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Polymorphisms in the Glucocorticoid Receptor Gene and Their Associations with Metabolic Parameters and Body Composition

Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands

	ABSTRACT

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

 
Most actions of glucocorticoids (GCs) are mediated by the glucocorticoid receptor (GR). The interindividual response to GCs varies considerably, as demonstrated by a variable suppressive response to 0.25-mg dexamethasone (DEX). Several polymorphisms in the gene coding for the GR have been described. It is unclear to what extent the observed response variability is due to GR polymorphisms or to other factors. However, at least three polymorphisms seem to be associated with altered GC sensitivity and changes in body composition and metabolic parameters. The N363S polymorphism has been associated with increased sensitivity to GCs, increased insulin response to DEX, a tendency towards lower bone mineral density, and increased body mass index (BMI). However, other reports found no associations with BMI. Another polymorphism, previously described as a BclI restriction fragment length polymorphism, recently was identified as a C G nucleotide change. The G allele also was associated with increased sensitivity to GCs. In middle-aged subjects, the G allele of this BclI polymorphism was associated with increased abdominal obesity, while at older age, a lower BMI was found, accompanied by a tendency towards lower lean body mass. A third polymorphism consists of two linked, single-nucleotide mutations in codons 22 and 23, of which the second mutation results in an amino acid change from arginine (R) to lysine (K). In contrast to the other polymorphisms, this ER22/23EK polymorphism was associated with a relative resistance to GCs. In line with this, ER22/23EK carriers had lower total cholesterol and low-density lipoprotein cholesterol levels as well as lower fasting insulin concentrations and a better insulin sensitivity. C-reactive protein levels were lower in ER22/23EK carriers, as was found in a different population of elderly males. In accordance with this healthy metabolic profile, we found in this population a significantly better survival in ER22/23EK carriers after a 4-year follow-up. GCs also affect the brain. Although a certain level of cortisol is essential for proper brain functioning, excessive GC levels have been shown to negatively affect brain morphology and functions. At older age, we found that the risk of dementia and white matter lesions was lower in ER22/23EK carriers. GCs are also important in the regulation of body fat distribution. At young age, we observed sex-specific differences in body composition. Male ER22/23EK carriers were taller, had more muscle mass, and were stronger than noncarriers. In young females, ER22/23EK carriers had tendencies towards smaller waist and hip circumferences and lower body weight. Another polymorphism (TthIIII) was not associated with altered GC sensitivity. In conclusion, these polymorphisms in the GR gene may contribute considerably to the observed variability in GC sensitivity. As a result, they are associated with several differences in body composition and metabolic factors.

	I. Introduction

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

 
Glucocorticoid (GC) secretion is regulated by the hypothalamus, which receives stimuli from the central nervous system (CNS) (Chrousos and Gold, 1992). This results in a diurnal profile of cortisol secretion, with high levels in the morning and low concentrations in the afternoon and evening, with a small peak after lunch. In obese individuals, cortisol secretion is elevated and its peripheral turnover rate is higher, which results in normal, or even lower, serum cortisol concentrations (Murphy, 1968; Streeten et al., 1969; Cheek et al., 1981). Dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis was found to be more pronounced in central obesity than in peripheral or gluteofemoral obesity (Marin et al., 1992; Pasquali et al., 1993; Rosmond et al., 1998).

The regulation of cortisol metabolism in humans is not only centrally determined. Two key enzymes in cortisol metabolism have been identified that influence the effects of cortisol at the peripheral level: 11beta-hydroxysteroid dehydrogenase (11ß-HSD) I and 11ß-HSD II. The latter enzyme inactivates cortisol by conversion into cortisone, particularly in the kidney but also in other aldosterone-selective target tissues. The other enzyme, 11ß-HSD I, is present predominantly in adipose tissue, liver, lung, vascular system, ovary, and CNS (Monder and White, 1993; Stewart and Krozowski, 1999). The function of 11ß-HSD I is to convert cortisone into the active form, cortisol. Interestingly, in obese humans, this enzyme has been shown to have tissue-specific actions. 11ß-HSD I activity is lower in the liver, while increased activity in vitro is observed in the subcutaneous adipose tissue of obese men. This results in higher local cortisol levels in adipose tissue, which is suggested to be an important factor in the mechanism leading to harmful metabolic consequences of obesity.

It is known that GC sensitivity, measured by a dexamethasone (DEX) suppression test, varies greatly between individuals (Huizenga et al., 1998b). However, within individuals, GC sensitivity is rather stable. This suggests that, in humans, a setpoint for DEX sensitivity with respect to the feedback action exists, which might be genetically determined. An important factor in the cascade of GC action, also at the pituitary level, is binding to the GC receptor (GR).

The GR belongs to the superfamily of nuclear receptors that are present in the cytoplasm and act as transcription factors to regulate gene expression. Following cortisol binding, a conformational change occurs that leads to dissociation of the receptor from a large complex of proteins, of which heat shock protein (HSP) 90 is the most important (Pratt and Toft, 1997; Toft, 1998). This activated, ligand-bound receptor translocates to the nucleus, where it can act in several ways (Schaaf and Cidlowski, 2003). The GR can initiate transcription through binding to GC-responsive elements of the target gene. The GR also can affect gene transcription through direct protein-protein interaction and can activate, as well as repress, target gene expression (Diamond et al., 1990; Yang-Yen et al., 1990; Yudt and Cidlowski, 2002). In mice in which a mutation was induced that impaired dimerization and DNA binding, these processes have been shown to be not critical for survival (Reichardt et al., 1998).

Previously, some rare mutations of the GR gene were described that led to clinical signs and symptoms of generalized cortisol resistance (Lamberts et al., 1992). Due to these receptor defects, cortisol has impaired actions through the GR. As a consequence, the central negative feedback of GCs is diminished, GC production by the adrenal is elevated, and cortisol binds with high affinity to the mineralocorticoid receptor (MR). Symptomatology in patients with cortisol resistance is the consequence of a compensatory hyperactivity of the HPA axis, which results in overproduction of mineralocorticoids, which, in turn, leads to hypertension, hypokalemic alkalosis, fatigue, and in females — due to higher adrenal production of androgens — hyperandrogenism. In normal conditions, organs that have an important mineralocorticoid function are protected from high cortisol levels by the enzyme 11ß-HSD II, which rapidly inactivates cortisol into cortisone. In the situation of cortisol resistance, cortisol levels are too high for the inactivational capacity of this enzyme. The number of patients diagnosed with cortisol resistance syndrome is low (i.e., nine) (Brufsky et al., 1990; Hurley et al., 1991; Karl et al., 1993,1996a; Malchoff et al., 1993; Ruiz et al., 2001; Mendonca et al., 2002; Vottero et al., 2002). Two mutations found in vitro could have been pre-existing acquired mutations in vivo, leading to Nelson syndrome and lupus nephritis (Karl et al., 1996b; Jiang et al., 2001). Most patients carried a mutation or defect in the ligand-binding domain; only one patient had a mutation in the DNA-binding domain (Lamberts, 2001). A possible explanation for the low number of patients is that a severe form of cortisol resistance is not compatible with life.

Hypersensitivity to endogenous cortisol has been described as well. Iida and colleagues (1990) reported a patient with symptoms of Cushing’s syndrome, despite hypocortisolemia. Newfield and coworkers (2000) described a second patient with serious symptoms of Cushing’s syndrome at peripubertal age but having normal cortisol levels. The lymphocytes of this second patient contained an increased number of GR per cell, with normal binding affinity. The molecular etiology of hyperreactivity to cortisol has not been clarified fully but two single-nucleotide polymorphisms of the GR gene seem to play an important role in determining hypersensitivity. Figure 1 shows a schematic overview of the GR gene, with locations of previously described mutations causing cortisol resistance and of polymorphisms shown to be associated with altered GC sensitivity. In contrast to the infrequent mutations, most polymorphisms are located in the N-terminal transactivation domain (Bray and Cotton, 2003). This review deals with these GR gene polymorphisms, which were not only associated with differences in GC sensitivity but also related to differences in body composition and metabolic parameters.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Schematic overview of the GR gene, showing polymorphisms (white arrows), which have been shown to alter glucocorticoid (GC) sensitivity and are associated with differences in body composition, as well as mutations (black arrows) leading to the syndrome of cortisol resistance.

	II. The N363S Polymorphism of the GR Gene

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

View larger version (34K): [in this window] [in a new window]   FIG. 2. Cortisol levels (nmol/l) after dexamethasone (DEX) suppression tests (graphs on the left: A, C, E) and absolute change in cortisol (nmol/l) after DEX (graphs on the right: B, D, F). Results from a 0.25-mg DEX suppression test are shown for N363S and BclI carriers. Data concerning a 1-mg test are shown for ER22/23EK carriers. (A & B) Noncarriers (white bars) were compared to N363S carriers (striped bars). Lower post-DEX cortisol and greater decrease in N363S carriers suggest a hypersensitivity to GCs. (C & D) Heterozygous and homozygous BclI G-allele carriers (black bars), also lower post-DEX cortisol and greater decrease in BclI G-allele carriers, suggesting that BclI G-allele carriers are hypersensitive to GCs. (E & F) ER22/23EK carriers (vertically striped bars), who had higher cortisol levels after 1-mg DEX and a smaller decrease in cortisol, which suggests that the ER22/23EK variant is associated with a relative resistance to GCs.

View larger version (8K): [in this window] [in a new window]   FIG. 3. Concentrations of DEX necessary to achieve 50% of the maximal inhibition (IC50) in mitogen-induced peripheral mononuclear cell proliferation assays in noncarriers and carriers of the N363S polymorphism. A trend was observed towards a lower mean IC50 in N363S carriers, compared to noncarriers. [Adapted with permission from Huizenga NA, Koper JW, De Lange P, Pols HA, Stolk RP, Burger H, Grobbee DE, Brinkmann AO, De Jong FH, Lamberts SW 1998 A polymorphism in the glucocorticoid receptor gene may be associated with and increased sensitivity to glucocorticoids in vivo. J Clin Endocrinol Metab 83:144–151. Copyright The Endocrine Society.]

	III. The BclI Polymorphism of the GR Gene

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

 
Murray et al. (1987) reported an intronic restriction fragment length polymorphism (RFLP) of the GR gene, which was described as consisting of a short fragment of 2.3 kb and a large fragment of 4.5 kb. Since then, several association studies have been performed to investigate the role of this variant in obesity, using Murray’s terminology and technique. Recently, we identified the exact nucleotide alteration: a C G mutation, 646 nucleotides downstream from exon 2, which results in fragments of 2.2 kb and 3.9 kb (van Rossum et al., 2003b). The C allele is the most-frequently occuring and thus can be considered the wild-type allele (Table II). Table III displays an overview of reports of the BclI polymorphism and its associations with body composition and metabolic parameters.

In an 100-day experiment conducted with 12 pairs of monozygotic twins at young-adult age, effects of the BclI variant were studied in relation to body composition and metabolic changes in response to overfeeding (Ukkola et al., 2001b). In this study, no homozygous G-allele carriers were found. In contrast with these findings, CC carriers experienced a greater increase in body weight, visceral fat, and cholesterol levels after overfeeding than CG carriers. However, another study in adolescents showed that female heterozygous CG-allele carriers experienced a greater increase in subcutaneous fat, as measured by skinfold, when compared to both homozygous CC carriers and GG carriers during a 12-year follow-up period (Tremblay et al., 2003). No differences were found in baseline or post-follow-up subcutaneous fat mass, total fat mass, or, importantly, trunk fat mass. The authors speculated that one mutated allele could have a different effect than two mutated alleles. In the latter state, an alternative pathway might be switched on to compensate for changes resulting from two polymorphic alleles. Mechanisms supporting this theory have been reported in mouse models involving cyclooxygenase-2 and glucose transporter-4 genes (Stenbit et al., 1997; Fain et al., 2001). The results of Trembley et al. are not in line with the allele-dosage associations between the BclI polymorphism and hypersensitivity to GCs and BMI that we observed in our large elderly populations (van Rossum et al., 2003b). However, at baseline, they show that female homozygous GG carriers tend to have more subcutaneous fat than CC carriers and CG carriers. Although this difference was not statistically significant, it might explain why they did not find an even greater increase in GG carriers than in CG carriers during follow-up. Thus, in this study, the GG carriers might already have been slightly fatter at preadolescent age.

The molecular mechanism of the BclI polymorphism has not been clarified. It is likely that this intronic polymorphism exerts its effects in a different way than the N363S polymorphism. No alterations in glucose and insulin metabolism have been observed in carriers of the BclI polymorphism within the normal-weight population, while N363S carriers clearly showed an increased insulin response to DEX. Only in obese carriers of the BclI polymorphism were hyperinsulinemia and relative insulin resistance observed. However, no transfection experiments are possible to elucidate the mechanism, since the BclI polymorphism is located in an intron. We cannot rule out the possibility that this intronic polymorphism is linked to another polymorphism in the promoter region of the GR gene, which could result in increased GR expression or a variant in the 3'-untranslated region, which could increase stability of mRNA. However, we did not observe any linkage to the polymorphisms reviewed here (data not shown). Another possibility could be linkage to another gene in the vicinity of the GR gene. Since in most studies, the BclI polymorphism shows clear associations with increased sensitivity to GCs, this possibility is less likely. It is also known that intronic variations can influence the splicing process. However, the point mutation in the BclI site is not located near a regulatory splice site.

In summary, contrasting data have been reported about the BclI polymorphism with respect to its association with body composition. A possible explanation is that hypersensitivity to GCs due to the BclI polymorphism has different consequences during life. It might be that early in life, fat mass — particularly abdominal fat — is predominantly affected (i.e., BclI G-allele carriers have more fat), whereas later in life, the most-pronounced effects are observed on lean mass (i.e., BclI G-allele carriers have lower lean mass).

	IV. The ER22/23EK Polymorphism of the GR Gene

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

 
In a previous report, we described a polymorphism consisting of two linked, single-nucleotide mutations in codons 22 and 23 (exon 2 of the GR gene) (Koper et al., 1997). The first mutation in codon 22 did not result in an amino acid change (GAG GAA, both coding for a glutamic acid (E)) but the mutation in codon 23 (AGG AAG) caused a change from arginine (R) to lysine (K). In a population of 202 randomly selected individuals from the Rotterdam study, a population-based cohort study in the elderly, we found an association with higher post-DEX cortisol levels (Figure 2E) as well as less cortisol suppression after a 1-mg DEX suppression test in ER22/23EK carriers (Figure 2F). This finding suggests a relative GC resistance (van Rossum et al., 2002). In the same group having a mean age of 67 years, we found that carriers of the ER22/23EK variant had lower fasting insulin levels and increased insulin sensitivity (Figure 4). Carriers of the ER22/23EK polymorphism also had lower total and low-density lipoprotein cholesterol levels (Figure 5).

View larger version (9K): [in this window] [in a new window]   FIG. 4. (A) Fasting insulin concentrations in noncarriers and ER22/23EK carriers at first examination. Insulin concentrations tended to be lower in ER22/23EK carriers compared to noncarriers (p = 0.06). On the right, fasting insulin concentrations in noncarriers and ER22/23EK carriers at second examination (2.5 years later). Fasting insulin concentrations were significantly lower in ER22/23EK carriers (p < 0.001). (B) Homeostasis model assessment-insulin resistance (HOMA-IR) scores (index of insulin resistance) at baseline and at second examination 2.5 years later in noncarriers and ER22/23EK carriers. At both measurements, ER22/23EK carriers were significantly less insulin resistant.

View larger version (10K): [in this window] [in a new window]   FIG. 5. Total cholesterol (A) and low-density lipoprotein (LDL) cholesterol concentrations (B) in noncarriers and ER22/23EK carriers at first examination. On the right side of each figure, cholesterol concentrations in noncarriers and ER22/23EK carriers at second examination (2.5 years later) are shown. At all measurements, ER22/23EK carriers had significantly lower total and LDL-cholesterol levels than noncarriers.

We also studied the effects of this polymorphism at a younger age by investigating a cohort of 350 male and female subjects, who were followed from the age of 13 until the age of 36 years. We studied whether anthropometric parameters and body composition differed between ER22/23EK genotypes (van Rossum et al., 2004b). In males aged 36 and 32 years, we found ER22/23EK carriers to be, on average, 5 cm taller. Although there were no differences in BMI or fat mass, lean body mass was significantly higher in male carriers of the ER22/23EK variant. In accordance, their muscle strength, as measured by arm pull tests and high jump from standing, also was significantly greater. These differences tended to be already present during puberty but reached statistical significance only at young-adult age.

In females, different associations were observed with the ER22/23EK variant. Waist circumferences tended to be smaller in female ER22/23EK carriers at young-adult age but no differences in BMI were found. It is known that GCs negatively affect muscle mass and induce abdominal obesity. Thus, at young-adult age, the ER22/23EK variant is associated with beneficial changes in body composition, which can possibly be explained by a relative resistance to GCs in these tissues. In addition, effects of the ER22/23EK polymorphism seem to be gender specific.

It is known that GCs influence important brain structures and a normal level of cortisol is critical for many cerebral functions. In humans, high cortisol levels have been found to result in decreased hippocampal formation volume and memory impaiment (Starkman et al., 1992; Lupien et al., 1997). Disturbances in the HPA axis have been found to be related to dementia disorders (Weiner and Lourie, 1968; Gottfries et al., 1994; Nasman et al., 1995) In a large, population-based study in the elderly, we studied whether the ER22/23EK polymorphism was associated with hippocampal volume, dementia, and white matter lesions. We found that ER22/23EK carriers had a lower risk of dementia as well as fewer white matter lesions in the brain (van Rossum et al., 2003a). In addition, the ER22/23EK polymorphism was associated with better performance on psychomotor speed tests. It has been shown that white matter lesions are associated with small vessel disease (Bots et al., 1993; Breteler et al., 1994). Thus, this association might be a direct result of the beneficial effects of the ER22/23EK polymorphism on metabolic risk factors for atherosclerosis. No associations were found between the ER22/23EK variant and hippocampal volume. This might be explained by the fact that, in basal conditions, most effects on the hippocampus are mediated by the MR, while the GR plays a major role only in the activated state (e.g., physical or psychological stress) (De Kloet and Reul, 1987). In other parts of the brain, the GR is more important for mediating effects of GCs, so the observed associations with dementia might be explained by a smaller direct effect of GCs on the brain due to a relative GC resistance.

The mechanism that explains the effects of this polymorphism is under study. Several possibilities exist through which this variation of the GR gene can lead to these effects. Since the ER22/23EK polymorphism is located in the transactivation domain, the arginine to lysine amino acid change might affect the receptor’s tertiary structure, influencing the transactivational and/or transrepressional activity on target genes (de Lange et al., 1997; Russcher et al., 2003). Recently, it has been shown that two different methionine (M) codons in GR mRNA may be used as the initiation codon: M1 and M27, resulting in two isoforms, GR-A and GR-B, respectively. The GR-B protein has a stronger transactivating effect in transient transfection experiments but no difference in transrepression (Yudt and Cidlowski, 2001). The nucleotide changes associated with the ER22/23EK polymorphism might affect the secondary structure of the GR mRNA, thus influencing the choice of initiation codon. Indeed, secondary structure prediction (M-fold) showed different structures for the wild-type and polymorphic mRNA. Another possible explanation for the decreased GC sensitivity might be that the GR transactivational activity is affected by a different GR-A/GR-B ratio (Russcher et al., 2003). A third option is that the ER22/23EK variant might change mRNA stability, which is maintained when proteins responsible for this stability bind to the mRNA molecule. If the polymorphic mRNA recruits proteins in a different way, mRNA stability is affected, which might be a clue for the decreased GC sensitivity in ER22/23EK carriers.

In summary, the ER22/23EK polymorphism of the GR gene is associated with a relative GC resistance and a healthier metabolic condition, as evidenced by lower cholesterol levels and increased insulin sensitivity. Furthermore, this variant is associated with a beneficial body composition at young age and leads to a lower risk of dementia and better survival in the elderly.

	V. The TthIIII Polymorphism of the GR Gene

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

 
In the GR gene promoter region, a TthIIII RFLP previously was reported by Detera-Wadleigh and colleagues (1991). Rosmond et al. (2000a) showed this polymorphism to be associated with elevated diurnal cortisol levels in a population of 284 Swedish men. No relationships were found between the TthIIII variant and anthropometry, glucose, and insulin metabolism or lipid spectrum. We recently identified the location of the nucleotide change: a C/T change, 3807 bp upstream of the GR mRNA start (Table II).

In the same subpopulation of the Rotterdam study in which we studied the relationship between the three other polymorphisms described in this review and feedback sensitivity to GCs, we investigated whether an association existed between the TthIIII polymorphism and GC sensitivity (E.F.C. van Rossum, P. Roks, F.H. de Jong, A.O. Brinkmann, H.A.P. Pols, J.W. Koper, S.W.J. Lamberts, unpublished data). In this group, we found 39.7% CC carriers, 44.5% CT carriers, and 15.8% TT carriers. No differences were found in cortisol levels between the TthIIII genotypes before and after 1-mg and 0.25-mg DEX suppression, nor in anthropometric parameters, glucose and insulin levels, or cholesterol concentrations. We also studied whether this TthIIII polymorphism interacted with the N363S, BclI, and ER22/23EK polymorphisms. No interactions with N363S or BclI were found. Interestingly, however, all carriers of the ER22/23EK polymorphism carried the TthIIII T variant. This T allele of the TthIIII polymorphism is very common and exists without the ER22/23EK variant being present. To study the effects of carrying the TthIIII T allele and the ER22/23EK polymorphism, we compared the following three groups: 1) noncarriers of both polymorphisms (TthIIII CC and ER22/23ER); 2) carriers of one variant allele of the TthIIII polymorphism (TthIIII CT/TT and ER22/23ER); and 3) carriers of both polymorphisms (TthIIII CT/TT and ER22/23EK). The latter group had a significantly reduced cortisol response to 1-mg DEX as well as lower insulin and cholesterol levels, compared to the two other groups. No differences were found between the group of noncarriers of both polymorphisms and the group of carriers of only the TthIIII T variant. This suggests that the TthIIII polymorphism is not functional by itself; it might be functionally relevant only in combination with ER22/23EK. We do not know whether the TthIIII variant at the 5'-flanking region of the GR gene is essential in the associations of the ER22/23EK polymorphism or if its presence at the same allele is coincidence and does not influence the effects of the ER22/23EK variant. Possibly, the associations Rosmond and coworkers (2000a) found between alterations in cortisol levels and the TthIIII polymorphism could be explained by the presence of the ER22/23EK variant. However, no data have been published on the ER22/23EK polymorphism in this Swedish population.

	VI. Discussion

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

 
This review focused on several GR gene polymorphisms that were associated with body composition and metabolic parameters. As shown in Figure 1, the three functional polymorphisms are located in exon 2 (transactivating domain) and intron 2. This is in contrast to the previously described rare mutations causing the syndrome of GC resistance, which are located predominantly in the ligand-binding domain. GCs are essential for many regulatory processes in the human body, so a mutation leading to an absolute resistance to GCs is not compatible with life. The previously described patients, carrying a mutation of the GR gene, have decreased negative feedback at the level of the pituitary gland, which leads to HPA axis hyperactivation. Many of the symptoms found in patients with GC resistance are the consequence of this compensatory increased HPA axis activity: hyperandrogenism (in particular, leading to symptoms in females and children before puberty) and increased mineralocorticoid effects. The latter are due to exposure of the MR to high concentrations of cortisol, which cannot be effectively inactivated by 11ß HSD II.

Polymorphisms, common variations at the DNA level occurring in the normal population with a frequency of more than 1%, have much more-subtle effects. However, because of their high frequency in the population, their impact may be much greater. In several — but not all — studies, polymorphisms in the GR gene described here seem to correlate significantly with variation of sensitivity to endogenous GCs in normal individuals. Table IV overviews the four discussed polymorphisms and their relation with altered GC sensitivity. The N363S and BclI polymorphisms both were associated with hypersensitivity to GCs, while the ER22/23EK polymorphism was associated with relative resistance to GCs. No associations were found with the TthIIII polymorphism. However, the ER22/23EK variant was found to be linked to the TthIIII polymorphism. In this respect, associations with GC resistance and beneficial metabolic profile (i.e., low insulin and cholesterol levels) were observed in carriers of both the ER22/23EK and TthIIII polymorphisms. Considering DEX suppression test outcomes in carriers of the three functional polymorphisms, it seems that the 0.25-mg DEX suppression test is most sensitive to detect hypersensitivity to GCs, while the 1-mg DEX suppression test may be more suitable to detect a relative resistance to GCs.

Observed associations with altered GC sensitivity may contribute to a better understanding of the variations in regulation of the HPA axis between normal individuals. Previous data suggest that the HPA axis setpoint in humans might be genetically determined, since the intra-individual variations in post-DEX cortisol concentrations are rather small (Huizenga et al., 1998b).

These GR gene polymorphisms may have modifying effects on conditions such as (hereditary) atherosclerosis. It is known that some individuals survive until a great age, although they have very high cholesterol levels (Weverling-Rijnsburger et al., 1997). Thus, they might be protected by a genetic variant such as the ER22/23EK. On the other hand, individuals who carry the N363S or the BclI polymorphism might be more at risk for cardiovascular disease. The N363S variant recently was found to be associated with coronary artery disease, independent of obesity, as well as with increased total cholesterol and triglyceride concentrations and an elevated total cholesterol/HDL ratio. Both the N363S and the BclI polymorphism may predispose to obesity. However, as is well known, environmental, dietary, and socioeconomic factors are also important determinants of the obesity phenotype. Associations with polymorphisms depend on many additional factors: differences in characteristics between populations, prevalence of the polymorphism, and interactions with other genetic polymorphisms. These factors, taken together, might explain the discrepancies between studies so far encountered.

In clinical practice, GCs are used widely to treat diseases (e.g., asthma, chronic inflammations, prevention of rejection of organ transplants) as well as replacement therapy. It is well known that effects of GC treatment vary considerably between patients. Some patients respond very well to therapeutical administration of GCs but also develop serious side effects, while others need a very high dose to establish any clinical effect and do not suffer from side effects. The response to GCs in the majority of patients lies between these extremes. It is likely that these polymorphisms are to some extent responsible for the variability in response to therapeutically used GCs. In the future, after appropriate additional research, it might be useful to screen for the presence of these GR gene variants, in order to determine an individual’s dose of GCs. This dose should be adjusted to a person’s need, taking into account the genetically determined GC sensitivity, in such a way that it is therapeutically effective but does not cause side effects. We do not know whether the altered sensitivity associated with these polymorphisms differs for various types of clinically used GCs and whether the manner of application (e.g., local, systemic) influences the effects of the polymorphisms.

During evolution, a selection process occurred in which some de novo mutations probably had beneficial effects and slowly became more frequent in the population. We found that the ER22/23EK variant in males was associated with greater lean mass and muscle strength. In this view, the ER22/23EK polymorphism could have resulted in strong individuals with a greater chance to survive due to an advantage in food-collecting and fighting ability. The N363S and BclI carriers may have had advantages for survival through their tendency to accumulate fat, which was especially favorable in times of food deficit. In this respect, the BclI polymorphism probably arose long ago, since the allele frequency in normal population is very high. However, in modern times of food abundance, combined with increased psychological stress and lack of exercise, the N363S and BclI polymorphisms may have turned into a disadvantage. An increased sensitivity to GCs, resulting in fat accumulation, is probably a risk factor in atherosclerosis. This is supported by the findings of increased risk of coronary artery disease and obesity in N363S carriers in an Australian population (Lin et al., 2003a,b).

In conclusion, the N363S, BclI, and ER22/23EK polymorphisms in the GR gene, but not the TthIIII polymorphism, are associated with altered GC sensitivity and result in a wide variety of phenotypic signs. These are not pathological per se but may partially explain an individual’s genetically determined tendency to a certain body composition and metabolic status (Figure 6). More research is needed to elucidate the mechanisms behind these associations at a molecular level.

View larger version (19K): [in this window] [in a new window]   FIG. 6. A tentative scheme of the N-terminal part of the glucocorticoid receptor gene in which three functional polymorphisms are indicated as well as a summary of their clinical associations.

	ACKNOWLEDGEMENTS

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

 
This research project was supported by a grant from the Dutch Organisation for Scientific Research (NWO) and a Research Institute of Diseases in the Elderly and NWO program grant entitled "Variations in Glucocorticoid Sensitivity."

	REFERENCES

TOP ABSTRACT I. Introduction II. The N363S Polymorphism of the GR... III. The BclI Polymorphism of the GR... IV. The ER22/23EK Polymorphism of the GR... V. The TthIIII Polymorphism of the GR... VI. Discussion ACKNOWLEDGEMENTS REFERENCES

 

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