tissue (Lee et al. 2010, 2011; Yang et al. 2012). One might thus speculate that a different trauma history, i.e. different lifetime exposure to glucocorticoids, could lead to epigenetic differences in the FKBP5 locus and thus differential genotype effects (Klengel et al. 2013). A limitation of our study is the absence of data on previous trauma exposure, so that we could not consider any possible gene × environment interactions impacting the endocrine and molecular phenotypes. Another limitation is that we enrolled only Caucasians in our study. However, the effects of FKBP5 polymorphisms on other endocrine parameters and gene × environment interactions have been observed in both African-American as well as Caucasian samples (Appel et al. 2011; Binder et al. 2004, 2008; Ising et al. 2008; Klengel et al. 2013; White et al. 2012; Xie et al. 2010; Zimmermann et al. 2011), and we have identified rs1360780 as a potential causal polymorphism, leading to differences in chromatin conformation, suggesting that these effects should be observed across different ethnicities (Klengel et al. 2013).
Clinical variables such as bipolar depression, recurrent depression or suicide attempts might also impact the HPA axis (Lok et al. 2012; Manenschijn et al. 2012; Mcgirr et al. 2011) and could thus confound the interaction result. The only FKBP5 genotype-dependent clinical difference was an increase in previous suicide attempts in risk allele carriers. However, this did not confound the observed interactions on endocrine and molecular measures.
In summary, FKBP5 polymorphisms seem to impact the extent of HPA-axis dysregulation in patients with major depression. While divergent results on the direction of the effects in controls, depressed patients and patients with PTSD have been reported, all studies indicate that the FKBP5 genotype, possibly in interaction with a stressor or trauma exposure, is associated with different patterns of neuroendocrine dysregulation in stress-related psychiatric disorders. Larger, independent studies characterizing the timecourse of these interactions and possible influences of epigenetic factors are needed for a better understanding of this phenomenon.
The authors would like to thank A. Sangl, M. Asmus, G. Ernst-Jansen, E. Kappelmann, M. Hartung and B. Siegel for their excellent technical assistance. The study was supported by a grant of the Exzellenz-Stiftung of the Max Planck Society. This work has also been funded by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN), FKZ 01GS0481.