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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 2 565-573
Copyright © 2004 by The Endocrine Society

Special Feature

Common Polymorphisms in the Glucocorticoid Receptor Gene Are Associated with Adrenocortical Responses to Psychosocial Stress

Stefan Wüst, Elisabeth F. C. van Rossum, Ilona S. Federenko, Jan W. Koper, Robert Kumsta and Dirk H. Hellhammer

Department of Psychobiology (S.W., I.S.F., R.K., D.H.H.), University of Trier, Johanniterufer 15, 54290 Trier, Germany; and Department of Internal Medicine (E.F.C.V.R., J.W.K.), Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Stefan Wüst, Ph.D., Department of Psychobiology, University of Trier, Johanniterufer 15, 54290 Trier, Germany. E-mail: wuest{at}uni-trier.de.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Chronic dysregulation of hypothalamus-pituitary-adrenal axis activity is related to several stress-related disorders. Evidence suggests that polymorphisms in the glucocorticoid receptor (GR) gene may have an impact on this neuroendocrine system.

In the present investigation, 112 healthy males were studied to estimate the impact of three GR gene polymorphisms (BclI RFLP, N363S, ER22/23EK) on cortisol and ACTH responses to psychosocial stress (Trier Social Stress Test) and pharmacological stimulation (1 µg ACTH1–24, 0.5 mg dexamethasone).

Because only four ER22/23EK heterozygotes were identified, these subjects were not statistically analyzed. Compared with subjects with the wild-type GR genotype (n = 36), 363S allele carriers (n = 10) showed significantly increased salivary cortisol responses to stress, whereas the BclI genotype GG (n = 18) was associated with a diminished cortisol response. BclI heterozygotes and homozygotes (GG) exhibited a trend toward lower ACTH responses, compared with wild-type subjects and 363S carriers. The cortisol response to ACTH1–24 administration was not significantly different between genotypes. After dexamethasone ingestion, 363S carriers showed a trend toward an enhanced cortisol suppression.

This is the first report documenting an impact of GR gene polymorphisms on cortisol (and perhaps ACTH) responses to psychosocial stress. These variants may contribute to the individual vulnerability for hypothalamus-pituitary-adrenal-related disorders.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
EXPOSURE TO PSYCHOLOGICAL or physiological stress causes a spectrum of autonomic, endocrine, and behavioral responses. A common endocrine feature of this stress response is the activation of the hypothalamus-pituitary-adrenal (HPA) axis including an increase of its end product cortisol, which is the most important glucocorticoid (GC) in humans. Cortisol affects a multitude of systems in the body, including the HPA axis itself, the cardiovascular system, the immune system, metabolism, and cell growth, and it also has an impact on behavior. Because chronic dysregulation of HPA activity seems to be associated with the onset and course of several psychosomatic and psychiatric disorders (1, 2, 3, 4, 5, 6, 7, 8), it is important to identify the determinants of the marked interindividual and intraindividual variation in HPA responses to challenge.

The glucocorticoid receptor (GR) is a member of the steroid receptor superfamily. It mediates many of the effects of GCs on target tissues via direct binding to hormone-responsive elements in the DNA or via interactions with other transcription factors resulting in a modulation of gene transcription (9, 10, 11). A cell’s response to GCs is predominantly determined by both the steroid level it is exposed to and by its GC sensitivity, i.e. the efficiency of GR-mediated signal transduction (9). Evidence from recent studies suggests that variants of the GR gene (located on chromosome 5, locus 5q31) that affect a cell’s sensitivity for GCs are important for rare clinical conditions like the generalized GC resistance syndrome (12, 13, 14, 15) but also may contribute significantly to the large interindividual variability of HPA activity and GC sensitivity of target tissues in normal, nonclinical populations (11).

A common BclI restriction fragment length polymorphism (RFLP) in the GR gene presumably located in intron 2 (16) has been found to relate to indices of insulin resistance in obese women (17), abdominal visceral fat areas in slightly obese women and men (18), dermal blanching after GC exposure (19), and body mass index (BMI), waist to hip ratio (WHR), leptin, and cortisol responses to a standardized lunch (20). Moreover, an interaction between the BclI RFLP and overfeeding has been reported on outcomes such as body weight, low-density lipoprotein cholesterol, and abdominal visceral fat (21). Another variant is the single nucleotide polymorphism N363S, which is an AAT-to-AGT point mutation in exon 2 causing an asparagine to serine amino acid change in codon 363 (22). The 363S allele has been associated with a higher BMI (23), a higher WHR (24), enhanced cortisol suppression, as well as an increased insulin response after dexamethasone administration and a tendency toward decreased bone mineral density in the lumbar spine (25). Yet another polymorphism has been described in exon 2 (22), and it comprises two point mutations in codons 22 and 23 that are separated by 1 bp. The mutation in codon 22 is silent (GAG to GAA change, both coding for glutamic acid), whereas the codon 23 change from AGG to AAG results in an amino acid change from arginine to lysine (ER22/23EK). In a recent study, the 22/23EK allele has been reported to relate to a decreased cortisol suppression in response to dexamethasone administration in elderly subjects (26). Interestingly, in a subgroup, aged 67–82 yr, the number of 22/23EK carriers was significantly higher than in younger subjects (53–67 yr).

Additional polymorphisms are the Tth111I RFLP in the 5' untranslated region of the GR gene (27) that has been associated with enhanced basal cortisol levels (28), and a recently identified polymorphism in exon 9ß has been found to relate to rheumatoid arthritis (29). Most of the remaining polymorphisms in the GR gene that have been detected are either silent, have a very low frequency, or did not show an association with relevant phenotypes in previous studies (30).

Although these findings document the functional relevance of several GR gene polymorphisms, knowledge on their influence on HPA responses to stimulation is very limited. For psychobiological research it is of particular interest to study the impact of these genetic variants on HPA responses to psychological stress.

Thus, the present study was conducted to estimate the impact of three GR gene polymorphisms, namely the BclI RFLP, the N363S, and the ER22/23EK, on cortisol and ACTH responses to pharmacological stimulation and psychosocial stress. The ACTH1–24 challenge and dexamethasone suppression tests provided the pharmacological stimuli, whereas the Trier Social Stress Test (TSST) (31) was repeatedly used for the application of acute psychosocial stress.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The present study sample consisted of 112 young males (mean age, 18.9 yr; SEM, ±23 yr), with a mean BMI of 21.84 ± .27. Females were not included in the study because the experimental procedure (see below) comprised three exposures to psychosocial stress within 3 wk and cortisol responses to the TSST in women are known to be modulated by the menstrual cycle (32). All 32 subjects who were smokers (mean cigarette consumption per day: 9.5 ± 1.07) agreed to refrain from smoking on test days, and all subjects reported to be medication free. Before the first experimental session, the absence of acute or chronic diseases was confirmed in a medical exam. Because one objective of the investigation was to perform a heritability analysis of HPA responses (not presented in this report) all subjects were twins. As confirmed by DNA fingerprint analysis, the sample consisted of 33 monozygotic and 23 dizygotic twin pairs. The protocol was approved by the ethics committee of the Rheinland-Pfalz State Medical Association, and written informed consent was obtained from all participants.

Experimental procedures

The study protocol comprised five test days. Test d 1–4 each were separated by a 1-wk interval, and subjects reported to the laboratory in the late afternoon. Subjects were instructed to refrain from physical exercise and larger meals at least 90 min before onset of the experiment. Forty-five minutes before the challenge tests, an iv catheter was inserted in an antecubital vein and kept patent with a lock. Each participant was exposed to four stimulation procedures at identical times across test days between 1600 and 1700 h. Daytimes were controlled to avoid baseline level or reactivity differences due to the circadian rhythm of HPA axis activity. On the first day, volunteers received an iv injection of 1 µg Synacthen (Novartis, Nuernberg, Germany, low-dose ACTH1–24 stimulation test). On test d 2, 3, and 4, the TSST was performed. The TSST mainly consists of a free speech and a mental arithmetic task of 15 min duration performed in front of an audience and a camera. This stress protocol has been found to induce significant cortisol, ACTH, and cardiovascular responses at the first exposure in 70–80% of all subjects. In a number of previous studies, the TSST has proven to be a valuable tool for psychobiological research. Adrenocortical responses to the TSST have been shown to relate to gender (33, 34), personality traits (35), early life stress (36), and atopic dermatitis (2, 37). The speech topics and arithmetic tasks were slightly modified at each TSST protocol administration. Last, a low-dose dexamethasone suppression test (DST) was performed on test d 5. The participants were instructed to ingest 0.5 mg dexamethasone (Fortecortin, Merck, Darmstadt, Germany) at 2300 h, and they reported to the laboratory the next morning between 0730 and 0800 h. During this visit one blood sample was collected. The time interval between test d 5 and d 1–4 was at least 48 h.

Blood and saliva sampling

On TSST days, blood samples were repeatedly obtained 2 min before and 15, 25, 35, and 105 min after onset of the TSST. Saliva samples (obtained with the Salivette sampling device, Sarstedt, Nümbrecht, Germany) were collected at the same times as blood samples, and in addition at 45, 60 and 75 min for the assessment of free cortisol levels. The low-dose ACTH1–24 stimulation test involved the collection of saliva samples 2 min before and 15, 30, 45, 60, and 90 min after drug injection. The day after dexamethasone ingestion, subjects were instructed to collect saliva samples at 0800, 1100, 1500, and 2000 h. Between 0730 and 0800 h, one blood sample was obtained to assess ACTH and dexamethasone levels. Blood samples were immediately stored on ice and they were spun within 30 min at 2000 x g and 4 C for 10 min. EDTA plasma was removed and stored at -20 degree C until analysis. Saliva samples were kept at room temperature throughout one test session and then stored at -20 C. After thawing for biochemical analysis, samples were spun at 2000 x g at 10 C for 10 min.

Biochemical analyses

Salivary cortisol was analyzed with a time-resolved immunoassay with fluorescence detection (DELFIA) as described in detail elsewhere (38), and ACTH was measured with a two-site chemiluminescence assay (Nichols, Bad Nauheim, Germany). Intra- and interassay variability of both assays was less than 10 and 12%, respectively. Plasma dexamethasone was assessed with an in-house RIA at the Institute of Pharmacology, University of Heidelberg. Intra- and interassay variability for this analysis was 15.6 and 21.6%, respectively.

Determination of genotypes

DNA of all subjects was extracted from 10 ml peripheral venous blood employing kits (Qiagen Inc., GmbH, Germany). In previous studies (20, 21), the BclI RFLP was detected by Southern blotting of BclI-digested total genomic DNA. The RFLP was assumed to be caused by a polymorphic BclI site in intron 2 of the GR gene. From the published sequence (GenBank NT_030707) we identified three BclI sites around exon 2 of the GR gene, located 392 base pairs upstream of exon 2, 646 bp downstream of exon 2 and 2301 bp downstream of exon 2, respectively. PCR-RFLP and sequence analysis showed that the second site (646 bp downstream of exon 2) was indeed polymorphic (TGATCA -> TGATGA), and showed allelic frequencies comparable with those reported previously for the BclI polymorphism (39). The C allele and the G allele correspond to the 2.3- and 4.5-kb alleles, respectively, as originally described (17). In view of the nomenclature of the BclI polymorphism in the recent literature, we refer to it here by that name, rather than by "IVS2 + 646". Genotyping for all three polymorphisms was performed using the allelic discrimination technique on a sequence detection system (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands) according to the protocol supplied by the manufacturer. Primers and probes that were used are shown in Table 1Go and were obtained, together with the Universal Master Mix, from Applied Biosystems.


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  		TABLE 1. Primers and probes used for allelic discrimination

 
Statistical analysis

Two-way ANOVAs for repeated measures (group by time) were computed to assess cortisol and ACTH response patterns as well as response differences between genotypes in the TSST, the ACTH1–24 stimulation test, and the DST. Greenhouse-Geisser corrections were applied where appropriate, and only adjusted results are reported. To receive indices for cortisol and ACTH increases, delta values were defined as the difference between individual peak values and baseline levels. One-way ANOVAs were performed to compare mean deltas, mean baseline levels, and mean plasma ACTH levels after dexamethasone administration between experimental groups. Post hoc tests (Tukey-honestly significant difference) were computed when ANOVA procedures revealed significant effects. A {chi}2 test was computed to assess whether the ratio of smokers to nonsmokers differed significantly between the experimental groups. All results shown are the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
For the BclI polymorphism in the GR gene, allelic discrimination analysis identified 48 subjects homozygous for the C allele, 18 homozygous for the G allele, and 46 heterozygotes (Table 2Go). The resulting allelic frequencies (number of concerned alleles divided by total number of alleles) of 0.63 for the C allele and 0.37 for the G allele correspond with previous reports (17, 18, 20). Ten CC carriers were additionally either heterozygous (AG, n = 8) or homozygous (GG, n = 2) 363S carriers. Furthermore, six subjects with the BclI genotype CG were also heterozygous for the N363S variant of the GR gene. With 0.08 the allelic frequency of the 363S allele was slightly higher than the frequency of 0.03 found by Huizenga et al. (25), whereas Lin et al. (23) observed a similar frequency (0.07). Only four of 112 participants were identified as heterozygotes for the ER22/23EK polymorphism (two BclI CC subjects and two BclI heterozygotes; allelic frequency: 0.02). Neither ER22/23EK homozygotes nor subjects homozygous for the BclI G allele who were also 363S or ER22/23EK carriers could be observed in the present study.


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  		TABLE 2. Frequencies of the BclI, the N363S, and the ER22/23EK polymorphism in the glucocorticoid receptor gene

 
Due to the small subgroup sizes, the four ER22/23EK carriers as well as the six subjects heterozygous for the BclI and heterozygous for the N363S polymorphism were not included into the statistical analyses except for some observations on group, BclI G heterozygote & N363S heterozygote, that are reported on a descriptive level. The eight heterozygous (AG) and the two homozygous (GG) 363S carriers (who showed the BclI genotype CC) were combined to one experimental group because they did not differ in any of the reported cortisol or ACTH responses. Thus, the following four GR genotypes were statistically compared: 1) BclI CC and N363S AA (wild type, n = 36; the term wild type was chosen to enhance the comprehensibility of the text, and this decision was solely based on the fact that the BclI C allele and the N363S A allele showed the highest frequencies); 2) BclI CC and N363S AG or GG (363S carrier, n = 10); 3) BclI CG and N363S AA (BclI G heterozygote, n = 38); and 4) BclI GG and N363S AA (BclI G homozygote, n = 18). All observed GR genotypes are shown in Table 2Go.

The ratio of smokers (n = 32) to nonsmokers was not significantly different between the experimental groups. Eleven of 36 subjects were smokers in group wild type, three of 10 in group 363S carrier, 11 of 38 in group BclI G heterozygote, and four of 18 in group BclI G homozygote ({chi}23 = 4.38, P > 0.90). The remaining three smokers belonged to the subjects who were not included into the statistical analyses.

Mean salivary cortisol responses to the first TSST exposure are shown in Fig. 1AGo. Cortisol baseline levels were not significantly different among the four experimental groups, and similar results were obtained for all cortisol and ACTH baseline level comparisons reported in the present paper (one-way ANOVAS; all P > 0.20). As expected, an overall increase of cortisol levels after application of psychosocial stress was observed; ANOVA results indicated a significant main effect for the repeated-measures factor time (F2.94; 197.0 = 28.61, P < 0.001). Furthermore, cortisol responses differed significantly between experimental groups (time by group: F8.82; 197.0 = 1.98, P < 0.05). In participants with the wild-type GR genotype, peak concentrations were observed 10 min after stress cessation and the delta (peak minus baseline level) was 7.50 ± 1.67 nmol/liter. 363S carriers, however, showed a considerably larger delta of 19.25 ± 6.2 nmol/liter, although baseline levels were almost identical (11.62 and 10.91 nmol/liter, respectively). On average, 363S carriers reached their cortisol peak levels 30 min after stress cessation. In contrast, a notably attenuated mean salivary cortisol response was observed in BclI G homozygotes. Their delta of 5.76 ± 1.67 nmol/liter was the smallest of all four groups. Participants who were genotyped as BclI G heterozygotes reached their peak cortisol levels 10 min after termination of the TSST and showed a mean delta of 7.95 ± 2.5 nmol/liter. One-way ANOVA revealed a significant difference between delta measures in the four groups (F3; 76 = 2.96, P < 0.05). Post hoc tests (Tukey-honestly significant difference) results indicated that 363S carriers had a significantly larger mean delta, compared with wild-type subjects and BclI G homozygotes, respectively (both P < 0.05); in addition, delta levels in group 363S carrier showed a trend toward higher values, compared with group BclI G heterozygote (P = 0.06).



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  		FIG. 1. Salivary cortisol responses (±SEM) to the first stress exposure (A) and average responses (±SEM) to all three stress exposures (B) in the four experimental groups.

 
Next, the average free cortisol response to all three TSST exposures was computed for each participant. The resulting mean cortisol reactions in the four experimental groups are shown in Fig. 1BGo. It should be noted that a separate analysis of each TSST session revealed the expected habituation of cortisol responses across exposures, resulting in significantly decreased responses to the TSST on d 2 and 3 (main effect day: F1.65; 293.57 = 10.28, P < 0.001; not shown in a figure). However, mean cortisol responses across three TSST exposures still showed a significant overall increase (main effect time: F2.66; 143.56 = 41.14, P < 0.001). Corresponding with the adrenocortical reactions to the first exposure, the averaged free cortisol responses varied significantly between groups (time by group interaction: F7.98; 143.56 = 2.54, P < 0.05). Furthermore, delta measures as well were again significantly different between genotypes (one-way ANOVA: F3; 69 = 3.47, P < 0.05). On one hand, compared with subjects with the wild-type GR genotype (delta: 5.28 ± 1.07 nmol/liter), the mean increase was not significantly different in BclI G heterozygotes (delta: 5.67 ± 1.52 nmol/liter) and BclI G homozygotes (2.87 ± .97 nmol/liter), respectively. However, on the other hand, with a delta of 12.18 ± 3.89 nmol/liter, the largest increase was again observed in 363S carriers, and this response was significantly larger than mean delta values in BclI G homozygotes (P < 0.05), whereas compared with wild-type subjects (P = 0.06) and BclI G heterozygotes (P = 0.09), a trend toward higher values could be detected.

Because only six subjects showed the genotype BclI G heterozygote and N363S heterozygote, their cortisol responses to the TSST were analyzed only on a descriptive level. Before the first TSST exposure, these subjects showed a mean baseline level of 11.92 ± 2.40 nmol/liter. The cortisol peak level of 16.57 ± 3.06 nmol/liter was slightly higher than in BclI G homozygotes (15.59 ± 1.62 nmol/liter), slightly lower than in wild-type subjects (19.28 ± 3.02 nmol/liter), and it was reached 20 min after stress cessation. Across all three TSST exposures group BclI G heterozygote and N363S heterozygote showed a mean baseline level of 11.01 ± 1.23 nmol/liter. The cortisol peak concentration of 14.82 ± 2.72 nmol/liter was observed 10 min after termination of the TSST and was again somewhat higher than in BclI G homozygotes (11.36 ± 1.37 nmol/liter) and marginally lower than in wild-type subjects (15.97 ± 1.63 nmol/liter).

Mean ACTH reactions to the first TSST exposure in the four experimental groups are shown in Fig. 2AGo, and responses across all three stress sessions are depicted in Figure 2BGo.



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  		FIG. 2. Plasma ACTH responses (±SEM) to the first stress exposure (A) and average responses (±SEM) to all three stress exposures (B) in the four experimental groups.

 
After the first TSST exposure, ACTH levels showed the expected significant rise (main effect time: F1.8; 158.27 = 96.58, P < 0.001), whereas the time by group interaction just missed statistical significance (F5.36; 158.27 = 2.03, P = 0.07). All four groups reached their ACTH peak concentrations immediately after stress cessation and in wild-type subjects, a mean delta of 31.51 ± 5.3 pg/ml was observed. Again, the largest delta could be detected in 363S carriers (43.61 ± 8.40 pg/ml), but although this increase was approximately twice as large as the deltas in BclI G heterozygotes and BclI G homozygotes (20.02 ± 4.27 pg/ml and 22.46 ± 4.33 pg/ml, respectively), ANOVA revealed only a trend toward higher values (F3; 90 = 2.51, P = 0.06).

In correspondence with free cortisol reactions, mean ACTH responses significantly decreased across the three TSST exposures (main effect day: F1.62; 384.53 = 10.56, P < 0.001; not shown in a figure), indicating a habituation of pituitary responses to the experimental procedure. But again, mean hormone levels still varied significantly over time after TSST exposures (F1.4; 116.15 = 111.09, P < 0.001 for the main effect time). Delta levels in wild-type subjects (15.74 ± 2.74 pg/ml) were again (slightly) lower than in 363S carriers (24.86 ± 4.57 pg/ml) and very similar to the increases in BclI G heterozygote (15.76 ± 3.88 pg/ml) and BclI G homozygotes (14.92 ± 2.61 pg/ml), respectively. Neither a significant time by group interaction [F4.2; 116.15 = 0.91, not significant (n.s.)] nor a significant difference between groups’ delta levels (F3; 84 = 0.86, n.s.) could be detected.

Interestingly, in BclI G homozygotes, mean ACTH levels showed a significant increase from baseline to peak concentrations (t17 = -5.7, P < 0.001; Fig. 2BGo), whereas no such significant response across all three TSST exposures was observed for salivary cortisol levels (t15 = -1.17, n.s.; Fig. 1BGo).

As shown in Fig. 3Go, the administration of 1 µg ACTH1–24 was followed by the expected significant increase of free cortisol levels (F2.61; 195.85 = 102.00, P < 0.001 for the main effect time). Mean peak concentrations were reached 60 min after injection in wild-type participants and 15 min earlier in the other three groups. A significant time by group effect could not be detected (F7.83; 195.5 = 0.85, n.s.) and one-way ANOVA comparison of delta levels did also not reveal a significant difference (F3; 80 = 0.48, n.s.). Nevertheless, it is worth mentioning that again 363S carriers showed the largest delta and BclI G homozygotes the smallest, with a mean difference of more than 10 nmol/liter.



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  		FIG. 3. Salivary cortisol responses (±SEM) to 1 µg ACTH1–24 in the four experimental groups.

 
After ingestion of 0.5 mg dexamethasone at 2300 h, plasma dexamethasone levels the following morning were not statistically different in the experimental groups and varied between 146.69 ± 11.60 ng/100 ml (363S carriers) and 168.37 ± 15.73 ng/100 ml (BclI G homozygotes; F3; 94 = 0.5, n.s.). Individual plasma levels confirmed that all subjects ingested the dexamethasone tablet. As shown in Fig. 4AGo, salivary cortisol levels in all four groups were distinctively suppressed at 0800 h with mean levels between 2.72 ± .73 and 4.06 ± .70 nmol/liter. Under similar conditions but without dexamethasone intake at 0800 h, a mean cortisol level of 19.05 nmol/liter was observed in 126 healthy males (Wüst, S., I. S. Federenko, and D. H. Hellhammer, unpublished data). Interestingly, in 363S carriers as well as in BclI G heterozygotes, mean free cortisol levels were still suppressed at 2000 h, whereas a trend toward a cortisol increase in the evening was detected in wild types and BclI G homozygotes. In the latter two groups, 2000 h mean cortisol values (5.59 ± 1.11 and 5.88 ± 1.13 nmol/liter, respectively) were almost identical with those in subjects without dexamethasone treatment (mean cortisol level of 5.97 nmol/liter in the above-mentioned 126 males). However, despite this difference in evening values, the time by group interaction was only marginally significant (F6.65; 124.04 = 1.90, P = 0.08). Furthermore, plasma ACTH levels in the morning were also not significantly different between groups (F3; 92 = 0.82, n.s.; Fig. 4BGo). On a descriptive level, however, it is worth noting that again the largest difference was observed between 363S carriers (10.69 ± 2.36 pg/ml) and BclI G homozygotes (16.53 ± 3.25 pg/ml).



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  		FIG. 4. Salivary cortisol levels (±SEM) at 0800, 1100, 1500, and 2000 h (A) and morning plasma ACTH levels (0730–0800 h, ±SEM, B) after ingestion of 0.5 mg dexamethasone in the four experimental groups.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The objective of this study was to contribute, from the perspective of psychobiological stress research, to the understanding of the relevance of GR gene polymorphisms that have previously been shown to be functional at the endocrine and metabolic level.

The most striking finding of the present investigation is the significant difference in salivary cortisol responses to psychosocial stress between the three genotypes. Compared with subjects with two wild-type alleles, 363S carriers showed a markedly larger cortisol response, whereas the mean response in BclI G homozygotes was attenuated. When average responses across all three stress exposures were computed, there was no significant elevation of cortisol levels in BclI G homozygotes, whereas the magnitude of the response in 363S carriers was even larger than in response to the first TSST in BclI G homozygotes.

This is, to the best of our knowledge, the first report documenting a significant impact of GR gene polymorphisms on cortisol responses to psychosocial stress. These results suggest that the 363S allele is related to increased responses to a psychosocial stress procedure, whereas the BclI genotype GG seems to be associated with a relative hyporeactivity. This finding is quite striking, given that on one hand a multitude of genetic and environmental factors influence a complex psychoneuroendocrine response, and that on the other hand, only subtle variations in a single gene were studied in a sample of healthy young men. Moreover, ACTH responses to the first psychosocial stress exposure were considerably larger in 363S carriers than in BclI G homozygotes. Although this difference was not statistically significant (P = 0.06; perhaps a function of the modest sample size), the trend is consistent with the cortisol findings.

Rosmond et al. (20) have reported elevated cortisol responses to a standardized lunch in BclI GG subjects, compared with CC subjects; however, in the present study, salivary cortisol responses to the TSST were not significantly different between these two genotypes. One possibility is that the impact of GR polymorphisms on cortisol responses may vary depending on the nature of the stimulus. This speculation is consistent with previous findings that have reported that HPA responses to different kinds of stimuli are not necessarily intra-individually consistent. For example, Kirschbaum et al. (40) observed no association between salivary cortisol responses to the TSST and responses to CRH administration, whereas ergometry-induced cortisol changes correlated significantly with the responses to both the pharmacological stimulation and the TSST. Intraindividually consistent HPA responses to physical challenge and psychological stress were also reported by Singh et al. (41), who found an association between ACTH responses to high-intensity exercise after pretreatment with dexamethasone and cortisol responses to the TSST. However, Kirschbaum et al. (42) did not find a significant association between ACTH responses to CRH administration, the TSST, and bicycle ergometry, whereas salivary cortisol responses were significantly correlated only between the CRH test and the ergometry test.

Compared with the striking cortisol differences in response to psychosocial stress, differences across genotypes in response to pharmacological stimulation were less evident. Although mean salivary cortisol responses to Synacthen were about 10 nmol/liter larger in 363S carriers than in BclI G homozygotes, this difference did not reach statistical significance.

Likewise, on a descriptive level, the difference of evening cortisol levels in response to dexamethasone between groups wild type and 363S carrier appears to be relatively large (although not statistically significant, P = 0.08). The speculation that the latter genotype may be associated with an increased sensitivity to dexamethasone is supported by the finding that this group also showed the lowest morning ACTH level. Possibly, this group difference may also occur during the day after administration of a lower dose of dexamethasone. In accordance with this argument, Huizenga et al. (25) found significantly lower morning cortisol levels in 363S carriers, compared with controls in a 0.25 mg but not in a 1 mg DST. This assumption may also explain why Rosmond et al. (43) did not detect an association between the N363S and cortisol suppression after dexamethasone administration because in this study the 0.5 mg DST was performed, and only morning cortisol levels were assessed.

The results of the DST remained inconclusive with respect to the BclI polymorphism. On one hand, homozygous carriers of the G allele showed a trend toward a decreased cortisol suppression in the evening compared with 363S carriers. On the other hand, mean evening cortisol levels in subjects heterozygous for the G allele were very similar to those in 363S carriers.

Although all the participants in this study were twin pairs, we would argue this is very unlikely to introduce any systematic bias to our present findings for the following reasons. First, there are no observable group differences for pituitary-adrenal responses to psychosocial stress or dexamethasone administration between our study population of twins and populations of nontwin individuals that we have previously investigated in other studies (44, 45, 46). Second, most of the twin pairs (i.e. all the monozygotic and 13 dizygotic twin pairs) were identical with respect to the GR polymorphisms assessed here and were consequently assigned to the same experimental group. Although there is greater degree of shared genetic and environmental influence on HPA function between twins than between nontwins, this could systematically bias the present findings only if the direction of the effects of the GR genotype are similar to those of other shared genetic and environmental influences on the measures of HPA function assessed here. However, there is no evidence to support a linkage between the GR genotype and other genetic and environmental influences on HPA function, thereby suggesting it is highly unlikely there is any form of systematic bias (either overestimation of underestimation of the effects).

Although several associations between HPA-related variables and the two GR polymorphisms BclI and N363S have been reported, the exact mechanisms through which they exert their effects are presently unknown. The BclI polymorphism is now identified as a C/G SNP in intron 2, 646 nucleotides downstream of exon 2 (39). It is located outside the coding region and has no obvious impact on processing of GR pre-mRNA. However, it is possible that this polymorphism is linked to functionally relevant variations in the promoter region or 3' untranslated region of the GR gene. Furthermore, the BclI (or N363S) polymorphism could also be linked to variations in the two previously unknown human GR exons (1A and 1B) or the two associated promotors that were recently discovered (47).

The N363S polymorphism is located within exon 2 and results in an asparagine to serine change. This N-terminal domain of the receptor modulates transcription activation, and hyperphosphorylation of serine residues could enhance glucocorticoid-regulated gene expression (48). However, in transfection assays, the capacity of the codon 363S variant to activate mouse mammary tumor virus promoter-mediated transcription in COS-1 cells was unaltered, when compared with the 363N variant (25). In addition, in the same study, GR number and ligand binding affinity in peripheral mononuclear leukocytes were not different between N363S heterozygotes and controls. However, there was a trend toward greater sensitivity to dexamethasone in the heterozygotes’ lymphocytes in a mitogen-induced cell proliferation assay.

The findings of the present study suggest that the genotypes 363S carrier and BclI GG have opposite effects on salivary cortisol (and perhaps ACTH) responses to the TSST. Other HPA-related variables, however, were not found to be inversely associated with these polymorphisms. For both genotypes no relation to basal cortisol secretion was detected and both were positively associated with BMI and WHR (20, 23, 24). One possible explanation for these seemingly inconsistent findings is the considerable tissue specificity of GR-mediated effects, which may result in different effects of GR polymorphisms in different cells. For example, Panarelli et al. (19) have reported an enhanced GC sensitivity of subdermal blood vessels (skin-blanching response to budenoside) in BclI GG subjects, whereas in the same group, peripheral leukocytes tended to have lower affinity and reduced sensitivity for dexamethasone. Moreover, in a recent study, no association between three bioassays for GC sensitivity was found, namely the skin-blanching response, the GC sensitivity of peripheral leukocytes and the cortisol suppression after dexamethasone administration (49). Although the molecular mechanisms underlying target tissue-specific effects are presently unclear, one very important observation is that through the usage of the three different promoters of the GR gene, three different exons 1 can be transcribed (1A, B, and C), and alternative splicing of exon 1A can result in three different versions of this exon. Alternative usage of these promoters could be an important mechanism for tissue specific expression of GR levels in different cell types (11, 47, 50). Another finding of the present study that could be related to the marked tissue specificity of GR-mediated effects is the significant increase of ACTH levels across all three stress exposures in BclI G homozygotes, whereas in this group no such significant response was observed for salivary cortisol levels. A highly speculative assumption could be that the effects mediated by the BclI polymorphism are more pronounced at the adrenal level and less distinct at the level of the pituitary.

The HPA axis is of major interest for psychobiological and psychiatric research. The regulation of this neuroendocrine system has been shown to relate to prenatal factors (51, 52); early life stress (36, 53); several cognitive functions (see Ref.7 for a recent review); and a variety of clinical disease states including chronic stress (1, 54, 55), posttraumatic stress disorder (3), depression (6, 56), psychosis (8), inflammatory diseases (2, 5, 37), and metabolic syndrome (4). Thus, it is important to further elucidate the sources of the marked interindividual variability of HPA (re)activity and identify subgroups with enhanced or decreased vulnerability for HPA-related disorders.

The present study adds strong evidence for an impact of common GR gene polymorphisms on HPA regulation. Our findings support the hypothesis that common polymorphisms in the GR gene may have modulating effects on the relation between psychological factors and HPA regulation. Moreover, they could contribute to the individual vulnerability for HPA-related clinical states. For instance, it was recently reported that short-term use of the GR antagonist mifepristone (RU 486) may be effective in the treatment of psychotic major depression (57). This finding is indicative of a pivotal role of GR functioning in the onset or course of psychotic major depression and suggests that polymorphisms in the GR gene could act as modulating factors.

Clearly, the HPA axis and the above-mentioned diseases are complex phenotypes, influenced by many genes and environmental conditions. Thus, most single genes probably account for only a small proportion of phenotype variability. However, in the present investigation, a significant impact of common polymorphisms of a single gene on a complex psychoneuroendocrine response was documented, despite the relatively modest sample size. Based on these encouraging results, several polymorphisms on different HPA-related genes are currently investigated in a larger sample to further elucidate their relevance from the perspective of psychobiological research.


   Acknowledgments
 
We are indebted to Doris Haack, Ph.D. (University of Heidelberg), for measuring plasma dexamethasone levels in this study. We thank Pathik Wadhwa, M.D., Ph.D. (University of California, Irvine), for his most helpful comments on the present manuscript.


   Footnotes
 
This work was supported by the German Research Foundation, Grant FOR 255/2-1.

Abbreviations: BMI, Body mass index; DST, dexamethasone suppression test; GC, glucocorticoid; GR, glucocorticoid receptor; HPA, hypothalamus-pituitary-adrenal; n.s., not significant; RFLP, restriction fragment length polymorphism; TSST, Trier Social Stress Test; WHR, waist to hip ratio.

Received July 3, 2003.

Accepted October 15, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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