# 2009 The Authors Journal compilation # 2009 Blackwell Munksgaard
Acta Neuropsychiatrica 2009: 21: 11–17 All rights reserved DOI: 10.1111/j.1601-5215.2008.00342.x
ACTA NEUROPSYCHIATRICA
Association of glucocorticoid receptor polymorphisms with the susceptibility to major depressive disorder and treatment responses in Korean depressive patients Lee H-Y, Kang R-H, Han S-W, Paik J-W, Chang HS, Jeong YJ, Lee M-S. Association of glucocorticoid receptor polymorphisms with the susceptibility to major depressive disorder and treatment responses in Korean depressive patients. Objective: Major depressive disorder (MDD) is closely related to stress reactions and serotonin probably underpins the pathophysiology of MDD. Alterations of the hypothalamic-pituitary-adrenal axis at the gene level have reciprocal consequences on serotonin neurotransmission. Glucocorticoid receptor (GR) polymorphisms affect glucocorticoid sensitivity, which is associated with cortisol feedback effects. Therefore, we hypothesised that GR polymorphisms are associated with the susceptibility to MDD and predict the treatment response. Method: Ninety-six subjects with a minimum score of 17 on the 21-item Hamilton Depression Scale (HAMD) at baseline were enrolled into the present study. The genotypes of GR (N363S, ER22/23EK, Bcl1, and TthIII1 polymorphisms) were analysed. The HAMD score was again measured after 1, 2, 4 and 8 weeks of antidepressant treatment to detect whether the therapeutic effects differed with the GR genotype. Results: Our subjects carried no N363S or ER22/23EK genetic polymorphisms and three types of Bcl1 and TthIII1 genetic polymorphisms. The C/C genotype and C allele at Bcl1 polymorphism were more frequent in MDD patients than in normal controls (p , 0.01 and p ¼ 0.01, respectively). The genotype distributions did not differ significantly between responders and non-responders. Conclusion: These results suggest that GR polymorphism cannot predict the therapeutic response after antidepressant administration. However, GR polymorphism (Bcl1) might play a role in the pathophysiology of MDD. Future studies should check this finding in larger populations with different characteristics.
Introduction
Hyperactivity of the hypothalamic-pituitaryadrenal (HPA) axis is one of the most consistent neuroendocrine abnormalities in major depressive disorder (MDD). Specifically, patients with MDD show increased concentrations of cortisol in the plasma, urine and cerebrospinal fluid (CSF) and an exaggerated cortisol response to adrenocorticotrophic hormone (ACTH) (1–3). The corticosteroid receptor hypothesis has been proposed for the pathogenesis of MDD, which focuses on impaired corticosteroid receptor signalling, leading to
Hwa-Young Lee1,2,3, Rhee-Hun Kang1,2,3, Sang-Woo Han4, Jong-Woo Paik5, Hun Soo Chang1,2, Yoo Jung Jeong1,2, Min-Soo Lee1,2,3 1 Clinical Research Center for Depression, Korea University, Seoul, Korea; 2Institute of Human Behavior and Gene, Korea University, Seoul, Korea; 3 Department of Psychiatry, College of Medicine, Korea University, Seoul, Korea; 4Department of Psychiatry, College of Medicine, Soonchunhyang University, Seoul, Korea; and 5Department of Psychiatry, College of Medicine, Kyunghee University, Seoul, Korea
Keywords: depression; glucocorticoid receptor; polymorphism; susceptibility; treatment response Min-Soo Lee, 126-1 Anamdong 5-ga, Seongbuk-gu, Seoul 136-705 Seoul, Korea. Tel: 182 2 920 5997; Fax: 182 2 923 8119; E-mail: [email protected]
a reduced negative feedback of cortisol, an increased production of corticotropin-releasing hormone (CRH) and hypercortisolism (2). Interestingly, cortisol and CRH affect the serotonin (5-HT) system (1,4). During the stress response, glucocorticoids (GCs) stimulate all these features of 5-HT transmission (5). Conversely, 5HT transmission is impaired and noradrenergic transmission in the hippocampus is suppressed during chronic psychosocial stress and hypercortisolism, which is similar to the series of events evident during depression (6). This makes the GC 11
Lee et al. receptor (GR) a prime candidate gene for associations with susceptibility for MDD. Moreover, initial studies of GR antagonist mifepristone and other HPA-axis-targeting agents in affective disorder have been encouraging (7). The human gene coding for the GR is referred to as nuclear receptor subfamily 3, Group C, Member 1 (NR3C1). It has been reported that the following polymorphisms of the GC receptor gene alter GC sensitivity (8–11); N363S: 11088A.G, ER22/ 23EK: 168G.A, Bcl1: 2646C.G, and TthIII1: 13807C.T polymorphisms. The Bcl1 polymorphism involves a Bcl1 restriction site in intron 2 and is a C-to-G nucleotide change that appears 646 base pairs (bp) downstream from exon 2 (Bcl1). The TthIII1 polymorphism was identified as the location of a C-to-T nucleotide change that appears 3807 bp upstream of the start of GR mRNA. The N363S polymorphism is exon 2, which results in an aminoacid change from asparagine (N) to serine (S). The ER22/23EK polymorphism, also located in exon 2, consists of two linked nucleotide changes in codons 22 and 23 (GAG AGG to GAA AAG). The ER23EK polymorphism consists of two linked point mutations in codon 22 [silent mutations changing codon 22 from GAG to GAA, coding for glutamic acid (E)] and in codon 23 [changes from AGG to AAG, resulting in an amino-acid change from arginine (R) to lysine (K) (12). The GR polymorphisms affect GC sensitivity, which is associated with cortisol feedback effects. The degree of cortisol feedback is closely related to the development of depression and may also be affected by antidepressants (13). Therefore, we hypothesised that the above four GR polymorphisms are associated with the susceptibility to MDD and predict the response to treatment with citalopram.
Material and methods Subjects
The subjects in our previous studies (14,15) were included in the present study. This study was performed on patients aged 18–65 years diagnosed with MDD who visited Korea University Anam Hospital between March 2003 and December 2005. Each patient was diagnosed with MDD by psychiatrists using the Structured Clinical Interview for Diagnostic and Statistical Manual-IV Axis I Disorders – Korean version (SCID) (16). Patients with a minimum baseline score of 17 on the 21-item Hamilton Depression Scale (HAMD) were eligible for inclusion in this study (17). None of the included patients had another psychiatric axis I diagnosis, such as anxiety disorder, bipolar 12
disorder, or alcohol or drug dependency, or neurological diseases such as stroke, epilepsy or chronic diseases (other than controlled diabetes and hypertension) that might have impeded their psychological evaluation. Of the 138 patients that were registered, 96 patients who took the experimental drug at least once and who had at least one efficacy measurement were classified as the intention-to-treat (ITT) group. Of 96 ITT patients, the genotypes of only 84 patients were successfully analysed. At first week, all subjects succeeded to follow-up. Seventy-seven subjects at second week, 73 subjects at fourth week and 65 subjects at eighth week remained. The symptoms and severity of depressive disorder were evaluated by the HAMD at baseline and after weeks 1, 2, 4 and 8. Participants showing a reduction in the HAMD score of more than 50% relative to baseline were defined as responders, with the other participants designated as non-responders (18). Before entry into the study, a 2-week washout was performed on those patients who were being treated with different drugs. During the study period, the patients self-administered 20 mg of the antidepressant citalopram daily as the starting dose, with a daily dose of 20–60 mg applied at 1, 2, 4 and 8 weeks at the discretion of the investigators according to clinical situation of each patient. After 1 week, the investigators were permitted to increase the dosage, if the patient’s symptoms were not responding and the patient was tolerable at the dosage, or to maintain the same regimen, if the patient’s symptoms were improved and the patient was tolerable at the dosage. If not tolerable at the dosage, in contrast, the dosage was decreased. Dose escalations were based on clinical response and tolerance of side-effects. Among the various types of antidepressants, citalopram is well tolerated, with the most common side-effects being gastrointestinal and including nausea, diarrhoea and dry mouth. Citalopram is the most highly selective serotonin reuptake inhibitor, with little or no affinity for a variety of receptor types. For this reason, we chose citalopram as a study drug. Benzodiazepine was allowed as a coprescribed drug. This study was approved by the Ethics Committee of Korea University Anam hospital and a patient consent form for the genetics study (by the Ministry of Health and Welfare) was prepared. One hundred and five control subjects consisted of randomly selected healthy individuals of Korean origins who visited the Korea University Anam hospital for regular health screenings. Subjects with a history of depressive disorder or psychotropic medication history were excluded from the control group by applying the Structured Clinical
Glucocorticoid receptor polymorphism and depression Interview for SCID. Control subjects were excluded if they had any familial psychiatric history, or if they had scores of 10 on the Beck Depression Inventory (19). Subjects in the control group also signed consent forms for participation in the genetics study (published by the Ministry of Health and Welfare of Korea). Genotyping
ER22/23EK polymorphism. DNA was extracted from the peripheral blood and a polymerase chain reaction (PCR) was performed with the sense primer 5#-GATTCGGAGTTAACTAAAAG-3# and the antisense primer 5#-ATCCCAGGTCATTTCCCATC-3#. The amplification mixture contained 1 ml of DNA, 3 ml of 103 PCR buffer, 2.5 ml of 2.5 mM dNTP, 1 ml of each primer, 6 ml of 53 Gcaqture solution, 15.4 ml of distilled water and 0.1 ml of Taq polymerase (5 U/ml; iNtRON Biotechnology, Seoul, Korea). Samples were amplified using a PCR thermal cycler (Takara, Kyoto, Japan) for an initial 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C. After a final 7 min at 72°C, the reaction was terminated at 4°C. The amplified DNA was digested with the restriction endonuclease Mnl I (New England Biolabs, Ipswich, MA, USA) at 37°C for 2 h. Fragments were electrophoresed in 3% agarose gels and stained with ethidium bromide. The wild type consisted of 163- and 142-bp fragments and homozygous EK22/23EK consisted of 177- and 163-bp fragments. N363S polymorphism. A PCR was performed with the sense primer 5#-AGTACCTCTGGAGGACAGAT-3# and the antisense primer 5#-GTCCATTCTTAAGAAACAGG-3#. Samples were amplified using a PCR thermal cycler (Takara) for an initial 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C. After a final 7 min at 72°C, the reaction was terminated at 4°C. The PCR products were digested with Tsp509 I (New England Biolabs) at 65°C for 2 h. Fragments were electrophoresed in 3% agarose gels and stained with ethidium bromide. The wild type consisted of 113-, 95- and 9-bp fragments and the homozygous mutant consisted of 122- and 95-bp fragments. Bcl1 polymorphism. A PCR was performed with the sense primer 5#-AGAGCCCTATTCTTCAAACTG-3# and the antisense primer 5#-GAGAAATTCACCCCTACCAAC-3#. Samples were amplified using a PCR thermal cycler (Takara) for an initial 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C. After a final 7 min at 72°C, the reaction was terminated at 4°C. The
amplified DNA was digested with the restriction endonuclease Bcl1 (New England Biolabs), which cuts at the 646C site downstream of exon 2. Fragments were electrophoresed in 3% agarose gels and stained with ethidium bromide. The 418C allele consisted of 151- and 257-bp fragments and the 418G allele consisted of a 418-bp fragment. Tth111i polymorphism. A PCR was performed with the sense primer 5#-GGCCACAACAATAACCCAGT-3# and the antisense primer 5#-TGAAAGTGTTTTATGCACAGTATCC-3#. Samples were amplified using a PCR thermal cycler (Takara) for an initial 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 61.8°C, and 30 s at 72°C. After a final 7 min at 72°C, the reaction was terminated at 4°C. The amplified DNA was digested with the restriction endonuclease Tth1111 (New England Biolabs), which cuts at the 3807C site upstream of the GR mRNA start. Fragments were electrophoresed in 3% agarose gels and stained with ethidium bromide. The 3807C allele consisted of 167- and 170-bp fragments and the 3807T allele consisted of a 337-bp fragment. Statistical analysis
A last-observation-carried-forward technique for missing data was used to analyse efficacy in the ITT group, which included all patients who received medication at least once and who had at least one efficacy or safety measurement taken. The presence of Hardy-Weinberg equilibrium and genotype differences was examined using the chi-squared test for categorical variables and Student’s t-test or one-way ANOVA for continuous variables. Repeated-measures ANOVAs were performed for each weekly HAMD score. A probability value of p , 0.05 was considered statistically significant. Probability values were corrected by multiplying the number of comparisons made (Bonferroni correction).
Results Demographic data of the subjects
The gender distribution did not differ significantly between control subjects (40 males/66 females) and MDD patients (26 males/70 females) (p ¼ 0.12). There was no significant difference in the age distribution between control subjects and the MDD patients (49.1 16.3 years, mean SD in MDD patients and 52.3 10.8 years in control subjects, p ¼ 0.10). 13
Lee et al. Genetic predisposition to MDD
Table 1 shows the genomic location, nomenclature and single nucleotide polymorphism (SNP) reference of four genetic variants in NR3C1. Our subjects carried no N363S polymorphisms (all GG, with no G/A or A/A), no ER22/23EK polymorphisms (all A/A, with no G/A or G/G), and three types of Bcl1 and Tth1111 genetic polymorphisms. The genotype distributions of Bcl1 polymorphism conformed to Hardy-Weinberg equilibrium in both normal controls and MDD patients (p ¼ 0.06 and 0.26, respectively) and the results of the Tth1111 polymorphism were within the equilibrium in these two groups (p ¼ 0.92 and 0.73, respectively). The Bcl1 genotype distributions differed significantly between control subjects and MDD patients (Table 2). The C/C genotype and C allele of Bcl1 polymorphism were more frequent in MDD patients than in normal controls (p , 0.01 and p ¼ 0.01, respectively). After Bonferroni correction, the association remained statistically significant. Although the C allele of Tth1111 polymorphism was more frequent in MDD patients than in normal controls (p ¼ 0.04), this difference lost significance after applying Bonferroni correction. Haplotype analysis was shown in Table 3. The distribution of the haplotype was significantly different between MDD patients and normal controls (p ¼ 0.001). The haplotype carrying the C allele of Bcl1 and C allele of Tth1111 was more frequent in MDD patients than in normal controls. Prediction of responses and clinical variables according to GR polymorphisms
The genotype distributions did not differ significantly between responders and non-responders (Table 4). The age at onset, duration of illness and number of previous episodes neither differed among the genotype in MDD patients (Table 3). The HAMD scores at baseline and 8 weeks did not differ significantly with the genotype in MDD patients (Table 5). When HAMD baseline scores were included to analysis as covariate, there was no significant difference in HAMD score at 8 weeks Table 1. Location, nomenclature, and single nucleotide polymorphism (SNP) reference of four genetic variants in NR3C1
Name
Genomic location
Nucleotide change
Amino-acid change
dbSNP reference
Bcl1 N363S ER22/23EK TthIII1
Promoter Exon 2 Exon 2 Promoter
2646C.G 11088A.G 168G.A 13807C.T
– Asn363Ser Arg23Lys –
rs41423247 rs1800445 rs6195 rs10052957
14
according to genotypes (both Bcl1 and Tth1111 polymorphism, data not shown). With a repeated-measures ANOVA for HAMD scores from baseline to week 8 against the Bcl1 and Tth1111 polymorphism, we observed a significant effect of the change in HAMD score over the 8 weeks of treatment and no genotype effect of the Bcl1 and Tth1111 polymorphism on change in HAMD score (data not shown). When baseline HAMD scores were included to analysis as a covariate, there was no significant difference in HAMD scores at 8 weeks between the Bcl1 or Tth1111 genotypes (data not shown).
Discussion
This study was designed to identify the role of GR polymorphisms on the genetic predisposition to MDD and on the therapeutic response to antidepressant. We showed that the C/C genotype and C allele in the Bcl1 polymorphism are more frequent in MDD patients than in normal controls. G allele of Bcl1 polymorphism has been reported to be associated with hypersensitivity to GCs, as indicated by an increased response to the ACTH- and cortisol-suppressive effects of low-dose dexamethasone (8). In other words, the C allele of Bcl1 polymorphism is more GC-resistant than G allele. Increased GR resistance has generally been described as one of the endocrine hallmarks of depression, which forms part of a vicious circle of HPA-axis dysregulation leading to an upregulation of central CRH (2). In line with the result of this study, C allele, which is more GC-resistant, could induce the development of depression. Conversely, van Rossum et al. reported that the frequency of the Bcl1 G/G genotype was significantly higher in MDD patients than in normal controls (20), explaining their result by the tissue specific effect of Bcl1 polymorphism. Contrary to the action in the hypothalamus, GC increases CRH expression in limbic regions such as the amygdala (21,22). G allele of Bcl1 polymorphism which is more GCsensitive might thus have an increased positive GC feedback effect on CRH in the limbic system, resulting in an increased production of CRH, and increased CRH in limbic regions has been linked to depression-like symptoms (2,21). We found few T/T genotypes in the TthIII1 polymorphism and no N363S and ER22/23EK polymorphisms. While the C allele in the TthIII1 polymorphism was more frequent in MDD patients than in control subjects (p ¼ 0.04), this difference lost significance after Bonferroni correction. Rosmond et al. (23) showed that the Tth1111 polymorphism was
Glucocorticoid receptor polymorphism and depression Table 2. Genotype distributions and allele frequencies in normal control subjects and MDD patients Bcl1 genotype Group Normal controls (%) MDD (%)
Bcl1 allele
C/C
G/C
G/G
p* (pcorr)
C
G
p† (pcorr)
50 (47.6) 58 (69.9)
50 (47.6) 21 (25.3)
5 (4.8) 4 (4.8)
,0.01 (,0.01)
150 (71.4) 137 (82.5)
60 (28.6) 29 (17.5)
0.01 (0.02)
TthIII1 genotype Group Normal controls (%) MDD (%)
TthIII1 allele
C/C
C/T
T/T
p*,‡
C
T
p*
86 (82.7) 74 (92.5)
17 (16.3) 6 (7.5)
1 (1.0) 0 (0)
0.13
189 (90.9) 154 (96.3)
19 (9.1) 6 (3.8)
0.04 (0.08)
*Comparison of genotype frequencies between MDD and normal control group. †Comparison of allele frequencies between MDD and normal control group. ‡Genotype comparisons are made by Fisher's exact test. pcorr indicates p-value after Bonferroni correction.
associated with increased diurnal cortisol levels. However, another study found no differences in cortisol levels between the TthIII1 genotypes before and after dexamethasone suppression test (11), and no interactions with N363S or Bcl1. However, all carriers of the ER22/23EK polymorphism carried the TthIII1 T variant. The cortisol response to 1 mg of dexamethasone was significantly lower in carriers of both TthIII1 CT/TT and ER22/23EK polymorphisms than in the two other groups (TthIII1 CC and ER22/23ER, and TthIII1 CT/TT and ER22/23ER). In other words, this haplotype is associated with a higher resistance to GC. The TthIII1 polymorphism might be functionally relevant only in combination with ER22/23EK. In the present study, the TthIII1 polymorphism existed without the ER22/23EK variant being present, in contrast with it being reported that the ER22/23EK polymorphism is without exception linked to the TthIII1 T polymorphism (11). Therefore, the absence of a ER22/23EK polymorphism could be responsible for the absence of an association between MDD and TthIII1 polymorphism in this study.
Table 3. Haplotype analysis of GR polymorphisms Haplotype
n (%)
(Bcl1, TthIII1)
Group
1/1
HT1
(C, C)
HT2
(G, C)
HT3
(G, T)
HT4
(C, T)
NC MDD NC MDD NC MDD NC MDD
50 (45.5) 58 (69) 2 (1.8) 3 (3.6) 1 (0.9) 0 (0) 0 (0) 0 (0)
*/2 60 26 108 81 109 84 110 84
(54.5) (31) (98.2) (96.4) (99.1) (100) (100) (100)
NC, normal control. *Obtained using chi-squared test and Fisher's exact test.
Total 110 84 110 84 110 84 110 84
(100) (100) (100) (100) (100) (100) (100) (100)
p* 0.001 0.654 1.000 –
The ER22/23EK polymorphism was shown to be associated with a higher resistance to GCs (9) and an increased risk of developing MDD (20,24). It has been shown that the N363S polymorphism is associated with hypersensitivity to GCs (10). We did not find these polymorphisms in our Korean population and hence are unable to comment on the association between these polymorphisms and MDD. We did not identify a relation between GR polymorphisms and the therapeutic response to citalopram in the present study. It has been reported that ACTH levels are higher in carriers of the Bcl1 polymorphism than in non-carriers (from the dexamethasone/CRH test), with a trend towards decreases in HAMD scores being smaller in carriers than in non-carriers (25). Also, ER22/ 23EK carriers showed a significantly faster clinical response to antidepressant therapy (20). It was proposed that antidepressants can inhibit steroid transporters localised on the endothelial cells of the blood-brain barrier(or blood-CSF barriers) and in neurons in humans, such as the multidrug resistance p-glycoprotein, thus increasing the access of cortisol to the brain and the GC-mediated negative feedback effects on the HPA axis. Enhanced cortisol effects in the brain might maximise the therapeutic effects of antidepressants (13). At baseline, the symptomatology (as quantified by HAMD scores), age at onset, duration of illness and number of previous episodes did not differ from the GR polymorphisms in this study. Similarly, van Rossum et al. found no association of GR polymorphisms with clinical characteristics (20). High HPA-axis activity has been related to the risk of relapse in MDD (26,27). Furthermore, previous depressive episodes and HPA-axis hyperactivity might permanently alter the reactivity of 15
Lee et al. Table 4. The prediction of response according to the GR polymorphisms in MDD patients Bcl1 genotype
Responder (%) Non-responder (%) Total (%)
Bcl1 allele
G/G
G/C
C/C
p*
G
C
p†
36 (62.1) 22 (37.9) 58 (100)
13 (61.9) 8 (38.1) 21 (100)
3 (75.0) 1 (25.0) 4 (100)
0.87
85 (62.0) 52 (38.0) 137 (100)
19 (65.5) 10 (34.5) 29 (100)
0.83
TthIII1 genotype
TthIII1 allele
C/C Responder (%) Non-responder (%) Total (%)
C/T & T/T
46 (62.2) 28 (37.8) 74 (100)
3 (50.0) 3 (50.0) 6 (100)
p*
C
T
p†
0.67
95 (61.7) 59 (38.3) 154 (100)
3 (50.0) 3 (50.0) 6 (100)
0.68
*Comparison of genotype frequencies between responder and non-responder group. †Comparison of allele frequencies between responder and non-responder group. Responder group is defined as those patients who show more than or equal to a 50% relative decrease of HAMD score at week 8 compared with that at baseline.
responses in clinically depressed patients. Fourth, because the plasma levels of cortisol and ACTH were not measured, it is not possible to investigate the functional role of GR polymorphism. Fifth, T/T genotypes in the TthIII1 polymorphism were present at below 1% and hence this polymorphism could not be considered to be a single-nucleotide polymorphism in our Korean population. In contrast, the distributions of the C/C, CT and TT genotypes have been reported at 39.7, 44.5 and 15.8%, respectively, in Caucasians (11). Our Korean subjects carried no N363S or ER22/ 23EK genetic polymorphisms. These results cannot be generalised because of genotype distributions varying with ethnicity. Finally, the relatively small sample limits the generalisability of our findings. Our sample provided power greater than 0.67 at p , 0.05 to detect effects of the size we observed in our sample. This indicates that our sample provides a moderate power, considering that a power cut-off of 0.5 and an a-value ,0.05 are often used. Notwithstanding the above limitations, this is the first study to examine the possible association between GR polymorphisms and MDD in a Korean population. Elucidating the role of GR polymorphisms in MDD requires further studies applying a
the axis because the activation of the GR has been shown to change the methylation pattern of specific genes (28). In the present study the number of previous episodes did not vary with the GR polymorphism. The value for linkage disequilibrium is D# ¼ 0.815, r2 ¼ 0.159. The linkage disequilibrium was not found between these two polymorphisms. This study was subject to several limitations. First, although Korean subjects are highly genetically homogeneous, the small number of subjects included represents a one limitation. Larger number of subjects are needed to investigate the role of GR polymorphism in MDD. Second, treatment periods (5.97 2.98 weeks, mean SD) were various in this study. Third, the same dosage of citalopram was not prescribed to each patient and the plasma levels of citalopram were not measured in this study. Because pharmacokinetic activities are different among the patients, it is not possible that the same dosage of antidepressant could be prescribed to each patient in clinical settings. It could be better that dose escalations would be based on clinical response and tolerance of side-effects. Moreover, a previous study (29) reported the absence of any significant relationships between citalopram plasma levels and clinical Table 5. Clinical characteristics according to the GR polymorphisms in MDD patients Bcl1 Genotype G/G HAMD (baseline) HAMD (8-week) Age at onset Duration of illness Number of previous episodes
23.1 10.0 47.2 6.4 1.1
5.5 7.2 14.8 7.2 0.78
G/C 22.0 11.4 42.1 8.9 1.0
C/C 3.6 7.8 17.7 10.3 0.0
Data represent mean SD. Genotype comparisons are made by *ANOVA or †Kruskal-Wallis test.
16
TthIII1 genotype
20.3 8.0 50.3 6.9 1.0
p 2.8 9.5 20.0 7.9 0.0
0.40 0.62 0.41* 0.44* 0.90†
C/C 22.5 10.2 46.3 7.1 1.0
4.9 7.3 15.5 8.2 0.52
C/T & T/T
p
0.30 0.80 0.82* 0.54* 1.0†
20.3 11.0 44.7 5.0 1.0
1.9 10.1 22.3 5.5 0.0
Glucocorticoid receptor polymorphism and depression biochemical approach and involving a larger number of subjects to decrease the risk for false positives of this study. Acknowledgements This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (03-PJ10-PG13-GD01-0002).
References 1. HOLSBOER F, BARDEN N. Antidepressants and hypothalamic-pituitary-adrenocortical regulation. Endocr Rev 1996;17:187–205. 2. HOLSBOER F. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 2000;23:477–501. 3. NEMEROFF CB. The corticotropin-releasing factor (CRF) hypothesis of depression: new findings and new directions. Mol Psychiatry 1996;1:336–342. 4. DE KLOET ER, VREUGDENHIL E, OITZL MS, JOELS M. Brain corticosteroid receptor balance in health and disease. Endocr Rev 1998;19:269–301. 5. MEIJER OC, DE KLOET ER. Corticosterone and serotonergic neurotransmission in the hippocampus: functional implications of central corticosteroid receptor diversity. Crit Rev Neurobiol 1998;12:1–20. 6. WOLKOWITZ OM, REUS VI, MANFREDI F, INGBAR J, BRIZENDINE L, WEINGARTNER H. Ketoconazole administration in hypercortisolemic depression. Am J Psychiatry 1993;150:810–812. 7. PORTER RJ, GALLAGHER P. Abnormalities of the HPA axis in affective disorders: clinical subtypes and potential treatments. Acta Neuropsychiatrica 2006;18:193–209. 8. VAN ROSSUM EF, KOPER JW, VAN DEN BELD AW et al. Identification of the BclI polymorphism in the glucocorticoid receptor gene: association with sensitivity to glucocorticoids in vivo and body mass index. Clin Endocrinol 2003;59:585–592. 9. VAN ROSSUM EF, KOPER JW, HUIZENGA NA et al. A polymorphism in the glucocorticoid receptor gene, which decreases sensitivity to glucocorticoids in vivo, is associated with low insulin and cholesterol levels. Diabetes 2002;51:3128–3134. 10. HUIZENGA NA, KOPER JW, DE LANGE P et al. A polymorphism in the glucocorticoid receptor gene may be associated with and increased sensitivity to glucocorticoids in vivo. J Clin Endocrinol Metab 1998;83:144–151. 11. VAN ROSSUM EF, LAMBERTS SW. Polymorphisms in the glucocorticoid receptor gene and their associations with metabolic parameters and body composition. Recent Prog Horm Res 2004;59:333–357. 12. KOPER JW, STOLK RP, DE LANGE P et al. Lack of association between five polymorphisms in the human glucocorticoid receptor gene and glucocorticoid resistance. Hum Genet 1997;99:663–668. 13. PARIANTE CM, THOMAS SA, LOVESTONE S, MAKOFF A, KERWIN RW. Do antidepressants regulate how cortisol affects the brain? Psychoneuroendocrinology 2004;29:423–447.
14. CHOI MJ, KANG RH, LIM SW, OH KS, LEE MS. Brainderived neurotrophic factor gene polymorphism (Val66Met) and citalopram response in major depressive disorder. Brain Res 2006;1118:176–182. 15. HAM BJ, LEE BC, PAIK JW et al. Association between the tryptophan hydroxylase-1 gene A218C polymorphism and citalopram antidepressant response in a Korean population. Prog Neuropsychopharmacol Biol Psychiatry 2007;31:104–107. 16. HAN OS, HONG JP. Structured clinical interview for DSMIV axis I disorder-Korean version. Seoul: Hana Medical Publishing Co, 2000. 17. HAMILTON M. Development of a rating scale for primary depressive illness. Br J Soc Clin Psychol 1967;6:278–296. 18. FRANK E, PRIEN RF, JARRETT RB et al. Conceptualization and rationale for consensus definitions of terms in major depressive disorder. Remission, recovery, relapse, and recurrence. Arch Gen Psychiatry 1991;48:851–855. 19. BECK AT, WARD CH, MENDELSON M, MOCK J, ERBAUGH J. An inventory for measuring depression. Arch Gen Psychiatry 1961;4:561–571. 20. VAN ROSSUM EF, BINDER EB, MAJER M et al. Polymorphisms of the glucocorticoid receptor gene and major depression. Biol Psychiatry 2006;59:681–688. 21. REUL JM, HOLSBOER F. Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression. Curr Opin Pharmacol 2002;2:23–33. 22. SCHULKIN J, GOLD PW, MCEWEN BS. Induction of corticotropin-releasing hormone gene expression by glucocorticoids: implication for understanding the states of fear and anxiety and allostatic load. Psychoneuroendocrinology 1998;23:219–243. 23. ROSMOND R, CHAGNON YC, HOLM G et al. A glucocorticoid receptor gene marker is associated with abdominal obesity, leptin, and dysregulation of the hypothalamicpituitary-adrenal axis. Obes Res 2000;8:211–218. 24. BROUWER JP, APPELHOF BC, VAN ROSSUM EF et al. Prediction of treatment response by HPA-axis and glucocorticoid receptor polymorphisms in major depression. Psychoneuroendocrinology 2006;31:1154–1163. 25. VAN WEST D, VAN DEN EEDE F, DEL-FAVERO J et al. Glucocorticoid receptor gene-based SNP analysis in patients with recurrent major depression. Neuropsychopharmacology 2006;31:620–627. 26. RIBEIRO SC, TANDON R, GRUNHAUS L, GREDEN JF. The DST as a predictor of outcome in depression: a metaanalysis. Am J Psychiatry 1993;150:1618–1629. 27. ZOBEL AW, NICKEL T, SONNTAG A, UHR M, HOLSBOER F, ISING M. Cortisol response in the combined dexamethasone/CRH test as predictor of relapse in patients with remitted depression. A prospective study. J Psychiatr Res 2001;35:83–94. 28. KRESS C, THOMASSIN H, GRANGE T. Local DNA demethylation in vertebrates: how could it be performed and targeted? FEBS Lett 2001;494:135–140. 29. ARIAS B, CATALAN R, GASTO C, GUTIERREZ B, FANANAS L. 5-HTTLPR polymorphism of the serotonin transporter gene predicts non-remission in major depression patients treated with citalopram in a 12-weeks follow up study. J Clin Psychopharmacol 2003;23:563–567.
17
Acta Neuropsychiatrica 2009: 21: 11–17 All rights reserved DOI: 10.1111/j.1601-5215.2008.00342.x
ACTA NEUROPSYCHIATRICA
Association of glucocorticoid receptor polymorphisms with the susceptibility to major depressive disorder and treatment responses in Korean depressive patients Lee H-Y, Kang R-H, Han S-W, Paik J-W, Chang HS, Jeong YJ, Lee M-S. Association of glucocorticoid receptor polymorphisms with the susceptibility to major depressive disorder and treatment responses in Korean depressive patients. Objective: Major depressive disorder (MDD) is closely related to stress reactions and serotonin probably underpins the pathophysiology of MDD. Alterations of the hypothalamic-pituitary-adrenal axis at the gene level have reciprocal consequences on serotonin neurotransmission. Glucocorticoid receptor (GR) polymorphisms affect glucocorticoid sensitivity, which is associated with cortisol feedback effects. Therefore, we hypothesised that GR polymorphisms are associated with the susceptibility to MDD and predict the treatment response. Method: Ninety-six subjects with a minimum score of 17 on the 21-item Hamilton Depression Scale (HAMD) at baseline were enrolled into the present study. The genotypes of GR (N363S, ER22/23EK, Bcl1, and TthIII1 polymorphisms) were analysed. The HAMD score was again measured after 1, 2, 4 and 8 weeks of antidepressant treatment to detect whether the therapeutic effects differed with the GR genotype. Results: Our subjects carried no N363S or ER22/23EK genetic polymorphisms and three types of Bcl1 and TthIII1 genetic polymorphisms. The C/C genotype and C allele at Bcl1 polymorphism were more frequent in MDD patients than in normal controls (p , 0.01 and p ¼ 0.01, respectively). The genotype distributions did not differ significantly between responders and non-responders. Conclusion: These results suggest that GR polymorphism cannot predict the therapeutic response after antidepressant administration. However, GR polymorphism (Bcl1) might play a role in the pathophysiology of MDD. Future studies should check this finding in larger populations with different characteristics.
Introduction
Hyperactivity of the hypothalamic-pituitaryadrenal (HPA) axis is one of the most consistent neuroendocrine abnormalities in major depressive disorder (MDD). Specifically, patients with MDD show increased concentrations of cortisol in the plasma, urine and cerebrospinal fluid (CSF) and an exaggerated cortisol response to adrenocorticotrophic hormone (ACTH) (1–3). The corticosteroid receptor hypothesis has been proposed for the pathogenesis of MDD, which focuses on impaired corticosteroid receptor signalling, leading to
Hwa-Young Lee1,2,3, Rhee-Hun Kang1,2,3, Sang-Woo Han4, Jong-Woo Paik5, Hun Soo Chang1,2, Yoo Jung Jeong1,2, Min-Soo Lee1,2,3 1 Clinical Research Center for Depression, Korea University, Seoul, Korea; 2Institute of Human Behavior and Gene, Korea University, Seoul, Korea; 3 Department of Psychiatry, College of Medicine, Korea University, Seoul, Korea; 4Department of Psychiatry, College of Medicine, Soonchunhyang University, Seoul, Korea; and 5Department of Psychiatry, College of Medicine, Kyunghee University, Seoul, Korea
Keywords: depression; glucocorticoid receptor; polymorphism; susceptibility; treatment response Min-Soo Lee, 126-1 Anamdong 5-ga, Seongbuk-gu, Seoul 136-705 Seoul, Korea. Tel: 182 2 920 5997; Fax: 182 2 923 8119; E-mail: [email protected]
a reduced negative feedback of cortisol, an increased production of corticotropin-releasing hormone (CRH) and hypercortisolism (2). Interestingly, cortisol and CRH affect the serotonin (5-HT) system (1,4). During the stress response, glucocorticoids (GCs) stimulate all these features of 5-HT transmission (5). Conversely, 5HT transmission is impaired and noradrenergic transmission in the hippocampus is suppressed during chronic psychosocial stress and hypercortisolism, which is similar to the series of events evident during depression (6). This makes the GC 11
Lee et al. receptor (GR) a prime candidate gene for associations with susceptibility for MDD. Moreover, initial studies of GR antagonist mifepristone and other HPA-axis-targeting agents in affective disorder have been encouraging (7). The human gene coding for the GR is referred to as nuclear receptor subfamily 3, Group C, Member 1 (NR3C1). It has been reported that the following polymorphisms of the GC receptor gene alter GC sensitivity (8–11); N363S: 11088A.G, ER22/ 23EK: 168G.A, Bcl1: 2646C.G, and TthIII1: 13807C.T polymorphisms. The Bcl1 polymorphism involves a Bcl1 restriction site in intron 2 and is a C-to-G nucleotide change that appears 646 base pairs (bp) downstream from exon 2 (Bcl1). The TthIII1 polymorphism was identified as the location of a C-to-T nucleotide change that appears 3807 bp upstream of the start of GR mRNA. The N363S polymorphism is exon 2, which results in an aminoacid change from asparagine (N) to serine (S). The ER22/23EK polymorphism, also located in exon 2, consists of two linked nucleotide changes in codons 22 and 23 (GAG AGG to GAA AAG). The ER23EK polymorphism consists of two linked point mutations in codon 22 [silent mutations changing codon 22 from GAG to GAA, coding for glutamic acid (E)] and in codon 23 [changes from AGG to AAG, resulting in an amino-acid change from arginine (R) to lysine (K) (12). The GR polymorphisms affect GC sensitivity, which is associated with cortisol feedback effects. The degree of cortisol feedback is closely related to the development of depression and may also be affected by antidepressants (13). Therefore, we hypothesised that the above four GR polymorphisms are associated with the susceptibility to MDD and predict the response to treatment with citalopram.
Material and methods Subjects
The subjects in our previous studies (14,15) were included in the present study. This study was performed on patients aged 18–65 years diagnosed with MDD who visited Korea University Anam Hospital between March 2003 and December 2005. Each patient was diagnosed with MDD by psychiatrists using the Structured Clinical Interview for Diagnostic and Statistical Manual-IV Axis I Disorders – Korean version (SCID) (16). Patients with a minimum baseline score of 17 on the 21-item Hamilton Depression Scale (HAMD) were eligible for inclusion in this study (17). None of the included patients had another psychiatric axis I diagnosis, such as anxiety disorder, bipolar 12
disorder, or alcohol or drug dependency, or neurological diseases such as stroke, epilepsy or chronic diseases (other than controlled diabetes and hypertension) that might have impeded their psychological evaluation. Of the 138 patients that were registered, 96 patients who took the experimental drug at least once and who had at least one efficacy measurement were classified as the intention-to-treat (ITT) group. Of 96 ITT patients, the genotypes of only 84 patients were successfully analysed. At first week, all subjects succeeded to follow-up. Seventy-seven subjects at second week, 73 subjects at fourth week and 65 subjects at eighth week remained. The symptoms and severity of depressive disorder were evaluated by the HAMD at baseline and after weeks 1, 2, 4 and 8. Participants showing a reduction in the HAMD score of more than 50% relative to baseline were defined as responders, with the other participants designated as non-responders (18). Before entry into the study, a 2-week washout was performed on those patients who were being treated with different drugs. During the study period, the patients self-administered 20 mg of the antidepressant citalopram daily as the starting dose, with a daily dose of 20–60 mg applied at 1, 2, 4 and 8 weeks at the discretion of the investigators according to clinical situation of each patient. After 1 week, the investigators were permitted to increase the dosage, if the patient’s symptoms were not responding and the patient was tolerable at the dosage, or to maintain the same regimen, if the patient’s symptoms were improved and the patient was tolerable at the dosage. If not tolerable at the dosage, in contrast, the dosage was decreased. Dose escalations were based on clinical response and tolerance of side-effects. Among the various types of antidepressants, citalopram is well tolerated, with the most common side-effects being gastrointestinal and including nausea, diarrhoea and dry mouth. Citalopram is the most highly selective serotonin reuptake inhibitor, with little or no affinity for a variety of receptor types. For this reason, we chose citalopram as a study drug. Benzodiazepine was allowed as a coprescribed drug. This study was approved by the Ethics Committee of Korea University Anam hospital and a patient consent form for the genetics study (by the Ministry of Health and Welfare) was prepared. One hundred and five control subjects consisted of randomly selected healthy individuals of Korean origins who visited the Korea University Anam hospital for regular health screenings. Subjects with a history of depressive disorder or psychotropic medication history were excluded from the control group by applying the Structured Clinical
Glucocorticoid receptor polymorphism and depression Interview for SCID. Control subjects were excluded if they had any familial psychiatric history, or if they had scores of 10 on the Beck Depression Inventory (19). Subjects in the control group also signed consent forms for participation in the genetics study (published by the Ministry of Health and Welfare of Korea). Genotyping
ER22/23EK polymorphism. DNA was extracted from the peripheral blood and a polymerase chain reaction (PCR) was performed with the sense primer 5#-GATTCGGAGTTAACTAAAAG-3# and the antisense primer 5#-ATCCCAGGTCATTTCCCATC-3#. The amplification mixture contained 1 ml of DNA, 3 ml of 103 PCR buffer, 2.5 ml of 2.5 mM dNTP, 1 ml of each primer, 6 ml of 53 Gcaqture solution, 15.4 ml of distilled water and 0.1 ml of Taq polymerase (5 U/ml; iNtRON Biotechnology, Seoul, Korea). Samples were amplified using a PCR thermal cycler (Takara, Kyoto, Japan) for an initial 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C. After a final 7 min at 72°C, the reaction was terminated at 4°C. The amplified DNA was digested with the restriction endonuclease Mnl I (New England Biolabs, Ipswich, MA, USA) at 37°C for 2 h. Fragments were electrophoresed in 3% agarose gels and stained with ethidium bromide. The wild type consisted of 163- and 142-bp fragments and homozygous EK22/23EK consisted of 177- and 163-bp fragments. N363S polymorphism. A PCR was performed with the sense primer 5#-AGTACCTCTGGAGGACAGAT-3# and the antisense primer 5#-GTCCATTCTTAAGAAACAGG-3#. Samples were amplified using a PCR thermal cycler (Takara) for an initial 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C. After a final 7 min at 72°C, the reaction was terminated at 4°C. The PCR products were digested with Tsp509 I (New England Biolabs) at 65°C for 2 h. Fragments were electrophoresed in 3% agarose gels and stained with ethidium bromide. The wild type consisted of 113-, 95- and 9-bp fragments and the homozygous mutant consisted of 122- and 95-bp fragments. Bcl1 polymorphism. A PCR was performed with the sense primer 5#-AGAGCCCTATTCTTCAAACTG-3# and the antisense primer 5#-GAGAAATTCACCCCTACCAAC-3#. Samples were amplified using a PCR thermal cycler (Takara) for an initial 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C. After a final 7 min at 72°C, the reaction was terminated at 4°C. The
amplified DNA was digested with the restriction endonuclease Bcl1 (New England Biolabs), which cuts at the 646C site downstream of exon 2. Fragments were electrophoresed in 3% agarose gels and stained with ethidium bromide. The 418C allele consisted of 151- and 257-bp fragments and the 418G allele consisted of a 418-bp fragment. Tth111i polymorphism. A PCR was performed with the sense primer 5#-GGCCACAACAATAACCCAGT-3# and the antisense primer 5#-TGAAAGTGTTTTATGCACAGTATCC-3#. Samples were amplified using a PCR thermal cycler (Takara) for an initial 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 61.8°C, and 30 s at 72°C. After a final 7 min at 72°C, the reaction was terminated at 4°C. The amplified DNA was digested with the restriction endonuclease Tth1111 (New England Biolabs), which cuts at the 3807C site upstream of the GR mRNA start. Fragments were electrophoresed in 3% agarose gels and stained with ethidium bromide. The 3807C allele consisted of 167- and 170-bp fragments and the 3807T allele consisted of a 337-bp fragment. Statistical analysis
A last-observation-carried-forward technique for missing data was used to analyse efficacy in the ITT group, which included all patients who received medication at least once and who had at least one efficacy or safety measurement taken. The presence of Hardy-Weinberg equilibrium and genotype differences was examined using the chi-squared test for categorical variables and Student’s t-test or one-way ANOVA for continuous variables. Repeated-measures ANOVAs were performed for each weekly HAMD score. A probability value of p , 0.05 was considered statistically significant. Probability values were corrected by multiplying the number of comparisons made (Bonferroni correction).
Results Demographic data of the subjects
The gender distribution did not differ significantly between control subjects (40 males/66 females) and MDD patients (26 males/70 females) (p ¼ 0.12). There was no significant difference in the age distribution between control subjects and the MDD patients (49.1 16.3 years, mean SD in MDD patients and 52.3 10.8 years in control subjects, p ¼ 0.10). 13
Lee et al. Genetic predisposition to MDD
Table 1 shows the genomic location, nomenclature and single nucleotide polymorphism (SNP) reference of four genetic variants in NR3C1. Our subjects carried no N363S polymorphisms (all GG, with no G/A or A/A), no ER22/23EK polymorphisms (all A/A, with no G/A or G/G), and three types of Bcl1 and Tth1111 genetic polymorphisms. The genotype distributions of Bcl1 polymorphism conformed to Hardy-Weinberg equilibrium in both normal controls and MDD patients (p ¼ 0.06 and 0.26, respectively) and the results of the Tth1111 polymorphism were within the equilibrium in these two groups (p ¼ 0.92 and 0.73, respectively). The Bcl1 genotype distributions differed significantly between control subjects and MDD patients (Table 2). The C/C genotype and C allele of Bcl1 polymorphism were more frequent in MDD patients than in normal controls (p , 0.01 and p ¼ 0.01, respectively). After Bonferroni correction, the association remained statistically significant. Although the C allele of Tth1111 polymorphism was more frequent in MDD patients than in normal controls (p ¼ 0.04), this difference lost significance after applying Bonferroni correction. Haplotype analysis was shown in Table 3. The distribution of the haplotype was significantly different between MDD patients and normal controls (p ¼ 0.001). The haplotype carrying the C allele of Bcl1 and C allele of Tth1111 was more frequent in MDD patients than in normal controls. Prediction of responses and clinical variables according to GR polymorphisms
The genotype distributions did not differ significantly between responders and non-responders (Table 4). The age at onset, duration of illness and number of previous episodes neither differed among the genotype in MDD patients (Table 3). The HAMD scores at baseline and 8 weeks did not differ significantly with the genotype in MDD patients (Table 5). When HAMD baseline scores were included to analysis as covariate, there was no significant difference in HAMD score at 8 weeks Table 1. Location, nomenclature, and single nucleotide polymorphism (SNP) reference of four genetic variants in NR3C1
Name
Genomic location
Nucleotide change
Amino-acid change
dbSNP reference
Bcl1 N363S ER22/23EK TthIII1
Promoter Exon 2 Exon 2 Promoter
2646C.G 11088A.G 168G.A 13807C.T
– Asn363Ser Arg23Lys –
rs41423247 rs1800445 rs6195 rs10052957
14
according to genotypes (both Bcl1 and Tth1111 polymorphism, data not shown). With a repeated-measures ANOVA for HAMD scores from baseline to week 8 against the Bcl1 and Tth1111 polymorphism, we observed a significant effect of the change in HAMD score over the 8 weeks of treatment and no genotype effect of the Bcl1 and Tth1111 polymorphism on change in HAMD score (data not shown). When baseline HAMD scores were included to analysis as a covariate, there was no significant difference in HAMD scores at 8 weeks between the Bcl1 or Tth1111 genotypes (data not shown).
Discussion
This study was designed to identify the role of GR polymorphisms on the genetic predisposition to MDD and on the therapeutic response to antidepressant. We showed that the C/C genotype and C allele in the Bcl1 polymorphism are more frequent in MDD patients than in normal controls. G allele of Bcl1 polymorphism has been reported to be associated with hypersensitivity to GCs, as indicated by an increased response to the ACTH- and cortisol-suppressive effects of low-dose dexamethasone (8). In other words, the C allele of Bcl1 polymorphism is more GC-resistant than G allele. Increased GR resistance has generally been described as one of the endocrine hallmarks of depression, which forms part of a vicious circle of HPA-axis dysregulation leading to an upregulation of central CRH (2). In line with the result of this study, C allele, which is more GC-resistant, could induce the development of depression. Conversely, van Rossum et al. reported that the frequency of the Bcl1 G/G genotype was significantly higher in MDD patients than in normal controls (20), explaining their result by the tissue specific effect of Bcl1 polymorphism. Contrary to the action in the hypothalamus, GC increases CRH expression in limbic regions such as the amygdala (21,22). G allele of Bcl1 polymorphism which is more GCsensitive might thus have an increased positive GC feedback effect on CRH in the limbic system, resulting in an increased production of CRH, and increased CRH in limbic regions has been linked to depression-like symptoms (2,21). We found few T/T genotypes in the TthIII1 polymorphism and no N363S and ER22/23EK polymorphisms. While the C allele in the TthIII1 polymorphism was more frequent in MDD patients than in control subjects (p ¼ 0.04), this difference lost significance after Bonferroni correction. Rosmond et al. (23) showed that the Tth1111 polymorphism was
Glucocorticoid receptor polymorphism and depression Table 2. Genotype distributions and allele frequencies in normal control subjects and MDD patients Bcl1 genotype Group Normal controls (%) MDD (%)
Bcl1 allele
C/C
G/C
G/G
p* (pcorr)
C
G
p† (pcorr)
50 (47.6) 58 (69.9)
50 (47.6) 21 (25.3)
5 (4.8) 4 (4.8)
,0.01 (,0.01)
150 (71.4) 137 (82.5)
60 (28.6) 29 (17.5)
0.01 (0.02)
TthIII1 genotype Group Normal controls (%) MDD (%)
TthIII1 allele
C/C
C/T
T/T
p*,‡
C
T
p*
86 (82.7) 74 (92.5)
17 (16.3) 6 (7.5)
1 (1.0) 0 (0)
0.13
189 (90.9) 154 (96.3)
19 (9.1) 6 (3.8)
0.04 (0.08)
*Comparison of genotype frequencies between MDD and normal control group. †Comparison of allele frequencies between MDD and normal control group. ‡Genotype comparisons are made by Fisher's exact test. pcorr indicates p-value after Bonferroni correction.
associated with increased diurnal cortisol levels. However, another study found no differences in cortisol levels between the TthIII1 genotypes before and after dexamethasone suppression test (11), and no interactions with N363S or Bcl1. However, all carriers of the ER22/23EK polymorphism carried the TthIII1 T variant. The cortisol response to 1 mg of dexamethasone was significantly lower in carriers of both TthIII1 CT/TT and ER22/23EK polymorphisms than in the two other groups (TthIII1 CC and ER22/23ER, and TthIII1 CT/TT and ER22/23ER). In other words, this haplotype is associated with a higher resistance to GC. The TthIII1 polymorphism might be functionally relevant only in combination with ER22/23EK. In the present study, the TthIII1 polymorphism existed without the ER22/23EK variant being present, in contrast with it being reported that the ER22/23EK polymorphism is without exception linked to the TthIII1 T polymorphism (11). Therefore, the absence of a ER22/23EK polymorphism could be responsible for the absence of an association between MDD and TthIII1 polymorphism in this study.
Table 3. Haplotype analysis of GR polymorphisms Haplotype
n (%)
(Bcl1, TthIII1)
Group
1/1
HT1
(C, C)
HT2
(G, C)
HT3
(G, T)
HT4
(C, T)
NC MDD NC MDD NC MDD NC MDD
50 (45.5) 58 (69) 2 (1.8) 3 (3.6) 1 (0.9) 0 (0) 0 (0) 0 (0)
*/2 60 26 108 81 109 84 110 84
(54.5) (31) (98.2) (96.4) (99.1) (100) (100) (100)
NC, normal control. *Obtained using chi-squared test and Fisher's exact test.
Total 110 84 110 84 110 84 110 84
(100) (100) (100) (100) (100) (100) (100) (100)
p* 0.001 0.654 1.000 –
The ER22/23EK polymorphism was shown to be associated with a higher resistance to GCs (9) and an increased risk of developing MDD (20,24). It has been shown that the N363S polymorphism is associated with hypersensitivity to GCs (10). We did not find these polymorphisms in our Korean population and hence are unable to comment on the association between these polymorphisms and MDD. We did not identify a relation between GR polymorphisms and the therapeutic response to citalopram in the present study. It has been reported that ACTH levels are higher in carriers of the Bcl1 polymorphism than in non-carriers (from the dexamethasone/CRH test), with a trend towards decreases in HAMD scores being smaller in carriers than in non-carriers (25). Also, ER22/ 23EK carriers showed a significantly faster clinical response to antidepressant therapy (20). It was proposed that antidepressants can inhibit steroid transporters localised on the endothelial cells of the blood-brain barrier(or blood-CSF barriers) and in neurons in humans, such as the multidrug resistance p-glycoprotein, thus increasing the access of cortisol to the brain and the GC-mediated negative feedback effects on the HPA axis. Enhanced cortisol effects in the brain might maximise the therapeutic effects of antidepressants (13). At baseline, the symptomatology (as quantified by HAMD scores), age at onset, duration of illness and number of previous episodes did not differ from the GR polymorphisms in this study. Similarly, van Rossum et al. found no association of GR polymorphisms with clinical characteristics (20). High HPA-axis activity has been related to the risk of relapse in MDD (26,27). Furthermore, previous depressive episodes and HPA-axis hyperactivity might permanently alter the reactivity of 15
Lee et al. Table 4. The prediction of response according to the GR polymorphisms in MDD patients Bcl1 genotype
Responder (%) Non-responder (%) Total (%)
Bcl1 allele
G/G
G/C
C/C
p*
G
C
p†
36 (62.1) 22 (37.9) 58 (100)
13 (61.9) 8 (38.1) 21 (100)
3 (75.0) 1 (25.0) 4 (100)
0.87
85 (62.0) 52 (38.0) 137 (100)
19 (65.5) 10 (34.5) 29 (100)
0.83
TthIII1 genotype
TthIII1 allele
C/C Responder (%) Non-responder (%) Total (%)
C/T & T/T
46 (62.2) 28 (37.8) 74 (100)
3 (50.0) 3 (50.0) 6 (100)
p*
C
T
p†
0.67
95 (61.7) 59 (38.3) 154 (100)
3 (50.0) 3 (50.0) 6 (100)
0.68
*Comparison of genotype frequencies between responder and non-responder group. †Comparison of allele frequencies between responder and non-responder group. Responder group is defined as those patients who show more than or equal to a 50% relative decrease of HAMD score at week 8 compared with that at baseline.
responses in clinically depressed patients. Fourth, because the plasma levels of cortisol and ACTH were not measured, it is not possible to investigate the functional role of GR polymorphism. Fifth, T/T genotypes in the TthIII1 polymorphism were present at below 1% and hence this polymorphism could not be considered to be a single-nucleotide polymorphism in our Korean population. In contrast, the distributions of the C/C, CT and TT genotypes have been reported at 39.7, 44.5 and 15.8%, respectively, in Caucasians (11). Our Korean subjects carried no N363S or ER22/ 23EK genetic polymorphisms. These results cannot be generalised because of genotype distributions varying with ethnicity. Finally, the relatively small sample limits the generalisability of our findings. Our sample provided power greater than 0.67 at p , 0.05 to detect effects of the size we observed in our sample. This indicates that our sample provides a moderate power, considering that a power cut-off of 0.5 and an a-value ,0.05 are often used. Notwithstanding the above limitations, this is the first study to examine the possible association between GR polymorphisms and MDD in a Korean population. Elucidating the role of GR polymorphisms in MDD requires further studies applying a
the axis because the activation of the GR has been shown to change the methylation pattern of specific genes (28). In the present study the number of previous episodes did not vary with the GR polymorphism. The value for linkage disequilibrium is D# ¼ 0.815, r2 ¼ 0.159. The linkage disequilibrium was not found between these two polymorphisms. This study was subject to several limitations. First, although Korean subjects are highly genetically homogeneous, the small number of subjects included represents a one limitation. Larger number of subjects are needed to investigate the role of GR polymorphism in MDD. Second, treatment periods (5.97 2.98 weeks, mean SD) were various in this study. Third, the same dosage of citalopram was not prescribed to each patient and the plasma levels of citalopram were not measured in this study. Because pharmacokinetic activities are different among the patients, it is not possible that the same dosage of antidepressant could be prescribed to each patient in clinical settings. It could be better that dose escalations would be based on clinical response and tolerance of side-effects. Moreover, a previous study (29) reported the absence of any significant relationships between citalopram plasma levels and clinical Table 5. Clinical characteristics according to the GR polymorphisms in MDD patients Bcl1 Genotype G/G HAMD (baseline) HAMD (8-week) Age at onset Duration of illness Number of previous episodes
23.1 10.0 47.2 6.4 1.1
5.5 7.2 14.8 7.2 0.78
G/C 22.0 11.4 42.1 8.9 1.0
C/C 3.6 7.8 17.7 10.3 0.0
Data represent mean SD. Genotype comparisons are made by *ANOVA or †Kruskal-Wallis test.
16
TthIII1 genotype
20.3 8.0 50.3 6.9 1.0
p 2.8 9.5 20.0 7.9 0.0
0.40 0.62 0.41* 0.44* 0.90†
C/C 22.5 10.2 46.3 7.1 1.0
4.9 7.3 15.5 8.2 0.52
C/T & T/T
p
0.30 0.80 0.82* 0.54* 1.0†
20.3 11.0 44.7 5.0 1.0
1.9 10.1 22.3 5.5 0.0
Glucocorticoid receptor polymorphism and depression biochemical approach and involving a larger number of subjects to decrease the risk for false positives of this study. Acknowledgements This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (03-PJ10-PG13-GD01-0002).
References 1. HOLSBOER F, BARDEN N. Antidepressants and hypothalamic-pituitary-adrenocortical regulation. Endocr Rev 1996;17:187–205. 2. HOLSBOER F. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 2000;23:477–501. 3. NEMEROFF CB. The corticotropin-releasing factor (CRF) hypothesis of depression: new findings and new directions. Mol Psychiatry 1996;1:336–342. 4. DE KLOET ER, VREUGDENHIL E, OITZL MS, JOELS M. Brain corticosteroid receptor balance in health and disease. Endocr Rev 1998;19:269–301. 5. MEIJER OC, DE KLOET ER. Corticosterone and serotonergic neurotransmission in the hippocampus: functional implications of central corticosteroid receptor diversity. Crit Rev Neurobiol 1998;12:1–20. 6. WOLKOWITZ OM, REUS VI, MANFREDI F, INGBAR J, BRIZENDINE L, WEINGARTNER H. Ketoconazole administration in hypercortisolemic depression. Am J Psychiatry 1993;150:810–812. 7. PORTER RJ, GALLAGHER P. Abnormalities of the HPA axis in affective disorders: clinical subtypes and potential treatments. Acta Neuropsychiatrica 2006;18:193–209. 8. VAN ROSSUM EF, KOPER JW, VAN DEN BELD AW et al. Identification of the BclI polymorphism in the glucocorticoid receptor gene: association with sensitivity to glucocorticoids in vivo and body mass index. Clin Endocrinol 2003;59:585–592. 9. VAN ROSSUM EF, KOPER JW, HUIZENGA NA et al. A polymorphism in the glucocorticoid receptor gene, which decreases sensitivity to glucocorticoids in vivo, is associated with low insulin and cholesterol levels. Diabetes 2002;51:3128–3134. 10. HUIZENGA NA, KOPER JW, DE LANGE P et al. A polymorphism in the glucocorticoid receptor gene may be associated with and increased sensitivity to glucocorticoids in vivo. J Clin Endocrinol Metab 1998;83:144–151. 11. VAN ROSSUM EF, LAMBERTS SW. Polymorphisms in the glucocorticoid receptor gene and their associations with metabolic parameters and body composition. Recent Prog Horm Res 2004;59:333–357. 12. KOPER JW, STOLK RP, DE LANGE P et al. Lack of association between five polymorphisms in the human glucocorticoid receptor gene and glucocorticoid resistance. Hum Genet 1997;99:663–668. 13. PARIANTE CM, THOMAS SA, LOVESTONE S, MAKOFF A, KERWIN RW. Do antidepressants regulate how cortisol affects the brain? Psychoneuroendocrinology 2004;29:423–447.
14. CHOI MJ, KANG RH, LIM SW, OH KS, LEE MS. Brainderived neurotrophic factor gene polymorphism (Val66Met) and citalopram response in major depressive disorder. Brain Res 2006;1118:176–182. 15. HAM BJ, LEE BC, PAIK JW et al. Association between the tryptophan hydroxylase-1 gene A218C polymorphism and citalopram antidepressant response in a Korean population. Prog Neuropsychopharmacol Biol Psychiatry 2007;31:104–107. 16. HAN OS, HONG JP. Structured clinical interview for DSMIV axis I disorder-Korean version. Seoul: Hana Medical Publishing Co, 2000. 17. HAMILTON M. Development of a rating scale for primary depressive illness. Br J Soc Clin Psychol 1967;6:278–296. 18. FRANK E, PRIEN RF, JARRETT RB et al. Conceptualization and rationale for consensus definitions of terms in major depressive disorder. Remission, recovery, relapse, and recurrence. Arch Gen Psychiatry 1991;48:851–855. 19. BECK AT, WARD CH, MENDELSON M, MOCK J, ERBAUGH J. An inventory for measuring depression. Arch Gen Psychiatry 1961;4:561–571. 20. VAN ROSSUM EF, BINDER EB, MAJER M et al. Polymorphisms of the glucocorticoid receptor gene and major depression. Biol Psychiatry 2006;59:681–688. 21. REUL JM, HOLSBOER F. Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression. Curr Opin Pharmacol 2002;2:23–33. 22. SCHULKIN J, GOLD PW, MCEWEN BS. Induction of corticotropin-releasing hormone gene expression by glucocorticoids: implication for understanding the states of fear and anxiety and allostatic load. Psychoneuroendocrinology 1998;23:219–243. 23. ROSMOND R, CHAGNON YC, HOLM G et al. A glucocorticoid receptor gene marker is associated with abdominal obesity, leptin, and dysregulation of the hypothalamicpituitary-adrenal axis. Obes Res 2000;8:211–218. 24. BROUWER JP, APPELHOF BC, VAN ROSSUM EF et al. Prediction of treatment response by HPA-axis and glucocorticoid receptor polymorphisms in major depression. Psychoneuroendocrinology 2006;31:1154–1163. 25. VAN WEST D, VAN DEN EEDE F, DEL-FAVERO J et al. Glucocorticoid receptor gene-based SNP analysis in patients with recurrent major depression. Neuropsychopharmacology 2006;31:620–627. 26. RIBEIRO SC, TANDON R, GRUNHAUS L, GREDEN JF. The DST as a predictor of outcome in depression: a metaanalysis. Am J Psychiatry 1993;150:1618–1629. 27. ZOBEL AW, NICKEL T, SONNTAG A, UHR M, HOLSBOER F, ISING M. Cortisol response in the combined dexamethasone/CRH test as predictor of relapse in patients with remitted depression. A prospective study. J Psychiatr Res 2001;35:83–94. 28. KRESS C, THOMASSIN H, GRANGE T. Local DNA demethylation in vertebrates: how could it be performed and targeted? FEBS Lett 2001;494:135–140. 29. ARIAS B, CATALAN R, GASTO C, GUTIERREZ B, FANANAS L. 5-HTTLPR polymorphism of the serotonin transporter gene predicts non-remission in major depression patients treated with citalopram in a 12-weeks follow up study. J Clin Psychopharmacol 2003;23:563–567.
17
