Original Article Open Access
Copyright ©2010 Baishideng. All rights reserved.
World J Gastroenterol. Aug 21, 2010; 16(31): 3888-3896
Published online Aug 21, 2010. doi: 10.3748/wjg.v16.i31.3888
Glucocorticoid receptor gene haplotype structure and steroid therapy outcome in IBD patients
Jessica Mwinyi, Christa Wenger, Jyrki J Eloranta, Gerd A Kullak-Ublick, Division of Clinical Pharmacology and Toxicology, University Hospital, CH-8091 Zurich, Switzerland
Author contributions: Kullak-Ublick GA, Eloranta JJ and Wenger C designed the study; Wenger C and Eloranta JJ performed the laboratory experiments; Mwinyi J performed the sequence and statistical analysis; Mwinyi J, Eloranta JJ and Kullak-Ublick GA wrote the manuscript.
Supported by The Swiss IBD Cohort Study (SNF Grant 33CSC0-108792); the Swiss National Science Foundation (Grant 32-120463/1); the Zurich University Research Priority Programme “Integrative Human Physiology” (ZIHP); the Center of Clinical Research at the University Hospital Zurich; and the Novartis Foundation for Biomedical Research
Correspondence to: Gerd Kullak-Ublick, Professor, Division of Clinical Pharmacology and Toxicology, University Hospital, CH-8091 Zurich, Switzerland. gerd.kullak@usz.ch
Telephone: +41-44-2552068 Fax: +41-44-2554411
Received: March 11, 2010
Revised: May 14, 2010
Accepted: May 21, 2010
Published online: August 21, 2010

Abstract

AIM: To study whether the glucocorticoid receptor (GR/NR3C1) gene haplotypes influence the steroid therapy outcome in inflammatory bowel disease (IBD).

METHODS: We sequenced all coding exons and flanking intronic sequences of the NR3C1 gene in 181 IBD patients, determined the single nucleotide polymorphisms, and predicted the NR3C1 haplotypes. Furthermore, we investigated whether certain NR3C1 haplotypes are significantly associated with steroid therapy outcomes.

RESULTS: We detected 13 NR3C1 variants, which led to the formation of 17 different haplotypes with a certainty of > 95% in 173 individuals. The three most commonly occurring haplotypes were included in the association analysis of the influence of haplotype on steroid therapy outcome or IBD activity. None of the NR3C1 haplotypes showed statistically significant association with glucocorticoid therapy success.

CONCLUSION: NR3C1 haplotypes are not related to steroid therapy outcome.

Key Words: Inflammatory bowel disease; Steroid therapy; Glucocorticoid receptor; Pharmacogenetics; Haplotype analysis



INTRODUCTION

Inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are multifactorial disorders, which are characterized by chronic recurrent inflammation of the gastrointestinal tract[1]. The molecular pathogenesis of IBD is not fully elucidated, although an exaggerated mucosal immune response triggered by intestinal bacteria in genetically susceptible individuals appears to play an important role[2]. The combined prevalence of CD and UC is estimated to be 100 to 200 per 100 000 individuals in developed countries[3]. IBD shows extensive variation in individual clinical presentation and outcomes, which is likely to be caused by differences in genetic susceptibility, environmental factors, intestinal bacteria and activation of the intestinal immune system[3].

Although great advances have been made in the management and therapy of IBD, curative therapy does not yet exist. The anti-inflammatory agents mesalazine (5-aminosalicylic acid, 5-ASA) and sulfasalazine, in combination with glucocorticoids (GCs), are common first line therapy options in induction and maintenance of UC remission. Severe cases of UC are treated intravenously with GCs or cyclosporine. CD is mainly treated with GCs and/or antibiotics, and azathioprine (AZA), 6-mercaptopurine (6-MP), or the anti-folate methotrexate (MTX) are often added to maintain the state of remission[4]. GC-resistant or -dependent disease courses can be treated with anti-TNF-α antibodies, such as infliximab and adalimumab. GCs are often used in the initial treatment of most cases of moderate to severe active UC or CD. However, 20% of patients develop GC resistance within one year of treatment[5,6]. Non-response to GCs often leads to the need for a surgical intervention as a result of a poor therapy outcome. For example, it has been reported that 38% and 29% of steroid-resistant CD and UC patients, respectively, required surgery within one year after beginning GC treatment[5].

Glucocorticoids are potent inhibitors of T cell activation and cytokine secretion, primarily via binding to the cytoplasmically located glucocorticoid receptor (GR) as ligands. Due to ligand binding, homodimers consisting of two activated GRs are formed that translocate into the nucleus. The complex subsequently binds to specific glucocorticoid response elements (GREs) within the regulatory regions of GR target genes[7]. The mechanisms by which GC resistance develops, are not fully understood. Three possible mechanisms have been proposed. First, decreased plasma levels of GCs through overexpression of the drug efflux system P-glycoprotein (MDR1). Second, an altered function of GR or, third, excessive synthesis of pro-inflammatory cytokines induced by activation of pro-inflammatory transcription factors may reduce the affinity of GR to its ligands and lead to the development of GC resistance[4].

GR is known to be expressed as several polymorphic variants[8]. Several mutations in the NR3C1 gene have been found to modulate individual GC sensitivity in in vitro investigations and in studies with healthy individuals[9,10]. In the present study we evaluated the association between the NR3C1 gene haplotypes and therapeutic outcome of GC administration in a well-sized cohort of 181 patients with IBD. The aim was to comprehensively determine abundant GR variants by sequencing all protein-coding NR3C1 exons (exons 2 to 9) and the first 50 bp of the neighbouring intronic regions in all individuals. We hypothesized that NR3C1 gene polymorphisms may influence GC sensitivity and thus might serve as predictive markers for treatment success with GCs in IBD patients.

MATERIALS AND METHODS
Patients

One hundred and eighty-five clinically diagnosed Swiss IBD patients were recruited at the centers participating in the Swiss Inflammatory Bowel Disease Cohort Study (SIBDCS)[11]. All patients gave their informed consent for inclusion into the study. An ethical approval was obtained from the Medical Ethical Committees of the University Hospital Lausanne, Switzerland, and all local study sites. All patients had been treated with steroids and past steroid therapy outcome had been recorded. The standard employed criteria for the steroid therapy success or failure are available on the website http://www.epact.ch. Briefly, an insufficient response upon appropriate treatment in terms of doses and duration was considered a steroid therapy failure. EDTA-blood samples were stored at the central tissue repository at the Institute of Pathology, University of Bern, Switzerland. The SIBDCS data center at the University Hospital of Lausanne, Switzerland, provided data on past and current disease characteristics and GC therapy outcome. Diagnosis of IBD (CD or UC) was confirmed by the study investigators based on clinical presentation, endoscopic findings and histology.

Sequencing reactions

DNA was extracted from EDTA-blood using the QIAcube robotic workstation and a standard procedure (QIAamp DNA Mini Kit, QIAGEN, Switzerland). The PCR and sequencing primer design was based on the NCBI reference sequence (GenBank accession number NT_029289). Primers for genomic DNA were designed to span all expressed exons (2 to 9) and at least 50 bp of flanking intronic sequences at both 5’- and 3’-ends. The DNA sequences of purified PCR fragments were obtained with an ABI 3730xl sequencing machine. Details of the PCR primers can be found in the Table 1. Optimized PCR conditions, and methods used for subsequent purification and sequencing of the fragments are available upon request.

Table 1 Oligonucleotides used as polymerase chain reaction primers to amplify the NR3C1 exons.
Primer namePrimer sequenceNested PCRPrimer namePrimer sequence
GR 2_FCACTTAGGTTGTCTACCTTTCCTACYGR 2_FaTTCAAAAGGCCACTTAAACTTATTC
GR 2_RGATAGAAACTACTCTTCTGGTAACYGR 2_RaCCTTGGAGATCAGACCTGTTG
YGR 2_FbCTGTGCCCAGTTTCTCTTGC
YGR 2_RbCAGCCAGATCTGTCCAAAGC
YGR 2_FcTTGGAAACTCCTTCTCTGTGG
YGR 2_RcAATGTGGCATGCTGAATGG
GR 3_FCATTAGAGGACCTAGGAGCCACN
GR 3_RGAAGTGAACCAGAACCACACCN
GR 4_FTGAATTCAGTGTGTGTAAGAAGAACN
GR 4_RTTGCACTGTTTTCAGTTTGTTGN
GR 5_FCACCTGTATTCACCTGACTCTCCN
GR 5_RTTTTTTCTCCTTTTCCATGTCACN
GR 6_FGCCCCAAGCACTCATAACTCN
GR 6_RTCAGATGACAGAAGAAAACTGTGTCN
GR 7_FAATCTTGGTGTCACTTACTGTGCN
GR 7_RCCAAGATGCAGGAAGTTTAAGGN
GR 8_FCACCAACATCCACAAACTGGYGR 8_FaTTGGTCAGTGGGAACATC
GR 8_RCCACCAGTTCTTCTTACACACACYGR 8_RaATGGTGGCTTGTGCCTAC
GR 9a_FTGATGACGACTCAACTGCTTCN
GR 9a_RATCTGGGGAATTCCAGTGAGN
GR 9b_FTCCTAAAAGGGCACAGCTTCN
GR 9b_RCAATCATTTGCTTTTTGAATGCN
Haplotype analysis

The PHASE software was used to calculate the haplotypes based on the detected single nucleotide polymorphisms (SNPs) and mutations in the NR3C1 gene. PHASE predicts in silico haplotypes on the basis of a Bayesian inference algorithm[12,13]. Haplotype calculations were performed on 181 individuals, from which sequence data of adequate quality were obtained. To allow referral to specific haplotypes, a frequency-based priority criterion was used to name them (e.g. GR_1 for the most often occurring haplotype, Table 2).

Table 2 Predicted haplotypes found to be in best reconstruction for 181 inflammatory bowel diseases patients.
Haplotype number1Haplotype composition2
Absolute haplotype frequency
Relative haplotype frequencyNumber of haplotypes not included3
Reference: GGGATGGCCATGT(n = 362)
GR_111111111111111650.456
GR_241111112111211900.249
GR_341111211111111720.1992
GR_44111211111111180.022
GR_5221111211122160.017
GR_6111111211121240.011
GR_7111111211111130.008
GR_8111111111121120.0061
GR_9111111212121220.006
GR_10111122111111120.0062
GR_11221111211121120.0061
GR_12111111111121210.0031
GR_13111111221121210.003
GR_14111121111211110.003
GR_15111121211111110.003
GR_16112111111111110.003
GR_17221111111111110.0031
Figure 2
Figure 2 NR3C1 haplotypes predicted by PHASE in the cohort of 181 inflammatory bowel diseases patients. 1Counter (a to y) for the 25 theoretically arising haplotypes in the inflammatory bowel diseases cohort. SNP: Single nucleotide polymorphism.
Calculation of linkage disequilibria

Linkage disequilibria (LD) were calculated using the r2 statistics. Calculations were performed using the software package Haploview (http://www.haploview.com).

Statistical analysis

To detect differences in haplotype distribution between groups with different GC therapy outcomes, the Chi-Square test or the Fisher’s exact test was used. It was analyzed whether one or two copies of a specific haplotype were associated with a particular therapy outcome compared to the GR wild-type carriers. If the number of subjects per group was large enough, heterozygous carriers with one wild-type allele and homozygous carriers of one distinct haplotype were analyzed together against homozygous wild-type carriers. The latter calculations were only performed for haplotypes which occurred in a reasonably large (> 40) number. The statistical analysis was performed using the software package SPSS 17 (SPSS Inc., Chicago, IL).

RESULTS
NR3C1 sequence variability

DNA samples from 185 IBD patients (CD or UC) were initially sequenced for the NR3C1 coding exons 2 to 9 and 50 bp of the neighbouring intronic sequences. The sequencing results of 181 individuals were of adequate quality and further used for SNP and haplotype analyses. The sequence data were screened for genetic variations in the NR3C1 gene, using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nih.gov) and the GenBank entry NT_029289 as the reference sequence.

In Table 3 we list the allele frequencies of all detected SNPs within the IBD cohort under study. Thirteen variants were detected, which were-with exception of one mutation (rs6196, P < 0.01)-in Hardy-Weinberg equilibrium. All variants were single nucleotide substitutions. Six variants were detected within the intronic regions, whereas seven variants were found in exons (Figure 1). Three of the seven variants detected within the coding regions of the NR3C1 gene resulted in non-synonymous amino acid exchanges, while four of them did not lead to changes in the GR amino acid sequence. Eight variants occurred with an allelic frequency of more than one percent (rs56149945, rs6189, rs6190, rs4986593, rs6188, rs258750, rs10482704, rs6196). All non-synonymous amino acid exchanges (R23K, A229T, N363S) were found in the N-terminal half of GR, flanking the N-terminal transactivation domain[14]. The intronic variant found at DNA position 3824968 has not been previously listed in the NCBI SNP database.

Figure 1
Figure 1 Most frequently occurring NR3C1 haplotypes and their single nucleotide polymorphisms composition. The localization of the variant nucleotides in the N3RC1 gene is indicated. All detected non-synonymous single nucleotide polymorphism (SNP) (R23K, A229T, N363S) flank the N-terminal transactivation domain. Only four out of 17 predicted haplotypes occur at a frequency higher than 2%.
Table 3 Frequencies of single nucleotide polymorphisms detected in the glucocorticoid receptor gene (NR3C1).
SNP numberAlternativename1Variant number2DNA position3DNA regioncDNA position4Nucleotide referenceNucleotide variantAmino acid exchangeAllele frequency(n = 362)Reported allele frequencies2
12.1rs61893943266Exon 2558GAE22E0.0250.002-0.034
22.2rs61903943264Exon 2560GAR23K0.0250.002-0.034
32.3rs725427423942647Exon 21177GAA229T0.0030.002
42.4rs561499453942244Exon 21580AGN363S0.0250.000-0.046
53.1rs49865933856773Intron 3TC0.2130.008-0.228
64.1rs617534843852751Intron 4GC0.0060.000-0.009
75.1rs61883843271Intron 5GT0.2900.000-0.500
86.1rs61943841288Exon 62256CTH588H0.0060.000-0.091
98.1rs2587513825207Exon 82526CTD678D0.0060.000-0.149
108.2novel SNP3824968Intron 8AG0.003NA
118.3rs2587503824816Intron 8TC0.3070.091-0.362
128.4rs104827043824690Intron 8GT0.0170.000-0.027
139.1rs61963824417Exon 92790TCN766N0.0220.058-0.325
Haplotype analysis

The 13 NR3C1 variants described above were included in the haplotype calculations using the computer program PHASE. All 181 individuals were included in the haplotype prediction analysis (Figure 2). Twenty-five NR3C1 haplotypes were predicted by PHASE to exist in the studied cohort. Furthermore, PHASE determined 17 different distinct haplotypes, which were found to be in best reconstruction for the cohort (Table 2). Six out of these 17 haplotypes occurred at a frequency higher than 1% (Figure 1). PHASE was only able to determine the haplotype structure of 174 individuals out of 181 subjects with a certainty of ≥ 95%. The data of one individual were excluded because of missing demographic data. Thus, the predicted haplotypes of 173 individuals were included in the subsequent association analysis.

The two SNPs E22E/R23K were found to be in complete linkage disequilibrium (Figure 3). This finding is in agreement with previous publications[15,16]. Furthermore, a strong but not complete linkage was found between the SNPs rs6188 and rs258750 (Figure 3).

Figure 3
Figure 3 Linkage disequilibrium calculations of single nucleotide polymorphisms in the NR3C1 gene. Linkage disequilibrium plot of r2 values of observed variants in the N3RC1 gene. Colour scheme: r2 = 0%, white, 0% < r2 < 100%, shades of grey, r2 = 100%, black.
Analysis of NR3C1 haplotypes in relation to steroid therapy outcome

An overview of the demographic data of the 173 subjects included in the association analysis is shown in Table 4 (further patient data on comedications and extraintestinal manifestations are given in Tables 5 and 6), and the haplotype combinations calculated for all patients are shown in Table 7. As the numbers of homozygous carriers of variant NR3C1 haplotypes were low, the subjects were analyzed as carriers of one or two copies of a distinct variant haplotype, irrespective of whether the other allele was determined to be wild-type or variant in the case of heterozygotes (Table 8). Furthermore, haplotypes GR_2 and GR_3 were analyzed by testing the heterozygous allele combinations GR_2 + GR_1 (wt) together with the homozygous GR_2 subjects and the allele combination GR_3 + GR_1 (wt) together with homozygous GR_3 carriers against wild-type carriers. For all individuals, prior success of GC therapy was documented, and for patients under GC therapy at the point of study entry the applied dosage was also noted.

Table 4 Demographic data of 173 inflammatory bowel diseases patients included in the association analysis.
CharacteristicsCrohn’s diseaseUlcerative colitisAll
Patients84 (49%)89 (51%)173 (100%)
Age (documented for 171 individuals)37.5 (± 15.3)41.7 (± 14.2)39.7 (± 14.9)
mean ± SD354239
Median161816
Minimum728282
Maximum
Known GC treatment outcome in the past5050100
No. of patients currently treated with GCs8360143
Male/female52 (58.4%)/ 37 (41.6%)40 (47.6%)/ 44 (52.4%)92 (53.2%)/ 81 (46.8%)
Wild-type carriers15 (16.9%)20 (23.8%)35 (20.2%)
Carriers of one variant haplotype48 (53.9%)39 (46.4%)87 (50.3%)
Carriers of two variant haplotypes26 (29.2%)25 (29.8%)51 (29.5%)
Table 5 Past and current additional medication of 173 inflammatory bowel diseases patients included in the association analysis.
Additional medicationn
5-Aminosalicylic acid142
6-Mercaptopurine33
Adalimumab3
Antibiotics64
Azathioprine122
Bisphosphonates8
Certulizumab1
Cholestyramine8
Cyclosporine8
Infliximab45
Methotrexate27
Sulfasalazine12
Ursodeoxycholic acid3
Table 6 Extraintestinal manifestations.
Extraintestinal manifestationsn (%)1
Peripheral arthritis46 (27.2)
Uveitis/iritis6 (3.6)
Pyoderma gangrenosum4 (2.4)
Erythema nodosum9 (5.3)
Aphthous oral ulcers10 (5.9)
Ankylosing spondylitis7 (4.1)
Primary sclerosing cholangitis6 (3.6)
Table 7 Predicted frequencies of haplotype combinations in 173 inflammatory bowel diseases patients.
Haplotype combinationnFrequency
GR_1 + GR_2 or GR_2 hom510.295
GR_1 + GR_3 or GR_3 hom410.237
GR_1 hom (wt)360.208
GR_2 + GR_3230.133
GR_1 + GR_4 or GR_4 hom60.035
GR_1 + GR_530.017
GR_3 + GR_620.012
GR_1 + GR_7 or GR_7 hom10.006
GR_1 + GR_16 or GR_16 hom10.006
GR_2 + GR_810.006
GR_2 + GR_610.006
GR_2 + GR_910.006
GR_6 + GR_1310.006
GR_7 + GR_1510.006
GR_2 + GR_410.006
GR_2 + GR_1110.006
GR_5 + GR_910.006
GR_9 + GR_1710.006
Table 8 Association between glucocorticoids therapy outcome and the haplotype GR_4.
HaplotypeCohortcompositionTherapy success rate in wt carriers (success/no success)Therapy success rate in het/hom variant carriers (success/ no success)P-valueOR (CI)
GR_4 mergedAll0.682 (15/7)0.4 (2/3)0.3263.214 (0.434-23.787)
Male0.733 (11/4)NA (0/0)NANA
Female0.571 (4/3)0.4 (2/3)1.0002.000 (0.194-20.614)

No significant associations were observed between haplotype GR_2 and success of GC therapy (Figure 4). Upon stratification of the patient cohort according to gender or disease subgroup (UC or CD), no significant association between therapy success or haplotype GR_2 was observed either. Similarly, when stratifying according to the subgroup of heterozygous GR_2 + GR_1 and homozygous GR_2, no statistically significant difference in therapy response compared to wild-type carriers could be observed.

Figure 4
Figure 4 Haplotype GR_2 and steroid therapy outcome. Odds ratios and confidence intervals for the number of GR_2 carriers vs wild-type carriers in the responder group compared with non-responders to glucocorticoid therapy. No significant associations were found. Statistical analysis was performed with Fisher’s exact test. OR: Odds ratio; CI: Confidence interval; CD: Crohn's disease; UC: Ulcerative colitis.

No significant associations were observed between either haplotype GR_3 (Figure 5) or GR_4 (Table 8) and GC therapy outcome, or between individual SNPs and therapy success (Figure 6). Similarly, we observed no significant associations between the severity of disease (active or inactive state of UC or CD) or currently taken GC dose levels and NR3C1 haplotypes (data not shown).

Figure 5
Figure 5 Haplotype GR_3 and steroid therapy outcome. Odds ratios and confidence intervals for the number of GR_3 carriers vs wild-type carriers in the responder group of responders compared with non-responders to glucocorticoid therapy. No significant associations were found. Statistical analysis was performed using Fisher’s exact test. CD: Crohn's disease; UC: Ulcerative colitis; NA: Not applicable.
Figure 6
Figure 6 NR3C1 variants and their influence on steroid therapy outcome. Odds ratios and confidence intervals for carriers of six single nucleotide polymorphisms against wild-type carriers in the group of glucocorticoid (GC) responders compared with GC non-responders. No significant associations were found. Statistical analysis was performed using chi-square or Fisher’s exact test.
DISCUSSION

Glucocorticoid receptor (GR) plays an important role in many physiological and pathological processes and is the main target of glucocorticoids, widely used as therapeutic agents to treat a variety of autoimmune diseases[8,17]. Two GR isoforms, GRα and GRβ, generated by alternative mRNA splicing exist[18]. Only GRα can be activated by glucocorticoid ligands, while GRβ does not bind glucocorticoids and may in fact act as an inhibitor of glucocorticoid action[19]. Genetic variation in the NR3C1 gene has been shown to affect both disease pathophysiology and response to glucocorticoid therapy[15,20-22], suggesting that SNPs might play a role in GR function and associated steroid therapy outcome also in IBD patients. GR is known to regulate the intestinal bile acid uptake transporter ASBT[23,24], the expression of which is altered in IBD patients[25]. While it has been reported that GR mRNA expression levels are not predictors of steroid response in IBD[26] and that the GR polymorphisms R23K and N363S are not associated with CD in a pediatric Caucasian population[27], no studies on the role of NR3C1 gene variants in steroid therapy success were previously available. The aim of the current study was to analyze sequence variation and haplotype structures in the coding parts of the NR3C1 gene in a cohort of 181 Swiss IBD patients. We investigated whether NR3C1 genetic variants or haplotypes may influence steroid therapy outcome in IBD patients.

We identified 13 variants in this study, of which 12 had already been previously submitted to the NCBI SNP database. We calculated the corresponding haplotypes in the IBD patient cohort and studied the association of the most prevalent SNP combinations with steroid therapy outcome, disease activity, and age of disease onset. Several NR3C1 SNPs have been previously associated with altered disease susceptibility or risk of disease progression in other autoimmune diseases, such as Guillain-Barré Syndrome or multiple sclerosis[15,20]. Most of these studies only analyzed the impact of a small number of pre-defined SNPs, such as the BclI polymorphism or the E22E/R23K polymorphisms[15,20,21]. Few reports have been published on the potential influence of NR3C1 SNPs on sensitivity to endogenous or exogenously given GCs[17,28], and only one significant association between the polymorphism E22E/R23K and sensitivity to exogenously administered GCs in elderly Dutch people has been reported[22]. So far no large cohort studies have been reported in which the influence of NR3C1 SNPs on GC therapy outcome in IBD patients has been investigated. Here, we describe five NR3C1 haplotypes occurring at a frequency > 1% and analyze the potential association of the three most common haplotypes GR_2, GR_3 and GR_4 with GC therapy outcome in IBD patients. While a large number of NR3C1 variants are already registered in the NCBI SNP database, we observed only eight variants that occurred at a frequency > 1%, and these were responsible for the composition of a relatively small group of commonly occuring haplotypes. The overall risk for a certain UC and/or CD activity state or for a different steroid therapy outcome was not altered in GR_2, GR_3 or GR_4 carriers, in comparison with the wild-type carriers. Furthermore, no significant associations were observed between individual SNPs and GC therapy success. In the case of certain SNPs/haplotypes (e.g. GR_4, E22E/R23K), a larger cohort would have been preferable in order to obtain more reliable results, as these variants occurred quite rarely in our patient group. Similarly to our observations, Dekker et al[20] could not detect any associations between distinct haplotypes and SNPs in a Guillain-Barré Syndrome cohort treated with methylprednisolone, although the authors noted that their study group was too small to obtain statistically reliable results. It remains to be seen whether the rare GR variants present in our study cohort will show significant associations in larger cohorts of IBD patients.

In conclusion, we have performed a comprehensive study analyzing the role of genetic variants in the NR3C1 gene in glucocorticoid sensitivity in a Swiss cohort of IBD patients. We show that NR3C1 haplotypes are not a general modulating factor in glucocorticoid therapy outcome.

COMMENTS
Background

Crohn’s disease and ulcerative colitis are two distinct types of inflammatory bowel disease (IBD), which is an increasingly prevalent disease condition worldwide. Wide variation is observed in clinical manifestation and therapy responses in IBD, partly due to individual genetic variation.

Research frontiers

Glucocorticoid therapy is commonly used in treatment of IBD, however the response to therapy varies between individuals. The authors hypothesized that genetic variation in the NR3C1 gene encoding the glucocorticoid receptor (GR) may affect the response to glucocorticoids in IBD patients.

Innovations and breakthroughs

In this comprehensive genetic analysis, all coding exons and exon-intron junctions of the NR3C1 gene were sequenced in 181 IBD patients, who had been treated with glucocorticoids and whose past responses to this treatment had been recorded. This is the first published study on the effects of genetic variation in GR on glucocorticoid therapy in IBD patients, in a modestly sized study cohort.

Applications

If significant associations between genetic GR variants and glucocorticoid therapy outcome had been observed, this could have allowed more considered design of the individual therapy options upon prior genotyping of the patients.

Terminology

The transcription factor of the steroid receptor family, GR, is proposed to be a major mediator of anti-inflammatory pathways elicited by therapeutically administered glucocorticoids.

Peer review

The genetic study investigates the predictive value of NR3C1 gene variants towards the clinical outcome of patients with Crohn’s disease and ulcerative colitis. Although the result of this study was negative, the study was meaningful in that abundant GR variants were determined and analyzed in IBD patients.

Footnotes

Peer reviewers: Uday C Ghoshal, MD, DNB, DM, FACG, Additional Professor, Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Science, Lucknow 226014, India; Stephan Johannes Ott, PhD, MD, Clinic for Internal Medicine I, University-Hospital Schleswig-Holstein (UK S-H), Campus Kiel, Arnold-Heller-Str. 3, Hs. 6, 24105 Kiel, Germany; Ferenc Sipos, MD, PhD, Cell Analysis Laboratory, 2nd Department of Internal Medicine, Semmelweis University, Szentkirályi u. 46., Budapest 1088, Hungary; Hoon Jai Chun, MD, PhD, AGAF, Professor, Department of Internal Medicine, Institute of Digestive Disease and Nutrition, Korea University College of Medicine, 126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, South Korea

S- Editor Wang JL L- Editor O’Neill M E- Editor Ma WH

References
1.  Ferguson LR, Huebner C, Petermann I, Gearry RB, Barclay ML, Demmers P, McCulloch A, Han DY. Single nucleotide polymorphism in the tumor necrosis factor-alpha gene affects inflammatory bowel diseases risk. World J Gastroenterol. 2008;14:4652-4661.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Glas J, Stallhofer J, Ripke S, Wetzke M, Pfennig S, Klein W, Epplen JT, Griga T, Schiemann U, Lacher M. Novel genetic risk markers for ulcerative colitis in the IL2/IL21 region are in epistasis with IL23R and suggest a common genetic background for ulcerative colitis and celiac disease. Am J Gastroenterol. 2009;104:1737-1744.  [PubMed]  [DOI]  [Cited in This Article: ]
3.  Weersma RK, Stokkers PC, van Bodegraven AA, van Hogezand RA, Verspaget HW, de Jong DJ, van der Woude CJ, Oldenburg B, Linskens RK, Festen EA. Molecular prediction of disease risk and severity in a large Dutch Crohn's disease cohort. Gut. 2009;58:388-395.  [PubMed]  [DOI]  [Cited in This Article: ]
4.  Pierik M, Rutgeerts P, Vlietinck R, Vermeire S. Pharmacogenetics in inflammatory bowel disease. World J Gastroenterol. 2006;12:3657-3667.  [PubMed]  [DOI]  [Cited in This Article: ]
5.  Faubion WA Jr, Loftus EV Jr, Harmsen WS, Zinsmeister AR, Sandborn WJ. The natural history of corticosteroid therapy for inflammatory bowel disease: a population-based study. Gastroenterology. 2001;121:255-260.  [PubMed]  [DOI]  [Cited in This Article: ]
6.  Munkholm P, Langholz E, Davidsen M, Binder V. Frequency of glucocorticoid resistance and dependency in Crohn›s disease. Gut. 1994;35:360-362.  [PubMed]  [DOI]  [Cited in This Article: ]
7.  Farrell RJ, Kelleher D. Glucocorticoid resistance in inflammatory bowel disease. J Endocrinol. 2003;178:339-346.  [PubMed]  [DOI]  [Cited in This Article: ]
8.  Russcher H, Smit P, van den Akker EL, van Rossum EF, Brinkmann AO, de Jong FH, Lamberts SW, Koper JW. Two polymorphisms in the glucocorticoid receptor gene directly affect glucocorticoid-regulated gene expression. J Clin Endocrinol Metab. 2005;90:5804-5810.  [PubMed]  [DOI]  [Cited in This Article: ]
9.  van Winsen LL, Hooper-van Veen T, van Rossum EF, Polman CH, van den Berg TK, Koper JW, Uitdehaag BM. The impact of glucocorticoid receptor gene polymorphisms on glucocorticoid sensitivity is outweighted in patients with multiple sclerosis. J Neuroimmunol. 2005;167:150-156.  [PubMed]  [DOI]  [Cited in This Article: ]
10.  van Rossum EF, Koper JW, Huizenga NA, Uitterlinden AG, Janssen JA, Brinkmann AO, Grobbee DE, de Jong FH, van Duyn CM, Pols HA. 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.  [PubMed]  [DOI]  [Cited in This Article: ]
11.  Pittet V, Juillerat P, Mottet C, Felley C, Ballabeni P, Burnand B, Michetti P, Vader JP. Cohort profile: the Swiss Inflammatory Bowel Disease Cohort Study (SIBDCS). Int J Epidemiol. 2009;38:922-931.  [PubMed]  [DOI]  [Cited in This Article: ]
12.  Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet. 2001;68:978-989.  [PubMed]  [DOI]  [Cited in This Article: ]
13.  Stephens M, Donnelly P. A comparison of bayesian methods for haplotype reconstruction from population genotype data. Am J Hum Genet. 2003;73:1162-1169.  [PubMed]  [DOI]  [Cited in This Article: ]
14.  Giguère V, Hollenberg SM, Rosenfeld MG, Evans RM. Functional domains of the human glucocorticoid receptor. Cell. 1986;46:645-652.  [PubMed]  [DOI]  [Cited in This Article: ]
15.  van Winsen LM, Hooper-van Veen T, van Rossum EF, Koper JW, Barkhof F, Polman CH, Uitdehaag BM. Glucocorticoid receptor gene polymorphisms associated with more aggressive disease phenotype in MS. J Neuroimmunol. 2007;186:150-155.  [PubMed]  [DOI]  [Cited in This Article: ]
16.  Koper JW, Stolk RP, de Lange P, Huizenga NA, Molijn GJ, Pols HA, Grobbee DE, Karl M, de Jong FH, Brinkmann AO. Lack of association between five polymorphisms in the human glucocorticoid receptor gene and glucocorticoid resistance. Hum Genet. 1997;99:663-668.  [PubMed]  [DOI]  [Cited in This Article: ]
17.  Russcher H, Smit P, van Rossum EF, van den Akker EL, Brinkmann AO, de Heide LJ, de Jong FH, Koper JW, Lamberts SW. Strategies for the characterization of disorders in cortisol sensitivity. J Clin Endocrinol Metab. 2006;91:694-701.  [PubMed]  [DOI]  [Cited in This Article: ]
18.  Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfeld MG, Evans RM. Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature. 1985;318:635-641.  [PubMed]  [DOI]  [Cited in This Article: ]
19.  Bamberger CM, Bamberger AM, de Castro M, Chrousos GP. Glucocorticoid receptor beta, a potential endogenous inhibitor of glucocorticoid action in humans. J Clin Invest. 1995;95:2435-2441.  [PubMed]  [DOI]  [Cited in This Article: ]
20.  Dekker MJ, van den Akker EL, Koper JW, Manenschijn L, Geleijns K, Ruts L, van Rijs W, Tio-Gillen AP, van Doorn PA, Lamberts SW. Effect of glucocorticoid receptor gene polymorphisms in Guillain-Barré syndrome. J Peripher Nerv Syst. 2009;14:75-83.  [PubMed]  [DOI]  [Cited in This Article: ]
21.  van Winsen LM, Manenschijn L, van Rossum EF, Crusius JB, Koper JW, Polman CH, Uitdehaag BM. A glucocorticoid receptor gene haplotype (TthIII1/ER22/23EK/9beta) is associated with a more aggressive disease course in multiple sclerosis. J Clin Endocrinol Metab. 2009;94:2110-2114.  [PubMed]  [DOI]  [Cited in This Article: ]
22.  van Rossum EF, Koper JW, van den Beld AW, Uitterlinden AG, Arp P, Ester W, Janssen JA, Brinkmann AO, de Jong FH, Grobbee DE. Identification of the BclI polymorphism in the glucocorticoid receptor gene: association with sensitivity to glucocorticoids in vivo and body mass index. Clin Endocrinol (Oxf). 2003;59:585-592.  [PubMed]  [DOI]  [Cited in This Article: ]
23.  Jung D, Fantin AC, Scheurer U, Fried M, Kullak-Ublick GA. Human ileal bile acid transporter gene ASBT (SLC10A2) is transactivated by the glucocorticoid receptor. Gut. 2004;53:78-84.  [PubMed]  [DOI]  [Cited in This Article: ]
24.  Eloranta JJ, Jung D, Kullak-Ublick GA. The human Na + -taurocholate cotransporting polypeptide gene is activated by glucocorticoid receptor and peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and suppressed by bile acids via a small heterodimer partner-dependent mechanism. Mol Endocrinol. 2006;20:65-79.  [PubMed]  [DOI]  [Cited in This Article: ]
25.  Wojtal KA, Eloranta JJ, Hruz P, Gutmann H, Drewe J, Staumann A, Beglinger C, Fried M, Kullak-Ublick GA, Vavricka SR. Changes in mRNA expression levels of solute carrier transporters in inflammatory bowel disease patients. Drug Metab Dispos. 2009;37:1871-1877.  [PubMed]  [DOI]  [Cited in This Article: ]
26.  Hausmann M, Herfarth H, Schölmerich J, Rogler G. Glucocorticoid receptor isoform expression does not predict steroid treatment response in IBD. Gut. 2007;56:1328-1329.  [PubMed]  [DOI]  [Cited in This Article: ]
27.  Sanchez R, Levy E, Costea F, Sinnett D. IL-10 and TNF-alpha promoter haplotypes are associated with childhood Crohn’s disease location. World J Gastroenterol. 2009;15:3776-3782.  [PubMed]  [DOI]  [Cited in This Article: ]
28.  van Rossum EF, Russcher H, Lamberts SW. Genetic polymorphisms and multifactorial diseases: facts and fallacies revealed by the glucocorticoid receptor gene. Trends Endocrinol Metab. 2005;16:445-450.  [PubMed]  [DOI]  [Cited in This Article: ]