DNA Sequence, June 2004 Vol. 15 (3), pp. 167–173
Full Length Research Paper
Identification of Polymorphisms in the Human Glucocorticoid Receptor Gene (NR3C1) in a Multi-racial Asthma Case and Control Screening Panel GREGORY A. HAWKINSa,*, PAMELA J. AMELUNGb, RICHARD S. SMITHa, HAJO JONGEPIERc, TIMOTHY D. HOWARDa, GERARD H. KOPPELMANc, DEBORAH A. MEYERSa, EUGENE R. BLEECKERa and DIRKJE S. POSTMAc a Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA; bUniversity of Maryland School of Medicine, Baltimore, MD, USA; cBeatrixoord Hospital, University of Groningen, Groningen, The Netherlands
(Received 11 November 2003)
The glucocorticoid receptor (GR) gene (NR3C1) maps to 5q31, a region genetically linked to asthma. In this study, NR3C1 exons 1A, 1B, and exons 1C to 9 (a and b) were sequenced in a screening panel of asthmatics and unaffected controls from US Caucasian, African American, US Hispanic, and Dutch Caucasian populations to identify polymorphisms for genetic association studies. Eight polymorphisms were identified in exon 1A, but none were located in putative transcription regulatory sites. Thirty-four polymorphisms were identified in exons 1B to 9 (a and b), 17 of which were novel. Eight coding polymorphisms were identified (4 non-synonymous). One novel mutation (Ala229Thr) was identified in a Hispanic individual. Linkage disequilibrium (LD) was strongest between polymorphisms spanning intron 2 to exon 9b. This data shows the variability of NR3C1 polymorphism frequencies between racial groups and confirms that NR3C1 nonsynonymous coding polymorphisms are generally rare in mild/moderate asthmatics and unaffected controls. Keywords: Glucocorticoid receptor; Glucocorticoid; Asthma; Polymorphism; Inflammation; DNA sequencing
INTRODUCTION Glucocorticoids are the most powerful anti-inflammatory agents known and are routinely used for the treatment of asthma. Glucocorticoids exert their anti-inflammatory effects by binding glucocorticoid receptor (GR) a and suppressing transcription of genes encoding pro-inflammatory mediators (Kellendonk et al., 1999). The GR was the first transcription factor isolated and studied in man
(Muller and Renkawitz, 1991). The gene for GR (NR3C1) maps to chromosome 5q31 (Francke and Foellmer, 1989; Gehring, 1993) and consists of 12 exons (Fig. 1) (Leung and Bloom, 2003) that are differentially spliced to produce three 80 kd isoforms: GRa, GRb, and GRg (Encio and DeteraWadleigh, 1991; Rivers et al., 1999). The GR a and b are produced by alternative splicing between exons 9a and 9b. The GR g is created by addition of a single amino acid as a result of variable splicing of exon 3 and 4 (Rivers et al., 1999). GR is active as a dimer, however, only GRa is capable of forming the active dimer complex. The exact function of GRb is still unclear, but it has been proposed that GRb acts as a dominant negative inhibitor of GRa transactivation (Bamberger et al., 1995; Oakley et al., 1996). A recent report (Hauk et al., 2002) has shown that induction of GRb expression in mouse hybridoma cells converts these cells to a corticosteroid-insensitive phenotype. In addition, it was shown that GRa and GRb are physically associated in these corticosteroid-insensitive cells. It was proposed that GRa and GRb heterodimers may interfere with GR a activity, thus contributing to corticosteroid resistance. In addition to exon 9 splice variants, there are three forms of exon 1: 1A, 1B, and 1C (Breslin et al., 2001; Nunez and Vedeckis, 2002). NR3C1 also contains at least three promoters adjacent to each form of exon 1 that control expression of up to five different mRNA splice variants, each with different 50 untranslated regions (Breslin et al., 2001). Exons 1B and 1C are
*Corresponding author. Tel.: þ 1-336-713-7500. Fax: þ1-336-713-7566. E-mail:
[email protected] ISSN 1042-5179 print/ISSN 1029-2365 online q 2004 Taylor & Francis Ltd DOI: 10.1080/10425170410001704517
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FIGURE 1 Map of glucocorticoid receptor gene (NR3C1) on chromsome 5q31 and polymorphisms.
separated by approximately 1 kb and lie in a very GC rich region of NR3C1 that is highly similar to the mouse sequence. Based on in vivo expression in cancer cell lines, exons 1B and 1C appear to be responsible for ubiquitous and tissue specific expression of NR3C1 (Nunez and Vedeckis, 2002). Exon 1A is the most distal form of exon 1 and lies approximately 31 kb 50 of exon 1B (Breslin et al., 2001; Nunez and Vedeckis, 2002; Neeck et al., 2002). Three mRNA splice variants of exon 1A (1A1, 1A2, and 1A3) have been identified, with exon 1A3 splice variant containing the complete transcript of exon 1A (2,056 bases). A putative interferon regulatory binding site (IRF-1) and glucocorticoid response element (GRE) were identified in the transcribed portion of exon 1A by DNA fingerprinting. The putative GRE was shown to bind dexamethasone and increase expression of exon 1A3 mRNA splice variant. This GRE was also shown to bind GRb, providing a potential negative feedback mechanism for controlling gene expression. From these expression studies, it appears that exon 1A may be primarily responsible for regulating NR3C1 expression in response to glucocorticoids, however, the presence of three forms of exon 1 indicates a very complex expression profile exists for NR3C1. Glucocorticoids and GR have been implicated as mediators in numerous immunologically related diseases including rheumatoid arthritis (Neeck et al., 2002), autoimmune encephalomyelitis (Marchetti et al., 2002), lupus nephritis (Jiang et al., 2001), and asthma (Leung et al., 1998). Multiple studies have linked the 5q31 region to asthma phenotypes
(Postma et al., 1995; Cookson, 2002), indicating that NR3C1 could be a potential candidate gene affecting these asthma phenotypes. In our lab, we are interested in determining if polymorphisms in NR3C1 are associated with severe asthma or asthma characterized by glucocorticoid resistance. Numerous NR3C1 mutations affecting receptor binding, GR structure, and transcriptional activity have been described (Malchoff et al., 1993; Strasser-Wozak et al., 1995; Huizenga et al., 1998; Rivers et al., 1999; Vottero et al., 2002; Bray and Cotton, 2003), especially in patients with rare familial glucocorticosteroid resistance. However, these mutations have not been useful in completely defining glucocorticoid resistance, and specifically not in the case of severe asthma. In order to lay the ground work for studying phenotypes affected by variations in GR response, we performed an extensive DNA sequence analysis on NR3C1 in subsets of four different populations, US Caucasian, African American, US Hispanic, and Dutch Caucasian (with each population consisting of subsets of asthma cases (mild/moderate) and non-asthmatic controls), to assess the frequency of and linkage disequilibrium (LD) between GR polymorphisms. MATERIALS AND METHODS Study Populations Our screening panel consisted of DNA samples collected in Dutch (Postma et al., 1995) and CSGA
GLUCOCORTICOID RECEPTOR GENETIC POLYMORPHISMS
(Collaborative Study on the Genetics of Asthma) (Xu et al., 2001) asthma studies. The DNA panel was composed of 96 individuals, including 24 Caucasians, 24 African Americans, 24 Hispanics, and 24 Dutch Caucasians. Each population subset was divided into 16 individuals with asthma and 8 unaffected controls. Dutch controls were classified as not having bronchial hyperresponsiveness to histamine challenge, atopy, or airway obstruction (Panhuysen et al., 1998). CSGA controls were classified as not having a history of asthma symptoms or any first degree relatives with asthma. Polymerase Chain Reaction Our general strategy for polymorphism verification and discovery was to generate 400– 600 bp overlapping PCR products covering the promoter regions, each of the exons and the 30 -UTRs. Each 30 ml PCR contained 30 ng of genomic DNA, 1 £ PCR buffer (Life Technologies, Gaithersburg, MD), 1.5 mM MgCl2, 200 mM dNTPs, 15 pmoles of each forward and reverse primer, and 0.5 U of Taq polymerase (Life Technologies). Depending on prior reaction optimization, general cycling conditions were: 948C 4 min, followed by 25 –30 cycles at 948C for 1 min, Tanneal for 1 min, and at 728C for 1 min; and finishing with a single extension cycle at 728C for 5 min. PCR products were purified using the Quickstep 96 well PCR purification kit (Edge Biosystems, Gaithersburg, MD) and stored in water at 2 208C. Each exon was amplified separately, with the larger exons (exons 1A, 1C, 2, 9a and 9b) were broken down into smaller overlapping fragments. Initial difficulty amplifying several regions of exon 1B and exon 1C were encountered due to high GC content. These segments were divided into smaller fragments and amplified using the MasterAmp PCR or FailSafe PCR kits (Epicentre Technologies, Madison, WI). PCR amplification was carried out in 10 ml volumes using 30 ng DNA and 5 ml of 2 £ Master Amp mix. Cycling conditions for these segments consisted of an initial denaturation at 958C for 3 min followed by 35 cycles at 958C for 30 s, 558C at 30 s, and 728C for 1 min, and a final extension at 728C for 7 min. DNA Sequencing DNA sequencing was performed using the ABI BigDye Terminator sequencing kit (Applied Biosystems, Inc., Foster City, CA). Each 10 ml sequencing reaction contained 10– 50 ng of purified PCR product, 1.5 pmoles of sequencing primer, 1 ml of BigDye Terminator mix, 1.5 ml of 5 £ sequencing dilution buffer (400 mM Tris pH 9.0, 10 mM MgCl2) and water to volume. Cycling conditions were 948C for 1 min; 25 cycles at 948C for 30 s, 508C for 30 s, and 608C for 4 min; and finishing with a single 728C extension step
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for 5 min. Sequencing products were ethanol precipitated, air-dried, resuspended in 10 – 25 ml ddH20, and analyzed on an ABI 3700 DNA Analyzer. DNA sequencing data was aligned and polymorphisms identified using Sequencher DNA analysis software (Gene Codes Corporation, Ann Arbor, MI). Statistical Methods LD tests were based on an exact test assuming multinomial probability of the multi-locus genotype, conditional on the single-locus genotype (Weir, 1996). A Monte Carlo simulation was used to assess significance by permuting the single-locus genotypes among individuals in the sample to simulate the null distribution. The empirical p-values of LD tests were based on 10,000 replicate samples.
RESULTS We re-sequenced the known published sequence (GenBank accession numbers: exon 1A, AF395116; mRNA, M10901 (alpha), M11050 (beta); genomic, AC091925, AC012634) for NR3C1 exons 1A, 1B, 1C, and 2–9 (a and b), 30 UTR regions, selected portions of putative promoter sequences, and exon flanking regions containing putative exon splice donor sites. Thirty-four polymorphisms were detected within and flanking exon 1B and exons 1C to 9 (a and b) (Fig. 1, Table I), 10 of which have not been previously reported. Many of these new polymorphisms were observed in a single individual and were re-sequenced in a separate PCR fragment to eliminate the possibility that the polymorphisms were introduced by PCR. Eight polymorphisms were located in coding regions, four of which coded for nonsynonymous changes. One new non-synonymous coding polymorphism (Ala229Thr) was identified. This coding polymorphism, found in a single Hispanic asthmatic, is located in the N-terminal of the GR and does not affect DNA binding domains. Eight polymorphisms were identified in exon 1A (Table II), seven of which have not been previously reported. No polymorphisms were located in the putative GRE or IRF-1 regulatory sites of exon 1A. Five exon 1A polymorphisms were found in only one population [2996 (Dutch Caucasian), 2645 (African American), 2226 (African American), þ747 (US Caucasian), IVS1A þ70 (African American)], while only one exon 1A polymorphism (þ554) was found in all four populations. Two polymorphisms (IVS3 246 and IVS4 216) were located near exon flanking regions that contain splice donor sites. However, based on consensus sequence comparisons, neither IVS3 246 nor IVS4 216 lie within putative splice donor sites. A test for LD was performed on nine common polymorphisms spanning from exon 1A to
rs6198 rs10482716
rs6193 rs6191
rs258751 rs258750 rs6196
rs6188
rs10482667
rs10482623
rs10482622 rs6195
rs10482620 rs6189 rs6190 rs6192
23725 23723 23556‡ 23341‡ 23117 22683‡ 22611‡ 22609‡ 22474‡ 2128‡ Glu22Glu Arg23Lys (36) Phe65Val Ala229Thr Lys293Lys Asn363Ser (21,36) IVS2 þ 147 IVS2 þ 221 IVS2 þ 646 (35) IVS2 þ 699 IVS2 þ 704 IVS3 þ 108 IVS3 246 (36) IVS4 216 (36) IVS7 232 Asp678Asp IVS8 2285 Asn766Asn (36) þ409 9a 30 UTR IVS9a 2290 þ773 9b 30 UTR{ þ1235 9b 30 UTR{ þ1308 9b 30 UTR{ þ1352 9b 30 UTR{
Position T/C T/G C In/Del C/T T/C T/G G In/Del C/T A In/Del C/T GAG . GAA AGG . AAG TTT . TGT GCG . ACG AAG . AAA AAT . AGT A/G A/G C/G G/T A/C A/G G/C G/T Complex In/Del GAC . GAT T/C AAT . AAC C In/Del A/G G/T A In/Del A/G C/T
Minor change C G Del T C G Del T Del T A A G A A G G G G T C G C T N/D T C C Del G T Del G T
Rare allele
Controls
8 2(14) 8 2(14) 0 2(14) 0 1 2(28) 3 1 0 0 0 0 0 0 0 0(24) 0(14) 0(22) 0(14) 0(22) 0(14) 0(22) 0(14) 0 0(12) 0 0(12) 0 1 0(30) 0(14) 3(30) 1(14) 13(30) 5(12) 0(30) 0(12) 0(30) 0(12) 1 0(14) 0 0 11(28) 5(12) All polymorphic 0 0 13 6 8 3 0 0 0 0(14) 10(22) 9 0 0(12) 4 3(12) 1 0(12)
Cases*
†
Controls
1 3(14) 1 3(14) 2(30) 1(14) 0(30) 0(14) 3 1(14) 0(30) 0(14) 4(30) 2(14) 4(30) 2(14) 1(22) 1(10) 3(30) 0(10) 0(30) 0(10) 0(30) 0(10) 1(30) 0(10) 0(30) 0(14) 3(30) 2(14) 0(26) 0(14) 0(30) 0 0(30) 0 2(30) 3(14) 0(30) 0(14) 0(30) 0(14) 0 0 0(26) 0(14) 7 5(14) All polymorphic 2(30) 3(14) 7 5(14) 1 4(14) 2 0(14) 1(30) 2(10) 11(24) 7(12) 1 0(14) 2(30) 1(14) 0(30) 0(14)
Cases*
African American †
Controls
6(30) 1(14) 6(30) 1(14) 4 1 0 0 1 2 0 0 0 0 0 0 0 0 0(30) 0 0(28) 0 0(28) 0 0(28) 0 0 1(14) 0 0(14) 0 0 1 0 0 1 17 2 1 0 1 0 0 0 0(30) 0(14) 7(30) 4 All polymorphic 0 0(14) 9(30) 3(14) 7 1 0(30) 1 0(30) 0(14) 15(28) 5 0 0(14) 2 2(14) 0 1(14)
Cases*
US Hispanic †
Controls† 6 2 6 2 1 1 0 0 6 4 0 0 0 0 0 0 0 0 0(28) 0 1(30) 0 1(30) 0 0(30) 0 0 0(14) 0 0(14) 1(30) 1 0(30) 0(12) 1(30) 0(12) 13(30) 3(12) 0(30) 0(12) 0(30) 0(12) 1 0 0 2(14) 12(26) 4(14) All polymorphic 0 0 16 6 7 2 0(30) 0(12) 0(32) 0(14) 22(30) 6(12) 0 0 9 4 0 0(14)
Cases*
Dutch Caucasian
* Out of 32 chromosomes unless indicated in ( ). † Out of 16 chromosomes unless indicated in ( ). ‡ Position relative to the ATG start codon in exon 2 based on the reference sequences Genbank accession AC091925 and AC012634. { Position relative to stop codon in reference sequences Genbank accession M10901, M11050, AC091925, and AC012634. ND, not determined.
UTR UTR UTR UTR
UTR
rs3806855 rs3806854
50 exon 1B 50 exon 1B Exon 1B Intron 1B Intron 1B Exon 1C Exon 1C Exon 1C Exon 1C Intron 1C Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Intron 2 Intron 2 Intron 2 Intron 2 Intron 2 Intron 3 Intron 3 Intron 4 Intron 7 Exon 8 Intron 8 Exon 9 a Exon 9 a 30 Intron 9 a Exon 9 b 30 Exon 9 b 30 Exon 9 b 30 Exon 9 b 30
rs10482605 rs10482609 rs10482610 rs10482611
dbSNP accession
Location
US Caucasian
Frequency of minor allele
TABLE I NR3C1 polymorphism locations and frequencies in exon 1B to 9 (a and b)
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rs6868190
* Position relative to the start of transcription in exon 1A based on reference sequence Genbank Accession AF395116. † Out of 32 chromosomes unless indicated in ( ). ‡ Out of 16 chromosomes unless indicated in ( ). N/D, not determined.
0 1(14) 1 1(14) 0 0 0 0 All polymorphic 0(28) 0(14) 0(28) 0(14) 0(28) 0(14) 0 0(12) 5 3(12) 0(30) 0(14) 0(30) 0(14) All polymorphic 0(30) 0(14) 1(30) 0(14) 0(30) 0(14) 0(26) 0(14) 0(26) 0(14) 4(30) 2(14) 1(20) 4(14) All polymorphic 0 0(14) 3 1(14) 2 3(14) 0 0(14) 4 1(14) 0(26) 0 0(26) 0(14) All polymorphic 1(30) 0 0(30) 0 0(30) 0 Del A Del T N/D C A C A In/Del A/G A In/Del C/T A In/Del C/G A/G C/T 2996 2921 2645 2226 þ 554 þ 747 IVS1A þ35 IVS1A þ70 Promoter 1A Promoter 1A Promoter 1A Promoter 1A Exon 1A Exon 1A Intron 1A Intron 1A
Controls‡ Cases† Controls‡ Cases† Controls‡ Cases† Controls‡ Cases† Rare allele Minor change Position dbSNP accession Location*
US Hispanic African American US Caucasian
Frequency (minor allele)
TABLE II NR3C1 exon 1A polymorphism locations and frequencies
Dutch Caucasian
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exon 9b 30 ;UTR (exon 1A 2 847, 2 3556, 2 2611, IVS2 þ 646, IVS4 2 16, IVS8 2 285, Asn766Asn, þ 773 9b, and þ 1308 9b) within each population. Table III shows the strongest LD between select pairs of the nine polymorphisms. Strongest LD was measured between polymorphisms IVS4 2 16 and IVS8 2 285 with p , 1026 in all four populations. Interestingly, although polymorphisms þ 773 9b and þ 1308 9b are separated by only 535 bases and allele frequencies of each polymorphisms were not low, LD between these two polymorphisms was low (p . 0:2 for all populations). No significant LD was measured between polymorphisms in exon 1A (2 847, 2 3556, and 2 2611) and remaining polymorphisms.
DISCUSSION The use of anti-inflammatory inhaled glucocorticoids is a mainstay in treating asthma, and these drugs have made a tremendous impact on increasing the quality of life of asthmatics by providing better symptom control and reducing hospitalizations. Despite positive effects of glucocorticoids in treating asthma, there remains considerable variability in steroid responsiveness (Kerstjens et al., 1995), which may be wholly or partially caused by genetic variations in genes that code for components of the GR complex, and specifically GR. This study is the first to detail and compare polymorphisms in NR3C1 in a screening panel of asthmatic cases and unaffected controls. While no definitive comparisons can be made between cases and controls due to small sample sizes, our results show that NR3C1 contains much genetic variation, particularly in the African American population. Many genetic variants were found at low frequencies, usually in a single individual, and are thus not useful for genetic association studies. However, these rare alleles could be informative in familial analysis of inherited diseases associated with NR3C1. Eight coding polymorphisms were identified, including four non-synonymous coding polymorphisms, one of which is novel. All non-synonymous coding polymorphisms occurred at low frequencies, thus if GR function is significantly affected by common genetic variants, mechanisms other than amino acid changes must be involved in regulating GR function. One possibility is that polymorphisms in NR3C1 could be associated with changes in transcriptional or translational activity. As an example, during a case/control study of Japanese diabetics, Ikeda and co-workers (Ikeda et al., 2001) identified an A . C polymorphism -22 bases of exon 1C that significantly affected in vitro transcriptional activity of NR3C1 and was proposed to have effects on glucocorticoid sensitivity. We did not find this polymorphism in our
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TABLE III LD between pairs of select NR3C1 polymorphisms +773 9b 30 UTR
+1308 9b 30 UTR
IVS8 2285
Asn766Asn
IVS2 þ646
– – – p ¼ 0:0014 DCC
p ¼ 0:0001 CC p ¼ 0:004 AA p ¼ 0:007 HIS p , 1026 DCC
p ¼ 0:01 CC – p ¼ 0:007 HIS
– – –
IVS4 216
p , 1026 p , 1026 p , 1026 p , 1026
– – p ¼ 0:0009 HIS –
– – – p ¼ 0:01 DCC
– – p ¼ 0:008 HIS –
CC AA HIS DCC
IVS8 2285
– – – –
– p ¼ 0:014 AA p ¼ 0:0002 HIS –
– – p ¼ 0:005 DCC –
– – – –
Asn766Asn
– – – –
– – – –
– – – p ¼ 0:004 DCC
– – – –
CC, Caucasian; AA, African American; HIS, US Hispanic; DCC, Dutch Caucasian.
screening panel, suggesting that this variant is unique to the Japanese population. In a previous small case/control study (30 cases and 24 controls), Derijk and co-workers (DeRijk et al., 2001) were able to determine that polymorphism þ1308 9b 30 UTR was associated with rheumatoid arthritis, a disease characterized by inflammation. Most interesting, this study was able to show that the rare G allele increases the stability of the GRb mRNA. This A . G transition is located within an ATTTA (ATTTA . GTTTA) sequence motif known to affect GRb mRNA stability (Chen and Shyu, 1994; Chen et al., 2002). More recently, Schaaf and Cidlowski (2002) were able to show that COS-1 cells transfected with a NR3C1 clone containing the GTTTA variant of the GRb cDNA produced more stable GRb mRNA in increased production of GRb protein. They postulated that stabilization of GRb mRNA and resulting increased production of the GRB could result in increased glucocorticoid resistance. If this model is true, asthmatics that inherit the G allele of þ1308 9b 30 UTR may be predisposed to increased inflammation resulting from corticosteroid resistance. Thus þ1308 9b 30 UTR could be a key component in modulating GRb mRNA stability and GRb production in individuals prone to corticosteroid insensitivity (Schaaf and Cidlowski, 2002). LD was measured between nine frequent NR3C1 polymorphisms and found to be high between polymorphisms spanning intron 2 (IVS2 þ646) to exon 9b (þ1308 9b). Polymorphisms in and near exon 1A, 1B, and 1C were not in LD with this cluster of polymorphisms. This observation was not unexpected considering the size of NR3C1 (.150 kb) and the large distance between polymorphisms. Four exon 1A polymorphisms were identified in the putative promoter region and two polymorphisms were identified in the 50 UTR of exon 1A. None of the polymorphisms were located in or near putative
regulatory binding domains, and the low frequency and the population specific nature of these exon 1A polymorphisms does not support a major role of theses polymorphisms in transcriptional regulation. In summary, we have sequenced and identified forty-two polymorphisms within and flanking exons 1A, 1B, and exons 1C to exon 9b of NR3C1. In general, most of these polymorphisms are rare and will probably have a minimal role in any functional association of GR to asthma susceptibility. However, these rare polymorphisms may be important in small subgroups of glucocorticoid resistant asthmatics. Therefore large numbers of asthmatics cases and unaffected controls will be required to assess the relevance of these rare polymorphisms. With these future goals in mind, this study has been important in providing a base-line of polymorphisms in non-severe asthma populations to compare to polymorphisms that may be unique to severe asthma subgroups. Most importantly, we have determined that non-synonymous coding polymorphisms are rare in mild/moderate asthmatics and unaffected controls. Acknowledgements We would like to thank all participants of the Dutch and CSGA studies. We are thankful to C.I.M. Panhuysen, B. Meijer, and G.G. Meijer, for their work in patient recruitment for the Dutch study. This work was supported by the Netherlands Asthma Foundation grant AF 95.09 and National Institutes of Health (NIH) grants R01HL/48341, U01 HL/49602, HL01-012, R01HL/66393 and the NIA Claude D. Pepper Older Americans Independence Center 1P30 AG21332-02. References Bamberger, C.M., Bamberger, A.M., de Castro, M. and Chrousos, G.P. (1995) “Glucocorticoid receptor beta, a potential
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